Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations
2020
An eye on the dog as a translational model for ocular pharmacology
Lionel Sebbag Iowa State University
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Recommended Citation Sebbag, Lionel, "An eye on the dog as a translational model for ocular pharmacology" (2020). Graduate Theses and Dissertations. 18223. https://lib.dr.iastate.edu/etd/18223
This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. An eye on the dog as a translational model for ocular pharmacology
by
Lionel Sebbag
A dissertation submitted to the graduate faculty
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
Major: Biomedical Sciences (Pharmacology)
Program of Study Committee: Jonathan P. Mochel, Major Professor Albert E. Jergens M. Heather West Greenlee Joshua R. Beck Ann M. Perera
The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this dissertation. The Graduate College will ensure this dissertation is globally accessible and will not permit alterations after a degree is conferred.
Iowa State University
Ames, Iowa
2020
Copyright © Lionel Sebbag, 2020. All rights reserved.
ii
DEDICATION
This dissertation is lovingly dedicated to my parents, Monique and Michel
For their endless love, support, and encouragement
Je vous aime très fort, Terbah!!
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TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ...... vii
ABSTRACT ...... ix
CHAPTER 1. GENERAL INTRODUCTION ...... 1 References ...... 2
CHAPTER 2. FLUOROPHOTOMETRIC ASSESSMENT OF TEAR VOLUME AND TURNOVER RATE IN HEALTHY DOGS AND CATS ...... 4 Abstract ...... 4 Introduction ...... 5 Materials and Methods ...... 6 Animals ...... 6 Fluorophotometry ...... 7 Data analysis...... 8 Results ...... 9 Discussion ...... 10 References ...... 14 Tables and Figures ...... 18
CHAPTER 3. KINETICS OF FLUORESCEIN IN TEAR FILM AFTER EYE DROP INSTILLATION IN BEAGLE DOGS: DOES SIZE REALLY MATTER? ...... 22 Abstract ...... 22 Introduction ...... 23 Materials and Methods ...... 25 Animals ...... 25 Tear film fluorescein following instillation of 1 vs. 2 drops ...... 25 Volumetric capacity of the palpebral fissure ...... 26 Tear turnover rate at various instilled volumes ...... 27 Data analysis...... 27 Results ...... 29 Volumetric capacity of the canine palpebral fissure ...... 29 Periocular spillage of instilled solution ...... 29 Tear turnover rate ...... 30 Tear film fluorescein concentrations following 1 vs. 2 drops ...... 31 Discussion ...... 31 References ...... 36 Tables and Figures ...... 41
iv
CHAPTER 4. HISTAMINE-INDUCED CONJUNCTIVITIS AND BREAKDOWN OF BLOOD-TEAR BARRIER IN DOGS: A MODEL FOR OCULAR PHARMACOLOGY AND THERAPEUTICS ...... 46 Abstract ...... 46 Introduction ...... 47 Materials and Methods ...... 49 Experimentally Induced Conjunctivitis in Dogs ...... 49 Naturally Acquired Conjunctivitis in Dogs ...... 52 Data Analysis ...... 53 Results ...... 54 Experimentally Induced Conjunctivitis in Dogs ...... 54 Naturally Acquired Conjunctivitis in Dogs ...... 57 Discussion ...... 57 References ...... 61 Tables and Figures ...... 65
CHAPTER 5. IMPACT OF ACUTE CONJUNCTIVITIS ON OCULAR SURFACE HOMEOSTASIS IN DOGS ...... 74 Abstract ...... 74 Introduction ...... 75 Materials and Methods ...... 76 Animals ...... 76 Procedures ...... 76 Data analysis...... 78 Results ...... 78 Discussion ...... 79 References ...... 83 Tables and Figures ...... 86
CHAPTER 6. TEAR FLUID PHARMACOKINETICS FOLLOWING ORAL PREDNISONE ADMINISTRATION IN DOGS WITH OR WITHOUT CONJUNCTIVITIS ...... 88 Abstract ...... 88 Introduction ...... 89 Materials and Methods ...... 90 Animals ...... 90 Procedures ...... 91 Preparation of tear samples for analysis ...... 92 Liquid chromatography – mass spectrometry ...... 93 Data analysis...... 94 Results ...... 94 Discussion ...... 95 References ...... 99 Tables and Figures ...... 102
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CHAPTER 7. ALBUMIN LEVELS IN TEAR FILM MODULATE THE BIOAVAILABILITY OF MEDICALLY-RELEVANT TOPICAL DRUGS ...... 107 Abstract ...... 107 Introduction ...... 108 Materials and Methods ...... 110 Animals ...... 110 Experiment ...... 111 Data analysis...... 112 Results ...... 113 Pupil dilation from tropicamide ...... 113 Pupil constriction from latanoprost ...... 114 IOP changes from latanoprost ...... 114 Discussion ...... 114 References ...... 121 Tables and Figures ...... 125
CHAPTER 8. GENERAL CONCLUSION - AN EYE ON THE DOG AS THE SCIENTIST’S BEST FRIEND FOR TRANSLATIONAL RESEARCH IN OPHTHALMOLOGY: FOCUS ON THE OCULAR SURFACE ...... 129 Abstract ...... 129 Introduction ...... 130 Comparative anatomy and physiology of the ocular surface ...... 132 Anatomy ...... 132 Tear film dynamics ...... 139 Tear film composition ...... 143 Tear collection for bioanalytical purposes ...... 145 Direct tear sampling ...... 146 Indirect tear sampling ...... 148 Proposed strategy for lachrymal determinations in dogs ...... 150 Spontaneous and experimental models of ocular surface disorders in dogs ...... 154 Spontaneous ocular surface diseases in dogs with translational applications to humans...... 155 Breakdown of the blood-tear barrier in dogs: A model for ocular pharmacology and therapeutics ...... 159 Corneal injury in dogs: in vivo and ex vivo models ...... 163 Conclusions ...... 164 References ...... 166 Tables and Figures ...... 191
APPENDIX A. MATHEMATICAL MODELING USING NONLINEAR MIXED- EFFECTS ...... 203
APPENDIX B. TEAR FILM THICKNESS AND THEORETICAL TEAR VOLUME ...... 206
APPENDIX C. MATHEMATICAL MODELING USING NONLINEAR MIXED-EFFECTS ...... 207 vi
APPENDIX D. HISTAMINE-INDUCED CONJUNCTIVITIS ...... 209
APPENDIX E. PREPARATION OF SCHIRMER STRIPS FOR LC-MS ANALYSIS ...... 214 vii
ACKNOWLEDGMENTS
‘You are not a reasonable man’ said my major professor during my PhD defense. I agree, there is
nothing rational in pursuing a graduate degree while working as an assistant professor and
raising a family. I owe this accomplishment to the overwhelming support I received over the last
4 years, for which I will be forever grateful.
To my beautiful wife, Alison, for your patience in surviving the cold winters of Iowa and letting
me escape to my office all too often to get work done. I love you deeply, you are an incredible
mother to Jacob and Netta and a wonderful person with a kind heart.
To my major professor, Dr. Jonathan Mochel, for pushing me to get out of my comfort zone,
both on a professional and personal level. “If I have seen further it is by standing on the
shoulders of giants.” — Isaac Newton
To other members of my graduate committee: Dr. Al Jergens, Dr. Heather Greenlee, Dr. Josh
Beck and Dr. Ann Perera, I am truly honored to have completed this journey by your side. Thank you for guiding me on the right path.
To my previous mentors and resident-mates at UC Davis, now close friends and colleagues, for
believing in me, and for shaping the clinician-scientist career I am pursuing today with passion and dedication.
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To the Ophthalmology service at Iowa State University, for providing me the flexibility to juggle between the student and the faculty hat, and for the fantastic times working together in such a great environment.
To the other co-workers and friends at Iowa State University, for the fun and memorable experiences we shared.
To my parents Monique and Michel, my brothers Jérôme and David, mamie Jeanette, Hillel, and all my family scattered throughout the world. Family is everything. You are my inspiration and
תצליחו בחיים .my strength in life
To my four-legged companions, Winston, Holly, Morgan and Dexter z”l, for your love that is mostly unconditional (as long as chicken is provided every now and then).
I realize the list of acknowledgements can only capture a small fraction of the people who supported me. I send my gratitude to all. ix
ABSTRACT
Today’s high failure rate in ophthalmic clinical trials can be largely explained by two
major shortcomings: (i) the animals routinely studied (rabbits, mice, rats) are not representative
of the affected population due to apparent anatomical and physiological differences with
humans; and (ii) studies conducted in healthy eyes do not account for physiological disturbances
in ocular homeostasis present in diseased eyes. Unlike traditional laboratory animals, diseases in
dogs better reflect the complex genetic, environmental, and physiological variation present in
humans; however, the translational potential of canine research is currently limited by scarce
information on normative data specific to dogs, and the limited means to mimic ocular disease in
a reliable and non-invasive manner in this species.
The work conducted in the dissertation provides a deeper understanding of the canine
ocular surface in health and disease states, investigating laboratory Beagle dogs and canine
patients of varied breeds and cephalic conformations. Tear fluid was collected from canine eyes
in successive experiments – primarily via Schirmer tear strips but also capillary glass tubes and
absorbent sponges – and subsequent bioanalytical tools included fluorophotometry (tear film
fluorescence), infrared spectroscopy (total protein content), immunoassays (serum albumin,
cytokines, chemokines) and liquid chromatography-mass spectrometry (corticosteroids). Data
analysis combined conventional statistical tests with nonlinear mixed-effects mathematical modeling to improve the robustness of the predictions.
The main research outcomes of the dissertation work are the following: (i) Normative data were established for canine tear film dynamics, including tear volume (65.3 µL), basal tear turnover rate (12.2%/min) and reflex tear turnover rate (50%/min). In both clinical and research settings, successive lacrimal tests should be spaced by ≥ 10 min in dogs to provide sufficient x time for the tear film to replenish. (ii) The volumetric capacity of the canine palpebral fissure was 31.3 µL, approximating the volume of a single eyedrop. Kinetic studies confirmed that a single drop is sufficient for topical administration in dogs, any excess being lost predominantly by blinking and spillage over the periocular skin. (iii) Topical histamine solutions of 1, 10, and
375 mg/mL induced mild, moderate, and severe conjunctivitis in dogs, respectively. The resulting disruption of the blood-tear barrier promoted leakage of plasma compounds (eg., albumin) into the tear film, a finding confirmed in dogs with naturally acquired ocular diseases.
This ‘large animal’ model was robust, non-invasive, and self-resolving, providing a unique opportunity to investigate the ocular surface in health and disease. (iv) Acute conjunctivitis increased tear quantity and decreases tear stability, although ocular surface homeostasis was rapidly restored. (v) Corticosteroid levels in the tear film did not change significantly between healthy vs. diseased eyes following oral prednisone administration, although findings may differ for drugs with other physicochemical properties. (vi) Albumin in tears lowered the ocular bioavailability of topically administered drugs, as shown for tropicamide and (to a lesser extent) latanoprost in dogs.
The thesis concludes with a comprehensive review of key ocular parameters in humans, dogs, and traditional laboratory species (rabbits, mice, rats), detailing species differences in ocular surface anatomy, physiology, tear film dynamics and tear film composition, and highlighting the benefits of integrating dogs into preclinical studies given striking resemblances between the canine and human eyes (One Health approach). 1
CHAPTER 1. GENERAL INTRODUCTION
Companion animals such as the dog constitute an underutilized resource for translational
research in biomedical sciences.1 Unlike traditional laboratory animals (eg., rabbits, mice, rats), naturally-occurring diseases in dogs better reflect the complex genetic, environmental, and physiological background present in humans; therefore, integrating canine subjects into preclinical studies can accelerate and improve the framework in which research is translated to the human clinic, and ultimately generate discoveries that will benefit the health of humans and animals. This ‘One Health’ approach is rapidly growing in several medical fields (eg., oncology, neurology, stem cells),2-4 yet the literature in comparative ophthalmology is very limited to date.
Dogs are routinely examined by veterinarians for the diagnosis and management of
diverse ocular pathologies, exploiting the expertise of general practitioners and veterinary
specialists across the world. Importantly, numerous canine ocular disorders share striking
phenotypical resemblances with their human clinical analogues – as exemplified by
keratoconjunctivitis sicca (‘dry eye’)5 – and could therefore serve as spontaneous animal models
of ocular diseases. To date, the paucity of canine research for translational purposes is generally explained by perceived limitations such as ethical and economic considerations, lack of transgenic dogs, and limited molecular tools compared to laboratory species.1,6,7 In the authors’
opinion, more scientifically relevant hindrances to the use of canines in ophthalmology research
include limited data on the physiology of the ocular surface in dogs, and the lack of non-invasive
experimental model to mimic disease pathophysiology in this species. Unfortunately, direct
extrapolation from other animals is not possible given notable species differences in ocular
anatomy and physiology.8,9 2
The first two chapters of this dissertation describe fundamental work to better understand the physiology of the canine ocular surface, establishing normative data for key parameters such as tear volume, tear turnover rate, volumetric capacity of the palpebral fissure, and tear film kinetics following topical eyedrop administration. The next two chapters describe the development of a robust in vivo model of conjunctivitis in dogs, a translational large animal model that provides a unique opportunity for scientists to investigate the ocular surface in health and disease states. Practical applications of the model are reported in the last two chapters of the thesis, highlighting the clinical significance of blood-tear barrier breakdown on ocular pharmacology and drug bioavailability. Finally, the last chapter of this manuscript provides a comprehensive review of the ocular surface in humans and selected animals, describing major pitfalls that tremendously limit the translational potential of conventional laboratory animals
(rabbits, mice, rats) in ophthalmic research. This chapter further highlights the benefits of leveraging information from canine pharmacokinetic, efficacy and safety preclinical studies given striking resemblances between the canine and human eyes.
References
1. Kol A, Arzi B, Athanasiou KA, et al. Companion animals: Translational scientist's new best friends. Sci Transl Med 2015;7:308ps321.
2. Garden OA, Volk SW, Mason NJ, et al. Companion animals in comparative oncology: One Medicine in action. Vet J 2018;240:6-13.
3. Partridge B, Rossmeisl JH. Companion animal models of neurological disease. J Neurosci Methods 2020;331:108484.
4. Hoffman AM, Dow SW. Concise Review: Stem Cell Trials Using Companion Animal Disease Models. Stem Cells 2016;34:1709-1729.
5. Kaswan RL, Salisbury MA, Ward DA. Spontaneous canine keratoconjunctivitis sicca. A useful model for human keratoconjunctivitis sicca: treatment with cyclosporine eye drops. Arch Ophthalmol 1989;107:1210-1216. 3
6. Barabino S, Dana MR. Animal models of dry eye: a critical assessment of opportunities and limitations. Invest Ophthalmol Vis Sci 2004;45:1641-1646.
7. Pennington MR, Ledbetter EC, Van de Walle GR. New Paradigms for the Study of Ocular Alphaherpesvirus Infections: Insights into the Use of Non-Traditional Host Model Systems. Viruses 2017;9.
8. Vézina M. Comparative Ocular Anatomy in Commonly Used Laboratory Animals In: Weir AB,Collins M, eds. Assessing Ocular Toxicology in Laboratory Animals. Totowa, NJ: Humana Press, 2013;1-21.
9. Gilger BC, Abarca E, Salmon JH. Selection of Appropriate Animal Models in Ocular Research: Ocular Anatomy and Physiology of Common Animal Models In: Gilger BC, ed. Ocular Pharmacology and Toxicology. Totowa, NJ: Humana Press, 2014;7-32.
10. Shah M, Cabrera-Ghayouri S, Christie LA, et al. Translational Preclinical Pharmacologic Disease Models for Ophthalmic Drug Development. Pharm Res 2019;36:58.
11. Shell JW. Pharmacokinetics of topically applied ophthalmic drugs. Surv Ophthalmol 1982;26:207-218.
4
CHAPTER 2. FLUOROPHOTOMETRIC ASSESSMENT OF TEAR VOLUME AND TURNOVER RATE IN HEALTHY DOGS AND CATS
Lionel Sebbag,1,2 Rachel A. Allbaugh,1 Rita F. Wehrman,1 Lisa K. Uhl,1
Gil Ben-Shlomo,1 Thomas Chen,3 Jonathan P. Mochel2
1 Department of Veterinary Clinical Sciences, Iowa State University, College of
Veterinary Medicine, IA, USA; 2 Department of Biomedical Sciences, SMART Pharmacology,
Iowa State University, College of Veterinary Medicine, IA, USA; 3 Department of Small Animal
Clinical Sciences, The University of Tennessee, College of Veterinary Medicine, TN, USA.
Modified from a manuscript published in the Journal of Ocular Pharmacology and
Therapeutics
Abstract
Purpose: The study establishes normative data of tear volume (TV) and tear turnover rate (TTR) in healthy dogs and cats, two species commonly used for translational research in ophthalmology. Methods: Thirty six dogs and 24 cats were enrolled, encompassing a variety of breeds with diverse skull conformations (brachycephalic, mesocephalic, dolichocephalic). Two
µL of 10% fluorescein were instilled onto the upper bulbar conjunctiva of both eyes, followed by tear collection with 2-µL capillary tubes at 0, 2, 4, 6, 10, 15 and 20 minutes. Fluorescein concentrations were measured with a computerized scanning ocular fluorophotometer. The TV and TTR were estimated based upon nonlinear mixed-effects analysis of fluorescein decay curves. Results: In dogs, median (interquartile range) TV, basal TTR and reflex TTR were 65.3
μL (42.3-87.9), 12.2%/min (3.7-22.1) and 50.0%/min (25.9-172.3), respectively. In cats, median
(interquartile range) TV, basal TTR and reflex TTR were 32.1 μL (29.5-39.9), 10.9%/min (3.0-
23.7) and 50.0%/min (28.4-89.4), respectively. Body weight (r=0.44) and age (r=0.30) were 5 positively correlated (P≤0.019) with TV in dogs. Age was negatively correlated (P≤0.018) with
TTR in dogs (r=-0.33) and cats (r=-0.24). However, TV and TTR were not associated with skull conformation in either species. Conclusions: Dogs have greater TV than cats but similar basal and reflex TTR. Tear parameters were impacted by body weight and age, but not skull conformation. In both clinical and research settings, successive lacrimal tests should be spaced by ≥10min to provide sufficient time for the tear film to replenish, as basal TTR is approximately
11-12%/min in both species.
Introduction
Tear fluid dynamics, or the balance between tear secretion, distribution, absorption, evaporation and drainage, are critical for the maintenance of ocular surface health.1 Tear volume
(TV) and tear turnover rate (TTR) are parameters that provide insight into these complex dynamics, and as such are valuable for numerous clinical and research applications: (i) TTR helps differentiate between aqueous-deficient dry eye and evaporative dry eye in human patients;2 (ii) TTR impacts the quantity of various tear components such as electrolytes and proteins;3-5 and (iii) determination of TV in horses highlights a large dilution effect of tear fluid on exogenously applied drugs, whereby over half of the drug concentration is diluted immediately upon topical instillation onto equine eyes.6
Dogs and cats, in addition to being the most common companion animals worldwide, are commonly used as animal models for translational ocular surface research, as exemplified by canine keratoconjunctivitis sicca7 and feline epithelial wound healing.7,8 However, information about tear fluid dynamics is lacking in these species, despite numerous reports in humans1,2,9 and various animals such as rabbits,10 horses6 and cows.11 Evaluation of tear dynamics in dogs and cats is likely confounded by their diversity in facial conformations. Indeed, brachycephaly 6
(foreshortening of the facial skeleton) can impact tear drainage in dogs12 and in cats,13 and the
associated lagophthalmos of some brachycephalic animals can impact tear distribution and
evaporation.
The present study establishes normative data of TV and TTR in dogs and cats using fluorophotometry, a method considered to be the gold standard in assessing tear dynamics.6,9 A
secondary objective is to determine the impact of cephalic conformation and other variables (age,
body weight, Schirmer values) on canine and feline tear dynamics.
Materials and Methods
Animals
Thirty-six dogs (n = 72 eyes) and 24 cats (n = 48 eyes) were enrolled in the study. Prior
to study participation, a consent form was signed by owners and each subject was confirmed to
be ophthalmoscopically healthy by slit-lamp examination, indirect ophthalmoscopy, rebound
tonometry (TonoVet, Icare Finland Oy, Espoo, Finland) and normal Schirmer test values (≥ 15
mm/min in dogs, ≥ 9 mm/min in cats).7,14 A soft measuring tape was used to measure the skull
width (widest interzygomatic distance), skull length (dorsal tip of the nose to occipital
protuberance) and muzzle length (dorsal tip of the nose to the stop). These values were used to
calculate the cephalic index (CI = skull width / skull length) and the craniofacial ratio (CFR =
muzzle length / skull length) in each animal.15-17 The CI was used to characterize canine subjects as brachycephalic (n = 10), mesocephalic (n = 16) or dolichocephalic (n = 10), as described by
Evans and De Lahunta.15 Since similar numerical features are lacking in cats, the CI was still
utilized for data analysis but feline subjects were classified as brachycephalic (n = 9; e.g. Persian,
Himalayan, Exotic Shorthair) or non-brachycephalic (n = 15; e.g. Domestic Shorthair, Bengal)
based on previous studies.13,17 The study was approved by the Institutional Animal Care and Use 7
Committee of Iowa State University, and was conducted in accordance with the Association for
Research in Vision and Ophthalmology statement for the use of animals in ophthalmic and vision
research.
Fluorophotometry
Fluorophotometry was performed as previously described with minor modifications.6
Briefly, 2µL of 10% sodium fluorescein (Akorn Inc., Buffalo Grove, IL) was instilled onto the dorsal bulbar conjunctiva of each eye using a pipette, with care not to touch the ocular surface.
Immediately following 3 manual eyelid blinks (0 min), a 2-µL capillary glass tube (Drummond
Scientific Co., Broomhall, PA) was placed in contact with the inferior tear lake for ≤ 2 seconds to collect a tear sample. Eyes were then allowed to blink naturally, and tear samples were collected in a similar fashion at 2, 4, 6, 10, 15 and 20 minutes. A maximum of 2 seconds was selected for tear collection as a compromise to obtain sufficient tear fluid for analysis while minimizing the risk of inadvertently touching the ocular surface and causing reflex tearing. This duration, however, was often insufficient to completely fill the 2-µL capillary tubes with tears.
Thus, following each collection, the length of fluid contained within the capillary tube was measured to the nearest mm using a ruler, a value extrapolated to the volume of tears collected given that the 2-µL tube is 32 mm in length. The content of each capillary tube was expelled into a 2-mL Eppendorf tube pre-filled with 1mL of phosphate-buffered saline (Gibco® PBS, pH 7.2,
Thermo Fisher Scientific, Rockford, IL). An additional 1mL of PBS was added to each tube,
followed by vortex mixing for 30 seconds and transfer to a glass cuvette. Fluorescein
concentration was measured in each sample with a computerized scanning ocular
fluorophotometer (Fluorotron MasterTM, Coherent Radiation, Palo Alto, CA). All tear sample
collection and experiments were performed in the morning (from 8 am to 12 pm) to reduce the 8
potential impact of circadian rhythm on TTR.18 All subjects were confirmed to be free of corneal
epithelial defects using a cobalt blue light evaluation at completion of the last tear collection. Of
note, both eyes of each animal received the same amount of fluorescein at baseline, except for
six beagle dogs in whom one random eye received 2 µL of 10% fluorescein6 while the other eye received 1 µL of 1% fluorescein19 - an experiment conducted to assess the effect of fluorescein
dosing on tear dynamics.
Data analysis
Since the fluorophotometer output is nonlinear at high fluorescein concentrations, a
calibration curve was established for the Fluorotron MasterTM by analyzing a dilution series of known fluorescein concentrations in triplicate (1 to 10,000 ng/mL).20,21 Fluorescein
concentrations in tear samples were corrected based on the resulting calibration equation (y = -
4E-05x2 + 0.9567x + 20.581).
Fluorescein data of each animal were inputted to Monolix® version 2018R2 (Lixoft,
Orsay, France). Selected data points were censored in Monolix® when a peak of fluorescence
could not be identified on the fluorophotometer reading, or if the tear fluorescein concentration
did not make physiologic sense (e.g. higher fluorescein at 2 min compared to baseline).6 Overall,
<10% (93/944) of all data points were left-censored.
Mathematical models of fluorescein disposition time-course were written as non-linear mixed effects (NLME) models as previously described and detailed in Appendix A.22-25 Data collection
from the left and the right eye practically constitutes a repeat sample from the same individual.
To account for this repeat sampling procedure, biological data collected from the right and left
eye of each study subject were modeled using a within-dog variability term in the statistical model structure. Inclusion of covariate relationships with the model parameters TV and TTR 9
(age, body weight, sex, skull type, cephalic index, craniofacial ratio, STT value, and amount of
fluorescein instilled onto the ocular surface) was assessed for statistical significance using a
Pearson correlation test (for continuous variables) or Fisher’s exact test (for categorical
variables) at a P < 0.05 threshold. Data modeling best fitted a biphasic decay curve, as
previously reported,1,19,26,27 allowing for the calculation of the following parameters (Figure 1):
(i) Tear volume (TV), calculated from the monophasic decay of fluorescence in the first
regression line after instillation of fluorescein;1,26 (ii) Reflex TTR (rTTR), calculated from the
slope of the first regression line;27 (iii) Basal TTR (bTTR), calculated from the slope of the
second regression line.1,19,26,27 Basal TTR represents tear drainage during non-stimulated physiologic conditions, while reflex TTR represents the faster drainage that occurs as a response to noxious stimulation or irritative conditions (e.g. foreign body, corneal abrasion, eyedrop administration). Data supporting the validity and robustness of the model are shown in Figure 2
and Appendix A. Lastly, theoretical TV was calculated based on tear film thickness measured in
a subset of dogs (Appendix B).6,28,29
Results
All eyes (n = 72 in dogs, n = 48 in cats) were deemed healthy on ophthalmic examination and were utilized for data analysis. Using the model, TV and TTR for the general canine and feline population were calculated, and results are presented as median and interquartile range
(25th-75th percentile) in Table 1. Several correlations were found between individual
characteristics and tear film parameters (Table 2). In particular, body weight (r = 0.44, P <
0.001; Figure 3) and age (r = 0.30, P = 0.019) were positively correlated with TV in dogs, and a
negative correlation was found between age and TTR in dogs (r = - 0.33, P = 0.007) and cats (r =
-0.24, P = 0.018). Further, a positive correlation was detected between the dose (amount of 10
fluorescein instilled onto the ocular surface) and the calculated TV in dogs (P < 0.001). Of note, a post-hoc sample size calculation (SigmaPlot version 14.0, Systat Software, Point Richmond,
CA) showed that n = 360 dogs and n = 130 cats would be required to detect statistical differences in TV and TTR among animals with diverse skull conformations, assuming a power of 80% and an alpha of 0.05.
Discussion
The present study establishes the tear film dynamics in healthy dogs and cats, accounting for the diversity of skull conformation occurring in these companion animals. Dogs and cats have a few advantages over current preclinical models of ocular surface disease: their ocular anatomy better resembles humans than rabbits or laboratory rodents,30 and both species develop
spontaneous diseases that share strong similarities with human pathologies (e.g. dry eye disease,
herpes keratitis).7,31 In pharmacology, for instance, extrapolation of findings from rabbits to
humans is compromised by significant differences in precorneal residence time of drugs between
these two species.32 In fact, the tear flow in rabbits is much slower than in humans,1,10 a difference likely explained by variability in tear film stability, mucin composition, and blink rate.20,33,34 Such differences would be minimized when working with companion animals, since it
takes approximately the same amount of time for the tear fluid to replenish in humans as in dogs
and cats (5-10 min); indeed, basal TTR in dogs (12.2%/min) and cats (10.9%/min) better mimics
the human’s ocular surface physiology (10-20%/min).1 On the other hand, the TV is different
among these species: TV in dogs (65.3 µL) and cats (32.1 µL) is larger than in humans (7.0-12.4
µL),9,27 a finding that could explain why Schirmer testing is recommended for 1 min in
companion animals vs. 5 min in people. Compared to humans, dogs and cats have a larger
corneal surface to lubricate (average corneal diameter in humans is 11.7 mm vs. 16.7 mm and 11
16.5 mm for dogs and cats, respectively),35-37 and they possess an additional secretory tissue
(gland of the third eyelid) to supplement the main lacrimal gland with aqueous tear production.
Differences in tear film thickness (3.4 µm in humans29 vs. 15.1 µm in dogs [Appendix B]) could also explain the larger canine TV. Such differences in lacrimal volume have important practical implications. When compared to humans, dogs and cats have a greater initial dilution of a drug administered onto their ocular surface. Conversely, they offer easier collection of sufficient tear fluid for analytical purposes; in fact, up to 106 µL and 43 µL can be easily collected within 1 min using ophthalmic sponges in dogs and cats, respectively.38
Fluorophotometry is often considered superior to other tear assessment methods, such as
fluorescein clearance test or lacrimal scintigraphy,1,39 to study tear film dynamics. However, one
must be cognizant of the initial amount of fluorescein used in fluorophotometry studies as it can
impact the calculated tear dynamics parameters,18,27 a finding verified in the present study. Here,
we combined this analytical method with detailed modeling of the data to improve the robustness
of our findings. Unlike previous studies in which t = 5 min is empirically selected as the
transition between reflex and basal tearing,1,9,19,26 mathematical modeling appreciates the
nuances of fluorescein decay between eyes and subjects, while incorporating individual
characteristics (such as age and body weight) into the final analysis. Another value of the NLME
approach is the ability to model both eyes simultaneously, thereby taking into account within-
subject (between eyes) variability (WSV) in the tear fluid dynamics. This is particularly relevant
as this variability was estimated to be fairly high, such that modeling of the data without
factoring in WSV could lead to significant model misfits.
Further, we purposely enrolled animals with a variety of cephalic conformations to be
representative of the diverse breeds examined by veterinarians and researchers. Brachycephalic 12
animals could theoretically have reduced TTR due to functional punctal occlusion caused by
medial entropion and/or altered anatomy of the nasolacrimal duct associated with the skull
conformation.13 The lack of statistical impact of cephalic conformation on TTR is likely due to
strict inclusion criteria (normal subjects without obvious pathological changes) and an overall
relatively low sample size. Recruiting brachycephalic animals with healthy ocular surface was
indeed rather challenging, particularly in cats. A few significant correlations were detected
between individual characteristics and tear dynamics parameters. Notably, lacrimal volume gets
larger with increasing body weight in dogs (Figure 3), a finding previously documented in
juvenile,40 but not adult dogs.41 Moreover, TTR decreases with age in both dogs and cats. In
humans, aging reduces eyelid kinematics (blinking amplitude and peak velocity),42 a physiologic
change that could result in a less efficient pump mechanism to drain the tear fluid through the
nasolacrimal duct; the same may be true in companion animals.
In both clinical and research settings, we recommend waiting 10 min between successive
lacrimal tests. Indeed, 10 min would be required to fully replenish the canine or feline tear film if
the initial lacrimal test did not cause ocular irritation (e.g. strip meniscometry), as the basal TTR
is approximately 11-12%/min in both species. However, 5 minutes may be sufficient if the
diagnostic test is causing reflex tearing (e.g. Schirmer test) given that the initial TTR is very fast
(rTTR = 50%/min). From a pharmacological standpoint, the concentration of drug instilled onto
the ocular surface is immediately diluted by 3-fold in dogs and 2-fold in cats upon mixing with the tear film, assuming an average drop size of 35 µL.39 The precorneal residence time of this
drug is expected to be < 10 min as TTR following eye drop administration is presumably faster
than under physiologic conditions.32 Further, fluorophotometry data from healthy animals can be
compared to clinical cases with ocular surface disease, helping to differentiate between 13
symptomatic and asymptomatic patients (TTR is significantly lower in symptomatic patients)43
– particularly in cats for whom clinical signs of aqueous tear deficiency are not as overt as in
dogs44 – and between aqueous-deficient and evaporative dry eye (TTR is significantly lower in
aqueous deficiency),45 a distinction well established in human patients but poorly characterized
in veterinary medicine.
The main limitation of our study is the relatively low sample size, which could explain
the lack of significant effect of the skull type (brachycephalic, mesocephalic, dolichocephalic) on
tear film dynamics. Further, in situ assessment of tear fluorescence — as described in most
investigations on human subjects9,19,21,27 — was not possible in our study, as dogs and cats would not tolerate the 20-min protocol without heavy sedation or general anesthesia, which in turn would affect tear film dynamics. Thus, tear fluid had to be collected with capillary tubes prior to analysis and this could have added another source of variability in the fluorophotometry measurements. The volume of fluid collected was calculated in each sample, but the exact duration of tear collection was not standardized among subjects. Since collection duration did not exceed 2 seconds to avoid reflex tearing, the potential impact of sampling duration on
fluorophotometry data is deemed negligible in the present study; however, this parameter should
be recorded in future studies should longer collection duration be necessary (i.e. higher risk of
inadvertent reflex tearing).
In conclusion, the normative data established in the present study has several implications
for both clinicians and researchers. In particular, successive lacrimal tests should be spaced by ≥
10 min to provide sufficient time for the tear film to replenish, as basal TTR is approximately 11-
12 %/min in both species. In both species, tear film parameters were impacted by body weight
and age, but not skull conformation. 14
References
1. Tomlinson, A., Khanal, S. Assessment of tear film dynamics: quantification approach. Ocul Surf. 2005;3:81-95.
2. Cerretani, C.F., Radke, C.J. Tear dynamics in healthy and dry eyes. Curr Eye Res. 2014;39:580-595.
3. Botelho, S.Y., Martinez, E.V. Electrolytes in lacrimal gland fluid and in tears at various flow rates in the rabbit. Am J Physiol. 1973;225:606-609.
4. Fullard, R.J., Tucker, D.L. Changes in human tear protein levels with progressively increasing stimulus. Invest Ophthalmol Vis Sci. 1991;32:2290-2301.
5. Sebbag, L., McDowell, E.M., Hepner, P.M., Mochel, J.P. Effect of tear collection on lacrimal total protein content in dogs and cats: a comparison between Schirmer strips and ophthalmic sponges. BMC Vet Res. 2018;14:61.
6. Chen, T., Ward, D.A. Tear volume, turnover rate, and flow rate in ophthalmologically normal horses. Am J Vet Res. 2010;71:671-676.
7. Kaswan, R.L., Salisbury, M.A., Ward, D.A. Spontaneous canine keratoconjunctivitis sicca. A useful model for human keratoconjunctivitis sicca: treatment with cyclosporine eye drops. Arch Ophthalmol. 1989;107:1210-1216.
8. Petznick, A., Madigan, M.C., Garrett, Q., Sweeney, D.F., Evans, M.D. Contributions of ocular surface components to matrix-metalloproteinases (MMP)-2 and MMP-9 in feline tears following corneal epithelial wounding. PLoS One 2013;8:e71948.
9. Mishima, S., Gasset, A., Klyce, S.D., Baum, J.L. Determination of tear volume and tear flow. Invest Ophthalmol. 1966;5:264-276.
10. Chrai, S.S., Patton, T.F., Mehta, A., Robinson, J.R. Lacrimal and instilled fluid dynamics in rabbit eyes. J Pharm Sci. 1973;62:1112-1121.
11. Slatter, D.H., Edwards, M.E. Normal bovine tear flow rates. Res Vet Sci. 1982;33:262-263.
12. Binder, D.R., Herring, I.P. Evaluation of nasolacrimal fluorescein transit time in ophthalmically normal dogs and nonbrachycephalic cats. Am J Vet Res. 2010;71:570-574.
13. Schlueter, C., Budras, K.D., Ludewig, E., et al. Brachycephalic feline noses: CT and anatomical study of the relationship between head conformation and the nasolacrimal drainage system. J Feline Med Surg. 2009;11:891-900.
15
14. Sebbag, L., Kass, P.H., Maggs, D.J. Reference values, intertest correlations, and test-retest repeatability of selected tear film tests in healthy cats. J Am Vet Med Assoc. 2015;246:426-435.
15. Evans, H.E., de Lahunta, A. The Skeleton. In: Evans HE, de Lahunta A (eds), Miller's Anatomy of the Dog. Philadelphia: Saunders; 2013.
16. Packer, R.M., Hendricks, A., Tivers, M.S., Burn, C.C. Impact of facial conformation on canine health: Brachycephalic obstructive airway syndrome. PLoS One 2015;10:e0137496.
17. Farnworth, M.J., Chen, R., Packer, R.M., Caney, S.M., Gunn-Moore, D.A. Flat feline faces: Is brachycephaly associated with respiratory abnormalities in the domestic cat (Felis catus)? PLoS One 2016;11:e0161777.
18. Webber, W.R., Jones, D.P., Wright, P. Fluorophotometric measurements of tear turnover rate in normal healthy persons: evidence for a circadian rhythm. Eye (Lond) 1987;1 ( Pt 5):615-620.
19. van Best, J.A., Benitez del Castillo, J.M., Coulangeon, L.M. Measurement of basal tear turnover using a standardized protocol. European concerted action on ocular fluorometry. Graefes Arch Clin Exp Ophthalmol. 1995;233:1-7.
20. Webber, W.R., Jones, D.P. Continuous fluorophotometric method of measuring tear turnover rate in humans and analysis of factors affecting accuracy. Med Biol Eng Comput. 1986;24:386- 392.
21. Eter, N., Göbbels, M. A new technique for tear film fluorophotometry. Br J Ophthalmol. 2002;86:616-619.
22. Riviere, J.E., Gabrielsson, J., Fink, M., Mochel, J.P. Mathematical modeling and simulation in animal health. Part I: Moving beyond pharmacokinetics. J Vet Pharmacol Ther. 2016;39:213- 223.
23. Mochel, J.P., Danhof, M. Chronobiology and pharmacologic modulation of the renin- angiotensin-aldosterone system in dogs: What have we learned? Rev Physiol Biochem Pharmacol. 2015;169:43-69.
24. Mochel, J.P., Fink, M., Peyrou, M., Soubret, A., Giraudel, J.M., Danhof, M. Pharmacokinetic/Pharmacodynamic modeling of renin-angiotensin aldosterone biomarkers following angiotensin-converting enzyme (ACE) inhibition therapy with benazepril in dogs. Pharm Res. 2015;32:1931-1946.
25. Fink, M., Letellier, I., Peyrou, M., et al. Population pharmacokinetic analysis of blood concentrations of robenacoxib in dogs with osteoarthritis. Res Vet Sci. 2013;95:580-587.
26. Kuppens, E.V., Stolwijk, T.R., de Keizer, R.J., van Best, J.A. Basal tear turnover and topical timolol in glaucoma patients and healthy controls by fluorophotometry. Invest Ophthalmol Vis Sci. 1992;33:3442-3448. 16
27. Shimizu, A., Yokoi, N., Nishida, K., Kinoshita, S., Akiyama, K. Fluorophotometric measurement of tear volume and tear turnover rate in human eyes. Nippon Ganka Gakkai Zasshi 1993;97:1047-1052.
28. Salgüero, R., Johnson, V., Williams, D., et al. CT dimensions, volumes and densities of normal canine eyes. Vet Rec. 2015;176:386.
29. Wang, J., Aquavella, J., Palakuru, J., Chung, S., Feng, C. Relationships between central tear film thickness and tear menisci of the upper and lower eyelids. Invest Ophthalmol Vis Sci. 2006;47:4349-4355.
30. Vézina, M. Comparative ocular anatomy in commonly used laboratory animals. In: Weir AB, Collins M (eds), Assessing Ocular Toxicology in Laboratory Animals. Totowa, NJ: Humana Press; 2013:1-21.
31. Maes, R. Felid herpesvirus type 1 infection in cats: a natural host model for alphaherpesvirus pathogenesis. ISRN Vet Sci. 2012;2012:495830.
32. Zaki, I., Fitzgerald, P., Hardy, J.G., Wilson, C.G. A comparison of the effect of viscosity on the precorneal residence of solutions in rabbit and man. J Pharm Pharmacol. 1986;38:463-466.
33. Leonard, B.C., Yañez-Soto, B., Raghunathan, V.K., Abbott, N.L., Murphy, C.J. Species variation and spatial differences in mucin expression from corneal epithelial cells. Exp Eye Res. 2016;152:43-48.
34. Wei, X.E., Markoulli, M., Zhao, Z., Willcox, M.D. Tear film break-up time in rabbits. Clin Exp Optom. 2013;96:70-75.
35. Salouti, R., Nowroozzadeh, M.H., Zamani, M., Ghoreyshi, M., Khodaman, A.R. Comparison of horizontal corneal diameter measurements using the Orbscan IIz and Pentacam HR systems. Cornea 2013;32:1460-1464.
36. Datiles, M.B., Kador, P.F., Kashima, K., Kinoshita, J.H., Sinha, A. The effects of sorbinil, an aldose reductase inhibitor, on the corneal endothelium in galactosemic dogs. Invest Ophthalmol Vis Sci. 1990;31:2201-2204.
37. Carrington, S.D., Woodward, E.G. Corneal thickness and diameter in the domestic cat. Ophthalmic Physiol Opt. 1986;6:385-389.
38. Sebbag, L., Harrington, D.M., Mochel, J.P. Tear fluid collection in dogs and cats using ophthalmic sponges. Vet Ophthalmol 2018;21:249-254. 39. Oriá, A.P., Rebouças, M.F., Martins Filho, E., Dórea Neto, F.A., Raposo, A.C., Sebbag, L. Photography-based method for assessing fluorescein clearance test in dogs. BMC Vet Res. 2018;14:269.
17
40. Broadwater, J.J., Colitz, C., Carastro, S., Saville, W. Tear production in normal juvenile dogs. Vet Ophthalmol. 2010;13:321-325.
41. Hartley, C., Williams, D.L., Adams, V.J. Effect of age, gender, weight, and time of day on tear production in normal dogs. Vet Ophthalmol. 2006;9:53-57.
42. Sun, W.S., Baker, R.S., Chuke, J.C., et al. Age-related changes in human blinks. Passive and active changes in eyelid kinematics. Invest Ophthalmol Vis Sci. 1997;38:92-99.
43. Sorbara, L., Simpson, T., Vaccari, S., Jones, L., Fonn, D. Tear turnover rate is reduced in patients with symptomatic dry eye. Cont Lens Anterior Eye 2004;27:15-20.
44. Sebbag, L,. Pesavento, P.A., Carrasco, S.E., Reilly, C.M., Maggs, D.J. Feline dry eye syndrome of presumed neurogenic origin: a case report. JFMS Open Rep 2018;4:2055116917746786.
45. Khanal, S., Tomlinson, A., Diaper, C.J. Tear physiology of aqueous deficiency and evaporative dry eye. Optom Vis Sci. 2009;86:1235-1240.
18
Tables and Figures
Table 1. Normative data of tear volume and tear turnover rate in dogs and cats, presented as median and interquartile range (25th-75th percentile). The relative standard errors (RSE) of parameters estimates from the model are listed for each species. For comparison, the human tear film parameters are presented in the right column.
Dog Cat Human1,9,27 RSE (%) RSE (%) (n = 72 eyes) (n = 48 eyes) (n = 31-74 eyes)
65.3 32.1 Tear volume (µL) 7.5 20.3 7.0-12.4 (42.3-87.9) (29.5-39.9)
Basal tear turnover rate 12.2 10.9 5.4 13.7 10-20 (%/min) (3.7-22.1) (3.0-23.7)
Reflex tear turnover rate 50.0 50.0 5.4 13.7 31.5-100 (%/min) (25.9-172.3) (28.4-89.4)
Table 2. Correlations between tear parameters and co-variates, using the Pearson test for continuous variables (age, body weight, STT, CI, CFR) and the Fisher’s exact test for categorical variables (gender, dose, skull type). STT = Schirmer tear test; CI = Cephalic index; CFR = Craniofacial ratio. Pearson’s correlation coefficients (r) are noted for continuous variables. N/A = Not assessed; — No correlation found.
Body Skull Age Gender STT Dose CI CFR weight type
r = 0.30 r = 0.44 TV — — P < 0.001 — — — (P = 0.019) (P < 0.001) Dog r = -0.33 TTR — P = 0.005 — — — — — (P = 0.007) r = 0.72 TV — — — N/A — — — (P < 0.001) Cat r = -0.24 TTR — — — N/A — — — (P = 0.018)
19
Figure 1. Representative fluorescein decay curve in tear fluid of a dog or a cat, allowing for calculation of tear volume and tear turnover rate (reflex and basal) using parameters calculated with non-linear mixed effects model. C0 represents the fluorescein concentration in tear fluid at t = 0 min, extrapolated from the fluorescein decay curve. NaFl = sodium fluorescein; TTR = tear turnover rate.
20
Figure 2. Comparison of predicted tear fluorescence over time (purple curve) to observed data (blue points) for a random sample of dogs (A) and cats (B). Censored data are shown as vertical red bars. 21
Figure 3. A positive association was found between canine body weight and tear volume (Pearson correlation test). Estimated tear volumes are described in the table for body weights ranging from 1 to 65 kg.
22
CHAPTER 3. KINETICS OF FLUORESCEIN IN TEAR FILM AFTER EYE DROP INSTILLATION IN BEAGLE DOGS: DOES SIZE REALLY MATTER?
Lionel Sebbag,1,2 Nicolette S. Kirner,3 Rachel A. Allbaugh,1 Alysha Reis,3 Jonathan P. Mochel2
1 Department of Veterinary Clinical Sciences, Iowa State University, College of
Veterinary Medicine, IA, USA; 2 Department of Biomedical Sciences, SMART Pharmacology,
Iowa State University, College of Veterinary Medicine, IA, USA; 3 Lloyd Veterinary Medical
Center, Iowa State University, College of Veterinary Medicine, IA, USA
Modified from a manuscript published in Frontiers in Veterinary Sciences
Abstract
The study aimed to determine the impact of drop size on tear film pharmacokinetics, and
assess important physiological parameters associated with ocular drug delivery in dogs. Two
separate experiments were conducted in 8 healthy Beagle dogs: (i) Instillation of 1 drop (35 µL)
or 2 drops (70 µL) of 1% fluorescein solution in each eye, followed by tear collections with
capillary tubes from 0 to 180 min; (ii) Instillation of 10 to 100 µL of 0.1% fluorescein in each
eye, followed by external photography with blue excitation filter (to capture periocular spillage
of fluorescein) and tear collections from 1 to 20 min (to capture tear turnover rate; TTR).
Fluorescein concentrations were measured in tear samples with a fluorophotometer. The TTR
was estimated based upon nonlinear mixed-effects analysis of fluorescein decay curves. Tear
film pharmacokinetics were not superior with instillation of 2 drops vs. 1 drop based on tear film
concentrations, residual tear fluorescence, and area under the fluorescein-time curves (P ≥
0.163). Reflex TTR varied from 20.2-30.5 %/min and did not differ significantly (P = 0.935)
among volumes instilled (10-100 µL). The volumetric capacity of the canine palpebral fissure
(31.3 ± 8.9 µL) was positively correlated with the palpebral fissure length (P = 0.023). Excess 23
solution was spilled over the periocular skin in a volume-dependent manner, predominantly in
the lower eyelid, medial canthus and lateral canthus. In sum, a single drop is sufficient for topical
administration in dogs. Any excess is lost predominantly by spillage over the periocular skin, as
well as accelerated nasolacrimal drainage.
Introduction
Topical administration is the route of choice for treating diseases that affect the anterior
segment of the eye.1,2 This route is simple, convenient, noninvasive, and allows for the use of
relatively high drug concentrations at the target tissue while minimizing systemic exposure.1,2
One of the main challenges associated with topical administration, however, remains the poor
bioavailability of therapeutic drugs to the inner tissues of the eye given rapid precorneal loss
from reflex blinking and efficient nasolacrimal drainage.3-5 Optimization of eyedrop delivery can
enhance therapeutic benefits for the patient,2,6 regardless of the underlying pathology (e.g. dry
eye, infectious keratitis, glaucoma), yet little consensus exists on fundamental concepts such as
the number of eyedrops to apply. The label of ophthalmic products often recommends to ‘apply
one to two drops’ (e.g. Optixcare®, Lotemax®), while diverse publications in veterinary and
human ophthalmology describe the use of either ‘1 drop’,7,8 ‘1 to 2 drops’,9,10 or ‘2 drops’.11,12
The volume of solution instilled through topical administration is known to influence the precorneal residence time, a key parameter in ocular pharmacology.5,13 A prolonged contact time
between the solution and the ocular surface is often desired, as it enhances drug bioavailability
and permits longer intervals between instillations.6 In humans, best practices for ocular delivery
often recommend a single drop of commercial preparations (~ 35 µL) per dosing session,2,5,6 as
the maximum volume that the human palpebral fissure can hold without overflowing is estimated
at 25-30 µL.1,14 Any excess is rapidly lost via nasolacrimal drainage and spillage over the 24
eyelashes and periocular skin;6,15 therefore, a second drop does not provide any therapeutic
advantage in humans and may in fact be counterproductive by increasing systemic absorption
and the risk of associated adverse effects. In rabbits, a single drop is also sufficient as the
lacrimal drainage rate is proportional to the volume of solution instilled (up to 50 µL), hence tear
film drug concentrations decrease less rapidly with lower instilled volumes.13,16 In fact, the
smaller the instilled volume, the greater the fraction of applied dose that is absorbed inside the
rabbit’s eye.13,16 Similar findings may be true in dogs, albeit direct extrapolation between species
is not possible given important differences in ocular anatomy and physiology. In particular, the
canine tear volume (65.3 µL)17 is nearly 9-fold larger than humans (7.0 µL)14 and rabbits (7.5
µL),13 while the canine tear turnover rate is comparable to humans (12.2 %/min vs. 10-20 %/min, respectively)17,18 but faster than rabbits (7.1 %/min).13
The primary objective of this study was to determine the influence of volume instilled via
topical administration (i.e. 1 vs. 2 drops) on tear film kinetics of fluorescein in dogs. Given the
aforementioned species differences in tear film dynamics, we originally hypothesized that the
kinetic profile would be superior following instillation of 2 drops in canine eyes, a hypothesis
that was proven to be wrong. Hence, to explain why a single eyedrop is deemed sufficient in
dogs, a secondary objective was to determine the maximal volume that the canine palpebral
fissure can hold, as well as the drainage rate relative to diverse volumes (10 to 100 µL) instilled
onto the canine ocular surface. The present work focuses on canine-specific ocular physiology,
providing valuable information to veterinary practitioners, pet owners, and scientists working
with dogs as a translational animal model for ocular surface diseases.
25
Materials and Methods
Animals
Eight Beagle dogs (4 neutered male, 4 spayed female) were included in the study, all
confirmed to be healthy based on physical and ophthalmic examinations performed by a board-
certified veterinary ophthalmologist (LS), including Schirmer tear test-1 (Eye Care Product
Manufacturing, LLC, Tucson, AZ, USA), rebound tonometry (TonoVet, Icare Finland Oy,
Espoo, Finland), slit-lamp biomicroscopy (SL-17; Kowa Company, Ltd., Tokyo, Japan) and indirect ophthalmoscopy (Keeler Vantage; Keeler Instruments, Inc., Broomall, PA, USA). All dogs were 3.0 – 3.5 years old and weighed 7.5 – 10 kg. The study was approved by the
Institutional Animal Care and Use Committee of Iowa State University (IACUC #18-398), and was conducted in accordance with the Association for Research in Vision and Ophthalmology statement for the use of animals in ophthalmic and vision research.
Tear film fluorescein following instillation of 1 vs. 2 drops
A 1% fluorescein concentration was obtained by mixing 10% fluorescein solution (Akorn
Inc., Lake Forest, IL, USA) with 1.4% polyvinyl alcohol lubricating eye drops (Artificial Tears,
Rugby, Rockville Center, NY, USA). On Day 1, one eye in each dog was randomly selected
(Excel software) to receive 35 µL (1 drop) of 1% fluorescein solution while the contralateral eye received 70 µL (2 drops) of the same solution, using a pipette (Eppendorf Reference® 2, 10-100
µL) for accuracy. On Day 2 (24 hours later), the order of eyes was reversed and the experiment was repeated. Of note, the volume chosen for a single drop (35 µL) approximates the average drop size of commercial ophthalmic preparations used in veterinary and human medicine (35-39
µL),19,20 and is routinely described in previous scientific publications.4,8,21,22 Following topical instillation, tear fluid was collected in each eye with a 2-µL capillary glass tube (Drummond 26
Scientific Co., Broomhall, PA, USA) at the following time points: 0 min (i.e. immediately after
instillation and spontaneous blinking), 1 min, 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60
min, 90 min, 120 min, and 180 min. The capillary tube was placed against the inferior tear lake
for ≤ 2 seconds, a duration sufficient to collect tear fluid by capillary action while minimizing
the risk of inadvertent ocular irritation and reflex tearing. Given the rapid collection time (< 2
sec), the lack of blinking during collection (eyelids manually opened) and the relatively large
tear volume in dogs (~65 µL)(Sebbag et al., 2019), the authors believe that it is unlikely for
reflex tearing, if any, to affect tear fluorescein concentrations in a significant manner. The length
of fluid contained within each capillary tube was measured to the nearest millimeter using a
ruler, a value used to calculate the volume of fluid collected (as 32 mm equate to 2 µL). The
collected fluid was then expelled into a 2-mL Eppendorf tube that contained 500 µL of
phosphate buffered saline (Gibco® PBS, pH 7.2, Thermo Fisher Scientific, Rockford, IL, USA), vortexed for 30 seconds, and transferred to a cuvette for analysis. Fluorescein concentrations
were measured (in ng/mL) with a computerized scanning ocular fluorophotometer (Fluorotron
MasterTM, Coherent Radiation, Palo Alto, CA, USA) as previously described,17 with the
exception that tear fluid was diluted with 0.5 mL of PBS herein (instead of 2 mL) to improve the
sensitivity of fluorescein detection (data not shown); the cuvette was slightly raised in the
device’s cuvette holder to account for the lower total volume.
Volumetric capacity of the palpebral fissure
A 0.1% fluorescein concentration was obtained by mixing 10% fluorescein solution with
1.4% polyvinyl alcohol lubricating eye drops. Each eye received the following volumes of 0.1%
fluorescein solution via pipette administration, the order being selected at random (Excel
software, Microsoft Corp., Redmond, WA, USA) in each dog: 10 µL, 20 µL, 30 µL, 40 µL, 50 27
µL, 60 µL, 70 µL, 80 µL, 90 µL, and 100 µL. To minimize any carry-over effect from one
session to another, the eyes and periocular skin were thoroughly rinsed with eye wash (Ocusoft®
Eye Wash, OcuSOFT Inc., Richmond, TX, USA) at completion of each experiment, and a 1-hour
break was provided between repeated instillations in each eye to allow ample time for the
physiological tear film dynamics to be restored.17 At each session, within 10 seconds of topical
instillation and spontaneous blinking, an external photograph was taken with a Nikon D90
camera to capture each eye and associated periocular skin. To enhance detection of fluorescence,
the camera was equipped with a screw-on Tiffen Wratten 15 deep yellow filter (Tiffen
Manufacturing, Hauppauge, NY, USA) as well as an external flash (Nikon Speedlight SB-700) covered with a blue excitation filter (SJ-4 blue color). Of note, this photographic method better highlighted 0.1% than 1% fluorescein, hence the choice of 0.1% solution for this experiment.
Tear turnover rate at various instilled volumes
In the experiment described above, following external photography (taken ~10 seconds after 0.1% fluorescein instillation), tear fluid was collected with 2-µL capillary glass tubes at the following time points in each eye: 1 min, 2 min, 4 min, 6 min, 10 min, 15 min, and 20 min.17
Tear film fluorescein concentrations were measured in all samples (see above for details) and
recorded in ng/mL.
Data analysis
Fluorophotometry – First, a fluorescein calibration curve was established by analyzing a
dilution series of known fluorescein concentrations in triplicate (1 to 10,000 ng/mL). Fluorescein
concentrations in tear samples were corrected based on the resulting calibration equation (y =
19.3 + 0.9 x – 3E-05 x2).17 Fluorescein data of each animal were inputted to Monolix® version 28
2019R1 (Lixoft, Orsay, France), and tear turnover rate (TTR) was derived from a non-linear
mixed effects model as previously described.17 Selected data points were censored in Monolix
when a peak of fluorescence could not be identified on the fluorophotometer reading, or if the
tear fluorescein concentration did not make physiologic sense (e.g. higher fluorescein at 2 min
compared with baseline).17 Overall, 11/640 (1.7%) of all data points were left censored.
External photography – The volumetric capacity of the palpebral fissure was calculated in each
eye as the average between the lowest instilled volume that led to periocular spillage of
fluorescein solution, and the highest instilled volume for which all fluorescence remained on the
ocular surface. For instance, a volumetric capacity of 35 µL was calculated for an eye that had
spillage first noted at 40 µL of instilled solution, but no spillage noted at 30 µL (Figure 1).
When present, the location of spillage was recorded (i.e. lower eyelid, upper eyelid, medial canthus, lateral canthus), and the area of fluorescence that extended beyond the eyelids margins was delineated with the ‘freehand selection’ tool in ImageJ 1.52a software (National Institutes of
Health, Bethesda, MD, USA). The area of fluorescein spillage was recorded in mm2 (Figure 2)
using a length bar specific to each eye (i.e. palpebral fissure length measured in mm with
calipers).
Statistical analysis – Normality of data was assessed with the Shapiro-Wilk test. A mixed model for repeated measures (MMRM) was fitted to the data using the R software version 3.6.0. In the model, fluorescein concentration was the response variable, the group (1 or 2 drops), time (0 to
180 min) and group-by-time interaction were treated as fixed effects, and the animal and animal-
by-group interaction were treated as random effects, using animal as block. After the model was
fit, the fixed effects were tested, and comparisons between 1 vs. 2 drops were made for the following outcomes: (i) fluorescein concentration in tears at each time point, and (ii) percent of 29
fluorescein remaining at each time point, using the baseline data of 1 drop for both groups in
order to account for the different volumes instilled in both eyes. The R software was also used to
calculate the area under the concentration-time curve (AUC), a parameter that was compared
between groups (1 vs. 2 drops) using the paired t-test. Differences among volume instilled in
fluorescein periocular spillage and tear turnover rate were assessed with a one-way ANOVA,
while the relationship between the volumetric capacity and the palpebral fissure length was
assessed with the Pearson’s correlation test. Statistical analyses were performed with SigmaPlot
14.0 (Systat software, Point Richmond, CA), and P values < 0.05 were considered significant.
Results
Data were normally distributed (P > 0.05), therefore results are presented as mean ± standard deviation (range).
Volumetric capacity of the canine palpebral fissure
Mean ± SD (range) volumetric capacity of the canine palpebral fissure was 31.3 ± 8.9 µL
(15-45 µL). A moderate positive correlation (r = 0.57, P = 0.023) was found between the length
(in mm) and the volumetric capacity (in µL) of the palpebral fissure (Figure 3). Further, mean palpebral fissure length and volumetric capacity were slightly larger in male dogs (22.5 mm and
35 µL) compared to female dogs (22 mm and 27.5 µL), although these differences were not statistically significant (P ≥ 0.090).
Periocular spillage of instilled solution
The lower eyelid represented the most common location (92%, Figure 1B, Figure 2, and
Figure 4A) covered by fluorescein spillage from the ocular surface, followed by the medial 30
canthus (73%, Figure 4B), the lateral canthus (68%, Figure 4C) and the upper eyelid (32%,
Figure 4E-F). Instillation of large volumes often resulted in excessive periocular spillage that
covered multiple skin locations, although the amount and distribution of spillage varied within
and between dogs; for instance, instillation of 100 µL of fluorescein onto the left eye of 3
different dogs resulted in either mild (Figure 4D) or pronounced (Figure 4E-F) periocular spillage. Overall, the area of periocular spillage increased as the volume of instilled solution increased (Figure 5), with statistical differences noted between 90-100 µL vs. 50-60 µL (P =
0.002), 90-100 µL vs. 30-40 µL (P < 0.001), and 70-80 µL vs. 30-40 µL (P = 0.003).
Tear turnover rate
Parameters estimation was performed using the stochastic approximation expectation maximization algorithm for nonlinear mixed-effects models implemented in the Monolix Suite, as previously described for analysis of canine pharmacokinetic data.23,24 Standard goodness-of-fit
diagnostics were used to assess the validity of the model, including visual predictive checks,
individual predictions vs. observations, individual weighted residuals plotted against tear
fluorescein concentrations, and simulations of fluorescein vs. time disposition from 500 Monte
Carlo simulations (Appendix C). Using the final mathematical model, a visual inspection of
individual fluorescein decay curves showed a tendency for a ‘steeper’ initial slope (i.e. a faster
tear drainage) with increasing volumes of instilled fluorescein, as seen in a representative animal
that received 10-100 µL of topical solution (Figure 6). However, the average rTTR (20.2-30.5
%/min) did not vary significantly among groups (P = 0.935), nor did the bTTR (1.1-1.4 %/min,
P = 0.988) observed a few minutes following fluorescein instillation (Table 1).
31
Tear film fluorescein concentrations following 1 vs. 2 drops
Tear film fluorescein concentrations in eyes receiving 1 vs. 2 drops are depicted in
Figure 7. Immediately following instillation of fluorescein (t = 0 min), tear film concentrations
were significantly higher (P = 0.046) in eyes receiving 2 drops (2345 ± 237 µg/mL) compared to
1 drop (2104 ± 403 µg/mL). However, no statistical differences in fluorescein concentrations
were noted at t = 1 min (P = 0.163) or any subsequent time points (P ≥ 0.293). In fact, the overall exposure of the ocular surface to fluorescein (AUC of fluorescein concentration-time curve) was slightly higher in eyes receiving 1 drop (30,513 ± 21530 µg*min/mL) compared to 2 drops
(28,975 ± 17,410 µg*min/mL). However, this difference was not statistically significant (P =
0.742), and the overall effect of volume instilled on tear film fluorescein was non-significant (P
= 0.619) when taking ‘time’ into account in the model.
The percentage of solution retained on the ocular surface following 1 vs. 2 drops is summarized
in Figure 8. At t = 1 min, the percent retained was higher in eyes receiving 2 drops (90.6 ± 16.7
%) compared to 1 drop (81.8 ± 8.2 %), a difference that approached statistical significance (P =
0.071). However, no significant differences were noted for any other time point (P ≥ 0.220), or
in the overall effect of volume instilled on the percentage of solution retained (P = 0.731) when
the variable ‘time’ was taken into account.
Discussion
The present study supports the use of a single eyedrop in Beagle dogs, whether used
therapeutically in canine patients with ocular disease, or experimentally in canine models of
translational research.25,26 A second drop achieved higher tear film concentrations immediately
after topical administration (t = 0 min), a finding that is partly explained by a lower dilution
effect for 2 drops (1.9-fold) than 1 drop (2.9-fold) from the tear fluid present on the canine ocular 32
surface (~65 µL).17 However, the benefit of instilling two drops was short-lived (<1 min) and unlikely to be clinically important., although the present study focuses on fluorescein and cannot be directly extrapolated to ophthalmic drugs such as antibiotics, corticosteroids or anti-glaucoma medications. A second drop is wasted from an economic perspective, and can potentially exacerbate local and/or systemic adverse effects by overflow on the periocular skin and drainage through the nasolacrimal duct, respectively. The latter was not evaluated herein as fluorescein is non-biologically active, albeit previous studies have shown that overwhelming the lacrimal system can increase the amount of drug that reaches the blood via the naso-buccal mucosa.27 In
sum, the kinetic profile of fluorescein in tears was not superior with 2 drops vs. 1 drop, a finding that is often explained by an accelerated lacrimal drainage with increasing volume in both rabbits13 and humans.3 However, this explanation is not valid in dogs as the rate of lacrimal
drainage did not change significantly in our canine subjects despite a 10-fold increase in instilled
volume (10 to 100 µL); rather, the present study shows that excessive periocular spillage is the
main culprit limiting the benefit of using 2 topical drops in canine ophthalmology. The amount
of solution that overflowed on the periocular skin was greater with larger volumes of instilled
solution, and primarily affected the lower eyelid, medial canthus and lateral canthus. Such
spillage can participate to local adverse effects, such as Malassezia sp. overgrowth in dogs
receiving topical medications,28 or skin hyperpigmentation and lengthening of eyelashes in
humans receiving topical prostaglandin analogues.29
The volumetric capacity of the canine palpebral fissure is 31.3 µL. This value is
somewhat similar to the volumetric capacity in humans (25-30 µL),1,14 and approximates the
average volume of a single eyedrop of commercial preparations (~35 µL).19,20 As such, a single
eyedrop is deemed sufficient in dogs and humans because their ocular surface is unable to 33
accommodate volumes larger than ~ 30 µL, yet therapy with a single drop is relatively inefficient
in both species given the short precorneal residence time and low ocular bioavailability.30
Several strategies can be implemented to enhance the benefits of eyedrop administration,
including: (i) Eyelid closure and/or nasolacrimal punctal occlusion for several minutes following
topical instillation;6,31,32 (ii) Higher drug concentration – Walters et al. showed that topical 1.5%
levofloxacin in humans achieved tear concentrations that were 3-10 times higher than those seen
with 0.3% ofloxacin at multiple time points over 24 hours;11 (iii) Higher solution viscosity and/or
use of mucoadhesive polymers;15,33 (iv) Administration of a second drop ≥ 1 min apart from the
first drop – Herring et al. showed that administration of 2 drops (1-min apart) of 0.5%
proparacaine in dogs achieved significantly greater and longer anesthetic effect compared to eyes
that received a single drop;34 and (v) Use of volumes smaller than the average commercial drop
size.16,35
Strategies to improve ocular drug delivery should ideally be investigated in each species
separately, as direct extrapolation between species is hindered by differences in ocular anatomy
and physiological parameters such as blink and tear turnover rates.13,14,17,36 Rabbits, for instance,
have a much slower blink rate (3-6 blinks/h)37,38 and a slower tear drainage (7 %/min)13
compared to humans (17 blinks/min and 10-20 %/min, respectively),18,39 as well as a different
expression of mucins on the ocular surface that could affect the retention of mucoadhesive
polymers.40 These differences explain why an instilled eyedrop is partially lost (20-30%) due to reflex blinking and periocular spillage in humans, but not in rabbits,15,37 or why a solution’s
viscosity has a great impact on precorneal retention and drug ocular bioavailability in humans,
but not in rabbits.15,41 In a study from over 4 decades ago, it was recognized that “considerable
reservations may be felt about comparing results from rabbits with those from humans because 34 of the differences between the physiology of tear flow and mixing and general anatomy”, yet
“the rabbit is the principal experimental animal in ophthalmology, so comparisons are needed”.42
Since then, rabbits continued to be the ‘species of choice’ for ophthalmic studies given their availability and easy handling, yet the present study shows that dogs likely represent a more relevant model for translational research. Indeed, dogs and humans share many similarities that are relevant to ophthalmic drug delivery, although important differences exist (e.g. tear volume)17 that should be accounted for in comparative studies. The similarities include the blink rate (14.2 vs. 17 blinks/min),39,43 basal TTR (12.2 %/min vs. 10-20 %/min),17,18 reflex TTR following eyedrop instillation (20-30 %/min vs. 30 %/min, respectively),44 volumetric capacity of the palpebral fissure (31.3 µL vs. 25-30 µL),1,14 and periocular spillage of excess solution. The aforementioned similarities justify the use of dogs as a translational model in ophthalmic research, especially given the presence of spontaneous canine diseases that closely resemble human conditions including keratoconjunctivitis sicca,45,46 herpetic keratitis47 and neurotrophic keratopathy.48
The main limitation of the study is the use of dogs from a single breed (Beagles), all being ophthalmoscopically healthy and relatively young (3-3.5 years). The tear film pharmacokinetics of 2 drops may be different in a larger canine breed, presumably due to differences in volumetric capacity and/or tear drainage, as shown in German Shepherd dogs using the fluorescein clearance test.20 Similarly, the ocular surface of older dogs may accommodate a larger volume due to laxity in the eyelids, and the instilled solution may be retained for longer durations due to reductions in tear volume, reflex tearing, and tear turnover rate.49,50 In addition, the present findings do not fully represent the physiology of eyes with ocular surface disease, in which chemosis can reduce the volumetric capacity of the palpebral 35
fissure,51 inflammation can affect tear drainage52 and ocular absorption,53 and excessive tearing from ocular irritation can further dilute the administered solution.54 In particular, patients with
inflamed nasolacrimal duct (dacryocystitis) may actually benefit from instillation of a second
drop, as greater nasolacrimal drainage would theoretically be beneficial in such cases. A second limitation of the study is related to the use of sodium fluorescein as a marker for tear film kinetics. Fluorescein was shown to overestimate tear turnover in human subjects, as a portion of instilled fluorescein can be lost by conjunctival permeation and not nasolacrimal drainage.55
However, a common alternative described by other investigators (i.e. gamma scintigraphy)56 is
not applicable to dogs, in whom the general anesthesia required to hold still for the procedure
would negatively impact the tear film dynamics.
The present study on drop size and tear film pharmacokinetics can be summarized as
follows. Instillation of 2 drops provided tear film fluorescein concentrations that were higher
than 1 drop at baseline, due to lower dilution effect from tears, although the benefits were short-
lived (<1 min) and not clinically important. The kinetic profile of fluorescein in tear film was not
superior in eyes receiving 2 drops vs. 1 drop, as determined by the residual tear fluorescence at
various time points, and the overall exposure of the ocular surface (AUC) to the solution
instilled. Therefore, a single standard size drop is sufficient for topical administration in dogs, a
finding supported by the volumetric capacity of the canine palpebral fissure (31.3 µL). Any
excess is lost predominantly by spillage over the periocular skin, as well as accelerated
nasolacrimal drainage.
36
References
1. Davies NM. Biopharmaceutical considerations in topical ocular drug delivery. Clin Exp Pharmacol Physiol 2000;27:558-562.
2. Bartlett JD. Chapter 3 - Ophthalmic Drug Delivery In: Bartlett JD, Jaanus SD, Fiscella RG, et al., eds. Clinical Ocular Pharmacology (Fifth Edition). Saint Louis: Butterworth-Heinemann, 2008;39-52.
3. Friedrich SW, Cheng YL, Saville BA. Theoretical corneal permeation model for ionizable drugs. J Ocul Pharmacol 1993;9:229-249.
4. Palakuru JR, Wang J, Aquavella JV. Effect of blinking on tear volume after instillation of midviscosity artificial tears. Am J Ophthalmol 2008;146:920-924.
5. Jünemann AGM, Choragiewicz T, Ozimek M, et al. Drug bioavailability from topically applied ocular drops. Does drop size matter? Ophthalmology Journal 2016;1:29-35.
6. Fraunfelder FT. Extraocular fluid dynamics: how best to apply topical ocular medication. Trans Am Ophthalmol Soc 1976;74:457-487.
7. Ledbetter EC, Nicklin AM, Spertus CB, et al. Evaluation of topical ophthalmic ganciclovir gel for the treatment of dogs with experimentally induced ocular canine herpesvirus-1 infection. Am J Vet Res 2018;79:762-769.
8. Seal JR, Robinson MR, Burke J, et al. Intracameral sustained-release bimatoprost implant delivers bimatoprost to target tissues with reduced drug exposure to off-target tissues. J Ocul Pharmacol Ther 2019;35:50-57.
9. Hyndiuk RA, Eiferman RA, Caldwell DR, et al. Comparison of ciprofloxacin ophthalmic solution 0.3% to fortified tobramycin-cefazolin in treating bacterial corneal ulcers. Ciprofloxacin Bacterial Keratitis Study Group. Ophthalmology 1996;103:1854-1862; discussion 1862-1853.
10. Simone JN, Pendelton RA, Jenkins JE. Comparison of the efficacy and safety of ketorolac tromethamine 0.5% and prednisolone acetate 1% after cataract surgery. J Cataract Refract Surg 1999;25:699-704.
11. Walters T, Rinehart M, Krebs W, et al. Tear concentration and safety of levofloxacin ophthalmic solution 1.5% compared with ofloxacin ophthalmic solution 0.3% after topical administration in healthy adult volunteers. Cornea 2010;29:263-268.
12. Leonardi A, Capobianco D, Benedetti N, et al. Efficacy and tolerability of ketotifen in the treatment of seasonal allergic conjunctivitis: Comparison between ketotifen 0.025% and 0.05% eye drops. Ocul Immunol Inflamm 2018:1-5.
13. Chrai SS, Patton TF, Mehta A, et al. Lacrimal and instilled fluid dynamics in rabbit eyes. J Pharm Sci 1973;62:1112-1121. 37
14. Mishima S, Gasset A, Klyce SD, et al. Determination of tear volume and tear flow. Invest Ophthalmol 1966;5:264-276.
15. Zaki I, Fitzgerald P, Hardy JG, et al. A comparison of the effect of viscosity on the precorneal residence of solutions in rabbit and man. J Pharm Pharmacol 1986;38:463-466.
16. Chrai SS, Makoid MC, Eriksen SP, et al. Drop size and initial dosing frequency problems of topically applied ophthalmic drugs. J Pharm Sci 1974;63:333-338.
17. Sebbag L, Allbaugh RA, Wehrman RF, et al. Fluorophotometric Assessment of Tear Volume and Turnover Rate in Healthy Dogs and Cats. J Ocul Pharmacol Ther 2019;35:497-502.
18. Tomlinson A, Khanal S. Assessment of tear film dynamics: quantification approach. Ocul Surf 2005;3:81-95.
19. Van Santvliet L, Ludwig A. Determinants of eye drop size. Surv Ophthalmol 2004;49:197- 213.
20. Oriá AP, Rebouças MF, Martins Filho E, et al. Photography-based method for assessing fluorescein clearance test in dogs. BMC Vet Res 2018;14:269.
21. Elibol O, Alçelik T, Yüksel N, et al. The influence of drop size of cyclopentolate, phenylephrine and tropicamide on pupil dilatation and systemic side effects in infants. Acta Ophthalmol Scand 1997;75:178-180.
22. Bandlitz S, Purslow C, Murphy PJ, et al. Time course of changes in tear meniscus radius and blink rate after instillation of artificial tears. Invest Ophthalmol Vis Sci 2014;55:5842-5847.
23. Mochel JP, Danhof M. Chronobiology and pharmacologic modulation of the renin- angiotensin-aldosterone system in dogs: What have we learned? Rev Physiol Biochem Pharmacol 2015;169:43-69.
24. Pelligand L, Soubret A, King JN, et al. Modeling of large pharmacokinetic data using nonlinear mixed-effects: A paradigm shift in veterinary pharmacology. A case study with robenacoxib in cats. CPT Pharmacometrics Syst Pharmacol 2016;5:625-635.
25. Gronkiewicz KM, Giuliano EA, Kuroki K, et al. Development of a novel in vivo corneal fibrosis model in the dog. Exp Eye Res 2016;143:75-88.
26. Sebbag L, Allbaugh RA, Weaver A, et al. Histamine-induced conjunctivitis and breakdown of blood-tear barrier in dogs: A model for ocular pharmacology and therapeutics. Front Pharmacol 2019;10:752.
27. Chavis RM, Welham RA, Maisey MN. Quantitative lacrimal scintillography. Arch Ophthalmol 1978;96:2066-2068. 38
28. Newbold GM, Outerbridge CA, Kass PH, et al. Malassezia spp on the periocular skin of dogs and their association with blepharitis, ocular discharge, and the application of ophthalmic medications. J Am Vet Med Assoc 2014;244:1304-1308. 29. Johnstone MA, Albert DM. Prostaglandin-induced hair growth. Surv Ophthalmol 2002;47 Suppl 1:S185-202.
30. Awwad S, Mohamed Ahmed AHA, Sharma G, et al. Principles of pharmacology in the eye. Br J Pharmacol 2017;174:4205-4223.
31. Gelatt KN, MacKay EO, Widenhouse C, et al. Effect of lacrimal punctal occlusion on tear production and tear fluorescein dilution in normal dogs. Vet Ophthalmol 2006;9:23-27.
32. Flach AJ. The importance of eyelid closure and nasolacrimal occlusion following the ocular instillation of topical glaucoma medications, and the need for the universal inclusion of one of these techniques in all patient treatments and clinical studies. Trans Am Ophthalmol Soc 2008;106:138-145; discussion 145-138.
33. Zhu H, Chauhan A. Effect of viscosity on tear drainage and ocular residence time. Optom Vis Sci 2008;85:715-725.
34. Herring IP, Bobofchak MA, Landry MP, et al. Duration of effect and effect of multiple doses of topical ophthalmic 0.5% proparacaine hydrochloride in clinically normal dogs. Am J Vet Res 2005;66:77-80.
35. Patton TF. Pharmacokinetic evidence for improved ophthalmic drug delivery by reduction of instilled volume. J Pharm Sci 1977;66:1058-1059.
36. Vézina M. Comparative ocular anatomy in commonly used laboratory animals In: Weir AB,Collins M, eds. Assessing Ocular Toxicology in Laboratory Animals. Totowa, NJ: Humana Press, 2013;1-21.
37. Maurice D. The effect of the low blink rate in rabbits on topical drug penetration. J Ocul Pharmacol Ther 1995;11:297-304.
38. Korb DR, Greiner JV, Glonek T, et al. Human and rabbit lipid layer and interference pattern observations In: Sullivan DA, Dartt DA,Meneray MA, eds. Lacrimal Gland, Tear Film, and Dry Eye Syndromes 2: Basic Science and Clinical Relevance. Boston, MA: Springer US, 1998;305- 308.
39. Bentivoglio AR, Bressman SB, Cassetta E, et al. Analysis of blink rate patterns in normal subjects. Mov Disord 1997;12:1028-1034.
40. Leonard BC, Yañez-Soto B, Raghunathan VK, et al. Species variation and spatial differences in mucin expression from corneal epithelial cells. Exp Eye Res 2016;152:43-48.
39
41. Saettone MF, Giannaccini B, Teneggi A, et al. Vehicle effects on ophthalmic bioavailability: the influence of different polymers on the activity of pilocarpine in rabbit and man. J Pharm Pharmacol 1982;34:464-466.
42. Chrai SS, Robinson JR. Ocular evaluation of methylcellulose vehicle in albino rabbits. J Pharm Sci 1974;63:1218-1223.
43. Carrington SD, Bedford PGC, Guillon JP, et al. Polarized light biomicroscopic observations on the pre-corneal tear film. 1. The normal tear film of the dog. Journal of Small Animal Practice 1987;28:605-622.
44. Shell JW. Pharmacokinetics of topically applied ophthalmic drugs. Surv Ophthalmol 1982;26:207-218.
45. Kaswan RL, Salisbury MA, Ward DA. Spontaneous canine keratoconjunctivitis sicca. A useful model for human keratoconjunctivitis sicca: treatment with cyclosporine eye drops. Arch Ophthalmol 1989;107:1210-1216.
46. Leonard BC, Stewart KA, Shaw GC, et al. Comprehensive clinical, diagnostic, and advanced imaging characterization of the ocular surface in spontaneous aqueous deficient dry eye disease in dogs. Cornea 2019.
47. Ledbetter EC, Kice NC, Matusow RB, et al. The effect of topical ocular corticosteroid administration in dogs with experimentally induced latent canine herpesvirus-1 infection. Exp Eye Res 2010;90:711-717.
48. Sebbag L, Crabtree EE, Sapienza JS, et al. Corneal hypoesthesia, aqueous tear deficiency, and neurotrophic keratopathy following micropulse transscleral cyclophotocoagulation in dogs. Vet Ophthalmol 2019.
49. Sun WS, Baker RS, Chuke JC, et al. Age-related changes in human blinks. Passive and active changes in eyelid kinematics. Invest Ophthalmol Vis Sci 1997;38:92-99.
50. Van Haeringen NJ. Aging and the lacrimal system. Br J Ophthalmol 1997;81:824-826.
51. Kang YK, Im JC, Shin JP, et al. Short-term analysis of the residual volume of an eye drop following 23-gauge microincision vitrectomy surgery. Korean J Ophthalmol 2017;31:439-445.
52. Sorbara L, Simpson T, Vaccari S, et al. Tear turnover rate is reduced in patients with symptomatic dry eye. Cont Lens Anterior Eye 2004;27:15-20.
53. Behrens-Baumann W. Absorption of topically administered ciprofloxacin, ofloxacin and gentamicin in the inflamed rabbit eye. Ophthalmologica 1996;210:119-122.
54. Lux A, Maier S, Dinslage S, et al. A comparative bioavailability study of three conventional eye drops versus a single lyophilisate. Br J Ophthalmol 2003;87:436-440. 40
55. Macdonald EA, Maurice DM. Loss of fluorescein across the conjunctiva. Exp Eye Res 1991;53:427-430.
56. Gurny R, Ryser JE, Tabatabay C, et al. Precorneal residence time in humans of sodium hyaluronate as measured by gamma scintigraphy. Graefes Arch Clin Exp Ophthalmol 1990;228:510-512.
41
Tables and Figures
Table 1. Mean ± standard deviation of reflex tear turnover rate (rTTR) and basal tear turnover rate (bTTR) in 8 Beagle dogs following topical instillation of 10-100 µL of 0.1% fluorescein solution in each eye. P values depict the results of one-way ANOVA testing.
Volume P- 10 20 30 40 50 60 70 80 90 100 (µL) value
20.6 20.2 22.7 24.3 30.3 22.3 24.6 24.6 30.5 22.2 rTTR ± ± ± ± ± ± ± ± ± ± 0.935 (%/min) 14.5 11.4 17.1 12.0 23.3 13.0 13.2 18.4 22.1 11.6
1.4 1.3 1.3 1.1 1.2 1.2 1.1 1.2 1.1 1.2 bTTR ± ± ± ± ± ± ± ± ± ± 0.988 (%/min) 0.4 0.4 0.5 0.5 0.6 0.6 0.6 0.5 0.6 0.6
Figure 1. Photographs of the right eye and eyelids in a representative Beagle dog. The volumetric capacity of the palpebral fissure was calculated as 35 µL in this eye, based on the lack or presence of periocular spillage of 0.1% fluorescein with instillation of either 30 µL (A) or 40 µL (B), respectively.
42
Figure 2. Photograph of the right eye and eyelids following topical instillation of 90 µL of 0.1% fluorescein in a representative Beagle dog. The area of periocular spillage was delineated with ImageJ software (version 1.52a, National Institute of Health), and recorded in mm2 based on a length bar (10 mm) specific to each eye.
Figure 3. A positive association was found between the length and the volumetric capacity of the palpebral fissure (Pearson’s correlation test).
43
Figure 4. Representative ocular images following instillation of 60 µL (A), 80 µL (B), 40 µL (C), or 100 µL (D-F) of 0.1% fluorescein solution in different Beagle dogs. Notice the periocular spillage that predominantly affects the lower eyelid (A), medial canthus (B), lateral canthus (C), or multiple locations including the upper eyelid (D-F).
Figure 5. Bar chart depicting the mean area (+SD) of periocular spillage of 0.1% fluorescein solution, instilled at various volumes (10-100 µL) in 8 Beagle dogs (n = 16 eyes). 44
Figure 6. Comparison of predicted tear fluorescence over time (purple curve) with observed data (blue points) following topical instillation of 0.1% fluorescein solution (10 to 100 µL) in a representative Beagle dog. Censored data are shown as vertical red bars.
Figure 7. Scatter plot depicting the mean + SD of tear film fluorescence over time in canine eyes receiving either 1 drop (35 µL; red circles) or 2 drops (70 µL; blue triangles) of 1% fluorescein solution. Differences in tear fluorescence were noted at t = 0 min (P = 0.046) but no other time points (P ≥ 0.163). 45
Figure 8. Bar chart depicting the mean + SD of residual tear film fluorescence at each time point in canine eyes receiving either 1 drop (35 µL; plain red bars) or 2 drops (70 µL; hatched white bars) of 1% fluorescein solution. For standardization, the residual fluorescence in both groups was compared to the tear fluorescence obtained at t = 0 min in eyes receiving a single drop of fluorescein. No statistical differences were noted between both groups at any time point (P ≥ 0.220). 46
CHAPTER 4. HISTAMINE-INDUCED CONJUNCTIVITIS AND BREAKDOWN OF BLOOD-TEAR BARRIER IN DOGS: A MODEL FOR OCULAR PHARMACOLOGY AND THERAPEUTICS
Lionel Sebbag,1,2 Rachel A. Allbaugh,1 Amanda Weaver,3
Yeon-Jung Seo,2 Jonathan P. Mochel2
1 Department of Veterinary Clinical Sciences, Iowa State University, College of Veterinary
Medicine, IA, USA; 2 Department of Biomedical Sciences, SMART Pharmacology, Iowa State
University, College of Veterinary Medicine, IA, USA; 3 Lloyd Veterinary Medical Center, Iowa
State University, College of Veterinary Medicine, IA, USA
Modified from a manuscript published in Frontiers in Pharmacology
Abstract
Conjunctival inflammation disturbs the blood–tear barrier and thus affects the tear film
stability and composition. We aimed to develop a non-invasive and reliable method to induce conjunctivitis in dogs, a large animal model for translational work on ocular surface disease in humans. Six beagle dogs underwent a randomized, vehicle-controlled, balanced crossover trial— on six separate days, one eye received topical artificial tears (vehicle), while the other eye received one of six concentrations of histamine solution (0.005–500 mg/ml). At sequential times after eyedrop administration, a conjunctivitis score was given to each eye based on the degree of palpebral and bulbar conjunctival hyperemia and chemosis, ocular pruritus, and discharge. Total protein content (TPC) and serum albumin were quantified in tear fluid at baseline and 20 min.
Additionally, 13 dogs presenting for various ophthalmic diseases with associated conjunctivitis were examined. Experimentally induced conjunctivitis developed rapidly (<1 min) following topical histamine administration and lasted for 1–3 h (four lowest doses) to 6–8 h (two highest 47
doses). The severity of conjunctivitis was dose-dependent. Histamine was overall well tolerated,
although transient blepharitis, aqueous flare, and ocular hypertension occurred in a few dogs
receiving histamine ≥375 mg/ml. TPC and serum albumin levels increased in tears of eyes
receiving histamine ≥1.0 mg/ml, being significantly higher than vehicle and baseline in eyes
receiving histamine ≥375 mg/ml. Lacrimal albumin levels were also increased in 13 dogs with
naturally acquired conjunctivitis, up 2.7–14.9 fold compared to contralateral healthy eyes.
Histamine-induced conjunctivitis represents a robust model for translational work on the ocular
surface given the low cost, non-invasiveness, self- resolving nature, ability to adjust the duration and severity of the disease, and shared features with naturally occurring ocular diseases.
Histamine solutions of 1, 10, and 375 mg/ml induce mild, moderate, and severe conjunctivitis in dogs, respectively. Leakage of serum albumin in tear fluid of eyes with conjunctivitis suggests a breakdown of the blood–tear barrier.
Introduction
Conjunctivitis, or inflammation of the vascularized mucous membrane lining the inside
of the eyelids, anterior sclera, and (when present) the nictitating membrane, is a common
ocular surface disease in both humans and veterinary patients.1,2 In addition to well- recognized etiologies (e.g., infectious, allergic, toxic/irritative, immune-mediated), conjunctivitis frequently develops as a bystander to most adnexal and ocular diseases, such as blepharitis, keratitis, uveitis, and glaucoma.1,2 Regardless of the cause, conjunctivitis is debilitating to patients due to
ocular discomfort, redness, and discharge, as well as the potential development of conjunctival
scarring, fornix foreshortening, or symblepharon in severe or untreated cases. Further,
conjunctivitis compromises the tear film homeostasis and thereby contributes indirectly to ocular
surface damage. Changes in tear composition result from a loss of secretory function and 48
numbers of mucin secreting goblet cells,3 but can also be linked to the disruption of the blood–
tear barrier — a critical yet poorly understood mechanism.4 Indeed, conjunctivitis increases
vascular permeability and results in leakage of plasma compounds into the tear film, as
exemplified by human patients with conjunctivitis and dry eye who were noted to have
significantly greater serum albumin in tears compared to healthycontrols,5-8 and dogs with spontaneous keratoconjunctivitis sicca for whom the clinical signs of conjunctivitis were positively correlated with tear levels of serum proteins.9
Despite the prevalence of conjunctivitis, there is limited knowledge about the disease
impact on the ocular surface in clinical patients. Are tear film composition and quality affected
by conjunctivitis-induced breakdown of the blood–tear barrier? Is there an impact on the
pharmacokinetic profile of drugs on the ocular surface? Do medications administered
systemically reach the tear film compartment at higher concentrations? Such knowledge can be
gained from experimentally induced models of conjunctivitis in animals, such as intraperitoneal
injection of ovalbumin in guinea pigs10 and topical administration of dust mite allergens in
dogs.11 However, a delayed response occurs with ovalbumin (6 h from antigen exposure to
conjunctival pathology), and dust mite allergens only cause conjunctivitis in individuals already
sensitized to this antigen. In contrast, the use of topical histamine is promising as it causes conjunctivitis in a nonspecific and rapid manner12 and the compound is a potent inflammatory mediator that is associated with various disorders such as allergy, inflammation, autoimmune conditions, and possibly cancer.13 Takahashi and colleagues used topical histamine in guinea
pigs and quantified the extravasation of Evans Blue to demonstrate vascular permeability in the
conjunctiva.12 The authors focused on a single dose of histamine and did not assess the safety of
the drug, disease severity, or disease duration. In the present study, the model of histamine- 49
induced conjunctivitis was fine-tuned and perfected: we investigated a diverse range of histamine
concentrations, performed serial ophthalmic and physical examinations to assess safety, and
described in detail the clinical and biochemical changes observed at each dose. The dog is a
preclinical species of choice for modeling human ocular diseases as the canine ocular anatomy is
more similar to humans than routine laboratory species;14 dogs share similar environmental
stressors with people, and spontaneously occurring ocular surface diseases are common in this
species. We are confident that this translatable in vivo model of conjunctivitis will guide future
studies to gain a deeper understanding of the disease, elucidating the impact of conjunctivitis on
drug pharmacokinetics, tear film dynamics, metabolomics, and proteomics, among others.
Materials and Methods
Experimentally Induced Conjunctivitis in Dogs
Animals
Six beagle dogs (1.5–2.0 years, 7.7–10.1 kg) were used in the study. The gender and
neuter status was the same for all subjects (female spayed), as sex hormones are known to be key
regulators of vascular tone in various organs, including the eye.15 All dogs were confirmed to be
healthy based on complete physical and ophthalmic examinations, including slit-lamp
biomicroscopy (SL-17; Kowa Company, Ltd., Tokyo, Japan), indirect funduscopy (Keeler
Vantage; Keeler Instruments, Inc., Broomall, PA, USA), rebound tonometry (TonoVet; Icare
Finland Oy, Espoo, Finland), Schirmer tear test-1 (STT-1; Eye Care Product Manufacturing
LLC, Tucson, AZ, USA), and fluorescein staining (Flu-Glo, Akorn, Inc., Buffalo Grove, IL,
USA). The dogs were group-housed in kennels with ambient temperature maintained at 18–24°C and lights automatically turned on/off at 06:00/18:00. The study was approved by the
Institutional Animal Care and Use Committee of Iowa State University (log # 2-18-8704-K) and 50
adhered to the Association for Research in Vision and Ophthalmology (ARVO) statement for the
Use of Animals in Ophthalmic and Vision Research.
Topical Histamine and Vehicle Solutions
Histamine ophthalmic solutions were formulated by mixing histamine powder (Histamine
dihydrochloride, FCC grade, Arcos® organics, Geel, Belgium) in 1.4% polyvinyl alcohol
lubricating eye drops (Artificial tears solution, Rugby, Rockville Center, NY, USA) using a
sterile manner under a laminar flow hood. The pH of each solution was tested with a pH meter
(B-212 Twin compact pH, Horiba, Kyoto, Japan) and adjusted to 6.5 by adding 1% sodium
hydroxide (prepared from granules mixed with sterile water), one drop at a time until the target
pH was reached. The following 12 concentrations of histamine solution were compounded into
15-ml sterile eyedropper bottles (Steri- dropper®, Medi-Dose®, Ivyland, PA, USA) by the pharmacist at Iowa State University’s Lloyd Veterinary Medical Center: 0.001, 0.005, 0.01,
0.0375, 0.1, 0.5, 1.0, 10, 100, 375, 500, and 1,000 mg/ml. The vehicle solution consisted of artificial tears solution adjusted to pH of 6.5 with a minute amount of 1% sodium hydroxide (< 5 drops in 15-ml bottle). Histamine and vehicle solutions were used within 24 h of preparation and kept in the dark at room temperature (18–24°C) before use and in between experiments.
Experimental Design
Pilot Study to Assess Tolerability and Appropriate Histamine Concentrations
A pilot study was conducted to assess the tolerability of histamine solutions in dogs (local and systemic) and the most appropriate concentrations to select for the crossover trial. Each of the 12 eyes (n = 6 dogs) was randomly allocated to one of the 12 histamine concentrations
(Excel, Microsoft, Redmond, WA, USA). A single drop of histamine solution was instilled onto 51
the ocular surface, followed by slit-lamp biomicroscopy and conjunctivitis scoring at 1, 3, 5, 7,
10, 15, 20, 25, 30, 40, 50, 60, 90, 120, 180, 240, 300, 360, and 420 min. Additionally, the
following ocular and physical parameters were recorded at 1, 10, 20, 30, 60, 120, and 240 min
post-histamine administration: intraocular pressure, aqueous flare grading, body temperature, heart rate, respiratory rate, respiratory efforts, capillary refill time, and indirect blood pressure
(Doppler model 811-B, Parks Medical Electronics, Las Vegas, NV, USA).
Balanced Crossover Vehicle-Controlled Trial
Six histamine solutions were selected for the crossover trial based on good tolerability
and diversity of conjunctivitis scoring (pilot study, data not shown). The six histamine solutions
are listed as follows: H1 = 0.005 mg/ml, H2 = 0.1 mg/ml, H3 = 1.0 mg/ml, H4 = 10 mg/ml, H5 =
375 mg/ml, and H6 = 500 mg/ml. For each dog, one eye received histamine solution (random
selection by coin-toss) while the other eye received vehicle solution, and this order was kept
consistent throughout the study. Each dog received all six histamine solutions over six
consecutive days, using one solution per day (once in the morning) in a balanced crossover trial
(Appendix D, Figure 1). In each dog, ocular and physical parameters were recorded at selected
times (similar to pilot study), while tear collection and conjunctivitis scoring were performed as
outlined below:
• Tear collection: At baseline and at 20 min post-eyedrop administration, tear fluid was collected from both eyes and total protein content (TPC) was analyzed as previously described.16
Briefly, a standardized Schirmer strip was inserted into the ventrolateral conjunctival fornix until
the 20-mm mark was reached. Each wetted Schirmer strip was placed into a 0.2-ml tube (pre- punctured at its bottom with an 18-gauge needle), secured into a 2-ml tube with adhesive tape, and centrifuged at 3,884 × g for 2 min (Mini Centrifuge, VWR International, Radnor, PA, USA).
After estimating the volume of extracted tear fluid with a pipette, each tear sample was diluted 52
1:3 with phosphate- buffered saline (PBS 1X, Gibco™, Thermo Fisher Scientific, Inc., Waltham,
WA, USA). TPC was calculated with Direct Detect™ infrared spectrometer and expressed in
mg/ml after adjusting for the threefold dilution of each sample. Subsequently, the residual tear
fluid was diluted fourfold with diluent provided with the albumin ELISA kit (Serum albumin
ELISA kit, Life Span Biosciences, Inc., Seattle, WA, USA). Serum albumin and various
cytokines/chemokines/growth factors (Canine Procarta Plex™ 11-plex immunoassay, Cat No
EPX11A-50511-901, Thermo Fischer Scientific, Waltham, MA, USA) were quantified in each tear sample following the manufacturer’s protocol.
• Conjunctivitis score: At baseline and at selected times post- eyedrop administration (similar to
pilot study), one examiner (LS) who was masked to which eye received histamine or vehicle
solution performed slit-lamp biomicroscopy of both eyes to determine a conjunctivitis score at
each time point. The conjunctivitis score was calculated as the sum of the following categories,
each graded on a three- to four-point scale (Appendix D, Table 1): hyperemia, chemosis and follicles of the palpebral and bulbar conjunctiva, conjunctival discharge, and ocular pruritus.17,18
Naturally Acquired Conjunctivitis in Dogs
Thirteen dogs presenting to Iowa State University’s Lloyd Veterinary Medical Center
(ISU-LVMC) for clinical ophthalmic disease with associated conjunctivitis were enrolled. A
verbal informed consent was obtained from all owners, which was sufficient for the hospital’s
ethics committee given that Schirmer tear test is part of routine clinical care. Dogs were
examined by a board-certified veterinary ophthalmologist (LS or RA) for diagnosis and
treatment of various ocular complaints including corneal ulceration, uveitis, and glaucoma.
Details of the dogs (breed, sex, age) and their clinical diagnosis are presented in Table 1. Tear
collection and albumin analysis were performed in both eyes of each dog as described above. 53
Data Analysis
Normality of the data was assessed with the Shapiro-Wilk test. The one-way ANOVA and Tukey post hoc test was used to compare the six histamine doses for i) duration of conjunctivitis, determined as the time for conjunctivitis score to return to zero, and ii) severity of conjunctivitis, determined by the area under the curve of conjunctivitis score from 0 to 180 min. Within each histamine dose, the one-way repeated measures ANOVA and Bonferroni post hoc test was used to assess differences in conjunctivitis scores between time points. The Student t-test was used to evaluate differences between vehicle and histamine-treated eyes for TPC and albumin levels, both at baseline and at 20 min following eyedrop administration. Further, a ratio was calculated for TPC and albumin levels for each histamine dose as follows: (Concentration histamine 20 min/histamine baseline)/(Concentration vehicle 20 min/ vehicle baseline). This ratio describes the percentage change in protein levels between baseline and 20 min post-induction of
conjunctivitis, taking into account the variability inherent to the tear collection method itself by
adding the vehicle-treated eyes as the denominator.16 The one-way ANOVA and Tukey post hoc test was used to assess differences in protein ratios and levels of cytokines/chemokines/growth factors in tears of different groups. In dogs with naturally acquired conjunctivitis, differences in lacrimal concentrations of albumin between affected and unaffected eyes were analyzed with the
Mann–Whitney test. Statistical analysis was performed using SigmaPlot version 14.0 (Systat
Software, Point Richmond, CA), and values P < 0.05 were considered statistically significant.
54
Results
Experimentally Induced Conjunctivitis in Dogs
Clinical Features of Conjunctivitis
All eyes receiving the vehicle solution were scored at zero for all time points. In eyes receiving histamine, the ocular surface was examined from different angles to evaluate the canine conjunctiva in a comprehensive manner, including a view from the front (Figure 1A),
side (Figure 1B), top (Figure 1C), and globe retropulsion with lower lid retraction (Figure 1D),
which facilitated assessment of the palpebral conjunctiva and nictitating membrane. Figure 2 shows representative photographs and conjunctivitis scoring of a canine eye at 30 min following
H4 administration (10 mg/ml histamine solution), while Figure 3 demonstrates the development
and progression of conjunctivitis from 0 to 420 min in a canine eye receiving H5 (375 mg/ml
histamine solution). Data were normally distributed such that results are presented as mean ±
standard deviation (range). Conjunctivitis developed rapidly (<1 min) for all doses (Figure 4A).
Of note, not a single eye developed conjunctival follicles. Details of conjunctivitis scoring for
each subsection (palpebral chemosis, bulbar hyperemia, etc.) is described in Appendix D (Table
2).
The duration of conjunctivitis was statistically different among histamine doses (P <
0.001), ranging from 61 ± 37 min (25–120 min) for H1, 110 ± 36 min (90–180 min) for H2, 115 ±
52 min (60–180 min) for H3, 190 ± 45 min (120–240 min) for H4, 390 ± 33 min (360–420 min)
for H5, and 400 ± 31 min (360–420 min) for H6. All pairwise comparisons were statistically
significant (P ≤ 0.029) except for H1–H3 (P = 0.201) and H5–H6 (P = 0.998).
The severity of conjunctivitis was significantly different among histamine doses (P <
0.001), with an AUC0–180min (in score x min) ranging from 59.2 ± 36.8 (21.5–119) for H1,
200.2 ± 53.9 (107.5–254.5) for H2, 277.2 ± 128.3 (144.5–495.5) for H3, 522.3 ± 131.8 (358–735) 55
for H4, 1,168.5 ± 266.4 (892.5–1,585) for H5, and 1,186.5 ± 242.4 (914–1,443.5) for H6. All
pairwise comparisons were statistically significant (P ≤ 0.037) except for H5–H6 (P = 1.000) and
H2–H3 (P = 0.794; Figure 4B).
A post hoc power analysis showed that n = 5 dogs were sufficient to detect a difference in
total clinical scores of 2.2 (as observed clinically in eyes with mild vs. moderate vs. severe conjunctivitis), a standard deviation of 0.9, a power of 80%, and an α value of 0.05.
Tolerance
Locally, histamine administration was very well tolerated in dogs except for transient adverse effects noted with H5 and H6: i) Blepharitis, manifested by mild blepharedema and
erythema (Figure 5A), was noted in 1/6 dogs receiving H5 and 2/6 dogs receiving H6,
developing within 10–30 min of histamine administration and self-resolving within 3 h. ii)
Aqueous flare, subtle in intensity (trace to 1+), was noted in 2/6 dogs receiving H5 and 6/6 dogs receiving H6, developing within 25–90 min and self-resolving within 1–3 h. Aqueous flare was commonly accompanied by miosis (Figure 5B). iii) Ocular hypertension, defined as IOP > 25
19 mmHg, was noted in 1/6 dogs receiving H5 at 30 min (IOP = 32 mmHg), although it was not statistically greater than baseline IOP (P = 0.529) and it self-resolved within 10 min. Ocular hypertension was also noted in 3/6 dogs receiving H6 in which IOP was significantly higher at
30 min (23.8 ± 5.5 mmHg; P = 0.011) and 60 min (23.2 ± 2.6 mmHg; P = 0.029) compared to
baseline (16.8 ± 2.6 mmHg). Importantly, no fluorescein stain uptake or corneal changes were
noted for any histamine dose.
Systemically, all the vital parameters monitored were stable and not a single dog
receiving H1 to H6 developed systolic hypotension (<90 mmHg). However, the systolic blood pressure dropped from 130 to 88 mmHg in a single dog receiving 1,000 mg/ml histamine during 56
the pilot phase, and the dog exhibited transient depression and weakness until blood pressure
returned to baseline 10 min later.
Total Protein Content and Serum Albumin Levels in Tears
At baseline, lacrimal TPC varied from 3.0 to 29.0 mg/ml (8.8 ± 4.0 mg/ml) and no
differences were noted between vehicle and histamine-treated eyes for any dose (P ≥ 0.365).
Twenty minutes following eyedrop administration, lacrimal TPC varied from 2.3 to 36.3 mg/ml
(9.9 ± 5.6 mg/ml) and was statistically greater in histamine vs. vehicle-treated eyes for H5 (P =
0.009) and H6 (P = 0.021) but no other doses (P ≥ 0.310, Figure 6A). Mean ± SD (range)
changes in TPC were 171 ± 112% (6–271%) and 170 ± 181% (13–499%) for H5 and H6,
respectively, values that were significantly greater than H1 and H2 (P ≤ 0.036, Figure 6B).
At baseline, albumin levels in tears varied from 0.015 to 1.431 mg/ml (0.413 ± 0.455
mg/ml) and no differences were noted between vehicle and histamine eyes for any dose (P ≥
0.394). Twenty minutes following eyedrop administration, albumin levels varied from 0.019 to
9.595 mg/ml (1.158 ± 2.098 mg/ml) and were statistically greater in histamine vs. vehicle-treated
eyes for H5 (P = 0.021) and H6 (P = 0.029) but no other doses (P ≥ 0.106; Figure 6C). Mean ±
SD (range) changes in albumin levels were 2,348 ± 1,454% (314–4,348%) and 4,031 ± 4,679%
(583–12,689%) for H5 and H6, respectively, values that were significantly greater than H1 and H2
(P ≤ 0.016; Figure 6D).
Canine Chemokines/Cytokines/Growth Factors in Tears
The levels of interferon-gamma (IFNγ), interleukin-2 (IL-2), and beta nerve growth
factor (NGF-β) were below limits of quantification in all tear samples. Tear concentrations of
other chemokines/cytokines/growth factors, described as mean ± standard deviation (range), 57
were as follows (Figure 7): 4.5 ± 19.4 pg/ml (0–147.6 pg/ml) for interleukin-6 (IL-6), 2,647.8 ±
5,680.4 pg/ml (0–25,640.2 pg/ml) for interleukin-8 (IL-8), 17.8 ± 16.3 pg/ml (0–86.8 pg/ml) for interleuekin-10 (IL-10), 30.6 ± 104.0 pg/ml (0–807.3 pg/ml) for interleukin-12 (IL-12), 532.5 ±
510.6 pg/ml (0–2,650.8 pg/ml) for vascular endothelial growth factor A (VEGF A), 0.80 ± 2.9 pg/ml (0–20.9 pg/ml) for tumor necrosis factor alpha (TNFα), 1.3 ± 4.3 pg/ml (0–24.8 pg/ml) for stem cell factor (SCF), and 5.4 ± 13.1 pg/ml (0–80.2 pg/ml) for chemokine monocyte chemoattractant protein-1 (MCP-1). Statistical differences among groups (vehicle histamine solutions) were noted for IL-8 (Figure 7B), IL-10 (Figure 7C), IL-12 (Figure 7D), and VEGF A
(Figure 7E).
Naturally Acquired Conjunctivitis in Dogs
Ocular disease was unilateral in all dogs, and spontaneous conjunctivitis was noted upon examination of all affected eyes (Figures 8–10). Lacrimal concentrations of albumin ranged from 1.1 to 17.95 mg/ml in affected eyes, representing a significant change by 2.67–14.86 fold
(P < 0.001) compared to lacrimal concentrations noted in contralateral unaffected eyes (0.13–
3.06 mg/ml). Re-examination of two dogs following therapy of the underlying ocular disease
(cases #12–13; Table 1, Figure 10) showed reduction in lacrimal albumin concentrations concomitant with a reduction in the conjunctivitis score (Table 1).
Discussion
The present study establishes a robust in vivo model of conjunctivitis in dogs, a translational large animal model that provides a framework for ocular surface studies in clinically relevant subjects, investigating the impact on conjunctivitis on tear film dynamics, pharmacokinetics, metabolomics, and other relevant fields. Dogs are an ideal large animal 58 species for such translational model: not only does the canine ocular anatomy better resemble humans than small laboratory animals do,14 but dogs also share similar environmental stressors to humans and conjunctivitis is a common and naturally occurring disease in this species.2 As a proof of concept, our study examined 13 dogs with naturally acquired conjunctivitis and confirmed the presence of elevated albumin levels in tears of affected eyes. Similar to histamine- induced conjunctivitis, eyes with naturally occurring disease exhibited a breakdown of the blood–tear barrier regardless of the underlying etiology (e.g., corneal ulceration, uveitis, glaucoma, and orbital cellulitis). Of note, the “blood–tear barrier” is not as well defined as other ocular barriers (e.g., blood–aqueous and blood–retinal barriers) and would benefit from future anatomical and physiological studies to confirm the terminology used in the present study and in previous work.4-7,20
Histamine-induced conjunctivitis is not novel. The ocular use of histamine has been described in humans, guinea pigs, and rabbits as the compound is inexpensive and triggers local inflammation in a non-specific manner.12,21,22 In dogs, we showed that histamine-induced conjunctivitis is non-invasive, self-resolving, and dose- dependent, allowing investigators to modulate the severity and duration of conjunctival inflammation by adjusting the dose of histamine solution administered. In fact, both parameters increased with histamine concentration until saturation in dose response observed at 375 mg/ml. Thus, we propose doses of 1, 10, and
375 mg/ml to induce a mild, moderate, or severe conjunctivitis in dogs, respectively (Figure 11).
Of note, histamine is not only associated with ocular allergies but also implicated in general inflammation, autoimmune conditions, and possibly cancer.13 Ocular allergy was not the scope of the present study, as many models and detailed descriptions of the condition already exist.11,23 59
The model presented herein provides a unique opportunity for scientists to investigate the ocular surface in health and disease. First, since topical histamine had no impact on the contralateral eye, the model is applicable for studies that compare efficacy between drug and placebo, allowing for precise measurements of a drug action given the rapid (1 min) and sustained (120–420 min) development of conjunctivitis. Second, the changes noted in tear fluid levels of TPC and albumin strongly suggest a breakdown of the blood–tear barrier. In particular, albumin represents a marker of vascular permeability and plasma leakage given it is not produced by the lacrimal gland or corneo-conjunctival tissue.20 Our findings are consistent with previous reports of conjunctivitis, whether experimentally induced or naturally occurring from dry eye,4 corneal ulcers, allergies, or others.20,24 The mechanism of histamine-induced disruption of the blood–tear barrier is unknown. Increased vascular permeability likely plays a role,6,12,25 combined with a disruption of tight junctions between conjunctival epithelial cells due to increased contractility of actin linked to these adhesion complexes.26 The breakdown of the blood–tear barrier could be exploited for assessing the impact of conjunctivitis on drug pharmacokinetics, tear film metabolomics, and other fields. In the field of pharmacology, for instance, the vast majority of studies to date are limited to healthy subjects with intact blood–tear barriers,27 28,29 in whom the lacrimal drug concentrations likely under-estimate the ones noted in actual clinical patients with ocular surface inflammation.30 Such discrepancies likely result in inappropriate dosing, thus affecting the drug efficacy and increasing the risk of toxicity or antimicrobial resistance.31
In addition to protein quantification, the present study investigated a panel of cytokines/chemokines/growth factors in tear samples of dogs. Similar to human subjects with ocular surface disease such as dry eye or vernal keratoconjunctivitis,32,33 an increase in VEGF A, 60
pro-inflammatory cytokines (IL-8, IL-12), and anti-inflammatory cytokine (IL-10) was detected
in tears of dogs with experimentally induced conjunctivitis. Analysis of other biomarkers such as
acidic mammalian chitinase34,35 would be beneficial in future studies, but the paucity of tear fluid
collected in each dog limited the number of compounds that could be analyzed.
The relatively low number of dogs enrolled in the histamine experiment is a clear
limitation of our work, although a post hoc power analysis showed that n = 5 dogs were
sufficient to detect significant differences between histamine doses for the main study outcome
(clinical severity of conjunctivitis). Furthermore, although the subjectivity of our clinical scoring
may be perceived as a drawback, given that photograph-based methods are mainstream in human
studies,36,37 the method described herein is purposely adapted to working with dogs. Indeed, i) a
handheld slit-lamp greatly facilitates examination of animals for whom the skull conformation
and behavior traits are poorly suited for table-mounted devices,18 and ii) the presence of the
nictitating membrane and the minimal bulbar conjunctival exposure in dogs require a “dynamic”
examination of the ocular surface that is not conducive to photographic scales. The ocular
adverse effects noted with high doses of histamine represent another potential limitation of the
proposed model. There was minimal to absent local irritation from all doses, likely favored by
adjusting the test solutions to more physiologic pH values, but histamine concentrations ≥375
mg/ml resulted in meaningful side effects in selected cases. A transient breakdown of the blood–
ocular barrier likely explains the anterior uveitis,25 while ocular hypertension maybe linked to the aforementioned acute uveitis and/or episcleral venous compression from the overlying swollen conjunctiva, causing an increased resistance to aqueous outflow.38,39 Future studies are required
to characterize these adverse effects in detail. Lastly, the study does not explain the mechanistic
reason of breakdown of the blood–tear barrier, as we mainly focused on documenting the safety 61 and clinical features of the model in dogs. Our group is now working on characterizing the biochemical and histological changes resulting from histamine-induced conjunctivitis in dogs.
In summary, histamine-induced conjunctivitis in dogs represents a robust and reliable model for translational research on the ocular surface. The model is particularly appealing given the low cost, non-invasiveness, self-resolving nature, ability to adjust the duration and severity of the disease, and shared features with naturally occurring diseases in human and veterinary medicine.
The model could be used to assess new therapeutics and to better understand the impact of conjunctivitis on drug pharmacokinetics–pharmacodynamics, tear film dynamics, and tear film composition, among many other applications.
References
1. Azari AA, Barney NP. Conjunctivitis: a systematic review of diagnosis and treatment. JAMA 2013;310:1721-1729.
2. Maggs DJ. Diseases of the Conjunctiva In: Maggs DJ, Miller PE,Ofri R, eds. Slatter's fundamentals of veterinary ophthalmology. 6th ed. St Louis, Missouri: Saunders, 2018.
3. Contreras-Ruiz L, Ghosh-Mitra A, Shatos MA, et al. Modulation of conjunctival goblet cell function by inflammatory cytokines. Mediators Inflamm 2013;2013:636812.
4. van Delft JL, Meijer F, van Best JA, et al. Permeability of blood-tear barrier to fluorescein and albumin after application of platelet-activating factor to the eye of the guinea pig. Mediators Inflamm 1997;6:381-383.
5. Zavaro A, Samra Z, Baryishak R, et al. Proteins in tears from healthy and diseased eyes. Doc Ophthalmol 1980;50:185-199.
6. van Bijsterveld OP, Janssen PT. The effect of calcium dobesilate on albumin leakage of the conjunctival vessels. Curr Eye Res 1981;1:425-430.
7. Janssen PT, Van Bijsterveld OP. Blood-tear barrier and tear fluid composition. The Precorneal Tear Film in Health, Disease and Contact Lens Wear, Holly FJ, editor Lubbock, TX, Dry Eye Institute 1986:471-475.
8. Li K, Liu X, Chen Z, et al. Quantification of tear proteins and sPLA2-IIa alteration in patients with allergic conjunctivitis. Mol Vis 2010;16:2084-2091. 62
9. Fullard RJ, Kaswan RM, Bounous DI, et al. Tear protein profiles vs. clinical characteristics of untreated and cyclosporine-treated canine KCS. J Am Optom Assoc 1995;66:397-404.
10. Shii D, Nakagawa S, Yoshimi M, et al. Inhibitory effects of cyclosporine a eye drops on symptoms in late phase and delayed-type reactions in allergic conjunctivitis models. Biol Pharm Bull 2010;33:1314-1318.
11. Lourenço-Martins AM, Delgado E, Neto I, et al. Allergic conjunctivitis and conjunctival provocation tests in atopic dogs. Vet Ophthalmol 2011;14:248-256.
12. Takahashi A, Sumi T, Tada K, et al. Evaluation of histamine-induced conjunctival oedema in guinea pigs by means of image analysis. Br J Ophthalmol 2010;94:1657-1661.
13. Branco ACCC, Yoshikawa FSY, Pietrobon AJ, et al. Role of Histamine in Modulating the Immune Response and Inflammation. Mediators Inflamm 2018;2018:9524075.
14. Vézina M. Comparative Ocular Anatomy in Commonly Used Laboratory Animals In: Weir AB,Collins M, eds. Assessing Ocular Toxicology in Laboratory Animals. Totowa, NJ: Humana Press, 2013;1-21.
15. Schmidl D, Schmetterer L, Garhöfer G, et al. Gender differences in ocular blood flow. Curr Eye Res 2015;40:201-212.
16. Sebbag L, McDowell EM, Hepner PM, et al. Effect of tear collection on lacrimal total protein content in dogs and cats: a comparison between Schirmer strips and ophthalmic sponges. BMC Vet Res 2018;14:61.
17. Uchio E, Kimura R, Migita H, et al. Demographic aspects of allergic ocular diseases and evaluation of new criteria for clinical assessment of ocular allergy. Graefes Arch Clin Exp Ophthalmol 2008;246:291-296.
18. Eaton JS, Miller PE, Bentley E, et al. The SPOTS System: An Ocular Scoring System Optimized for Use in Modern Preclinical Drug Development and Toxicology. J Ocul Pharmacol Ther 2017;33:718-734. 19. Sebbag L, Allbaugh RA, Strauss RA, et al. MicroPulseTM transscleral cyclophotocoagulation in the treatment of canine glaucoma: Preliminary results (12 dogs). Vet Ophthalmol 2018.
20. Runström G, Mann A, Tighe B. The fall and rise of tear albumin levels: a multifactorial phenomenon. Ocul Surf 2013;11:165-180.
21. Hagedoorn WG, Maas ER. The Effect of Histamine on the Rabbit's Cornea*. American Journal of Ophthalmology 1956;42:89-93.
63
22. Bonini S, Schiavone M, Centofanti M, et al. Conjunctival hyperresponsiveness to ocular histamine challenge in patients with vernal conjunctivitis. J Allergy Clin Immunol 1992;89:103- 107.
23. Bundoc VG, Keane-Myers A. Animal models of ocular allergy. Curr Opin Allergy Clin Immunol 2003;3:375-379.
24. de Freitas Campos C, Cole N, Van Dyk D, et al. Proteomic analysis of dog tears for potential cancer markers. Res Vet Sci 2008;85:349-352.
25. Ashton N, Cunha-Vaz J. Effect of histamine on the permeability of the ocular vessels. Arch Ophthalmol 1965;73:211-223.
26. Guo Y, Ramachandran C, Satpathy M, et al. Histamine-induced myosin light chain phosphorylation breaks down the barrier integrity of cultured corneal epithelial cells. Pharm Res 2007;24:1824-1833.
27. Hirosawa M, Sambe T, Uchida N, et al. Determination of nonsteroidal anti-inflammatory drugs in human tear and plasma samples using ultra-fast liquid chromatography-tandem mass spectrometry. Jpn J Ophthalmol 2015;59:364-371.
28. Sebbag L, Showman L, McDowell EM, et al. Impact of Flow Rate, Collection Devices, and Extraction Methods on Tear Concentrations Following Oral Administration of Doxycycline in Dogs and Cats. J Ocul Pharmacol Ther 2018;34:452-459.
29. Sebbag L, Thomasy SM, Woodward AP, et al. Pharmacokinetic modeling of penciclovir and BRL42359 in the plasma and tears of healthy cats to optimize dosage recommendations for oral administration of famciclovir. Am J Vet Res 2016;77:833-845.
30. Grumetto L, Cennamo G, Del Prete A, et al. Pharmacokinetics of cetirizine in tear fluid after a single oral dose. Clin Pharmacokinet 2002;41:525-531.
31. Craig WA. Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men. Clin Infect Dis 1998;26:1-10; quiz 11-12. 32. Leonardi A, Sathe S, Bortolotti M, et al. Cytokines, matrix metalloproteases, angiogenic and growth factors in tears of normal subjects and vernal keratoconjunctivitis patients. Allergy 2009;64:710-717.
33. Enríquez-de-Salamanca A, Castellanos E, Stern ME, et al. Tear cytokine and chemokine analysis and clinical correlations in evaporative-type dry eye disease. Mol Vis 2010;16:862-873.
34. Bucolo C, Musumeci M, Maltese A, et al. Effect of chitinase inhibitors on endotoxin-induced uveitis (EIU) in rabbits. Pharmacol Res 2008;57:247-252.
35. Musumeci M, Bellin M, Maltese A, et al. Chitinase levels in the tears of subjects with ocular allergies. Cornea 2008;27:168-173. 64
36. Schulze MM, Jones DA, Simpson TL. The development of validated bulbar redness grading scales. Optom Vis Sci 2007;84:976-983.
37. Macchi I, Bunya VY, Massaro-Giordano M, et al. A new scale for the assessment of conjunctival bulbar redness. Ocul Surf 2018.
38. Allbaugh RA, Roush JK, Rankin AJ, et al. Fluorophotometric and tonometric evaluation of ocular effects following aqueocentesis performed with needles of various sizes in dogs. Am J Vet Res 2011;72:556-561.
39. Carreon T, van der Merwe E, Fellman RL, et al. Aqueous outflow - A continuum from trabecular meshwork to episcleral veins. Prog Retin Eye Res 2017;57:108-133
Tables and Figures
Table 1. Patient information and tear albumin concentrations in the affected and unaffected eyes of dogs presented to the Ophthalmology service at Iowa State University’s Lloyd Veterinary Medical Center with various ocular diseases. * Cases 12 and 13 were examined twice: findings at the initial visit are shown in the 1st row while findings following treatment of the ocular disease are depicted in the 2nd row. yo = year old; FS = female spayed; MC = male castrated; FI = female intact.
Conjunctivitis score (affected Tear albumin concentration in Tear albumin concentration in Ratio affected vs. Case # Patient information Ocular disease eye / unaffected eye) affected eye (mg/mL) unaffected eye (mg/mL) unaffected eye
1 9 yo FS Yorkshire terrier Keratoconjunctivitis sicca 4 / 0 3.50 1.22 2.87 Corneal ulcer (superficial), 2 9 yo MC French Bulldog 6 / 1 2.44 0.80 3.05 Eyelid mass Corneal ulcer (superficial), 65 3 4 yo MC Shih Tzu 3 / 1 1.10 0.38 2.89 Distichia 6 yo MC English Spontaneous chronic 4 8 / 0 9.98 1.67 5.99 Bulldog corneal epithelial defect 5 12 yo FS Shih Tzu Corneal ulcer (stromal) 5 / 0 3.51 0.84 4.18 12 yo FS English Cataract, 6 5 / 1 3.51 0.94 3.72 Springer Spaniel Lens-induced uveitis 7 3 yo MC Border Collie Uveitis (blastomycosis) 7 / 0 13.96 3.06 4.57 Uveitis, 8 12 yo FS mixed breed 5/ 0 1.80 0.24 7.52 Glaucoma (secondary) 9 4 yo MC Japanese Chin Glaucoma (primary) 6 / 1 1.82 0.68 2.67
10 8 yo FS Beagle Orbital cellulitis 5 / 0 1.93 0.13 14.86 Glaucoma (primary), 11 7 yo FI Coonhound 6 / 0 17.95 1.72 10.42 end-stage
Spontaneous chronic 7 / 1 6.30 1.77 3.55 12 * 5 yo MC Pitbull corneal epithelial defect 1 / 1 1.74 1.74 1.0
6 / 0 1.52 0.42 3.60 13 * 4 yo FS Beagle Uveitis (blastomycosis) 2 / 0 0.37 0.34 1.09 66
Figure 1. A comprehensive evaluation of the canine conjunctiva is facilitated by examination of the ocular surface at different angles, including afront view (A), side view (B), top view (C), and globe retropulsion with lower lid retraction (D). The latter permits visualization of the palpebral conjunctiva and nictitating membrane.
Figure 2. Photographs of the left eye in a dog, 30 min following topical administration of 10 mg/ml histamine solution. The overall conjunctivitis score was 6, based on the absence/presence and degree of follicles, hyperemia, and chemosis of both palpebral and bulbar conjunctiva, conjunctival discharge, and ocular pruritus.
67
Figure 3. Development and progression of conjunctivitis in a representative canine eye receiving topical 375 mg/ml histamine solution. Conjunctivitis developed rapidly (< 1 min), with progression of conjunctival hyperemia and chemosis for 20–30 min, and subsequent slow improvement and self-resolution by 420 min.
68
Figure 4. (A) Graphs depicting the mean + SD of conjunctivitis score over time in dogs receiving H1 (0.005 mg/ml; white circles), H2
(0.1 mg/ml; black circles), H3 (1.0 mg/ml; white triangles), H4 (10 mg/ml; black triangles), H5 (375 mg/ml; white squares), and H6 (500 mg/ml; black squares). Within the same dose, a blue asterisk (*) depicts statistical significance of conjunctivitis scoring compared to baseline (for readability, only the first and last significant time points are depicted). (B) Box-and-whiskers plots depicting the area under the curve of conjunctivitis score from 0 to 180 min in dogs receiving topical histamine of various concentrations. Mean and median values are shown by horizontal dotted and solid lines, respectively. First and third quartiles (25th and 75th percentiles) are represented by the lower and upper limits of the box, respectively, while the 2.5th and the 97.5th percentiles are shown as the lower and upper whiskers, respectively. The conjunctivitis severity of histamine doses with different letters differ significantly (P < 0.05). 69
Figure 5. Blepharitis (A) and miosis (B) in a dog that received high dose of histamine ophthalmic solution (500 mg/ml).
Figure 6. Bar charts depicting the total protein content (A, B) and serum albumin levels (C, D) in tears of six beagle dogs receiving vehicle or histamine eyedrops. An asterisk (*) indicates statistical significance (P < 0.05) between vehicle and histamine (A, C) or among histamine doses (B, D). 70
Figure 7. Bar charts depicting mean + SD of various chemokines, cytokines and growth factors quantified in tears of six beagle dogs receiving vehicle (vehicle) or histamine solutions: interleukin-6 (IL-6; A), interleukin-8 (IL-8; B), interleukin-10 (IL-10; C), interleukin-12 (IL-12; D), vascular endothelial growth factor A (VEGF A; E), tumor necrosis factor alpha (TNFα; F), stem cell factor (SCF; 1.3 ± 4.3 pg/ml; G), and chemokine monocyte chemoattractant protein-1 (MCP-1; H). Differences among groups are depicted by an asterisk (*) if statistically significant (P < 0.05). 71
Figure 8. Clinical photographs of canine eyes from patients presented to the Ophthalmology Service at Iowa State University’s Lloyd Veterinary Medical Center. Conjunctivitis is present in all eyes, a condition noted concurrently to various ocular diseases. Patient case numbers are listed in the top left corner of each photograph (see Table 1 for additional patient information).
72
Figure 9. Clinical photographs of a 7-year-old female intact Coonhound dog (A) diagnosed with end-stage glaucoma in the right eye (case #11). Tear concentrations of albumin were much higher in the affected right eye (B) compared to the contralateral healthy left eye (C).
Figure 10. Clinical photographs of a 5-year-old male castrated Pitbull (A, B) diagnosed with a spontaneous chronic corneal epithelial defect (case #12) and a 4-year-old female spayed Beagle (C, D) diagnosed with uveitis secondary to blastomycosis (case #13). Following treatment of each ocular disease (B, D), the severity of conjunctivitis was reduced and the concentration of albumin in tears was lower compared to the initial visit (A, C). 73
Figure 11. Representative clinical pictures of mild conjunctivitis (A; score = 3), moderate conjunctivitis (B; score = 6), and severe conjunctivitis (C; score = 9) following topical administration of histamine in dogs. 74
CHAPTER 5. IMPACT OF ACUTE CONJUNCTIVITIS ON OCULAR SURFACE HOMEOSTASIS IN DOGS
Lionel Sebbag,1,2 Elizabeth Soler,1 Rachel A. Allbaugh,1 Jonathan P. Mochel2
1 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa
State University, Ames, IA 50011, USA; 2 Department of Biomedical Sciences, College of
Veterinary Medicine, Iowa State University, Ames, IA 50011, USA
Modified from a manuscript under review in Veterinary Ophthalmology
Abstract
Objective: To investigate the effects of acute conjunctivitis on tear film characteristics and corneal sensitivity in dogs. Animals studied: Eight female spayed Beagle dogs (1.5-2 years old, 7.5-10 kg). Procedures: On two consecutive days, one randomly selected eye in each dog received 1 or 375 mg/mL histamine solution to induce mild or severe conjunctivitis, while the contralateral eye served as control. Diagnostic tests were performed in the following order: fluorescein instillation and repeated tear collection over 20 minutes (to determine tear volume
[TV] and turnover rate [TTR] by fluorophotometry), Schirmer tear test-1 (STT-1), tear ferning, corneal esthesiometry and tear film breakup time (TFBUT). Results: Results are presented as median values for severe conjunctivitis, mild conjunctivitis, and control eyes. Eyes with severe conjunctivitis had significantly higher STT-1 (24, 19.5, 17.5 mm/min) and significantly lower
TFBUT (10.5, 13.5, 15.5 sec), but no changes were noted in corneal tactile sensation (2, 2.5, 2.5 cm) or tear ferning (grades 2, 2, 2.5). Severe conjunctivitis significantly increased TV by nearly
10-fold (631, 97, 65 µL) initially (reflex tearing), although basal TV returned rapidly (<5 minutes) in all eyes (46, 58, 48 µL). Finally, there was a non-significant trend for higher reflex
TTR in the conjunctivitis vs. control eyes (68, 58, 43 %/min). Conclusions: Acute conjunctivitis 75 increases tear quantity and decreases tear quality in dogs, but has no impact on corneal sensitivity. Changes in tear film dynamics could affect ocular pharmacology (e.g. precorneal retention time), although homeostasis of lacrimal volume and drainage is rapidly restored.
Introduction
A thin layer of tears covers the ocular surface and serves vital functions such as lubrication, nutrition, removal of debris, and defense against microbes. Thus, ocular surface health is compromised when tear film homeostasis is disturbed, as noted with changes in tear quantity, tear quality, and tear film dynamic.1,2 Quantitative and qualitative tear film deficiencies are well recognized in humans and veterinary species,3,4 but changes in tear film dynamics (i.e. production/distribution/drainage) could also play an important role in the pathophysiology of ocular surface diseases. For instance, excess lacrimal production dilutes the levels of endogenous
(e.g. IgA) and exogenous (e.g. medication) compounds in tears,5 while faster drainage reduces their contact time with the ocular surface.6 Equally important, diseases of the ocular surface such as blepharitis and conjunctivitis often result in, or exacerbate tear film instability.3,7,8
Conjunctivitis is a common ocular surface disease and is known to lower tear film stability in many species, including humans,9 dogs10,11 and cats.12,13 Primarily, the loss of tear film homeostasis results from altered mucin secretion onto the ocular surface by the conjunctival goblet cells.14 However, the relationship between conjunctivitis and the tear film is more complex and also involves neurosensory stimulation from ocular irritation,15,16 changes in tear volume, and variations in tear clearance.9,17,18
In the present study, conjunctivitis was experimentally induced in dogs and a series of diagnostic tests were conducted to assess tear quantity, tear quality, tear dynamics, and corneal sensitivity. A deeper understanding of the interaction between conjunctivitis and tear film will 76
help clinicians break the vicious cycle whereby ocular surface disease leads to tear film
instability, which subsequently exacerbates inflammation, and so forth.3
Materials and Methods
Animals
Eight Beagle dogs were enrolled in the study. All dogs were female spayed, aged 1.5-2.0
years, weighed 7.5-10 kg, and confirmed to be healthy based on a complete physical and
ophthalmic examination, including Schirmer tear test-1 (STT-1; Eye Care Product
Manufacturing LLC, Tucson, AZ, USA), tonometry (TonoVet, Icare Finland Oy, Espoo,
Finland), slit-lamp biomicroscopy (SL-17; Kowa Company, Ltd., Tokyo, Japan) and indirect ophthalmoscopy (Keeler Vantage; Keeler Instruments, Inc., Broomall, PA, USA). The study was approved by the Institutional Animal Care and Use Committee of Iowa State University.
Procedures
A series of procedures were conducted in both eyes of each dog in a specific order, as described in Figure 1. All measurements were completed in the morning (to reduce diurnal variability), in the same examination room under controlled temperature (70-72°F) and ambient humidity (25-30%).
• Induction of conjunctivitis: Histamine powder (Histamine dihydrochloride, FCC grade,
Arcos® organics, Geel, Belgium) was sterily mixed with 1.4% polyvinyl alcohol lubricating
eye drops (Artificial tears solution, Rugby, Rockville Center, NY, USA) to compound 1
mg/mL and 375 mg/mL ophthalmic solutions.19 A single drop (35 µL)20 of histamine
solution was applied onto one randomly selected eye of each dog (Excel software, Microsoft
Corp., Redmond, WA), while the other eye received artifical tears and served as control. 77
Both mild (1 mg/mL) and severe (375 mg/mL) conjunctivitis were induced in the selected
eye, one day apart, the order of which was randomized for each dog (Excel software).
• Fluorophotometry: Tear film fluorophotometry was performed in each eye as previously
described.21 Briefly, 2 µL of 10% fluorescein (Akorn Inc., Buffalo Grove, IL, USA) was
instilled onto the dorsal bulbar conjunctiva using a pipette, followed by 3 manual blinks (0
minutes) and tear collection with 2-µL capillary tubes (Drummond Scientific Co.,
Broomhall, PA) at times 0, 2, 4, 6, 10, 15 and 20 minutes. Fluorescein concentrations were
measured in each sample with a computerized scanning ocular fluorophotometer (Fluorotron
MasterTM, Coherent Radiation, Palo Alto, CA), and tear fluorescence was recorded in ng/mL.
• Schirmer tear test-1 (STT-1): A Schirmer strip (Eye Care Product Manufacturing LLC,
Tucson, AZ, USA) was placed in the ventrolateral conjunctival fornix of each eye, and tear
quantity was recorded at 1 minute (mm/min) using a stopwatch. Strips were left until the ≥
20-mm mark was reached, followed by centrifugation for 2 minutes at 3,884 g in a punctured
0.2-mL tube (secured to 2-mL tube) to extract tear fluid for the ferning test.22
• Tear ferning test: For each sample, 2 µL of tear fluid was deposited on a glass slide inside
a 2-mm circular area delineated with a marking pen. Following air drying at room
temperature for 10 minutes,23 each sample was examined with light microscopy (10 X
magnification) and classified as grade 1, grade 2, grade 3, or grade 4 according to specific
criteria described by Oria and colleagues.24
• Corneal sensitivity: A nylon filament (0.12 mm, Cochet-Bonnet esthesiometer, Luneau
Ophtalmologie, Chartres, France) was extended to 6 cm and advanced until it contacted the
central cornea, creating a slight deflection in the filament. The filament length was shortened 78
by 0.5-cm increments until a blink response was consistently noted in at least 3 out of 5
attempts, recorded in cm as the corneal tactile sensation (CTS).25
• Tear film breakup time (TFBUT): Fluorescein 10% solution was diluted 1:5 with eyewash
(OCuSOFT® eye wash, OCuSOFT Inc., Richmond, TX), and 5-µL of the resulting 2%
fluorescein solution was instilled onto the dorsal bulbar conjunctiva of each eye.26 After 3
manual blinks, the eyelids were kept open and the dorsotemporal corneal surface was
observed at 16X magnification with a cobalt blue filter (SL-17). The TFBUT was recorded
with a stopwatch (to the nearest tenth of a second) as the time from eyelid opening to the
appearance of ≥ 1 dark spot(s) within the fluorescent green tear film. The average of 2
measurements per eye was used for data analysis.
Data analysis
Tear volume (TV) and tear turnover rate (TTR) were estimated based upon nonlinear mixed-effect analysis of fluorescein decay curves in tear samples (Monolix® version 2018R2;
Lixoft, Orsay, France).21,27,28 The biphasic decay of fluorescein allowed for calculation of reflex
(first phase) and basal (second phase) values for both tear parameters. Normality of data was
assessed with the Shapiro-Wilk test. For all parameters, differences among control vs. mild conjunctivitis vs. severe conjunctivitis eyes were assessed with the Kruskal-Wallis test and post hoc Dunn’s. Statistical analysis was performed using SigmaPlot 14.0 (Systat Software Inc., San
Jose, CA, USA), and P values < 0.05 were considered statistically significant.
Results
Results are presented below as median (interquartile range) as the data from tear film diagnostic tests and fluorophotometry were not normally distributed. 79
No statistical differences (P = 0.582) were noted in baseline values of STT-1 between eyes selected for control [19 (4.8) mm/min], mild conjunctivitis [20 (8.8) mm/min] and severe conjunctivitis [20 (2) mm/min]. However, when STT-1 was repeated 20 min after eyedrop administration (Fig. 1), tear values were higher in eyes with conjunctivitis [mild = 19.5 (5.5) mm/min; severe = 24 (3.3) mm/min] compared to control eyes [15.5 (9.8) mm/min], with statistical differences noted between control and severe conjunctivitis groups (P = 0.002) (Fig.
2A).
Differences in tear production were also noted with fluorophotometry data (Table 1).
Compared to control, median reflex TV was 1.5-fold and 9.7-fold higher in eyes with mild and moderate conjunctivitis, respectively, a difference that was statistically significant for the severe conjunctivitis group (P = 0.015). However, no statistical differences were noted among groups for basal TV (P = 0.965), basal TTR (P = 0.402) or reflex TTR (P = 0.201).
While tear quantity increased with conjunctivitis, the tear quality was reduced (Fig. 2B).
Median (interquartile range) TFBUT was lower in eyes with conjunctivitis [mild = 13.5 (2.6) seconds; severe = 10.5 (2.3) seconds] compared to control eyes [15.5 (5.1) seconds], with statistical differences noted between control and severe conjunctivitis groups (P = 0.002). Lastly, tear ferning (Fig. 2C) and corneal sensitivity (Fig. 2D) were not affected by conjunctivitis (P ≥
0.322).
Discussion
The present study demonstrates that acute conjunctivitis increases tear quantity and decreases tear quality in dogs, but has no significant impact on corneal sensitivity or tear ferning.
These results provide useful information for clinicians who manage canine patients with an acute injury to the ocular surface (e.g. corneal ulcer, foreign body or chemical burn with secondary 80 conjunctivitis), and for basic scientists who utilize histamine-induced conjunctivitis as a model to investigate ocular pharmacology and therapeutics.19
It is well established that corneal irritation causes reflex tearing across species,29-32 with greater corneal nociceptive stimulation leading to greater tear production.32 In contrast, little is known about the impact of conjunctival irritation on lacrimal secretion. Here, we showed that tear production was increased in the presence of conjunctivitis in dogs – irrespective of corneal stimulation (no changes noted in corneal tactile sensation) – as demonstrated by STT-1 values and TV calculation (fluorophotometry). In fact, the quantity of tears increased with the severity of conjunctivitis, a finding likely explained by increasing noxious stimulation to the conjunctival nerves that contribute to the afferent pathway of lacrimation, activating the efferent parasympathetic and sympathetic nerves to the lacrimal gland.33 Similar findings were noted in humans with acute catarrhal conjunctivitis9 and in cats with experimentally induced herpetic conjunctivitis, in whom STT-1 doubled (from 8 to 16 mm/min) concurrently with conjunctival disease scoring.29 Further, the amplitude of STT-1 increase with conjunctivitis (15.5 to 19.5-24 mm/min) is similar to changes noted in the presence of corneal ulceration; Williams and Burg showed that dogs with a unilateral corneal ulcer had significantly greater STT-1 values in the affected eye compared to the non-ulcerated fellow eye (20.2 vs. 16.7 mm/min, respectively).30
Importantly, the present study demonstrates the tremendous capacity of the canine ocular surface to restore tear homeostasis in a rapid manner. Indeed, the volume of tears quickly stabilized (<5 min) after induction of conjunctivitis, despite ongoing conjunctival inflammation
(lasting ≥ 1h with topical histamine),19 and no significant differences were noted in basal TV among control, mild conjunctivitis or severe conjunctivitis eyes. 81
This finding could be explained by rapid increase in the drainage of tears through the nasolacrimal duct, as shown in rabbit eyes that were instilled various volumes of eyedrops.6
However, tear drainage (assessed with TTR) did not differ significantly in dogs with or without conjunctivitis (present study), nor did it change in a recent report of dogs receiving artificial tears at a volume of 10 to 100 µL.27 Discrepancies between rabbits and dogs are likely due to notable differences in blinking rates (3-6 blinks/h vs. 14.2 blinks/min, respectively),34,35 an important physiologic response that promotes spillage of excessive tearing onto the periocular skin.27
The quality of tears, or tear film stability, was reduced in canine eyes with acute conjunctivitis. Shortened TFBUT is an established feature of dogs,11 cats29 and humans9 with chronic conjunctivitis, as chronicity of inflammation reduces the density of goblet cells that secrete pre-ocular mucins, although this finding is not well documented in eyes with acute conjunctivitis. The authors hypothesize that excessive aqueous secretion onto the ocular surface
(reflex tearing) changes the relative abundance of lipids and mucins in the tear film, resulting in faster tear evaporation and shorter TFBUT, although rapid loss of conjunctival goblet cells could also be an explanation; future studies assessing tear composition and conjunctival impression cytology are therefore needed to support that hypothesis. Regardless of the underlying cause, a short TFBUT is important to recognize in practice as it can participate to ocular discomfort experienced by the patient, necessitating mucinomimetic supplementation to relieve irritation and prevent exacerbation of conjunctival inflammation;3,29 in fact, rapid TFBUT results in the same degree of ocular discomfort than patients with aqueous tear deficiency, likely due to development of neuropathic pain.16,36
Evaluation of tear ferning can provide valuable information in patients with ocular surface disease.37 In fact, a recent study showed that all dogs with keratoconjunctivitis sicca had 82
abnormal ferning patterns (grades 3-4) while the majority of healthy canine eyes had a normal
ferning pattern.38 In contrast, our study did not find significant differences in tear ferning
between control eyes and eyes with acute conjunctivitis. This finding may be physiologically
true, although it could also be skewed by the low sample size, possible technical issues (e.g.
inconsistent drying times among samples), relative inexperience of the investigators with tear
ferning, or lack of standardization of grading. The latter was addressed in a recent equine study
by using a computerized stereology tool to characterize the tear crystallization in an objective
manner.23
The present study is limited to the short-term effects of conjunctivitis on ocular surface
homeostasis, in a relatively small population of dogs of a single breed. Topical histamine
provides a robust method to induce conjunctivitis in a rapid and non-invasive manner in dogs,19
however the duration of conjunctivitis is limited in time as the disease is self-resolving (average
duration of 115 and 390 min with 1 mg/mL and 375 mg/mL solutions, respectively).19 One could
consider repeating administration of topical histamine to prolong the duration of conjunctivitis,
as recently performed in a pharmacokinetic study of prednisone in dogs,39 although the safety
profile of consistent extended dosing with histamine is currently unknown. Nevertheless, the
present study shows that conjunctivitis alone is enough to activate the lacrimal functional unit
and cause reflex tearing, irrespective of noxious stimulation to the cornea, and that the
homeostasis of the ocular surface is rapidly restored. This is important because tear film
disturbances noted with acute conjunctivitis likely affect the comfort level of canine patients, and
might justify the use of hyaluronic acid-based lacrimomimetics to improve tear stability and reduce discomfort; further, the kinetics of solutions delivered to the ocular surface might be 83
affected in the short-term (e.g. dilution factor, precorneal retention time), influencing the
bioavailability of topical drugs.
References
1. Khanal S, Tomlinson A, Diaper CJ. Tear physiology of aqueous deficiency and evaporative dry eye. Optom Vis Sci 2009;86:1235-1240.
2. Craig JP, Nelson JD, Azar DT, et al. TFOS DEWS II report executive summary. Ocul Surf 2017;15:802-812.
3. Rolando M, Zierhut M. The ocular surface and tear film and their dysfunction in dry eye disease. Surv Ophthalmol 2001;45 Suppl 2:S203-210.
4. Ribeiro AP, Brito FLdC, Martins BdC, et al. Qualitative and quantitative tear film abnormalities in dogs. Ciência Rural 2008;38:568-575.
5. Fullard RJ, Tucker DL. Changes in human tear protein levels with progressively increasing stimulus. Invest Ophthalmol Vis Sci 1991;32:2290-2301.
6. Chrai SS, Patton TF, Mehta A, et al. Lacrimal and instilled fluid dynamics in rabbit eyes. J Pharm Sci 1973;62:1112-1121.
7. Tseng SC, Tsubota K. Important concepts for treating ocular surface and tear disorders. Am J Ophthalmol 1997;124:825-835.
8. Dartt DA, Willcox MD. Complexity of the tear film: importance in homeostasis and dysfunction during disease. Exp Eye Res 2013;117:1-3.
9. Khurana AK, Moudgil SS, Parmar IP, et al. Tear film flow and stability in acute and chronic conjunctivitis. Acta Ophthalmol (Copenh) 1987;65:303-305.
10. Moore CP. Qualitative tear film disease. Vet Clin North Am Small Anim Pract 1990;20:565- 581.
11. Moore C, L C. Ocular surface disease associated with loss of conjunctival goblet cells in dogs. J Am Anim Hosp Assoc 1990;26:458-466.
12. Lim CC, Cullen CL. Schirmer tear test values and tear film break-up times in cats with conjunctivitis. Vet Ophthalmol 2005;8:305-310.
13. Sebbag L, Pesavento PA, Carrasco SE, et al. Feline dry eye syndrome of presumed neurogenic origin: a case report. JFMS Open Rep 2018;4:2055116917746786.
84
14. Mantelli F, Argüeso P. Functions of ocular surface mucins in health and disease. Curr Opin Allergy Clin Immunol 2008;8:477-483.
15. Mcmonnies CW. The potential role of neuropathic mechanisms in dry eye syndromes. J Optom 2017;10:5-13.
16. Zhang J, Begley CG, Situ P, et al. A link between tear breakup and symptoms of ocular irritation. Ocul Surf 2017;15:696-703.
17. Prabhasawat P, Tseng SC. Frequent association of delayed tear clearance in ocular irritation. Br J Ophthalmol 1998;82:666-675.
18. Chang SW, Chang CJ. Delayed tear clearance in contact lens associated papillary conjunctivitis. Curr Eye Res 2001;22:253-257.
19. Sebbag L, Allbaugh RA, Weaver A, et al. Histamine-induced conjunctivitis and breakdown of blood-tear barrier in dogs: A model for ocular pharmacology and therapeutics. Front Pharmacol 2019;10:752.
20. Oriá AP, Rebouças MF, Martins Filho E, et al. Photography-based method for assessing fluorescein clearance test in dogs. BMC Vet Res 2018;14:269.
21. Sebbag L, Allbaugh RA, Wehrman RF, et al. Fluorophotometric assessment of tear volume and turnover rate in healthy dogs and cats. J Ocul Pharmacol Ther 2019;35:497-502.
22. Sebbag L, Harrington DM, Mochel JP. Tear fluid collection in dogs and cats using ophthalmic sponges. Vet Ophthalmol 2018;21:249-254.
23. Silva LR, Gouveia AF, de Fátima CJ, et al. Tear ferning test in horses and its correlation with ocular surface evaluation. Vet Ophthalmol 2016;19:117-123.
24. Oriá AP, Raposo ACS, Araújo NLLC, et al. Tear ferning test in healthy dogs. Vet Ophthalmol 2018;21:391-398.
25. Sebbag L, Crabtree EE, Sapienza JS, et al. Corneal hypoesthesia, aqueous tear deficiency, and neurotrophic keratopathy following micropulse transscleral cyclophotocoagulation in dogs. Vet Ophthalmol 2019 (Epub ahead of print).
26. Abelson MB, Ousler GW, Nally LA, et al. Alternative reference values for tear film break up time in normal and dry eye populations. In: Sullivan DA, Stern ME, Tsubota K, et al., eds. Lacrimal Gland, Tear Film, and Dry Eye Syndromes 3: Basic Science and Clinical Relevance Part A and B. Boston, MA: Springer US, 2002;1121-1125.
27. Sebbag L, Kirner N, Allbaugh R, et al. Kinetics of fluorescein in tear film after eye drop instillation in Beagle dogs: Does size really matter? Front Vet Sci 2019
85
28. Pelligand L, Soubret A, King JN, et al. Modeling of large pharmacokinetic data using nonlinear mixed-effects: A paradigm shift in veterinary pharmacology. A case study with robenacoxib in cats. CPT Pharmacometrics Syst Pharmacol 2016;5:625-635.
29. Lim CC, Reilly CM, Thomasy SM, et al. Effects of feline herpesvirus type 1 on tear film break-up time, Schirmer tear test results, and conjunctival goblet cell density in experimentally infected cats. Am J Vet Res 2009;70:394-403.
30. Williams DL, Burg P. Tear production and intraocular pressure in canine eyes with corneal ulceration. Open Vet J 2017;7:117-125.
31. Knickelbein KE, Scherrer NM, Lassaline M. Corneal sensitivity and tear production in 108 horses with ocular disease. Vet Ophthalmol 2018;21:76-81.
32. Situ P, Simpson TL. Interaction of corneal nociceptive stimulation and lacrimal secretion. Invest Ophthalmol Vis Sci 2010;51:5640-5645.
33. Dartt DA. Neural regulation of lacrimal gland secretory processes: relevance in dry eye diseases. Prog Retin Eye Res 2009;28:155-177.
34. Maurice D. The effect of the low blink rate in rabbits on topical drug penetration. J Ocul Pharmacol Ther 1995;11:297-304.
35. Carrington SD, Bedford PGC, Guillon JP, et al. Polarized light biomicroscopic observations on the pre-corneal tear film. 1. The normal tear film of the dog. J Small Anim Pract 1987;28:605- 622.
36. Tsubota K. Short tear film breakup time-type dry eye. Invest Ophthalmol Vis Sci 2018;59:DES64-DES70.
37. Masmali AM, Purslow C, Murphy PJ. The tear ferning test: a simple clinical technique to evaluate the ocular tear film. Clin Exp Optom 2014;97:399-406.
38. Williams D, Hewitt H. Tear ferning in normal dogs and dogs with keratoconjunctivitis sicca. Open Vet J 2017;7:268-272.
39. Sebbag L, Yan Y, Smith JS, et al. Tear fluid pharmacokinetics following oral prednisone administration in dogs with and without conjunctivitis. J Ocul Pharmacol Ther 2019;35:341- 349.
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Tables and Figures
Table 1. Median (interquartile range) of basal and reflex tear film dynamics in healthy eyes (control) and eyes with acute conjunctivitis. TV = Tear volume; TTR = Tear turnover rate. † Kruskal-Wallis test. * Significant difference between control and severe conjunctivitis eyes (post hoc Dunn’s test).
Mild Severe Control P value † conjunctivitis conjunctivitis
Basal TV (µL) 48.5 (121.2) 53.7 (73.1) 46.1 (3.8) 0.965
Reflex TV (µL) 65.1 (314.5) 97.2 (257.3) 630.8 (891.3)* 0.015
Basal TTR 5.8 (10.2) 7.0 (4.9) 3.9 (6.1) 0.402 (%/min) Reflex TTR 43.2 (46.4) 57.7 (32.9) 65.7 (30.8) 0.201 (%/min)
Figure 1. Flow chart of the ophthalmic procedures performed in 8 healthy Beagle dogs, before and after experimental induction of conjunctivitis with topical histamine. STT-1 = Schirmer tear test-1; TFBUT = Tear film breakup time.
87
Figure 2. Box-and-whiskers plots depicting test results of Schirmer tear test-1 (A), tear film breakup time (B), tear ferning (C) and corneal tactile sensation (D) in 8 healthy Beagle dogs receiving artificial tears (control, white), 1 mg/mL ophthalmic histamine (mild conjunctivitis, light gray) or 375 mg/mL ophthalmic histamine (severe conjunctivitis, dark gray). Each plot represents the mean (dashed line), median (solid line), 2.5th percentile (lower whisker), 25th percentile (lower limit of box), 75th percentile (upper limit of box), and 97.5th percentile (upper whisker).
88
CHAPTER 6. TEAR FLUID PHARMACOKINETICS FOLLOWING ORAL PREDNISONE ADMINISTRATION IN DOGS WITH OR WITHOUT CONJUNCTIVITIS
Lionel Sebbag,1,2 Yuqi Yan,3 Joe S. Smith,2,4 Rachel A. Allbaugh,1 Larry W. Wulf,5
Jonathan P Mochel2
1 Department of Veterinary Clinical Sciences, Iowa State University, College of Veterinary
Medicine, Ames, IA 50011; 2 Department of Biomedical Sciences, Iowa State University, College
of Veterinary Medicine, Ames, IA 50011; 3 Lloyd Veterinary Medical Center, Iowa State
University, College of Veterinary Medicine, Ames, IA 50011; 4 Department of Veterinary
Diagnostic & Production Animal Medicine, Iowa State University, College of Veterinary
Medicine, Ames, IA 50011; 5 PhAST Laboratory, Iowa State University, College of Veterinary
Medicine, Ames, IA 50011.
Modified from a manuscript published in the Journal of Ocular Pharmacology and
Therapeutics
Abstract
Purpose: To describe the pharmacokinetics of prednisone and prednisolone in tear fluid
of dogs receiving oral prednisone at anti-inflammatory to immunosuppressive doses, and to
assess the impact of induced conjunctivitis on lacrimal drug levels. Methods: Six healthy Beagle
dogs were administered 4 courses of prednisone at 0.5, 1.0, 2.0 and 4.0 mg/kg given orally once
a day for 5 days. At steady state, topical histamine was applied to induce mild (1 mg/mL) or
severe (375 mg/mL) conjunctivitis in one eye of each dog and tear samples were collected from
both eyes at selected times. Prednisone and prednisolone were quantified in tears by liquid
chromatography-mass spectrometry. Results: Lacrimal prednisone and prednisolone 89
concentrations ranged from 2-523 ng/mL and 5-191 ng/mL, respectively. Drug concentrations
were overall greater in dogs receiving higher doses of prednisone, but were not correlated with
tear flow rate. Eyes with conjunctivitis often had larger amounts of prednisone and prednisolone
in tear fluid compared to control eyes (up to +64%), but differences were not statistically
significant. Significantly greater, but clinically insignificant, levels of prednisolone were found
in eyes with severe vs. mild conjunctivitis for oral prednisone doses ≥ 1.0 mg/kg. Conclusions:
Disruption of the blood-tear barrier with conjunctivitis did not significantly affect drug levels in tears. Based on drug pharmacokinetics in tears, oral prednisone is likely safe for the management of reflex uveitis and ocular surface diseases. However, further prospective trials using systemic corticotherapy in diseased animals are warranted to confirm findings from this preclinical study.
Introduction
Prednisone is a corticosteroid with a wide range of pharmacological indications that is commonly used for the treatment of inflammatory and immune-mediated diseases in human and veterinary medicine. In ophthalmology, corticosteroids can alleviate ocular inflammation and help prevent devastating sequelae that could be painful or vision threatening.1 Of the various
routes of administration, systemic therapy is generally recommended when the target tissue
cannot be reached with topical ophthalmic corticosteroids (e.g. eyelids, posterior segment, orbit),
or as a complement to topical medications in cases of anterior uveitis.2
Systemically administered medications readily distribute to the vascular tissues of the
eye, but can also affect the ocular surface if the drug reaches the tear compartment. For instance,
oral doxycycline can be used as adjunctive therapy for keratomalacia in dogs and horses,3,4 while oral famciclovir is highly effective in managing herpetic keratoconjunctivitis in cats.5,6 For oral prednisone, detection of steroid levels in the tear film could support the use of systemic 90
corticotherapy for adjunctive treatment of inflammatory diseases such as chronic superficial
keratitis, immune-mediated keratitis, and keratoconjunctivitis sicca. Conversely, lacrimal levels
of corticosteroids could inhibit corneal wound healing and potentiate infection.7,8 This therapeutic dilemma is exemplified in patients with ulcerative keratitis and concurrent reflex uveitis: systemic steroids are superior to nonsteroidal anti-inflammatory medications for controlling severe uveitis and preventing devastating sequelae,9 yet ulceration could worsen and
result in corneal perforation if wound healing is compromised and infection is potentiated.
The main goal of the study was to describe the PK of prednisone and its active metabolite
prednisolone in tear fluid of dogs following oral administration at doses ranging from anti-
inflammatory to immunosuppressive use (0.5 to 4 mg/kg/d). We hypothesized that prednisone
and prednisolone would be quantifiable in canine tear fluid and concentrations would be greater
with increasing oral dosing. In an effort to make the findings of this study more clinically
relevant, a secondary objective was to determine the impact of conjunctivitis on drug
concentrations in tears. Indeed, conjunctivitis, a common bystander of most ocular diseases,
increases conjunctival vascular permeability and therefore enhances vascular leakage of plasma
compounds onto the ocular surface.10,11 We hypothesized that tear concentrations would be
greater in eyes with conjunctivitis (i.e. compromised blood-tear barrier) compared to healthy
eyes.
Materials and Methods
Animals
Six Beagle dogs were included in the study. All were spayed females of 1.5-2 years old
and weighing 7.5-10 kg. Prior to study inclusion, dogs were confirmed to be healthy based on
physical and ophthalmic examination, complete blood count, serum chemistry and urinalysis. 91
The study was approved by the Institutional Animal Care and Use Committee of Iowa State
University, and adhered to the Association for Research in Vision and Ophthalmology (ARVO)
statement for the Use of Animals in Ophthalmic and Vision Research.
Procedures
Over a period of 2 months, all dogs received 4 successive dosing regimens of oral
prednisone (CadistaTM predniSONE tablets, Jubilant Cadista Pharmaceuticals Inc., Salisbury,
MD, USA), characterized by 5 days of drug administration interrupted by 9 days washout period, which included: (i) 0.5 mg/kg once daily for 5 days; (ii) 1.0 mg/kg once daily for 5 days; (iii) 2.0 mg/kg once daily for 5 days; and (iv) 4.0 mg/kg once daily for 5 days. The following procedures were performed on Day 4 of each dosing regimen, a time chosen to ensure steady state drug levels were reached:12
• Induction of conjunctivitis in one eye: Histamine ophthalmic solutions were formulated by
mixing histamine powder (Histamine dihydrochloride, FCC grade, Arcos® organics, Geel,
Belgium) with 1.4% polyvinyl alcohol lubricating eye drops (Artificial tears solution, Rugby,
Rockville Center, NY, USA) in a sterile manner under a laminar flow hood. Twenty minutes
prior to prednisone administration, a single drop of histamine solution was applied to one
randomly selected eye in each dog, while the other eye received artificial tears (Control).
This ocular selection was kept constant throughout the study. Histamine rapidly induced
conjunctivitis (<1 min) that was either mild (n = 3 dogs, 1.0 mg/mL histamine solution;
Figure 1A) or severe (n = 3 dogs, 375 mg/mL histamine solution; Figure 1B), as previously
described.11 Conjunctivitis was maintained throughout the 12 h collection period by repeating
topical histamine administration every 1-4 h as needed to maintain effect. Topical 0.035%
ketotifen fumarate (Zaditor®, Novartis Pharmaceuticals Corporation, East Hanover, NJ, 92
USA) was instilled onto each eye at the end of the day to control any residual conjunctival
swelling.
• Tear collection: Tear fluid was sampled simultaneously in both eyes before prednisone
administration (t = 0 min) and at 15, 30, 60, 90, 120, 240, 480, and 720 min following drug
administration. The bent tip of a Schirmer tear strip (Eye Care Product Manufacturing, LLC,
Tucson, AZ, USA) was placed in the ventrolateral conjunctival fornix of each eye. While
recording test duration with a stopwatch, each Schirmer strip was removed and transferred
into a 2-mL Eppendorf tube when the 20-mm mark of wetness was reached, as to standardize
the volume of tears collected in each sample. The distal portion of each strip (25-35 mm
marks, not wetted with tears) was spiked with 5 μL internal standard (prednisone-d7, Toronto
Research Chemicals, North York, Canada) prepared as 10 ng/µL solution in 1:1
acetonitrile:water, and samples were stored at -80°C until analysis.
Preparation of tear samples for analysis
The details of tear fluid extraction are described in Appendix E. Briefly, both
centrifugation and elution in solvent were used as complementary methods to extract the drug
from Schirmer strips.13 Wetted strips containing tear fluid and internal standard were first centrifuged to retrieve the majority of absorbed tear fluid, followed by cutting and shredding the
strips into small pieces and eluting them in methyl tert-butyl ether (MTBE). Of note, this
particular solvent was chosen based on its superior ability to extract prednisone from Schirmer
strips as compared to methanol, acetonitrile and water (small pilot study, data not shown).
93
Liquid chromatography – mass spectrometry
Prior to study initiation, blank tears were collected from the same Beagle dogs using
polyvinyl ophthalmic sponges as previously described.14 Eight standard curve solutions were
prepared by spiking blank canine tears with stock solutions of prednisone/prednisolone
(Cerriliant, Round Rock, Texas, USA) to obtain the following concentrations: 1, 2, 5, 10, 20, 50,
100 and 200 ng/mL. Calibration curve samples were processed in a similar fashion to biological
tear samples, which involved wetting Schirmer strips with standard solutions until the 20-mm mark was reached, spiking prednisone-d7 internal standard onto the distal (dry) portion of the strips, centrifugation and elution in MTBE, etc. (see Appendix D for details). Concentrations of prednisolone and prednisone in canine tears were determined using high-pressure liquid chromatography (Agilent 1100 Pump, Column Compartment, and Autosampler, Santa Clara,
CA, USA) with ion trap mass spectrometry detection (LTQ, Thermo Scientific, San Jose, CA,
USA). The injection volume was set to 20 μL. The mobile phases consisted of A: 0.1% formic acid in water and B: 0.1% formic acid in acetonitrile at a flow rate of 0.25 mL/min. The mobile phase began at 20% B with a linear gradient to 95% B in 5.0 minutes, which was maintained for
2 minutes at 0.325 mL/min, followed by re-equilibration to 20% B at 0.325 mL/min for 3.5 min.
Separation was achieved with an ACE Ultracore C18 column, 100 mm x 2.1 mm , 2.5 µm particles (Mac-Mod Analytical, Chadds Cord, PA, USA) maintained at 40°C. The chromatographic peaks for the internal standard, prednisone and prednisolone (each eluted at
4.69± 0.05 min) were integrated using Xcalibur software (Thermo Scientific, San Jose, CA,
USA). Drug quantitation was based on linear regression analysis of calibration curves (weighted
1/X) using the analyte to internal standard area ratio. Calibration curves exhibited a correlation coefficient (r2) exceeding 0.995 across the concentration range. The limits of quantitation for 94
prednisone and prednisolone were 2 ng/mL and 5 ng/mL, respectively, while the limits of
detection for prednisolone and prednisolone were 0.5 ng/mL and 1 ng/mL, respectively.
Data analysis
Noncompartmental analysis of prednisone and prednisolone pharmacokinetics was
conducted with Phoenix software (WinNonlin, version 8.0, Pharsight Corporation, CA, USA) to
determine the maximum concentration (Cmax), time to maximum concentration (Tmax), and area
under the curve from time zero to time of last measurable concentration (AUClast).
The Shapiro-Wilk test was used to assess data for normality. Non-normally distributed
data were expressed as median and 95% central range (2.5-97.5th percentiles) and were analyzed
with nonparametric statistics. Normally distributed data were expressed as mean ± standard
deviation (95% central range), and were analyzed with parametric statistics. Associations
between tear flow rate and lacrimal concentrations of prednisone or prednisolone were assessed
with the Spearman’s correlation test. The Student t-test was used to assess differences in AUClast between eyes with mild or severe conjunctivitis. For each oral dose and for each PK parameter
(AUClast, Cmax, Tmax), differences between control and conjunctivitis eyes (mild + severe) were
assessed with the Student t test or Mann-Whitney test. For each PK parameter, differences
among oral doses (0.5, 1.0, 2.0 and 4.0 mg/kg) were assessed with the one-way ANOVA or
Kruskal Wallis test. Statistical analysis was performed using SigmaPlot 14.0 (Systat Software
Inc., San Jose, CA, USA), and values P < 0.05 were considered statistically significant.
Results
Following oral administration of prednisone, both prednisone and prednisolone were quantifiable in tear fluid, with concentrations ranging from 2-523 ng/mL and 5-191 ng/mL, 95
respectively. Lacrimal concentrations were not correlated with tear flow rate for either
prednisone (P ≥ 0.412) or prednisolone (P ≥ 0.388). However, higher doses of oral prednisone
resulted in higher tear concentrations of both steroids, as depicted by the individual
concentration-time curves in Figure 2. In fact, the overall drug exposure in tears (depicted by
AUClast) was statistically different among the 4 doses for both prednisone (P ≤ 0.002; Figure
3A) and prednisolone (P ≤ 0.001; Figure 3B). Similarly, statistical differences were detected among oral doses for prednisone Cmax (P = 0.008) and for prednisolone Cmax (P = 0.003) and
Tmax (P = 0.039; Table 1). In general, eyes with conjunctivitis showed a trend for larger
concentrations of prednisone and prednisolone in tear fluid compared to control eyes (Figures 2 and 3), with differences in average concentrations ranging from +5% to +64%. However, differences in AUClast between control and conjunctivitis eyes were not statistically significant
for either prednisone (P ≥ 0.095; Figure 3A) or prednisolone (P ≥ 0.485; Figure 3B). The
severity of conjunctivitis did have an impact on lacrimal concentrations, as significantly greater
levels of prednisolone were found in eyes with severe vs. mild conjunctivitis for oral doses 2-4 mg/kg/d (P ≤ 0.042; Figure 4), although these changes were not considered to be large enough to be clinically relevant.
Discussion
The present study describes, for the first time, the tear film pharmacokinetics of prednisone and prednisolone in veterinary medicine. Tear film concentrations of prednisone and prednisolone varied from 2-523 ng/mL and 5-191 ng/mL respectively, with higher doses of oral prednisone leading to higher lacrimal levels of both steroids. The question remains whether concentrations of the active metabolite (prednisolone) are relevant in clinical patients, that is potentially beneficial or detrimental to managing ocular surface diseases. Overall, tear film 96
prednisolone levels were ≥ 10-9 M (i.e. 0.4 ng/mL) in all dogs throughout the 12-hour sampling time, a concentration shown to decrease the expression of deleterious cytokines (TNF-α, IL-6) and matrix metalloproteinases in a rat model of keratitis.15 Therefore, oral prednisone might have
therapeutic benefits in managing corneal inflammation, assuming similar exposure-response
between species. In fact, although the use of corticosteroid in infectious keratitis remains
controversial,16 some authors believe that a judicious use of corticosteroids (combined with the
appropriate antimicrobial) could improve the outcome of keratitis as it reduces damage caused
by the host’s inflammatory response, decreases corneal scarring, and inhibits
neovascularization.17,18 From a safety viewpoint, the use of corticosteroids is known to
potentially delay corneal wound healing and exacerbate signs of ocular infection. In vitro,
inhibition of corneal wound healing in dogs is only reported for prednisolone concentrations that
are much higher (≥ 620 µg/mL)19 than the ones reported herein. In vivo, topical corticosteroid
use can be detrimental in patients with ulcerative keratitis2 although tear film concentrations
following topical 1% prednisolone acetate are unknown to date in any species. Data
extrapolation from pharmacokinetics of 0.3% ciprofloxacin in dogs20 shows that (i) topical 1%
prednisolone acetate could reach concentrations as high as 909 µg/mL, which is 1,000 to 10,000- fold greater than drug levels noted in the present study; and (ii) topical 1% prednisolone acetate
(applied every 6h) could result in drug exposure over 12h that is 14,000-27,000 fold and 3,300-
5,300 fold greater than oral prednisone given at anti-inflammatory dose (0.5-1 mg/kg/d) or immuno-suppressive dose (2-4 mg/kg/d), respectively. Of note, drug exposure over time is more relevant than single lacrimal concentrations (e.g. Cmax) given differences in pharmacological
disposition between topical and oral routes. While the ocular bioavailability of topical
administration is <10-20% given efficient washout by tears,21 oral administration could be 97
considered as a form of sustained-release at the ocular surface through lacrimal gland diffusion
and conjunctival leakage. As for the negative impact on the immune system, there is no
consensus on what concentration is considered harmful. In one study, prednisolone levels as low
as 0.005 µg/mL were shown to reduce the phagocytosis function of human leucocytes,22 while
prednisolone concentrations as high as 4.32 µg/mL did not impact leucocyte phagocytosis or
bactericidal activity in another study.23
Conjunctivitis is a common disorder in dogs that develops concurrently to most ocular
diseases, whether affecting the adnexa (e.g. blepharitis), ocular surface (e.g. corneal ulcer) or
intraocular tissues (e.g. uveitis). With conjunctivitis, plasma constituents tend to ‘leak’ into the
tear compartment as the permeability of conjunctival vessels is typically increased.10,11 This breakdown of the blood-tear barrier explains the large quantities of albumin in tears of diseased eyes, regardless of the underlying etiology of conjunctivitis (e.g. dry eye, corneal ulcer, allergies).24,25 Thus, to make the present PK findings more clinically relevant, conjunctivitis was
experimentally induced in selected canine eyes using a recently described model.11 Lacrimal
levels of prednisone and prednisolone were overall higher in conjunctivitis vs. control eyes, with
greater disease severity leading to generally greater drug levels in tears, especially for
prednisolone. However, differences between control vs. conjunctivitis eyes were not statistically
significant, and were fairly minimal (up to 64% increase) when compared to plasma albumin (up
to 12,000%).11 Unlike albumin, a very large molecule (66,500 Da) that does not permeate
through intact conjunctival tissue,24 we suspect that smaller molecules like prednisone (358 Da)
and prednisolone (360 Da) readily cross the blood-tear barrier under normal conditions, and are
therefore not significantly impacted by conjunctival inflammation. However, we cannot exclude
that larger amounts of corticosteroids actually reach the lacrimal fluid in eyes with conjunctivitis; 98
although the concurrent leakage of albumin binding to free prednisolone would probably reduce
its bioavailability at the ocular surface.26 Further, physicochemical properties other than
molecular weight may explain differences in lacrimal distribution between prednisone,
prednisolone, and other drugs reported in the veterinary literature3, 5, 13 – namely protein binding,
lipophilicity and degree of ionization.27 Here, the competitive nature of plasma protein binding
between prednisone and prednisolone could justify the slightly higher lacrimal concentrations of
prednisone in canine tears.28
The present study has a few limitations. First, the sample size of our experiment was
relatively small, and only females from a single dog breed were evaluated. Tear film PK could
theoretically differ in male vs. female dogs,29 or breeds other than Beagle, especially in
brachycephalic dogs in whom the lacrimal lipid layer is thin and corneal exposure is large.30 Yet,
previous studies did not find significant differences between mesocephalic and brachycephalic
dogs in regards to tear film dynamics (tear volume, tear turnover rate)31 or tear film drug
concentrations.20 Second, it is possible that we did not find statistical differences in tear film concentrations between healthy vs. conjunctivitis eyes because of the conjunctivitis model itself.
Experimental induction of conjunctivitis, although rapid and non-invasive,11 could have falsely
lowered lacrimal concentrations by causing reflex tearing and accelerated tear turnover. We
minimized this risk by inducing conjunctivitis ≥ 20 min before drug administration and sample
collection, but a small degree of ocular irritation could have lingered. Regardless, increased
tearing should not have affected drug levels in a notable manner, as we did not find a significant
correlation between tear flow rate and prednisone/prednisolone levels in tears. Last, the present
study used Schirmer strips to collect tears in dogs, and although this method yielded sufficient
tear fluid for analysis, the method has several disadvantages that could partly explain the large 99
variability in tear concentrations noted among subjects. Not only do Schirmer strips absorb tear
fluid but they also retain a certain amount of tear components (adsorption), the degree of which
can vary depending on the concentration.13 Here, we minimized the impact of adsorption by
incorporating two important steps in our sample preparation: (i) the internal standard was spiked
onto the distal end of Schirmer strips before tear extraction, and (ii) standard curves were
processed in a similar fashion to biological samples. We also maximized the amount of drug
extracted from Schirmer strips by combining centrifugation with solvent elution.13 Such
combination may improve the assay sensitivity (i.e. able to detect lower concentrations), but the process is very labor intensive and may not be necessary for all drugs. Future studies should consider a pilot experiment to assess the extraction efficacy of the combination method vs. centrifugation or solvent elution alone.
In conclusion, our data indicate that oral prednisone might be safe and beneficial as adjunctive therapy for reflex uveitis, ulcerative keratitis or other ocular surface disease in dogs.
However, these preliminary pharmacokinetic findings need to be complemented with prospective controlled studies using systemic corticotherapy in diseased animals. Similarly, future anatomical and physiological studies are needed to better understand the role of conjunctivitis in diffusion of systemically administered drugs into the tear film.
References
1. Dinning, W.J. Steroids and the eye--indications and complications. Postgrad Med. J 1976;52:634-638.
2. Holmberg, B.J., Maggs, D.J. The use of corticosteroids to treat ocular inflammation. Vet Clin North Am Small Anim Pract. 2004;34:693-705.
3. Collins, S.P., Labelle, A.L., Dirikolu, L., Li, Z., Mitchell, M.A., Hamor, R.E. Tear film concentrations of doxycycline following oral administration in ophthalmologically normal dogs. J Am Vet Med Assoc 2016;249:508-514. 100
4. Baker, A., Plummer, C.E., Szabo, N.J., Barrie, K.P., Brooks, D.E. Doxycycline levels in preocular tear film of horses following oral administration. Vet Ophthalmol 2008;11:381-385.
5. Sebbag, L., Thomasy, S.M., Woodward, A.P., Knych, H.K., Maggs, D.J. Pharmacokinetic modeling of penciclovir and BRL42359 in the plasma and tears of healthy cats to optimize dosage recommendations for oral administration of famciclovir. Am J Vet Res 2016;77:833-845.
6. Thomasy, S.M., Shull, O., Outerbridge, C.A., et al. Oral administration of famciclovir for treatment of spontaneous ocular, respiratory, or dermatologic disease attributed to feline herpesvirus type 1: 59 cases (2006-2013). J Am Vet Med Assoc 2016;249:526-538.
7. Petroutsos, G., Guimaraes, R., Giraud, J.P., Pouliquen, Y. Corticosteroids and corneal epithelial wound healing. Br J Ophthalmol. 1982;66:705-708.
8. Lee, E.J., Truong, T.N., Mendoza, M.N., Fleiszig, S.M. A comparison of invasive and cytotoxic Pseudomonas aeruginosa strain-induced corneal disease responses to therapeutics. Curr Eye Res 2003;27:289-299.
9. LeHoang P. The gold standard of noninfectious uveitis: corticosteroids. Dev Ophthalmol 2012;51:7-28.
10. van Delft, J.L., Meijer, F., van Best, J.A., van Haeringen, N.J. Permeability of blood-tear barrier to fluorescein and albumin after application of platelet-activating factor to the eye of the guinea pig. Mediators Inflamm 1997;6:381-383.
11. Sebbag L, Allbaugh RA, Weaver A, et al. Histamine-induced conjunctivitis and breakdown of blood-tear barrier in dogs: A model for ocular pharmacology and therapeutics. Front Pharmacol 2019;10:752.
12. El Dareer, S.M., Struck, R.F., White, V.M., Mellett, L.B., Hill, D.L. Distribution and metabolism of prednisone in mice, dogs, and monkeys. Cancer Treat Rep 1977;61:1279-1289.
13. Sebbag, L., Showman, L., McDowell, E., Perera, A., Mochel, J.P. Impact of flow rate, collection devices and extraction methods on tear concentrations following oral administration of doxycycline in dogs and cats. J Ocul Pharmacol Ther; 2018;34:452-459.
14. Sebbag, L., Harrington, D.M., Mochel, J.P. Tear fluid collection in dogs and cats using ophthalmic sponges. Vet Ophthalmol 2018;21:249-254.
15. Yan, H., Wang, Y., Shen, S., Wu, Z., Wan, P. Corticosteroids effects on LPS-induced rat inflammatory keratocyte cell model. PLoS One 2017;12:e0176639.
16. Wilhelmus, K.R. Indecision about corticosteroids for bacterial keratitis: an evidence-based update. Ophthalmology 2002;109:835-842.
101
17. Hindman, H.B., Patel, S.B., Jun, A.S. Rationale for adjunctive topical corticosteroids in bacterial keratitis. Arch Ophthalmol 2009;127:97-102.
18. Palioura, S., Henry, C.R., Amescua, G., Alfonso, E.C. Role of steroids in the treatment of bacterial keratitis. Clin Ophthalmol 2016;10:179-186.
19. Hendrix, D.V., Ward, D.A., Barnhill, M.A. Effects of anti-inflammatory drugs and preservatives on morphologic characteristics and migration of canine corneal epithelial cells in tissue culture. Vet Ophthalmol 2002;5:127-135.
20. Hendrix, D.V., Cox, S.K. Pharmacokinetics of topically applied ciprofloxacin in tears of mesocephalic and brachycephalic dogs. Vet Ophthalmol 2008;11:7-10.
21. Agrahari, V., Mandal, A., Trinh, H.M., et al. A comprehensive insight on ocular pharmacokinetics. Drug Deliv Transl Res. 2016;6:735-754.
22. Jones, C.J., Morris, K.J., Jayson, M.I. Prednisolone inhibits phagocytosis by polymorphonuclear leucocytes via steroid receptor mediated events. Ann Rheum Dis 1983;42:56- 62.
23. Losito, A., Williams, D.G., Cooke, G., Harris, L. The effects on polymorphonuclear leucocyte function of prednisolone and azathioprine in vivo and prednisolone, azathioprine and 6-mercaptopurine in vitro. Clin Exp Immunol 1978;32:423-428.
24. Runstrom, G., Mann, A., Tighe, B. The fall and rise of tear albumin levels: a multifactorial phenomenon. Ocul Surf 2013;11:165-180.
25. Zavaro, A., Samra, Z., Baryishak, R., Sompolinsky, D. Proteins in tears from healthy and diseased eyes. Doc Ophthalmol 1980;50:185-199.
26. Mikkelson, T.J., Chrai, S.S., Robinson, J.R. Altered bioavailability of drugs in the eye due to drug-protein interaction. J Pharm Sci 1973;62:1648-1653.
27. Prausnitz, M.R., Noonan, J.S. Permeability of cornea, sclera, and conjunctiva: a literature analysis for drug delivery to the eye. J Pharm Sci 1998;87:1479-1488.
28. Boudinot, F.D., Jusko, W.J. Plasma protein binding interaction of prednisone and prednisolone. J Steroid Biochem 1984;21:337-339.
29. Chang, T.K., Waxman, D.J. Sex differences in drug metabolism. In: Lyubimov, A.V., ed. Encyclopedia of Drug Metabolism and Interactions. New York: John Wiley & Sons, Inc.; 2012; 4736 pp
30. Carrington, S.D., Bedford, P.G., Guillon, J.P., Woodward, E.G. Biomicroscopy of the tear film: the tear film of the pekingese dog. Vet Rec 1989;124:323-328.
31. Sebbag L, Allbaugh RA, Wehrman RF, et al. Fluorophotometric assessment of tear volume and turnover rate in healthy dogs and cats. J Ocul Pharmacol Ther 2019;35:497-502.
Tables and Figures
Table 1. Mean ± standard deviation of the maximal concentration (Cmax) and time to reach Cmax (Tmax) for prednisone and prednisolone in control and conjunctivitis eyes. Within the same dose, comparisons between control and conjunctivitis eyes are described with P values above the plots and an asterisk (*) if statistically significant (P < 0.05). Within the same group (control or conjunctivitis), differences among doses are depicted with symbols to demonstrate statistically greater values compared to dose 1 (#), dose 2 (†) and dose 3 (‡).
Prednisone Prednisolone 102
Cmax (ng/mL) Tmax (min) Cmax (ng/mL) Tmax (min)
Dose 1 Control 23.2 ± 13.4 77.5 ± 89.7 19.2 ± 19.5 115.0 ± 99.3 (0.5 mg/kg) Conjunctivitis 55.1 ± 68.1 87.5 ± 85.1 22.4 ± 26.7 130.0 ± 86.3
Dose 2 Control 47.7 ± 26.4 140.0 ± 187.3 29.2 ± 24.9 215.0 ± 257.0 (1.0 mg/kg) Conjunctivitis 48.1 ± 25.5 87.5 ± 85.1 37.1 ± 32.1 145.0 ± 166.9
# Dose 3 Control 91.7 ± 74.7 127.5 ± 97.6 75.3 ± 64.3 420.0 ± 211.3 (2.0 mg/kg) Conjunctivitis 137.2 ± 115.5 65.0 ± 22.6 62.7 ± 31.3 320.0 ± 196.0
# # † Dose 4 Control 116 ± 67.2 127.5 ± 93.8 123.6 ± 47.8 160.0 ± 92.3 (4.0 mg/kg) Conjunctivitis 204.3 ± 173.5 120.0 ± 180.7 120.0 ± 38.1# † ‡ 160.0 ± 92.3
103
Figure 1. Topical histamine rapidly induced conjunctivitis (<1 min) that was either mild [(A); 1.0 mg/mL solution] or severe [(B); 375 mg/mL solution]. Color images are available online.
104
Figure 2. Tear film concentrations (mean + standard deviation) of prednisone (A, B) and prednisolone (C, D) in dogs receiving oral prednisone at 0.5 mg/kg once daily (black line, circles), 1.0 mg/kg once daily (red lines, down triangles), 2.0 mg/kg once daily (green lines, squares), and 4.0 mg/kg once daily (blue lines, up triangles). The concentrations are depicted for control eyes (A, C) and eyes with experimentally induced conjunctivitis (B, D). Of note, the conjunctivitis group includes eyes with mild and severe conjunctivitis. 105
Figure 3. Box-and-whiskers plots depicting the area under the tear concentration–time curve from time zero to time of last measurable concentration (AUClast). Each plot depicts the mean (dotted line), median (solid line), 2.5th percentile (lower whisker), 25th percentile (lower limit of box), 75th percentile (upper limit of box), and 97.5th percentile (upper whisker). Data for prednisone (A) and prednisolone (B) are shown for all 4 oral doses of prednisone (0.5–4.0 mg/kg) in both control eyes (white boxes) and eyes with experimentally induced conjunctivitis (dark gray). Of note, the conjunctivitis group includes eyes with mild and severe conjunctivitis. Within the same drug dose, comparisons between control and conjunctivitis eyes (t test) are described with P values above the plots. Within the same ocular group (control or conjunctivitis), differences among doses (one-way analysis of variance) are depicted with symbols to demonstrate statistically greater AUClast compared to dose 1 (#), dose 2 (†) and dose 3 (‡). 106
Figure 4. Bar charts depicting mean + standard deviation of area under the tear concentration- time curve from time zero to time of last measurable concentration (AUClast). Data for tear concentrations of prednisone (A) and prednisolone (B) are shown for all 4 oral doses of prednisone (0.5–4.0 mg/kg) in eyes with experimentally induced mild conjunctivitis (light gray) or severe conjunctivitis (dark gray). Within the same dose, statistical comparisons between mild and severe conjunctivitis (t test) are described with P values above the plots, and statistical significance (P < 0.05) is depicted with an asterisk (*). 107
CHAPTER 7. ALBUMIN LEVELS IN TEAR FILM MODULATE THE BIOAVAILABILITY OF MEDICALLY-RELEVANT TOPICAL DRUGS
Lionel Sebbag,1,2 Leah M. Moody,1 Jonathan P. Mochel2
1 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa
State University, Ames, IA 50011, USA; 2 Department of Biomedical Sciences, College of
Veterinary Medicine, Iowa State University, Ames, IA 50011, USA
Modified from a manuscript published in Frontiers in Pharmacology
Abstract
The breakdown of blood-tear barrier that occurs with ocular pathology allows for large
amounts of albumin to leak into the tear fluid. This process likely represents an important restriction to drug absorption in ophthalmology, as only the unbound drug is transported across the ocular tissue barriers to exert its pharmacologic effect. We aimed to investigate the effects of albumin levels in tears on the bioavailability of two commonly used ophthalmic drugs: tropicamide, an antimuscarinic that produces mydriasis and cycloplegia, and latanoprost, a
PGF2α analog used for the treatment of glaucoma. Eight female beagle dogs underwent a randomized, vehicle-controlled crossover trial. For each dog, one eye received 30 µL of artificial tears (control) or canine albumin (0.4 or 1.5%) at random, immediately followed by 30 µL of 1% tropicamide (2 days, 24h washout) or 0.005% latanoprost (2 days, 72h washout) in both eyes.
Pupil diameter (digital caliper) and intraocular pressure (IOP; rebound tonometry) were recorded at various times following drug administration (0 to 480 min) and compared between both groups with a mixed model for repeated measures. Albumin in tears had a significant impact on pupillary diameter for both tropicamide (P≤0.001) and latanoprost (P≤0.047), with no differences noted between 0.4% and 1.5% concentrations. Reduction in the maximal effect (pupil 108 size) and overall drug exposure (area under the effect time-curve of pupil size over time) were significant for tropicamide (6.2-8.5% on average, P≤0.006) but not for latanoprost (P≥0.663).
The IOP, only measured in eyes receiving latanoprost, was not significantly impacted by the addition of either 0.4% (P = 0.242) or 1.5% albumin (P = 0.879). Albumin in tear film, previously shown to leak from the conjunctival vasculature in diseased eyes, may bind to topically administered drugs and reduces their intraocular penetration and bioavailability. Further investigations in clinical patients and other commonly used ophthalmic medications are warranted.
Introduction
Topical instillation is the most common route of drug administration in ophthalmology, especially for the treatment of anterior segment diseases.1 This mode of administration is convenient and non-invasive, although drug bioavailability is generally poor (typically < 10%) due to physiological, structural, and biochemical barriers to drug penetration into the eye.1-3
Upon instillation, an eyedrop is immediately diluted in the tear film and a large portion of the drug is lost through reflex tearing, nasolacrimal drainage, and systemic absorption. Residual drug has to cross ocular tissue barriers (i.e. cornea, sclera and conjunctiva) to reach targets within the globe.1,3,4 In general, small lipophilic drugs permeate through the cornea while larger or hydrophilic compounds permeate through the conjunctiva and sclera.4 Protein binding in tear film represents another important restriction to drug absorption, as only the unbound drug is transported across the tissue barriers.5 In fact, the presence of albumin in tears can dramatically reduce the bioavailability of topical drugs via protein-drug interactions, as previously shown for pilocarpine in rabbit eyes.5 109
Albumin is a relatively large (66 kDa) and negatively charged protein that is widely
distributed in the body. Given the protein’s remarkable capacity for binding ligands,6 albumin
serves as a reservoir and transporter for drugs and other molecules such as hormones,
metabolites, and nutrients. At the level of the eye, plasma-derived albumin leaks onto the ocular surface from conjunctival vessels and mixes with the tear film.7 Albumin concentration in tears is generally low in healthy state but increases substantially in diseased eyes.7 In fact, albumin is
often considered a biomarker of ocular insult or inflammation as the breakdown of blood-tear
barrier noted with ocular pathology allows for large amounts of albumin to leak into the lacrimal
fluid.7-9 A recent study by Sebbag et al. showed that canine eyes with diverse ocular diseases
(e.g. corneal ulcer, uveitis, glaucoma) had lacrimal albumin levels that were up to 14.9-fold greater than contralateral healthy eyes.10
The impact of albumin binding on the drug’s pharmacological activity is extensively
studied in blood,11 yet little is known about the physiology and function of albumin in tears or
other biological fluids. In the present study, we examined the bioavailability of topically
delivered drugs in the presence of clinically relevant levels of albumin in tears.10 We
hypothesized that the drugs’ intraocular effect will be reduced by lacrimal albumin given the
inability of protein-bound drugs to permeate through ocular tissue barriers. Two ophthalmic
medications were investigated as a proof-of-concept experiment: 0.005% latanoprost and 1% tropicamide. These drugs are commonly used in human and veterinary practice, and possess different physicochemical properties (e.g. solution pH, drug concentration) that could influence protein-drug interactions. Latanoprost, a PGF2α analog, is used for the treatment of glaucoma
and ocular hypertension in human and veterinary patients.12,13 Tropicamide, an antimuscarinic
drug, is used to achieve short-acting mydriasis for enhanced visualization of the lens, vitreous 110
body and fundus, as well as cycloplegia to control accommodation during the assessment of
refractive error.14 Pupil response to tropicamide was also suggested as a noninvasive
neurobiological test for Alzheimer’s disease and other neurodegenerative disorders,15,16 although
this diagnostic test fell out of favor given large inter- and intra-individual variations and
subsequent poor test specificity.17 Drug binding to proteins in tear fluid could partly explain the
aforementioned variability in pupil size, a phenomenon our group investigated in the present
study to help guide future diagnostic and therapeutic applications in ophthalmology. The present
work was conducted in dogs, a species that represents an excellent large animal model for
translational research in humans given similarities in ocular anatomy18 and physiologic parameters pertinent to topical route of drug administration,19 as well as spontaneous disease
development such as glaucoma,20 dry eye21 and conjunctivitis.10
Materials and Methods
Animals
Eight female spayed Beagle dogs (1.5-2.0 years, 7.5-10 kg) were recruited. Prior to study enrollment, dogs were part of a teaching colony and underwent weekly physical and ophthalmic examinations, including tonometry (TonoVet, Icare Finland Oy, Espoo, Finland). At study inclusion, dogs were confirmed to be healthy based on a complete physical and ophthalmic examination, including tonometry (TonoVet), Schirmer tear test-1 (STT-1; Eye Care Product
Manufacturing LLC, Tucson, AZ, USA), slit-lamp biomicroscopy (SL-17; Kowa Company, Ltd.,
Tokyo, Japan) and indirect ophthalmoscopy (Keeler Vantage; Keeler Instruments, Inc.,
Broomall, PA, USA). The study was approved by the Institutional Animal Care and Use
Committee of Iowa State University (protocol # 19-049), and conducted in accordance with the
Association for Research in Vision and Ophthalmology guidelines for animal use. 111
Experiment
Two canine albumin ophthalmic solutions (0.4% and 1.5%) were formulated by mixing
canine albumin lyophilized powder (Animal Blood Resource International, Stockbridge, MI)
with lubricating eye drops (Artificial tears solution, Rugby, Rockville Center, NY, USA) in a
sterile manner under a laminar flow hood. Of note, albumin concentrations selected herein (0.4%
and 1.5%) aimed to achieve tear film albumin levels of ~ 1 to 5 mg/mL (i.e., after dilution of the
instilled drop with the canine tear film, accounting for ~ 3-fold dilution)19 thus representing a
spectrum of albumin levels in tears of dogs with spontaneous or experimentally-induced
conjunctivitis.10 Artificial tears solution without albumin (vehicle only) was used as control for
the experiment. Albumin and vehicle solutions were kept in the refrigerator (4 °C) and used
within 7 days of preparation.
For each dog, one eye was randomly selected to receive albumin solutions while the
contralateral eye served as control (vehicle solution); this choice was kept constant throughout
the study. Tropicamide 1% (Sandoz Inc., Princeton, New Jersey, USA) and latanoprost 0.005%
(Sandoz Inc., Princeton, New Jersey, USA) were each investigated over two separate days. To
allow pupil size to return to baseline and avoid a carry-over effect, the washout between
experimental days was 24h for tropicamide22 and 72h for latanoprost.23 For each drug, the eye allocated to albumin was randomly assigned to receive either 4 mg/mL or 15 mg/mL albumin solution on the first experimental day, and vice versa on the second day.
The experiments took place in a quiet and uniformly illuminated room (500 lux) under controlled temperature (70-72°F) and ambient humidity (25-30%). Measurements of pupil
diameter (PD) were obtained with a digital Vernier caliper (± 0.01 mm, Ultratech No. 1433,
General Tools & Instruments, Secaucus, NJ) held adjacent to the cornea, while measurements of 112 intraocular pressure (IOP) were obtained with rebound tonometry (TonoVet, Icare Finland Oy,
Espoo, Finland). Baseline PD and IOP were recorded in both eyes of each dog at the beginning of each study day.
Using a pipette, 30 µL of experimental solution were delivered topically: albumin in one eye, vehicle in the other. This was immediately followed (<10 sec) by topical instillation of 30
µL of the drug (tropicamide or latanoprost) in both eyes. Then, PD (tropicamide and latanoprost) and IOP (latanoprost only) were recorded in both eyes at the following time points: 2, 4, 6, 8, 10,
15, 20, 30, 45, 60, 90, 120, 180, 240, and 480 min.
Data analysis
Normality of data was assessed with the Shapiro-Wilk test. Differences in pupil diameter
(tropicamide, latanoprost) and IOP (latanoprost) between eyes receiving saline (control) or albumin (0.4% or 1.5%) were assessed with a mixed model for repeated measures (MMRM)24 using the R software version 3.6.0. In the model, PD or IOP were the response variable, the group (control or albumin), time (0 to 480 min) and group-by-time interaction were treated as fixed effects, and the animal and animal-by-group interaction were treated as random effects, using animal as block. After the model was fit, the fixed effects were tested, and comparisons between control and albumin eyes at baseline and each time point were made. The R software was also used to calculate the area under the effect-time curve (AUETC) and the maximal effect on pupil diameter (maximal dilation for tropicamide, maximal constriction for latanoprost).
Paired t-tests were conducted with SigmaPlot 14.0 (Systat Software Inc., San Jose, CA, USA) to assess the following parameters: (i) AUETC for tropicamide and latanoprost, (ii) PDmax for tropicamide, and (iii) PDmin for latanoprost. P values < 0.05 were considered statistically significant. 113
Results
Results from the Shapiro-Wilk test confirmed that the experimental data were normally distributed. Results are therefore presented as mean ± standard deviation (SD).
Pupil dilation from tropicamide
Taking the variable ‘time’ into account, albumin had a significant effect on pupil diameter post- tropicamide administration for both 0.4% (P = 0.001) and 1.5% concentrations (P
= 0.001). Compared to the contralateral eye (control), pupillary dilation was significantly
reduced in eyes receiving 0.4% albumin as early as 8 min (P = 0.043) and as late as 240 min (P =
0.009)(Figure 1A), and in eyes receiving 1.5% albumin as early as 8 min (P = 0.027) and as late
as 240 min (P = 0.021)(Figure 1B) following instillation of 1% tropicamide. A representative
clinical image is depicted in Figure 2, showing a lower degree of mydriasis in the dog’s left eye
(0.4% albumin and 1% tropicamide) compared to the right eye (artificial tears and 1%
tropicamide) at 45 min following eyedrop administration. Further, the cumulative effect of
tropicamide on pupillary dilation (from 0 to 480 min) was significantly reduced with the addition
of 0.4% or 1.5% albumin (P < 0.001), representing an average reduction in biological response
of 7.1% and 7.2% compared to controls, respectively (Figure 3); however, no differences were
noted in AUETC (pupil size over time) between both albumin concentrations (P ≥ 0.625). Last,
mean ± SD maximal pupillary dilation in eyes receiving tropicamide and 0.4% albumin (11.9 ±
0.7 mm) or 1.5% albumin (11.7 ± 1.3 mm) was significantly lower (P ≤ 0.006) compared to
contralateral controls (12.7 ± 0.8 mm, and 12.8 ± 1.1 mm, respectively), representing a reduction
in biological response of 6.2% and 8.5%, respectively (Figure 4).
114
Pupil constriction from latanoprost
Taking the variable ‘time’ into account, albumin had a significant effect on pupil
diameter post- latanoprost administration for both 0.4% (P = 0.016) and 1.5% concentrations (P
= 0.047). Compared to the contralateral eye (control), pupillary constriction was overall reduced
in eyes receiving 0.4% albumin (Figure 5A) or 1.5% albumin (Figure 5B), although differences
in pupil diameter were not statistically significant at any time point following instillation of
0.005% latanoprost (P ≥ 0.158 and P ≥ 0.416, respectively). No differences were noted in
AUETC (pupil size over time) between groups (P ≥ 0.663), nor in maximal pupillary constriction
(P = 1.000) obtained with 0.4% albumin (1.5 ± 0.1 mm), 1.5% albumin (1.5 ± 0.2 mm) and their
respective contralateral controls (1.5 ± 0.1 mm and 1.5 ± 0.2 mm).
IOP changes from latanoprost
Compared to the contralateral eye (control), IOP was significantly lower in eyes
receiving 0.4% albumin at 10 min (P = 0.010), 20 min (P = 0.010), 45 min (P = 0.010) and 240
min (P = 0.010) following instillation of 0.005% latanoprost (Figure 6A), while no significant
changes were noted at any time point (P ≥ 0.262) for the 1.5% albumin group (Figure 6B).
However, after taking ‘time’ into account in the mixed effects model, it is important to note that
the impact of albumin on IOP was not statistically significant for either 0.4% (P = 0.242) or
1.5% concentration (P = 0.879). Further, no differences were noted in AUETC (IOP over time) between control and albumin groups (P ≥ 0.351) or between both albumin groups (P = 0.979).
Discussion
The bioavailability of ophthalmic drugs can be reduced by the presence of albumin in
tears, a protein that leaks onto the ocular surface in large amounts in the diseased eye. A deeper 115
understanding of albumin-drug interactions in tears is critical to basic researchers in
pharmacology and vision science, but also physicians and veterinarians. Indeed, drug binding to
albumin may partly explain the challenge of treating certain diseases in ophthalmology. For
instance, a poor response of uveitis to topical corticosteroid may be due to high affinity of the
drug to albumin in tears, while a poor response of infectious keratitis to topical antibiotics may
be explained by the fact that only the unbound portion of an antimicrobial is microbiologically
active.25 Here, we showed a differential impact of albumin in tears on the ocular response of
tropicamide and latanoprost, two common ophthalmic drugs in human and veterinary patients,
and the same could be investigated in the canine model for other relevant drug classes. Dogs are
particularly suited for translational research in ocular pharmacology as – unlike small laboratory
animals – dogs share similar anatomical and physiological features to humans, similar
environmental stressors and genetic variation, and a range of naturally occurring ophthalmic
diseases that resemble the ones diagnosed in human patients.
Ocular response of tropicamide and latanoprost in the presence of albumin
The biological effect of tropicamide (i.e. mydriasis) was significantly reduced in canine
eyes that received concurrent topical administration of serum albumin, regardless of the protein’s
concentration. Albeit minimal (6.2-8.5 %), the impact of albumin on tropicamide-induced pupillary dilation is likely underestimated compared to clinical patients given limitations inherent to the study design (described below). Interestingly, this lower degree of tropicamide- induced mydriasis was also noted in canine eyes covered with a soft contact lens, a physical barrier to drug penetration.26 Similar findings were noted by Mikkelson et al. in rabbit eyes
receiving pilocarpine,5 although the magnitude of drug-response reduction was much greater in 116
rabbits (75-100 fold) compared to the present study in dogs (6-8%). Such discrepancy is likely explained by two important differences in study designs. In the present experiment, the concentrations of albumin (0.4% and 1.5%) were specifically chosen based on clinically-relevant
albumin levels detected in canine patients with diverse ocular diseases,10 taking into account the
3-fold dilution effect from resident tears.19 to better account for the true binding constant seen in
dialysis experiments.27 In contrast, the concentrations of albumin used in rabbits were higher
(1% and 3%) and may not reflect the range of biological concentrations of albumin at the ocular
surface. Another key difference is the way albumin was delivered to the ocular surface. While
albumin was pre-mixed with pilocarpine solution in the rabbit study, allowing for protein-drug
binding to occur ex situ (i.e. away from the ocular surface) over an extended duration, albumin
and tropicamide were delivered separately in the present study (albumin first, tropicamide within
<10 seconds) so that protein-drug interactions would occur in-situ (i.e. in the tear film) over a short duration. From a physiological standpoint, the latter method is more appropriate as the interaction time of a topical drug to albumin in tears is generally short, limited by the rapid tear turnover rate that occurs following eyedrop administration in dogs19 or other species.28,29 Of
note, tonometry was not performed in dogs receiving tropicamide (with or without albumin) in
the present study given the lack of effect of tropicamide on IOP in healthy canine eyes,30 but this
parameter could be considered in future studies as IOP can vary from tropicamide in dogs
receiving sedation30 or dogs with glaucomatous eyes.
The pharmacological activity of latanoprost (i.e. miosis) was also reduced in the presence of albumin in tears, although not to the same extent as for tropicamide. Indeed, differences in pupil size between albumin and control eyes were limited in duration (up to 30 min, compared to
240 min for tropicamide) and somewhat limited in magnitude (1% non-significant change in 117
AUETC). These findings are likely explained by the high sensitivity of the iris sphincter muscle
to the drug.31 The minimum amount of PGF2α required to generate contraction of the iris
sphincter in dogs is 10-10 M,31 while the concentration of latanoprost applied topically is
approximately 106 higher (0.005% ~ 10-4 M). The amount of drug lost to albumin in tears is
therefore insignificant, as only a small fraction of intraocular drug penetration is sufficient to
cause miosis. Furthermore, once latanoprost reaches the anterior chamber, the drug acts directly
on the iris sphincter muscle but also indirectly through the release of endogenous
prostaglandins.32,33 Endogenous PGF2α, which further acts on the prostanoid FP receptors and
contributes to the sphincter muscle’s tone in dogs,31 is released inside the anterior chamber and is
thereby not affected by albumin levels in the tear film.
Following latanoprost administration, the overall effect of lacrimal albumin on IOP
values was non-significant for either albumin concentration. Compared to control eyes, a
significantly lower IOP was noted at selected times (10, 20, 45 and 240 min) in eyes receiving
0.4% albumin concurrently to latanoprost, although it is important to note that IOP readings
displayed a large variability within -and between-subjects in all groups. To reduce IOP variability, a recent study in healthy Beagle dogs34 recommended a minimum of 5 training days immediately prior to the start of the study, and collecting IOP readings in triplicate at each time.
In absence of such precautions, the IOP results of the present study are likely confounded by a large measurement noise.
Factors affecting the impact of albumin on drug bioavailability
The present findings provide evidence that albumin levels in tears does not affect all drugs in a uniform manner. Rather, the mechanism of action of a drug and/or potency for its 118
biological target can modulate the impact of lacrimal albumin on the drug’s pharmacological
activity. The dose-response relationship, a cornerstone of pharmacology/toxicology, defines the
role of a dose for a chemical (e.g. drug, toxic agent) in evoking biological response.35 Figure 7A
depicts two drugs (A & B) with different dose-response profiles. In this scenario, if lacrimal
albumin reduces the amount of free drug available inside the eye by 50%, the response observed
(e.g. pupillary dilation) will be greatly reduced for drug B but minimally affected for drug A.
Along the same line, the amount of drug applied topically is an important factor to take into
consideration. For a given drug, if the dose administered topically falls in the ‘far right’ of the
drug’s dose-response curve, a reduction in drug available after albumin binding would only
minimally affect the observed response (dose X, Figure 7B); in contrast, if the dose delivered
produces an effect that falls within the steep portion of the dose-response curve, the impact of
dose reduction from albumin would be more pronounced (dose Y, Figure 7B). Mikkelson and
colleagues showed that the entire biological response (pilocarpine-induced miosis) could be
suppressed when low drug concentrations were used in the presence of serum albumin.5 Future
studies leveraging this work should assess the ocular response obtained from different
concentrations of the same drug (e.g. tropicamide 0.05%, 0.5%, 1%, 2%) in the presence of
albumin in tears.
A number of other factors can influence the drug-protein interactions on the ocular surface, including albumin concentrations in tears and physicochemical properties of the individual drug. In plasma, higher levels of albumin can further reduce the biological response of a drug, as exemplified by lower antimicrobial activity of fluoroquinolones with increasing levels of serum albumin.36 In tears, however, the present study did not find statistical differences between 0.4% and 1.5% albumin for either tropicamide or latanoprost. It is possible that the 119
magnitude of albumin levels is not as critical in tears as it is in plasma, as lacrimal concentrations
of albumin are relatively small (< 2%)10 in comparison to blood (≥ 4%).36 Similarly, the
influence of molecular weight on drug-albumin affinity may be minimal in tears, as most ophthalmic drugs have a relatively small and narrow range of molecular weights (e.g. 284 Da for tropicamide, 432 Da for latanoprost). In contrast, the pH of ophthalmic drugs is likely more impactful: pH varies from one ophthalmic solution to another, and albumin is known to change its binding affinity and conformation when exposed to changes in solution pH.37 Here,
tropicamide solution is slightly more acidic (pH 5.8) than latanoprost solution (pH 6.7), although
both solutions are near physiologic pH for the ocular surface and may have minimal impact on
albumin affinity compared to other ophthalmic drugs such as dorzolamide (pH 4.5). Future
studies should investigate the importance (or lack thereof) of other relevant biological factors on
albumin-drug interactions in tear fluid, such as drug lipophilicity, viscosity, temperature at the
ocular surface, fatty acids levels in tears, and drug-drug interactions.38
The study may underestimate drug-proteins interactions and their impact on bioavailability
The present work likely underestimates the true impact of proteins on ocular bioavailability of drugs, as the study design has two noteworthy limitations. First, although the lag time between albumin and drug instillation was short (<10 s), it may be sufficient for a portion of the administered albumin to be washed out of the ocular surface by the time the drug mixes with the tear film, as most of an administered eyedrop is lost to drainage in the first 15 to
30 seconds.28,29 Second, although albumin is a major actor of drug-protein binding on the ocular surface, other proteins play a critical role too. Using equilibrium dialysis, Chrai and Robinson showed that sulfisoxazole primarily binds to albumin in tears, but also α-globulin and (to a lesser 120
extent) γ-globulin and lysozyme,27 all of which are normal components of tears in dogs and other
species. Ultimately, the authors recommend that future investigations be conducted with
experimental models of blood-tear barrier breakdown, such as histamine-induced conjunctivitis
in dogs.10 With such models, albumin and other key proteins are already present on the ocular
surface when the drug is administered topically, although individual protein concentrations may
vary from one eye to another, and this variability should be accounted for in the interpretation of
study results. Furthermore, such models would account for other important changes that occur
with ocular surface inflammation and that could affect drug-protein interactions, such as altered
tear volume and turnover rate, tear film instability, and variations in mucin composition.39
Strategies to minimize drug-proteins interactions and enhance ocular bioavailability
A few strategies can be used to minimize the effects of drug-protein interactions on the ocular surface, and thereby maximize the drug pharmacological action by enhancing intraocular bioavailability. First, a higher drug concentration should be considered, especially if available commercially (e.g. tropicamide 1% instead of 0.5%), as the resulting concentration gradient of unbound drug will be higher (Fick’s first law of diffusion). Second, the amount of protein leakage into the tear film can be reduced by stabilizing the blood-tear barrier in the diseased eye, a process achieved by treating the underlying ocular disease10 and/or using vasoprotective drugs such as calcium dobesilate.40 Last, drug-protein interactions can be reduced by using competitive inhibitors of protein binding; for instance, Mikkelson and colleagues showed that the biological activity of pilocarpine (a miotic agent) increased 10-fold in the presence of the competitive inhibitor cetylpyridinium chloride.41 However, the use of competitive inhibition of albumin
binding is discouraged until the importance of albumin on the ocular surface is fully elucidated. 121
In fact, albumin in tear film may serve as a double-edged sword, being detrimental to the ocular bioavailability of topically administered medications, but also beneficial for symptomatic relief of dry eye,42,43 corneal wound healing,42 and anti-oxidative and anti-inflammatory activities.44
Conclusion
Albumin in tears modulate the ocular bioavailability of topically administered drugs, as observed in a ‘large animal’ model that shares similar anatomical and physiological features with humans. The effect of albumin depends on the medication (e.g. drug concentration and inherent physicochemical properties) and was overall mild (<10%) in the present work on healthy canine eyes, albeit likely underestimated given the rapid tear turnover rate following eyedrop administration. Models of ocular surface inflammation could enable future pharmacological studies to gain a deeper understanding of protein-drug interactions, accounting for albumin leakage in tears as well as other relevant factors that affect ocular surface homeostasis.
References
1. Gaudana R, Ananthula HK, Parenky A, et al. Ocular drug delivery. AAPS J 2010;12:348-360.
2. Järvinen K, Järvinen T, Urtti A. Ocular absorption following topical delivery. Advanced Drug Delivery Reviews 1995;16:3-19.
3. Bucolo C, Drago F, Salomone S. Ocular drug delivery: a clue from nanotechnology. Front Pharmacol 2012;3:188.
4. Prausnitz MR, Noonan JS. Permeability of cornea, sclera, and conjunctiva: a literature analysis for drug delivery to the eye. J Pharm Sci 1998;87:1479-1488.
5. Mikkelson TJ, Chrai SS, Robinson JR. Altered bioavailability of drugs in the eye due to drug- protein interaction. J Pharm Sci 1973;62:1648-1653.
6. de Wolf FA, Brett GM. Ligand-binding proteins: their potential for application in systems for controlled delivery and uptake of ligands. Pharmacol Rev 2000;52:207-236.
122
7. Runström G, Mann A, Tighe B. The fall and rise of tear albumin levels: a multifactorial phenomenon. Ocul Surf 2013;11:165-180.
8. Anderson JA, Leopold IH. Antiproteolytic activities found in human tears. Ophthalmology 1981;88:82-84.
9. Woodward DF, Ledgard SE. Effect of LTD4 on conjunctival vasopermeability and blood- aqueous barrier integrity. Invest Ophthalmol Vis Sci 1985;26:481-485.
10. Sebbag L, Allbaugh RA, Weaver A, et al. Histamine-Induced Conjunctivitis and Breakdown of Blood-Tear Barrier in Dogs: A Model for Ocular Pharmacology and Therapeutics. Front Pharmacol 2019;10:752.
11. Zhivkova ZD. Studies on drug-human serum albumin binding: the current state of the matter. Curr Pharm Des 2015;21:1817-1830.
12. Stjernschantz JW. From PGF(2alpha)-isopropyl ester to latanoprost: a review of the development of xalatan: the Proctor Lecture. Invest Ophthalmol Vis Sci 2001;42:1134-1145.
13. Willis AM, Diehl KA, Robbin TE. Advances in topical glaucoma therapy. Vet Ophthalmol 2002;5:9-17.
14. Manny RE, Hussein M, Scheiman M, et al. Tropicamide (1%): an effective cycloplegic agent for myopic children. Invest Ophthalmol Vis Sci 2001;42:1728-1735.
15. Scinto LF, Daffner KR, Dressler D, et al. A potential noninvasive neurobiological test for Alzheimer's disease. Science 1994;266:1051-1054.
16. Gómez-Tortosa E, del Barrio A, Jiménez-Alfaro I. Pupil response to tropicamide in Alzheimer's disease and other neurodegenerative disorders. Acta Neurol Scand 1996;94:104-109.
17. Reitner A, Baumgartner I, Thuile C, et al. The mydriatic effect of tropicamide and its diagnostic use in Alzheimer's disease. Vision Res 1997;37:165-168.
18. Vézina M. Comparative Ocular Anatomy in Commonly Used Laboratory Animals In: Weir AB,Collins M, eds. Assessing Ocular Toxicology in Laboratory Animals. Totowa, NJ: Humana Press, 2013;1-21.
19. Sebbag L, Allbaugh RA, Wehrman RF, et al. Fluorophotometric Assessment of Tear Volume and Turnover Rate in Healthy Dogs and Cats. J Ocul Pharmacol Ther 2019;35:497-502.
20. Grozdanic SD, Kecova H, Harper MM, et al. Functional and structural changes in a canine model of hereditary primary angle-closure glaucoma. Invest Ophthalmol Vis Sci 2010;51:255- 263.
123
21. Kaswan RL, Salisbury MA, Ward DA. Spontaneous canine keratoconjunctivitis sicca. A useful model for human keratoconjunctivitis sicca: treatment with cyclosporine eye drops. Arch Ophthalmol 1989;107:1210-1216.
22. Rubin LF, Wolfes RL. Mydriatics for canine ophthalmoscopy. J Am Vet Med Assoc 1962;140:137-141.
23. Pirie CG, Maranda LS, Pizzirani S. Effect of topical 0.03% flurbiprofen and 0.005% latanoprost, alone and in combination, on normal canine eyes. Vet Ophthalmol 2011;14:71-79.
24. Lee SS, Burke J, Shen J, et al. Bimatoprost sustained-release intracameral implant reduces episcleral venous pressure in dogs. Vet Ophthalmol 2018;21:376-381.
25. Dalhoff A. Seventy-Five Years of Research on Protein Binding. Antimicrob Agents Chemother 2018;62.
26. Hatzav M, Bdolah-Abram T, Ofri R. Interaction with therapeutic soft contact lenses affects the intraocular efficacy of tropicamide and latanoprost in dogs. J Vet Pharmacol Ther 2016;39:138-143.
27. Chrai SS, Robinson JR. Binding of sulfisoxazole to protein fractions of tears. J Pharm Sci 1976;65:437-439.
28. Shell JW. Pharmacokinetics of topically applied ophthalmic drugs. Surv Ophthalmol 1982;26:207-218.
29. Wilson CG. Assessing ocular drug delivery with lachrimal scintigraphy. Pharm Sci Technolo Today 1999;2:321-326.
30. Jugant S, Grillot AE, Lyarzhri F, et al. Changes in pupil size and intraocular pressure after topical application of 0.5% tropicamide to the eyes of dogs sedated with butorphanol. Am J Vet Res 2019;80:95-101.
31. Yoshitomi T, Ito Y. Effects of indomethacin and prostaglandins on the dog iris sphincter and dilator muscles. Investigative Ophthalmology & Visual Science 1988;29:127-132.
32. Yousufzai SYK, Ye ZHI, Abdel-Latif AA. Prostaglandin F2αand its Analogs Induce Release of Endogenous Prostaglandins in Iris and Ciliary Muscles Isolated from Cat and Other Mammalian Species. Experimental Eye Research 1996;63:305-310.
33. Bergh K, Wentzel P, Stjernschantz J. Production of prostaglandin e(2) by iridial melanocytes exposed to latanoprost acid, a prostaglandin F(2 alpha) analogue. J Ocul Pharmacol Ther 2002;18:391-400.
124
34. Fentiman KE, Rankin AJ, Meekins JM, et al. Effect of topical ophthalmic administration of 0.005% latanoprost solution on aqueous humor flow rate and intraocular pressure in ophthalmologically normal adult Beagles. Am J Vet Res 2019;80:498-504.
35. Snyder R. Basic Concepts of the Dose-Response Relationship. Assessment and Management of Chemical Risks: American Chemical Society, 1984;37-55.
36. Zeitlinger M, Sauermann R, Fille M, et al. Plasma protein binding of fluoroquinolones affects antimicrobial activity. J Antimicrob Chemother 2008;61:561-567.
37. Kochansky CJ, McMasters DR, Lu P, et al. Impact of pH on plasma protein binding in equilibrium dialysis. Mol Pharm 2008;5:438-448.
38. Zeitlinger MA, Derendorf H, Mouton JW, et al. Protein binding: do we ever learn? Antimicrob Agents Chemother 2011;55:3067-3074.
39. Rolando M, Zierhut M. The ocular surface and tear film and their dysfunction in dry eye disease. Surv Ophthalmol 2001;45 Suppl 2:S203-210.
40. van Bijsterveld OP, Janssen PT. The effect of calcium dobesilate on albumin leakage of the conjunctival vessels. Curr Eye Res 1981;1:425-430.
41. Mikkelson TJ, Chrai SS, Robinson JR. Competitive inhibition of drug-protein interaction in eye fluids and tissues. J Pharm Sci 1973;62:1942-1945.
42. Shimmura S, Ueno R, Matsumoto Y, et al. Albumin as a tear supplement in the treatment of severe dry eye. Br J Ophthalmol 2003;87:1279-1283.
43. Seki JT, Sakurai N, Moldenhauer S, et al. Human albumin eye drops as a therapeutic option for the management of keratoconjunctivitis sicca secondary to chronic graft-versus-host disease after stem-cell allografting. Curr Oncol 2015;22:e357-363.
44. Merlot AM, Kalinowski DS, Richardson DR. Unraveling the mysteries of serum albumin- more than just a serum protein. Front Physiol 2014;5:299.
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Tables and Figures
Figure 1. Mean + SD pupil diameter from 0 to 480 min in dogs receiving 1% tropicamide in both eyes, immediately preceded by topical instillation of artificial tears (control, white circles) in one randomly selected eye, and either 0.4% albumin (A, black triangles) or 1.5% albumin (B, black triangles). Statistical differences (P < 0.05) obtained with mixed model for repeated measures are depicted by gray asterisks (*).
Figure 2. Clinical image of a Beagle dog at 45 min following topical instillation of 1% tropicamide in both eyes, immediately preceded by topical artificial tears (right eye, control) and 0.4% albumin (left eye). Note the lower degree of mydriasis in the left eye. 126
Figure 3. Box-and-whisker plots depicting the area under the effect-time curve (AUETC) of pupil diameter over time (0 to 480 min) in dogs receiving 1% tropicamide in both eyes, immediately preceded by topical instillation of artificial tears (control, white boxes) in one randomly selected eye, and either 0.4% albumin (light gray box) or 1.5% albumin (dark gray box) in the other eye. Mean and median values are shown by horizontal dotted and solid lines, respectively. First and third quartiles (25th and 75th percentiles) are represented by the lower and upper limits of the box, respectively, while the 2.5th and the 97.5th percentiles are shown as the lower and upper whiskers, respectively.
Figure 4. Box-and-whisker plots depicting the maximal pupillary diameter in dogs receiving 1% tropicamide in both eyes, immediately preceded by topical instillation of artificial tears (control, white boxes) in one randomly selected eye, and either 0.4% albumin (light gray box) or 1.5% albumin (dark gray box) in the other eye. Mean and median values are shown by horizontal dotted and solid lines, respectively. First and third quartiles (25th and 75th percentiles) are represented by the lower and upper limits of the box, respectively, while the 2.5th and the 97.5th percentiles are shown as the lower and upper whiskers, respectively. 127
Figure 5. Mean + SD pupil diameter from 0 to 480 min in dogs receiving 0.005% latanoprost in both eyes, immediately preceded by topical instillation of artificial tears (control, white circles) in one randomly selected eye, and either 0.4% albumin (A, black triangles) or 1.5% albumin (B, black triangles). No statistical differences were noted between groups at any time point (mixed model for repeated measures, P ≥ 0.05).
Figure 6. Mean + SD intraocular pressure from 0 to 480 min in dogs receiving 0.005% latanoprost in both eyes, immediately preceded by topical instillation of artificial tears (control, white circles) in one randomly selected eye, and either 0.4% albumin (A, black triangles) or 1.5% albumin (B, black triangles). Statistical differences (P < 0.05) obtained with mixed model for repeated measures are depicted by gray asterisks (*). 128
Figure 7. Hypothetical scenarios highlighting the importance of dose-response relationship in understanding the impact of albumin in tears on the biological activity of an ophthalmic drug. (A) A 50% reduction in the amount of drug that can penetrate inside the eye will have a minimal effect on the biological effect of drug A (dotted line), but a profound effect on drug B (solid line). (B) For the same drug, a 50% reduction in the amount of drug that can penetrate inside the eye will have a minimal effect on the biological response if the initial drug concentration was high (X), but a profound effect if the initial drug concentration was relatively low (Y).
129
CHAPTER 8. GENERAL CONCLUSION - AN EYE ON THE DOG AS THE SCIENTIST’S BEST FRIEND FOR TRANSLATIONAL RESEARCH IN OPHTHALMOLOGY: FOCUS ON THE OCULAR SURFACE
Lionel Sebbag,1,2 Jonathan P. Mochel1
1 Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State
University, Ames, IA 50011, USA; 2 Department of Veterinary Clinical Sciences, College of
Veterinary Medicine, Iowa State University, Ames, IA 50011, USA
Modified from a manuscript under review in Medicinal Research Reviews
Abstract
Preclinical animal studies provide valuable opportunities to better understand human diseases and contribute to major advances in medicine. This review provides a comprehensive overview of ocular parameters in humans and selected animals, with a focus on the ocular surface, detailing species differences in ocular surface anatomy, physiology, tear film dynamics and tear film composition. We describe major pitfalls that tremendously limit the translational potential of traditional laboratory animals (ie., rabbits, mice and rats) in ophthalmic research, and highlight the benefits of integrating companion dogs with clinical analogues to human diseases into preclinical pharmacology studies.
This One Health approach can help accelerate and improve the framework in which ophthalmic research is translated to the human clinic. Studies can be conducted in canine subjects with naturally occurring or non-invasively induced ocular surface disorders (eg., dry eye disease, conjunctivitis), reviewed herein, and tear fluid can be easily retrieved from canine eyes 130
for various bioanalytical purposes. In this review, we discuss common tear collection methods,
including capillary tubes and Schirmer tear strips, and provide guidelines for tear sampling and
extraction to improve the reliability of analyte quantification (drugs, proteins, others).
Introduction
Preclinical animal models provide critical information to better understand human diseases’ characteristics, identify biomarkers, develop diagnostic tools and novel therapeutics.
Rabbits and laboratory rodents (mice, rats) are widely used for ophthalmic research as they are economical and easy to handle;1 however, serious drawbacks limit the translational usefulness of
data obtained in these species, notably due to the need to artificially induce pathology in these
animals (eg., through genetic manipulation or experimental surgery), as well as apparent differences in ocular anatomy and physiology compared to humans. For instance, precorneal residence time of topically applied solutions is much prolonged in rabbits owing to their low blink rate, resulting in 3-fold overestimation of ocular drug exposure if findings were directly extrapolated from rabbits to humans.2 Another example is topical nepafenac, a potent nonsteroidal anti-inflammatory drug (NSAID) that reaches therapeutic levels in the posterior segment of mice (owing to their thin cornea and small globe size), inhibiting choroidal neovascularization by decreasing production of VEGF3 – in contrast, humans require intravitreal
injections of anti-VEGF compounds to achieve the same outcome. Multiple other examples exist
in the scientific literature, together participating to the unacceptably low success rate of
ophthalmic clinical trials to date, and resulting in substantial economic loss and burden for
scientists, consumers, and society overall.4 In fact, the main cause for clinical trial failure is 131
either lack of safety or efficacy,5 two components that are supposedly ‘validated’ in initial
preclinical animal studies.
Under the umbrella of the One Health Initiative, a growing number of investigations have
integrated companion animals into preclinical studies to complement and expand the knowledge
gained from studies in other animal models, accelerate and improve the framework in which
research is translated to the human clinic, and ultimately generate discoveries that will benefit
the health of humans and animals.6 Over the last few years, several review articles have
highlighted the benefits of using dogs for translational research in oncology,7 neurology8 and
other biomedical fields,6 yet such information is not available in ophthalmology.
The present review provides a comprehensive comparison of key ocular parameters in
humans, dogs and traditional laboratory animals (ie., rabbits, mice, rats), highlighting selected
strengths and important pitfalls that must be addressed when ocular research is conducted in
animal models. This review is focusing on the ocular surface, a critical element of vision that includes the secreted tear film, lacrimal gland(s), eyelids, meibomian glands, cornea, conjunctiva, sclera, and nasolacrimal drainage apparatus. The ocular surface dictates the bioavailability of medications administered topically to the eye,9 and is a common site of
pathology in both human and veterinary medicine. Methods of tear fluid collection for
bioanalytical purposes are also being discussed, with special consideration on the safety and
efficiency of the collection technique at hand. Lastly, this review highlights on spontaneous and
experimental ocular surface disorders in dogs, providing a tool for researchers to better model
disease pathophysiology in clinical patients suffering from ocular surface disorders.
132
Comparative anatomy and physiology of the ocular surface
Anatomy
The anatomy of the ocular surface is depicted in Figure 1 for dogs, and its parameters are being summarized in Table 1 for all species discussed in this review (ie., humans, dogs, rabbits, mice, rats).
Lacrimal glands - Four types of lacrimal glands can be distinguished in mammals: (i) the orbital lacrimal gland (glandulae lacrimales superior), located in the dorsolateral orbit just caudal to the orbital rim, with secretory ducts that open into the upper conjunctival fornix
(humans, dogs, rabbits); (ii) the gland of the third eyelid, located in the ventromedial orbit at the base of the nictitating membrane with secretory ducts than open into the nictitans’ bulbar conjunctiva (dogs); (iii) the infraorbital gland (glandulae lacrimales inferior), located either intraorbital and ventromedial to the globe (rabbits) or extraorbital and caudal to the globe
(rodents), with a single secretory duct that opens into the lower conjunctival fornix; and (iv) the
Harderian gland, or Harder’s gland, extending from the base of the third eyelid into the caudal orbit, with secretory ducts opening at the nictitating membrane (rabbits, rodents).10,11 The histomorphology of lacrimal glands varies with age and sex of the individual.12 In dogs, an orbital lacrimal gland and gland of the third eyelid contribute to 60-70% and 30-40% of the overall tear secretion, respectively.13 The morphological and histological features of the canine glands resemble the human lacrimal gland including distinct lobules and acini that provide serous and mucous secretions, as well as intralobular ducts that drain into small excretory tubules.14,15 Likewise, an Harderian gland is not present in the canine or human orbit.14-16
However, two notable differences exist between species: (i) the combined volume of the two canine glands is smaller than the main lacrimal gland in humans (0.24 vs. 0.60 cm2)17,18; and (ii) 133
the accessory lacrimal glands of Krause and Wolfring are absent in dogs (or not yet reported),
presumably being consolidated through evolution into the single gland of the third eyelid.19
These accessory glands account for 10% of the total lacrimal secretory mass in humans but their
contribution to the overall tear secretion is negligible (1-2%).20,21 In rabbits, the histoarchitecture
of the main lacrimal gland is comparable to humans with loosely packed acini and round/oval
lumen; in contrast, mice and rats have densely packed acini with small pleiomorphic lumen and
numerous intercellular tight junctions.22 Like humans, rabbits also possess accessory lacrimal
glands of Wolfring in the tarsal portion of the palpebral conjunctiva.23 However, the Harderian
gland present in rabbits and rodents is a unique anatomical feature that has important
repercussions for comparative studies; in fact, the gland’s lipid secretions in the tear film have
profound effects on the ocular surface physiology (eg., tear composition, tear film dynamics,
blink rate) and pharmacology of topically applied medications (see sections 2.2 and 2.3).
Nasolacrimal apparatus - The morphology of the canine lacrimal drainage system is
remarkably similar to that of humans, except for a longer nasolacrimal duct (notably in long-
nosed dogs), and the presence of accessory duct openings into the nasal cavity.24 In both species,
tear drainage begins with the lower and upper nasolacrimal puncta and canaliculi in the medial
canthus, joining into a lacrimal sac in the bony lacrimal fossa, and extending into the
nasolacrimal duct that runs through an osseous channel towards the nasal cavity.25 Species similarities are also evident on a microscopic level, including an epithelial lining with microvilli and mucin-secreting goblet cells, sub-epithelial seromucous glands, and mucosal-associated lymphoid tissue.24 In contrast, the nasolacrimal apparatus of rabbits has distinct differences
compared to humans. Rabbits only have a single nasolacrimal punctum/canaliculus (medial
lower eyelid) and the nasolacrimal duct has two very distinct flexures due to the ventral 134
deflection of the snout, a unique feature that results in a convoluted path for tear drainage.25,26
The fetal development of the rabbit’s nasolacrimal apparatus is also unique in mammals, more
closely resembling reptiles vs. humans.27 At an ultrastructural level, the epithelium lining the
duct is double-layered (similar to humans) but there are no goblet cells or subepithelial
seromucous glands.28 Nonetheless, the use of the rabbit is still recommended as a practical model
to characterize the nasolacrimal apparatus,29 albeit this choice is described as ‘less than ideal’ by the authors. Mice and rats have a well-developed nasolacrimal apparatus that shares similar ontogenetic origin to humans,27 although the histological features are different. The duct lining is
covered by a multi-layered stratified squamous epithelium with goblet cells but without sub-
epithelial seromucous glands.25
Third eyelid - The nictitating membrane (third eyelid) is a large fold of the conjunctiva
that protrudes from the medial canthus over the anterior surface of the globe in many animals,
including dogs, rabbits and rodents. The counterpart in humans is the plica semilunaris, a
vestigial remnant in the form of a crescent-like conjunctival fold in the medial canthus.11,30
Despite gross differences, both structures have important physio-morphological similarities such
as the presence of goblet cells and lymphoid follicles, contributing to the lubrication and immune
protection of the ocular surface.30 Nonetheless, the presence of a third eyelid should be
considered in comparative studies as it could impact ocular examinations (eg., third eyelid
protrusion from ocular irritation) or ocular drug delivery (eg., altered retention time of a contact lens),31 among others. If required for ease of experimentation, a simple fixation of the nictitating
membrane can be performed31 as an alternative to complete surgical removal,13,32,33 as the latter
negatively impacts ocular surface homeostasis.13,34 135
Eyelids - Similar to humans, the canine upper and lower eyelids are comprised of an
outer dermis, tarsus, orbicularis oculi muscle, palpebral conjunctiva and secretory tissues
including meibomian glands (20-40 per eyelid), glands of Zeis and Moll.35,36 The main
anatomical difference is the tarsal plate, which is comprised of dense fibrous tissue and cartilage-
specific components in humans37 – providing a rigid internal support to the eyelids – compared
to a much thinner and poorly-developed fibrous tissue in dogs.38 Also, the interpalpebral fissure
area is approximately 20% larger in dogs (2.2 vs. 1.8 cm2)39,40, although the measurements of the
palpebral fissure width depend on the dog’s size and body weight.40
The palpebral opening in the rabbit is relatively small (10-16 mm),38,41,42 albeit much larger than mice (3.7-5 mm)43 and rats (6-9 mm)44,45, with a shorter and thicker upper eyelid
compared to the inferior palpebrae; consequently, the interpalpebral fissure area is 20% smaller
in rabbits than in man (1.44 vs. 1.8 cm2).39 The meibomian gland ducts and acini are also larger
in rabbits than mice and rats,46 but the overall volume and distribution of meibomian glands is
different than in humans: the total meibomian gland volume in the human (39.5 mm3) is twice
that of the rabbit (18.8 mm3), with a larger volume in the upper eyelid (man) compared to similar
volumes in the upper and lower eyelids (rabbit).39
Conjunctiva - The conjunctiva is a thin mucous membrane that serves important roles on
the ocular surface including mucin secretion and immune surveillance. The anatomical
subdivision of the conjunctiva is the same in humans, dogs, and common laboratory species
(rabbits, rodents): the palpebral conjunctiva – lining the inside of the eyelids – reflects back at
the level of the conjunctival fornix to form the bulbar conjunctiva, a region that covers the
anterior portion of the sclera and attaches to the corneoscleral limbus.16 However, the amount of
bulbar conjunctiva exposed (‘scleral show’) is notably larger in humans compared to animals 136
given differences in eyelid opening and/or corneal diameter. Another important species
difference is the presence of a nictitating membrane in animals (but not man), as the third eyelid
is covered by conjunctiva on its anterior and posterior surfaces. As such, animals have two
conjunctival fornices in the inferonasal region – one on each side of the third eyelid – and the
overall conjunctival surface is generally larger in animals compared to humans. In dogs, the
conjunctival area is supposedly larger than in humans given the depth of the canine conjunctival
fornices and the amount of conjunctiva covering the canine nictitating membrane,47 although no
objective data exist to date. In rabbits, the upper conjunctival fornix depth (20.36 mm)48 is larger
than in humans (15 mm),49 while the conjunctival area is reportedly comparable (13.34-18.48 vs.
17.65 cm2, respectively), although the measurements did not include the rabbit’s third eyelid
(surgically removed by investigators).50
Conjunctival goblet cells are distributed individually in humans, dogs and rabbits, in
contrast to clustered organization in mice and rats.51,52 The distribution of goblet cells is overall
similar in dogs and humans, with high density in the canine third eyelid and human plica
semilunaris, relatively high density in the conjunctival fornices and palpebral conjunctiva, and
lower density in the bulbar conjunctiva.30,52-56 In rabbits, the highest density is noted at the lid
margin of both upper and lower palpebral conjunctivae,57,58 while the density in the bulbar
conjunctiva is generally higher than in humans (399-1576 cells/mm2 vs. 7-979 cells/mm2).56,59 In addition to mucin-secreting goblet cells, the conjunctiva also contains an organized immune network termed conjunctiva-associated lymphoid tissue (CALT), a structure that plays a key role
in protecting the ocular surface by initiating and regulating immune responses.60 The presence of
lymphoid follicles was confirmed in the conjunctiva of most mammals studied by Chodosh and
colleagues – including humans, dogs, rabbits – with the exception of mice and rats,61 although a 137
later report detected lymphoid tissue in the nictitating membrane of BALB/c mice.62 At an ultrastructural level, specialized M cells are present in the epithelium overlying the conjunctival follicles in dogs63 and rabbits,64 similar to humans.65
Cornea - The anatomy of the cornea is unique to each species with important differences in corneal dimensions and ultrastructural features (eg., thickness, collagen arrangement, nerve supply).11,38,50,66-70 First, the cornea is generally larger in dogs and rabbits compared to humans, while the dimensions are much smaller in mice and rats.41,44,71-77 As such, the relative amount of cornea and conjunctiva exposed on the ocular surface varies among species, an anatomical fact that has important implications in ocular pharmacology and other research fields; for instance, the surface area ratio of conjunctiva to cornea is two times smaller in rabbits (8.6-8.9) than humans (17.1), a finding that could largely explain species differences in drug penetration into the anterior chamber.50 Second, the corneal thickness varies among mammals and is generally correlated to the size of the animal.68 From highest to lowest, the mean central corneal thickness is 497-594 µm in dogs,68,78 505-563 µm in humans,79 354-407 µm in rabbits,41,79,80 159-170 µm in rats68,80 and 90-137 µm in mice.68,74,80 The average canine cornea is only slightly thicker than in humans. In contrast, the thinner cornea in rabbits and rodents can limit the use of these laboratory species for selected experiments; for instance, cross-linking is discouraged in corneas thinner than 400 µm due to potential damage to the corneal endothelium or intraocular tissues.81
On a structural level, the main layers of the cornea are the same in humans and animals
(epithelium, stroma, Descemet membrane, endothelium) with the notable exception of the
Bowman’s membrane.11,70 Bowman’s membrane is present in nearly all primates (including humans) and selected animals (eg., sheep, deer, giraffe),82 but is absent in dogs and common laboratory species.68,70,82 The number of layers and overall thickness of the corneal epithelium 138
vary among species: humans (5-7 layers, 44-55 µm),79,83 dogs (6-9 layers, 52-64 µm),70,71,78
rabbits (5-7 layers, 45-49 µm),38,79,83 rats (10-14 layers, 26-33 µm),84,85 and mice (13 layers, 37-
46 µm).74 The corneal stroma, comprising nearly 90% of the total corneal thickness in most
mammals, is primarily composed of collagen fibrils arranged in lamellae. While extensive
collagen intertwining is noted in the majority of the corneal stroma in humans, it is only present
in the anterior most-aspect of the cornea in dogs and rabbits.86,87 Differences in collagen intertwining, along with the absence of Bowman’s membrane in laboratory species, explain the vast disparity in stiffness of the anterior stroma (16.2, 1.3 and 1.1 kPa) and posterior stroma (2.5,
0.5 and 0.4 kPa) in humans, dogs and rabbits, respectively.86,87 The elastic modulus of the cornea is reportedly higher in rodents, although the methodology used was different.88,89 Corneal rigidity
should be considered in comparative studies in which the biophysical attributes of the cornea are
important (eg., wound healing, keratoprosthesis). The corneal endothelium shares a similar morphological blueprint among species (single cell layer, honey-comb pattern), while the
cellular density varies from 3233 cells/mm2 in rabbits, 2875 cells/mm2 in mice, 2818 cells/mm2
in dogs, 2732 cells/mm2 in humans, and 2242 cells/mm2 in rats.68,90
The mammal cornea is the most densely innervated tissue in the body. Corneal nerves
play important roles to maintain ocular surface health and homeostasis, including sensory
functions (touch, pain, temperature), release of trophic neuropeptides, maintenance of the limbal
stem cell niche, and activation of brainstem circuits to promote reflex blinking and lacrimation.
From highest to lowest, the sensitivity of the cornea to mechanical stimulus is as follows:
humans (0.2-1.0 g/mm2), rats (0.42-0.47 g/mm2), mice (0.59 g/mm2), dogs (2.16-2.9 g/mm2), and
rabbits (6.21-10 g/mm2).2,91-95 The murine model is the most extensively studied of all laboratory
species given gross similarities between mice and humans in corneal sensitivity and nerve 139
architecture.67,96 The canine model is also studied in detail given shared features with humans in several spontaneous diseases such as diabetes mellitus, herpetic keratitis, and non-healing corneal ulcers;97-100 importantly, investigators should account for the canine breed selected for the experiment as corneal sensitivity depends on the dog’s cephalic conformation.95 In regard to rabbits, two striking species differences exist: (i) Corneal sensitivity in rabbits is much lower than in humans, dogs and rodents;2,92 and (ii) Morphology of the rabbit subbasal plexus is unique, with nerve fibers sweeping horizontally across the corneal surface in a temporal-to-nasal direction compared to a typical whorl-like or spiraling pattern in other species.67
Sclera - Humans have a widely exposed white sclera, a feature that is unique when compared to other primate species (Kobayashi, 1997). In contrast, the scleral exposure is minimal in dogs and routine laboratory species. The thickness of the sclera also differ among species: at the ocular surface (limbal sclera), recorded measurements vary from 0.8 mm in dogs,101 0.5 mm in humans,102 0.29 mm in rabbits,103 0.1 mm or less in rats,104 and 0.05-0.06 mm in mice.105
Tear film dynamics
Effective tear dynamics, combined with well-balanced composition of the tear film
(discussed in the next section), are critical for the maintenance of ocular surface homesostasis and physiology. Tear fluid dynamics – or the balance between tear secretion, distribution, absorption, evaporation, and drainage – are closely regulated by the lacrimal functional unit. The lacrimal functional unit is unique to each species (see aforementioned anatomical differences), comprised of secreting glands (orbital, accessory, third eyelid, Harder’s, meibomian), eyelids, conjunctival goblet cells, corneo-conjunctival surface, and their interconnecting innervation.106 140
Key physiological parameters provide insight into the complex tear dynamics – highlighted in
Figure 2 and Table 2 – and are therefore important to account for in translational studies that involve the ocular surface:
• Basal tear turnover rate: Tear turnover rate is considered a global measure of the tear
dynamics and integrity of the lacrimal functional unit.107,108 The basal tear turnover rate is
reportedly 13.1-17.5 %/min in humans,109,110 12.1 %/min in dogs,111 6.2-7.1 %/min in
rabbits112 and 5.2 %/min in mice;113 no information was available in rats. In other words, it
takes approximately the same time for the tear film to replenish in dogs and humans (~6-8
min) but the duration is longer in rabbits (~14-16 min) and mice (~20 min). The slow tear
turnover of rabbits and rodents has important repercussions in translational research,
including a longer precorneal retention time of instilled eyedrops (see next subsection), or
exaggeration of ocular surface disease due to delayed clearance of inflammatory mediators
from the tear film.114
• Tear volume: The volume of tears on the ocular surface is highest in dogs (65.3 µL),111
followed by humans (7-12.4 µL),109,115 rabbits (1.9-7.5 µL)112,116 rats (4.6 µL)117 and mice
(0.06-0.2 µL).113,118 Canine tear volume depends on the subject’s body weight but not the
dog’s cephalic conformation.111 Differences in study methodology notwithstanding, the
canine tear volume is approximately 5 to 9-fold larger than in humans. This discrepancy can
be partly explained by the additional secretory tissue in dogs (third eyelid gland) and the
larger corneal surface to lubricate in dogs (1.2-2.1 cm2 vs. 1.04-1.3 cm2).50,71,77 The canine
tear film may also be thicker than in humans (15.1 µm vs. 2.3-11.5 µm), although
measurements of tear thickness were only obtained in 6 dogs111 and the calculation of tear
thickness is reportedly highly variable within and between species.119 141
• Spontaneous blink rate: The blink action distributes fresh tears on the ocular surface in a
uniform layer, promotes secretion of tears from the accessory tear glands, and pumps excess
tears (or instilled drop) into the nasolacrimal drainage system. Spontaneous blinking is
triggered by higher centers in response to corneo-conjunctival nerve stimulation, presumably
due to changes in ocular surface temperature that result from thinning and evaporation of
unstable tear film.120 The spontaneous blink rate is very similar between dogs (14.2
blinks/min)121 and humans (8.5-17.6 blinks/min),122-124 although it is lower in rodents (< 5.3
blinks/min)91,124-126 and much lower in rabbits (0.05-0.19 blinks/min).2,123,127 In other words,
humans and dogs blink approximately every 4-7 seconds, while mice/rats blink every 11
seconds (or more) and rabbits only blink every 313-1200 seconds. This large disparity in
mammals’ blink rate can be explained by species differences in (i) ocular surface sensitivity,
(ii) tear composition, and (iii) the inherent stability of the animal’s tear fluid. In fact, (i) the
corneo-conjunctival sensitivity is higher in humans > rodents > dogs >> rabbits, a key
parameter that is linked to spontaneous blinking as well as reflex secretion of tear
components from the lacrimal glands, conjunctival goblet cells and meibomian glands;128 (ii)
tear composition is unique to each species (see next section), for instance large discrepancies
exist in the tear lipidomic profile of rabbits vs. man;129 and (iii) tear film stability is strongly
associated with the maximum blink interval, as recently shown in humans.130 Tear stability is
often measured with the tear film breakup time (TFBUT), defined as the interval between the
last complete blink and the first appearance of a dry spot in the tear film. Results of TFBUT
and other tear film diagnostics are summarized in Table 2, with care given to discard or
highlight values obtained in anesthetized or sedated animals (eg., TFBUT of 29.8 min in 142
sedated rabbits)131 as chemical intervention negatively impacts ocular surface homeostasis
(ie., abolished blinking, reduced tear secretion).
Importantly, investigators should account for additional parameters (and their species differences) in any study that involves topical drug administration. In fact, an eyedrop can be considered as a transient ocular irritant – especially if the solution’s pH or osmolarity is different than the tear film – thereby stimulating reflex blinking and lacrimation upon contact with the ocular surface.132
• Reflex blinking (or lack thereof): In dogs, a blink occurs immediately after eyedrop
administration and is responsible for removal of any excess solution onto the periocular skin
and nasolacrimal drainage system.40 The same is true in humans, in whom an instilled
eyedrop is partially lost (20-30%) due to reflex blinking and spillage onto the eyelids and
eyelashes.133 Blinking in response to eyedrop instillation is also reported in mice134 and
rats.135 In contrast, rabbits rarely blink following eyedrop administration, or do so
infrequently. In one study, rabbits did not blink for 20-30 minutes after instillation of an
eyedrop, and this alone could result in overestimating ocular drug exposure by 3-fold if
findings were to be extrapolated to humans.2
• Reflex tear turnover rate: Eyedrop administration abruptly increases the volume of fluid in
the conjunctival sac and ocular surface. The sudden disruption in homeostasis promotes a
faster nasolacrimal drainage until baseline conditions return. This physiologic response is
prominent in dogs (50 %/min)111 and humans,109,115 but is minimal in rabbits (6.1-6.9
%/min).112 In fact, the tear turnover rate in rabbits is mostly unchanged whether a small (1-5
µL) or large volume (25-50 µL) of eyedrop is instilled on the ocular surface,112 a finding 143
likely related to the poor corneal sensitivity and inexistent/minimal reflex blinking in this
species.2,92 No available report in mice or rats can be found in the literature.
• Volumetric capacity of the palpebral fissure: The surface of the canine eye can ‘hold’ on
average 31.3 µL of fluid,40 nearly identical to the volumetric capacity of the human eye (25-
30 µL)109,136 and the volume of a single ophthalmic drop (35 µL).137 Of note, the volumetric
capacity of the canine eye is positively correlated with the length of the palpebral fissure,40
and may be larger in breeds larger than Beagles (eg., German Shepherd dogs).137 The exact
volumetric capacity of the eye is not reported in laboratory species, but is presumably around
10-25 µL in rabbits (based on drug quantification in tears at various instilled volumes),112,138
≤ 5 µL in mice139,140 and ≤ 20 µL in rats.141
Tear film composition
The tear film is a complex biological fluid containing thousands of compounds of diverse structures and functions, including proteins, lipids and mucins, as well as minor constituents such as electrolytes, vitamins, and growth factors.36,142 The integrated interactions of these constituents are responsible for the promotion of a stable tear film and, ultimately, the homeostasis of the ocular surface. Species differences in tear film components are summarized in
Table 3.
Proteins – The total protein content is generally similar in dogs (5.2-14.6 mg/mL)143 and humans (6.0-11.0 mg/mL),144 although qualitative and quantitative differences exist.
Specifically, the three major constituents of the human tear proteome (lactoferrin, lysozyme, lipocalin)144 are only detected at low levels in dogs,145-150 although the relative abundance of other common proteins (eg., lacritin, secretory IgA, serum albumin) is generally similar between 144
the two species. Importantly, homologous proteins have been described in canine tears and may
play similar functions to their human counterparts – for instance, transferrin is an iron-binding
protein with similarities to lactoferrin, while major canine allergen is an abundant protein in
canine tears with similarities to lipocalin.148-150 From a qualitative aspect, a recent in-depth proteomic study showed that 25 out of 125 proteins detected in canine tears were common to humans.149 In rabbits, Wei et al. found that the total protein content was two-fold higher in rabbits compared with humans (20.6 mg/mL vs. 9.4 mg/mL), although the number of different proteins detected in tear samples was lower in rabbits.151 Other differences in tear proteins
among species are summarized in Table 3.
Mucins – Ocular mucins are large glycoproteins expressed by conjunctival goblet cells,
the corneal epithelium and the lacrimal gland(s), playing important roles on the ocular surface in
lubrication, wettability and barrier function.152 The main secretory mucin, MUC5AC, is
described at large levels on the ocular surface of humans and animals.11,55,152 The expression of
membrane-associated mucins, however, differs among species. In a recent study by Leonard et
al., dogs were found to have a very similar pattern of mucin expression to that of humans and
rhesus macaques, with MUC16 being the most abundant mucin transcript.153 In contrast, the
rabbit had a unique mucin expression pattern with all mucin transcripts expressed at relatively
similar levels; as such, the authors concluded that the predictive value of the rabbit as a model in
ocular surface studies should be called into question.153 In another study, the majority of ocular
mucins detected in dogs and rabbits were neutral fucosylated glycans, while the ones in humans
were mainly negatively charged sialylated glycans;154 however, the experiment lysed the ocular
surface epithelium and could not discriminate between mucins of differing origin. 145
Lipids – In a comprehensive lipidomic study comparing the meibum collected in several species, Butovich et al. found that the highest degree of biochemical similarity with humans was observed in mice, closely followed by the dog.129 An earlier study by Butovich et al. also reported the close resemblance of the tear lipid composition between dogs and humans.155 In these 3 species (humans, dogs, mice), the major lipid classes included wax esters, cholesterol esters, and o-acyl-ω-hydroxy fatty acids (OAHFA). In contrast, the major lipid classes in rabbit tears were DiHL esters (24,25-dihydro--lanosterol esters), diacylated diols, and OAHFA, with low to trace amounts of wax and cholesterol esters.129 Such discrepancy between rabbits and humans was confirmed in a separate study by Wei et al, who noted significant differences in the tear film concentrations of triglycerides (higher in rabbits), free cholesterol (lower in rabbits), phosphatidylcholine (higher in rabbits) and phosphatidylethanolamine (higher in rabbits).151
Taken together, the authors of these two studies argued that the rabbit is too different to serve as a valid animal model for humans, at least from a biochemical standpoint.
Tear collection for bioanalytical purposes
The tear film, a complex body fluid uniquely exposed to both internal and external environments, contains numerous endogenous and exogenous molecules (eg., proteins, lipids, mucins, xenobiotic) that can be assayed for clinical or research purposes. Topically and systemically administered drugs can be quantified in tear fluid to determine the clinical efficacy and dosing frequency from fitting of kinetic data.156-160 Multiple ‘omics’ approaches can also be utilized for analysis of the tear fluid including proteomics,149,150,161-166 lipidomics129,155,167,168 and metabolomics,168 providing valuable information for the development of novel diagnostics and therapeutics in ophthalmology, as well as biomarkers identification for various ocular and 146
systemic diseases.169-171 However, collecting tears and obtaining reproducible analytical results
in ophthalmology is challenging; in particular, the volume of tear fluid is limited (unlike other
biological fluids, such as blood or urine), and the biochemical profile of a tear sample is
intimately affected by the collection, storage, extraction, handling, and analytical methods used
by the investigator.
In this section, we review the main sampling methods reported in the scientific literature
and discuss their respective advantages and limitations. Further, based on the authors’ experience
with dogs in clinical and research settings (board-certified veterinary ophthalmologist [LS] and
pharmacologist [JPM]), the section provides recommendations specific to canine subjects and
their use in translational research (Figure 3).
Direct tear sampling
A microcapillary glass tube (1-10 µL) placed in contact with the inferior lacrimal lake is the most commonly reported technique to collect tear fluid. This method directly samples tear fluid by capillary action and is extensively described in humans,144,161,166,172-185, dogs,147,150,186-189 rabbits,183,184,187,190,191 mice,192,193 and rats.147,183,194,195 Other direct techniques (seldom reported)
involve micropipettes,196 polypropylene tubing147 or polytetrafluoroethylene tubing.172 With
capillary glass tubes, it is possible to obtain unaltered tear samples by avoiding reflex tearing
from ocular irritation, especially if the collection is performed by an experienced operator on a
cooperative patient. The minimal binding of tear compounds to glass is another reported
advantage of capillary tubes. However, the main limitation of microcapillary collection is the
long collection time, generally ≥ 5 min174,180,184,185,190,193 – this is particularly true in small
laboratory animals, with up to 15-30 minutes and 15-60 min required to collect sufficient tear 147
fluid in rabbits184,190 and rodents (mice and rats),193,194 respectively. Another critical limitation of
direct sampling is the low volume of tear fluid retrieved, generally ≤ 5 µL144,147,166,172,175,177-
179,182,183,185,191-193 – as such, the small sample collected may be grossly insufficient in some
individuals,144,188 may require excessive dilution that renders the target analyte undetectable,189
and does not take into account possible losses (eg., transfer, storage) or the need to repeat certain
assays in duplicates.
Several strategies can be used to overcome current obstacles with the volume of the tear
volume; however, each come with its own set of drawbacks (listed in parentheses): (i) Sedate or
anesthetize the animal to extend collection duration and obtain a larger volume (altered lacrimal
functional unit and ocular surface homeostasis);147,184,190,192-195 (ii) Pool tear samples from several
subjects (reduced statistical power and loss of information regarding inter-individual variability);161,187,192 (iii) Induce reflex tearing with a stimulant – either physical (eg.. irritation to
nasal mucosa or cornea), chemical (eg., parenteral pilocarpine or ammonium fumes) or physiological (eg., yawn or sneeze reflex) – thereby accelerating tear flow and shortening collection time (diluted tear sample, unable to control flow rates);180,193,194,197 (iv) Instill fluid
(eg., saline) on the ocular surface immediately prior to tear collection, a process called ‘flush’ or
‘washout’ that yields a larger tear sample in a shorter amount of time (diluted tear sample, non-
standardized instilled volume, non-homogenous mixing of fluid with tears).174,180,192,193,195,197,198
In particular, the diluting effect of reflex tearing or flush methods may drop the concentration of
low-abundant compounds below the analytical limit of quantification, and potentially mask
differences between groups due to reduced variance in tear composition.174,199 A third limitation
of microcapillary tubes is the technical difficulty associated with the collection method. In fact,
it is nearly impossible (or very challenging) to avoid reflex lacrimation in a consistent manner, 148
even with cooperative patients and experienced personnel;175,176,180,197,200 for instance, capillary
tear collection by Markoulli et al. resulted in tear secretion that was approximately 4-fold faster
than basal tear flow in humans (4.6 vs. 1.2 µL/min, respectively).109,180 Of note, sampling itself
may act as a stimulant due to environmental factors (air movement, light)200 and the
stress/anxiety experienced by patients when capillary tubes are used.173,176,179,197 Importantly, the
technical challenge of capillary tubes is amplified in animals given their uncooperative nature,
and in any patient with aqueous tear deficiency given the low tear volume; tear sampling can be
extra slow in these cases, possibly impeded/interrupted if an air bubble or mucinous material
enters the capillary lumen.185
Taken together, although direct tear collection remains the preferred method of some
investigators given the ‘undisturbed’ tear sample retrieved,199 the serious drawbacks listed above
have prompted a growing number of clinicians and researchers to consider indirect tear sampling
in humans as suitable alternatives.149,176 It is the authors’ opinion that indirect tear sampling is
also preferred in dogs, cats and laboratory animals. Ultimately, the patient’s safety and comfort
during tear collection is paramount and, as suggested by Berta, ‘it is better to use well-controlled methods than to try to cause as little irritation as possible’.200
Indirect tear sampling
Indirect techniques involve tear fluid absorption with either Schirmer tear strips or absorbent sponges, followed by extraction of tear compounds by centrifugation and/or solvent elution.
Schirmer tear strips are routinely used to measure tear volume for clinical assessment of dry eye disease in humans and veterinary species.165,201-206 The strips are made of Whatman no. 149
41 cellulose filter paper and possess specific characteristics to promote tolerance (5mm width x
35mm length, 0.22 mm thick, 20-25 µm porosity, foldable extremity for ease of insertion).197,207
In addition to their conventional use for aqueous tear assessment, Schirmer strips can retrieve tear fluid for bioanalytical purposes in humans,144,161,165,166,173,176,208 dogs,143,149,156,159,160,186,209 and
small laboratory species.191,210,211 For instance, Schirmer strips were used for in-depth
characterization of proteomics in human162 and canine tears,149 and can also successfully recover
specific analytes such as cytokines,208,212 clusterin,144 and xenobiotics.156,158-160,191,213
Absorbent sponges exist in different material types such as cellulose,179,181,214,215
polyvinyl acetal,143,159,173,181,191,214,216,217 polyester,185,188,217 and polyrurethane.181 A material with hydrophilic and hydrophobic properties (eg., polyvinyl acetal, polyurethane)181,214 is generally
preferred in order to optimize the amount of fluid absorbed and the amount of fluid retrieved
from the sponge. For tear fluid collection, the sponge is held against the lacrimal lake by the
operator (to minimize reflex tearing),179,185,188,191,217 or placed beneath the lower eyelid for a
given period of time.159,181,214 Tear fluid recovered from absorbent sponges can be assayed for
selected tear compounds, similar to Schirmer strips.
Indirect tear collection is superior to direct capillary sampling in many aspects, namely:
(i) Improved tolerance and acceptability by patients;144,176,217,218 (ii) Ease of use and operator
safety, especially for Schirmer strips, allowing non-specialists to perform the procedure with minimal training;176,181,217 and (iii) Larger volume of tears collected in a shorter
duration.179,181,185,191 Absorbent materials collect tears but can also pick up cellular and
extracellular ‘debris’, an attribute considered beneficial by some as the sample obtained is more
representative of the dynamic microenvironment at the ocular surface,219 but also perceived as a
limitation by others as the fluid retrieved is not ‘pure’ tears.199 On this note, the main limitation 150
of indirect sampling is the ‘invasiveness’ of the technique, at risk of promoting reflex tearing and
altering the composition of the tear fluid; indeed, several studies showed variable tear
composition between directly- and indirectly- collected samples, with notable differences in the
qualitative and quantitative profiles obtained for tear lipids167 and tear proteins.161,166,182,220
Another important drawback is related to the adsorptive properties of Schirmer strips or absorbent sponges, ie. incomplete release of tear compounds following extraction;143,159,207,208,216
however, the authors believe this limitation can be minimized/controlled with adequate
precautions (see section 3.3).
Proposed strategy for lachrymal determinations in dogs
Schirmer strips vs. absorbent sponges – Sponges can rapidly absorb up to 106 µL of tear
fluid in dogs,214 while the maximum absorptive capacity of Schirmer strips is ~ 31 µL (ie., 35 mm wetness).221 With sponges, however, the operator can only control the duration of tear
collection and not the volume of tears soaked up in each individual. The resulting variability in
tear volume absorbed often translates into large intra- and inter-subject variability in the
concentration of the compound(s) of interest, as shown for protein content143 and various drugs such as doxycycline,159 minocycline,222 voriconazole,223 and ofloxacin.191 On the other hand, the
ability to control the volume of tears absorbed with Schirmer strips (ie., same mm mark)
generally improves the reproducibility of the results.143,159,160,224
As such, the authors prefer (i) absorbent sponges for collecting large volumes of tears in
canine subjects – ie., for further use as blank tears in bioanalytical assays for example – and (ii)
Schirmer strips for collecting known amounts of tears in any scenario where reproducibility of
the data is important (ie., for group comparisons, or follow-up of the same individual over time). 151
Schirmer strips for protein quantification – For consistency purposes, the authors recommend the use of dye-free Schirmer tear strips, being consistent with the manufacturer and lot number (given the reported variability in absorptive and adsorptive properties among
Schirmer strips),225,226 as well as the time of collection (eg., morning)176, because of known diurnal variability in lacrimal protein composition in humans182,197 and dogs.224 The distal end of the Schirmer strips should remain in position (ventrolateral conjunctival fornix) until 20 mm, 25 mm or 30 mm wetness is reached.160,186,209,221,224 Strip wetness < 20 mm is discouraged given the potential ‘concentrating effect’ of the absorbent filter with low tear volumes,143 while complete wetness of the strip (35 mm) should be avoided as the total protein content is significantly greater with 35 mm compared to 20-30 mm mark,221 likely due to vascular fragility and excessive irritation ensued by the prolonged test duration. Importantly, investigators should be consistent with the selected mm- mark (strip wetness) within and between patients in order to standardize the volume of tears collected among subjects. This strategy provides a lower coefficient of variability in tear protein content compared to ophthalmic sponges143 and capillary glass tubes224 in dogs, thereby improving the reliability and reproducibility of the data. Tear extraction and protein analysis can be done directly after tear collection, or can be postponed to a future date as long as Schirmer strips are stored immediately at -80°C and the stability of the compound(s) of interest is verified.197 Following tear extraction with centrifugation,143,176,221,224 elution in solvent,144,149,161,162,186 or a combination of both,208,227 total protein content (TPC) should be quantified in order to standardize the amount of sample used for subsequent analyses.149,162 The authors’ preferred method is infrared spectroscopy with Direct DetectTM
(EMD Millipore, Danvers, MA) as the technique utilizes merely 2 µL of tear sample, without 152
any of the drawbacks of colorimetric protein assays (eg., Bradford, Lowry), including variability with specific protein composition and potential contamination from the absorbent material.179,228
Schirmer strips for drug quantification – The following steps should be considered to
optimize drug quantification in pharmacological studies:
• Study design: In studies that assess tear film pharmacokinetics following topical drug
administration, one must consider a potential limitation associated with Schirmer strips
which remove most of the tear fluid in early collection times, thereby negatively impacting
the ‘true’ tear concentration at later time points.197 For this reason, the authors recommend to
conduct pharmacokinetic studies in tears over several days (eg., 10 days for 10 collection
time points), repeating topical administration each day with a standardized volume and
limiting the collection to a single time point per day. An alternative is to use a larger sample
size and randomly allocate each time point to a subset of individuals or eyes (eg., 40 eyes
with n = 5 eyes for 8 separate time points),156 although this method should account for
differences between subjects such as greater tear volumes in dogs of larger body weight.111
Another aspect to consider in the study design is the assessment of drug kinetics in diseased
eyes, rendering the study results more clinically applicable (see section 4.2); in fact, tear film
concentrations and ocular bioavailability are likely to differ in healthy vs. diseased eyes (eg.,
excessive lacrimation, increased absorption into congested conjunctival vessels, albumin
binding),160,229 yet the majority of ocular studies to date are conducted in healthy individuals
which is a clear limitation for translation of research findings from bench to bedside.
• Tear collection with Schirmer strips: It is important to homogenize the volume collected
within and between subjects by standardizing the extent of strip wetness (≥ 20 mm mark) –
this approach limits the variability in tear concentrations related to the collection method 153
itself.159,160 The amount of wetness is then converted to a volume (µL) in order to calculate
actual tear film concentrations;221 data reporting is otherwise limited to µg/g of strip.156,191 In
dogs, the median volume absorbed by Schirmer strips is 18 µL (20 mm), 22 µL (25 mm), 26
µL (30 mm) and 31 µL (35 mm), information obtained from hundreds of in vivo collections
with pre- and post- weighing of Schirmer strips).221 This method is preferred over in vitro use
of phosphate buffered saline208 given differences in fluid viscosities and the inability of an in
vitro experiment to mimic the complex dynamics of tear absorption noted in vivo (eg., rapid
initial uptake,204 tear evaporation). In parallel, investigators should record the duration of tear
collection (eg., 50 seconds to reach 20 mm) in order to calculate a flow rate (µL/min) for
each sample obtained.159,180 In one of our experiments with doxycycline in dogs, flow rate
did not influence tear concentrations,159 but this finding might not be generalizable to other
drugs and/or other species of interest.
• Extraction protocol optimization: A drug can be extracted from Schirmer strips via
centrifugation, solvent elution, or a combination of both methods. However, a single
extraction protocol cannot be generalized to all pharmacological studies as the specific
physicochemical properties of each drug (eg., molecular weight, lipophilicity) can affect the
extraction efficiency from the filter papers.207 As such, investigators should consider
conducting a preliminary experiment to determine the optimal extraction protocol for the
drug studied, and report specific recovery rates (mean ± standard, range). For instance, the
recovery of prednisone and prednisolone was maximized with a combination of
centrifugation and elution in methyl tert-butyl ether, a solvent chosen over methanol and
acetonitrile based on superior drug extraction from Schirmer strips (Figure 4).160 Of note, a
comprehensive review of all reported protocols is beyond the scope of the present work, and 154
further research is warranted to assess the potential benefits (or lack thereof) of extraction
steps reported in the literature, such as cutting Schirmer strips into small pieces162,165,213 or
using ultrasonic agitation.160,165,197 Ultimately, an optimized extraction protocol is important
as it enhances the reliability of the data at hand, improving the sensitivity of the bioassay, and
providing drug concentrations closer to ‘true’ biological levels in the tear film.
• Bioanalytical method optimization: First, internal standard should be applied directly onto
the dry portion of the Schirmer strip (Figure 4),160 ie. before tear extraction instead of post-
elution with solvent as routinely described;156,213 this step allows for drug quantification to
account for potential variability in extraction efficiency between samples. Second, the
standard calibration curve solutions should be constructed by spiking known drug
concentrations and internal standard onto Schirmer strips, followed by the same extraction
protocol as for biological samples; this step is equivalent to ‘spike and recover’ experiments
recommended for other analytes such as cytokines216 and proteins.207 Third, actual tear fluid
should be used whenever possible as the selected matrix for standard calibration curve and
quality control solutions,159,160,223 as the reported surrogates (eg., artificial tear solution)230 do
not account for chemical interferences and matrix effects that typically occur with a complex
biological fluid (eg., ionization suppression).223 Blank tears can be collected with absorbent
materials prior to study initiation, retrieving up to 84 µL in 1 min with ophthalmic sponges in
dogs214 and 132 µL in 12 min with successive substitutions of polyurethane mini-sponges in
humans.181
Spontaneous and experimental models of ocular surface disorders in dogs
155
Spontaneous ocular surface diseases in dogs with translational applications to humans
Spontaneous ocular surface disorders are common in dogs and represent one of the major causes for referral visits to veterinary practitionners.231 In contrast, naturally-acquired ocular
surface pathology is much less common in rabbits232,233 and is rare in mice and rats.234-23
Keratoconjunctivitis sicca
Keratoconjunctivitis sicca (KCS), or ‘dry eye’, represents one of the most common ocular
diseases in humans with an estimated prevalence ranging from 5 to 50% in different regions
worldwide.237 The disease is also very common in dogs (prevalence 1.5 to 35%),231 although not a single report of spontaneous KCS case exists in laboratory animals such as rabbits, mice and rats.
The pathogenesis of KCS is very complex, involving diverse physio-anatomical factors such as lacrimal gland integrity, meibomian glands function, hormonal balance and neuronal input.237 Numerous models of dry eye have been established in animals over the years,238-240
helping to elucidate complex pathological mechanisms involved in KCS and develop novel
therapeutics for humans. However, the major drawbacks of most animal models are the acute
nature of the induced pathology (vs. chronic disease in humans) and the focus on a single
component of the lacrimal functional unit, such as surgically removing the lacrimal gland in
mice to reduce tear secretion,32 cauterizing the lid margin in rats to induce meibomian gland
dysfunction,241 or instilling topical 1% atropine in rabbits to disrupt the efferent neural input.242
These experimental models can be generally improved by increasing the number of interventions in the study animals, for instance combining lacrimal glands removal with chemical destruction of the conjunctiva in rabbits,243 or combining scopolamine administration with desiccating 156
environmental stress in mice; yet, these complex models remain suboptimal at best given the
acute nature and the inability to fully encompass the complexity of KCS pathophysiology.244
Dogs, on the other hand, develop KCS in a spontaneous manner and do not require invasive
procedures to disrupt the lacrimal functional unit.201,205 Most importantly, the disease is clinically
and immunopathologically similar to dry eye in humans, and possesses several attributes that are
beneficial for translational research:
• Canine KCS is typically bilateral, develops in middle-aged animals, is more common in
female dogs and in certain breeds (eg., American Cocker spaniel, English Bulldog),
mimicking the diversity of dry eye in humans related to sex and race.201,237
• Immune-mediated dacryoadenitis is the most common etiology of KCS in dogs – similar to
human patients with Sjögren’s syndrome – in which progressive lymphocytic infiltration of
the lacrimal gland(s) damages the secretory tissues and reduces aqueous tear production.201
• Meibomian gland dysfunction is recognized in many canine patients with ocular surface
disorders, affecting tear film stability in a similar manner than evaporative dry eye in human
patients.245
• Spontaneous symptoms of ocular irritation, conjunctival hyperemia and corneal scarring
correlate directly with aqueous tear production, a parameter that is easily
measured/quantified using a standard Schirmer tear test strip.
• Multiple diagnostic tools used in humans can easily be applied in dogs (but not rodents)
given the large size of the canine globe,205,239 including tear osmometry, vital staining, strip
meniscometry test, infrared meibography and corneo-conjunctival impression cytology.
• Dogs and humans display a similar responses to common therapeutics for dry eye disease; in
fact, the two FDA approved anti-inflammatory drugs for dry eye disease in humans 157
(cyclosporine, lifitegrast) were first developed in canine patients with spontaneous
KCS.201,246
The main limitation to consider in dogs is the tendency for clinical signs to be more
pronounced in that species vs. humans (eg., tenacious mucoid discharge, corneal melanosis, neovascularization), in part because canine KCS is often diagnosed at a later stage when owners fail to recognize more subtle clinical signs early on.
Allergic conjunctivitis
Allergic conjunctivitis is a common disorder in humans with an approximate prevalence of 40% in the North American population.247 The disease is characterized by an
immunopathological reaction of the ocular surface to the external environment, resulting in
clinical symptoms that range from mild conjunctivitis (seasonal or perennial) to the more severe,
vision-threatening vernal keratoconjunctivitis and atopic keratoconjunctivitis.247 Over the past few decades, extensive research on small laboratory species (mice, rats, guinea pigs) has helped elucidate some of the complex molecular and cellular processes involved in the pathogenesis of ocular allergies.248,249 However, these experiments primarily relied on a relatively small selection
of allergens (eg., ovalbumin, compound 48/80, ragweed pollen), using an experimental design
that merely mimics acute forms of the disease – not chronic allergen exposure over months to
years – therefore limiting the long-term clinical significance of these findings. On the other hand,
dogs possess notable benefits for the comparative study of allergic conjunctivitis, especially
when considering companion animals rather than laboratory Beagles: (i) these animals share the
same environment (and related allergens) as their human owners, unlike commonly used species
who are housed in a laboratory setting; (ii) companion dogs are outbred, providing a genetic 158
diversity background that better reflects the human population than inbred laboratory species;
and (iii) dogs develop a spontaneous form of allergic conjunctivitis. Spontaneous allergic
conjunctivitis is relatively common in dogs, often associated with other allergic disorders such as
canine atopic dermatitis.250 Similar to humans, the clinical signs of allergic conjunctivitis involve
conjunctival hyperemia, chemosis, pruritus and ocular discharge, the disease can be diagnosed
with high sensitivity and specificity using the conjunctival provocation test,250 and similar
therapeutics are used in both species including topical antihistamines, mast-cell stabilizers,
NSAIDs and immunomodulators.247
Microbial keratitis
It is well recognized that a natural host is best suited for studying infection, as several
species-specific factors (eg., anatomical, physiological, genetic, immune) closely influence the
host-pathogen interactions and subsequent clinical response.251 These factors likely explain why
rabbits and rodents – traditionally used to model ocular surface infections in humans – cannot
fully recapitulate the disease presentation and progression that occur in human patients.252,253 As such, there is an emerging appreciation for the translational advantage of studying spontaneous
(and not experimental) ocular infections in dogs:
• Herpetic keratitis: Recent work has highlighted the robustness and reproducibility of the
canine model to study ocular herpesvirus infections and disease,253,254 showing striking
similarities in the pathogenesis of canine herpesvirus-1 and herpes simplex virus-1, both
members of the alphaherpesvirinae subfamily with a seroprevalence of 21-98% in dogs
(CHV-1) and 67-90% in humans (HSV-1).253 159
• Bacterial keratitis: The most common bacterial genera isolated from canine patients overlap
with the ones recognized in human patients (Staphylococcus, Streptococcus,
Pseudomonoas).255,256 In fact, the major culprit in canine bacterial keratitis (Staphylococcus
pseudintermedius) is now considered an emerging zoonosis in humans.257
Others
Dogs can serve as models for other ocular surface diseases such as corneal endothelial
dystrophy (analogous to Fuch’s dystrophy in humans),258 limbal stem cell deficiency,259 ocular
surface squamous neoplasia260 and neurotrophic keratopathy,261 among others.
Breakdown of the blood-tear barrier in dogs: A model for ocular pharmacology and therapeutics
Histamine-induced conjunctivitis
To date, the vast majority of preclinical ocular studies for evaluation of candidate drug efficacy and safety are conducted in healthy eyes – in part for simplicity, but at a higher risk of
treatment failure rate when translating these findings to clinical studies. Indeed, healthy eyes do
not account for the disruption of ocular homeostasis that occurs with inflammatory diseases,
including (but not limited to) changes in tear film dynamics, tear composition and permeability
of ocular tissues. To address this shortcoming, the authors have recently established a robust in
vivo model of conjunctivitis in dogs, a translational large animal model that provides a unique
opportunity for scientists to investigate the ocular surface in health and disease states.212 The
model specifically focused on conjunctivitis as this condition is frequently encountered in
humans and dogs,262,263 developing either as a primary condition (eg., bacterial, viral), or as a bystander to common ophthalmic diseases such as blepharitis, keratitis, uveitis and glaucoma. 160
This model is particularly appealing given the low cost, non-invasiveness, self-resolving nature,
ability to adjust the duration and severity of the disease, and shared features with naturally
occurring diseases in human and veterinary medicine. The main highlights of the translational
‘large animal’ model are as follows:
• The selected compound (histamine) is inexpensive and triggers local inflammation in a non-
specific manner.
• Disease severity is dose-dependent, allowing investigators to induce mild (1 mg/ml),
moderate (10 mg/ml) or severe (375 mg/ml) conjunctivitis (Figure 5).
• Disease duration is dose-dependent, self-resolving within an average of 115 min (1 mg/ml),
190 min (10 mg/ml) or 390 min (375 mg/ml). The duration of conjunctivitis can be
lengthened by repeating topical histamine administration at set intervals.160
• Topical histamine is safe and generally well-tolerated, although selected eyes receiving the
highest dose of histamine (375 mg/ml) can develop mild ocular irritation (lasting < 1 min),
blepharitis or miosis.
• Tear film composition changes in eyes with experimentally-induced conjunctivitis (eg.,
higher levels of serum albumin and inflammatory cytokines), mimicking clinical patients
with ocular surface inflammation.
• A transient increase in tear quantity and decrease in tear quality occur, although tear film
homeostasis is rapidly restored in ≤ 5 min.264
Levels of serum albumin are increased in tear film of canine eyes with experimentally-
induced or naturally-acquired conjunctivitis,212,224 a physiological variation caused by the
breakdown of the blood-tear barrier (Figure 6). Disruption of the blood-tear barrier is also
described in human patients with spontaneous ocular surface disorders (eg., dry eye, allergic 161
conjunctivitis)182,265-267 and other animal species (rabbits, horses, guinea pigs).268-270 Increased
vascular permeability and disruption of tight junctions between conjunctival epithelial cells
likely play a role (Figure 6),271,272 although the exact etiopathogenesis is unknown and require
further investigation. A few noteworthy limitations are listed here: (i) the model is not adequate
to study ocular allergy given the lack of characteristic features noted in canine patients with
allergies (eg., follicular conjunctivitis); (ii) pro-inflammatory mediators other than histamine are also responsible for triggering conjunctival inflammation in clinical patients (eg., leukotrienes, cytokines); (iii) conjunctival inflammation is relatively short-lived (115-390 min) and cannot mimic the physiological changes noted in patients with chronic conjunctivitis (eg., reduced goblet cell density).
Clinical relevance of serum albumin leakage in tear film
Elevated serum albumin levels in the tear film represents a biomarker for ocular insult or inflammation in humans, dogs and other species.182,212,265,270 In brief, plasma-derived albumin
leaks onto the ocular surface from congested conjunctival vessels and mixes with the tear film;
as such, albumin concentration in tears is generally low in healthy state but increases
substantially in diseased eyes.182 For instance, a recent study showed that canine eyes with
diverse ocular diseases (eg., corneal ulcer, uveitis, glaucoma) had lacrimal albumin levels that
were up to 14.9-fold greater than contralateral healthy eyes.212 Albumin is a relatively large
protein that has a remarkable capacity for binding ligands.273 At the level of the eye, protein
binding represents an important restriction to drug absorption as only the unbound fraction of the
drug diffuses across the ocular tissue barriers.268 Combined with the rapid drainage of tears
following eyedrop administration (in humans/dogs, not true in rabbits), any portion of drug that 162
binds to albumin in tear film can be considered as ‘lost’ from a pharmacological standpoint.
Broader implications of the blood-tear barrier breakdown on ocular drug pharmacokinetics are
listed below:
• Reduced bioavailability for intraocular targets: The inability of bound therapeutic drugs
to penetrate the cornea lowers the amount of drug available inside the eye to exert its
pharmacological action. The physiological effects of increased albumin levels in tears was
recently demonstrated with tropicamide (and to a lesser extent latanoprost) in dogs,229 as well
as pilocarpine in rabbits.268 Of note, the impact of lacrimal albumin on the pharmacological
activity of a given drug is likely modulated by various factors, the concentration of the
formulation, the mechanism of action and the potency of the drug for its biological target.229
• Reduced bioavailability for ocular surface targets: Drug-albumin interactions in the tear
film could also be detrimental for management of ocular surface disorders, for instance
reducing the efficacy of therapeutics for bacterial keratitis as only the unbound portion of an
antibiotic is microbiologically active.274 Preliminary experiments conducted by the authors
showed that the presence of albumin results in higher minimal inhibitory concentrations (ie.,
reduced susceptibility) for various antibiotics against common bacterial isolates in dogs (in-
house unpublished data).
• Tear film concentrations of systemically administered drugs: Drug in the plasma
compartment can access the tear film by active secretion from the lacrimal gland, or passive
diffusion through the conjunctival vessels. The latter is theoretically enhanced when the
blood-tear barrier is disrupted. In humans, this physiological feature could explain why the
concentration-time profiles of cetirizine were similar in serum and tears in patients with
allergic conjunctivitis.157 In dogs, tear film corticosteroid levels were generally higher in 163
conjunctivitis vs. control eyes following oral prednisone administration (up to +64%),
although differences were not statistically significant.160 The degree of conjunctival
permeation is likely to vary among therapeutic drugs given differences in their physico-
chemical properties.275,276
These findings highlight the importance of conducting pharmacological studies in clinically relevant preclinical species that are able to recapitulate leaky conjunctival vessels and elevated albumin levels in the tear film of clinical patients with ocular diseases. For topical drug administration, the authors recommend using an experimental model of blood-tear barrier breakdown (eg., histamine-induced conjunctivitis or alkali burn models)212,277 so that albumin and other relevant proteins are already present on the ocular surface at the time the drug mixes with the tear film.229 For systemic drug administration, the authors suggest conducting a preliminary experiment to assess whether conjunctival inflammation affects tear film concentrations to a significant extent. If not, pharmacological assessment in healthy eyes should be sufficient. Incidentally, the physico-chemical properties of some drugs (eg., size, lipophilicity, polar surface area)275,276 may allow for high conjunctival permeation under normal conditions, thereby rendering differences between healthy vs. diseased eyes insignificant.
Corneal injury in dogs: in vivo and ex vivo models
Corneal injury is common in human and veterinary patients – whether due to trauma, surgery, or other causes – and the resulting corneal scar remains one of the leading causes of blindness in animals and people worldwide.278 Although small laboratory animal species are commonly used in corneal scarring research,279 results derived from these models have several limitations. The corneal thickness is much smaller in rabbits and rodents compared to humans 164
(Table 1). In addition to thin corneas, mice and rats have corneas that are much smaller in
diameter compared to people; consequently, it is often difficult to isolate the central cornea when
performing the experimental procedure (eg., chemical burn) and the damage caused to
surrounding limbal stem cells negatively impacts the wound healing process. Using the dog as an
animal model is therefore more appropriate, not only due to closer resemblances in ocular
surface anatomy and physiology with humans, but also the relatively high prevalence of
naturally-acquired corneal pathology in the canine species. In that regard, Gronkiewicz et al.
recently developed a novel in vivo corneal fibrosis model in canines;277 the authors induced corneal scarring with an alkali burn and investigated the ability of suberanilohydroxamic acid
(SAHA) to inhibit fibrosis using this large animal model. The availability of such a model presents a clear opportunity for translational research (ie., intact innervation, tear film, blood supply), although experimentally-induced corneal wounding (at risk of secondary infection) and subsequent corneal scar in dogs represent potential ethical challenges. As an alternative, other authors have established ex vivo canine corneal cultures that can be used to model wound healing and assess anti- fibrotic compounds,280-282 or better understand the pathophysiology of herpesvirus in a virus-natural-host environment;283 in that study, the authors established an air-liquid canine corneal organ culture model to study acute herpetic keratitis, showing important similarities in the response to CHV-1 to what has been described for HSV-1.283
Conclusions
“Considerable reservations may be felt about comparing results from rabbits with those
from humans because of the differences between the physiology of tear flow and mixing and
general anatomy. Nevertheless, the rabbit is the principal experimental animal in ophthalmology, 165
so comparisons are needed”.284 Sadly, this quote published over 45 years ago is still
representative of today’s state of ophthalmic research. Rabbits and small laboratory rodents
continue to be used primarily (if not exclusively) in most areas of ophthalmic research,1 a concerning fact given the vast anatomical and physiological differences that exist with humans.
Of note, such differences should not be regarded as merely ‘weaknesses’ for translational research, but rather evolutionary adaptations optimally suited to the environment and behavior of each species; for instance, rabbits likely developed a very stable tear film to limit intermittent blindness that occurs with each blink,285 thereby reducing the risk of predation. Noteworthily,
recent innovations have helped mitigate some of the drawbacks of traditional laboratory species
– for instance providing manual blinking and supplementary tear flow in anesthetized rabbits,286
or reverse engineering the ocular surface using human cells in vitro287 – however the authors
believe the complexity of the ocular surface and integrated lacrimal functional unit cannot be
fully recreated without in vivo conditions in awake subjects.
The comparative work presented throughout this review provides evidence that dogs are
best suited for translational research in ophthalmology. Unlike small laboratory animals, dogs
share similar anatomical and physiological features to humans, similar environmental stressors
and genetic variation, and a range of naturally occurring ophthalmic diseases that closely
resemble clinical phenotypes in human patients. The resemblance between dogs and humans is
particularly relevant in the field of ocular pharmacology, with notable similarities in blink rate,
tear turnover rate (basal, reflex), volumetric capacity of the palpebral fissure, and other factors
pertinent to drug diffusion (eg., globe volume, corneal thickness); nonetheless, a few differences
should be accounted for in comparative studies, such as the presence of a nictitating membrane,
greater tear volume, larger corneal size and lower corneal elastic modulus in dogs. Similar to 166
other fields of medicine, preclinical studies in ophthalmology could involve canine patients with
spontaneous ocular diseases – many of which share striking resemblances with their human
counterparts – integrating the expertise of veterinarians, physicians and basic science researchers
under the umbrella of the One Health Initiative.6,288 Alternatively, or complementarily,
preclinical animal work could be performed in laboratory dogs in whom ocular disease is
experimentally-induced, making sure to account for the blood-tear barrier breakdown (noted in
clinical patients with ‘red eyes’). In all cases, tear fluid can be easily collected from canine eyes
for various bioanalytical purposes, favoring Schirmer tear strips over other collection methods
given the excellent safety profile and enhanced reliability in analyte quantification (eg., proteins, drugs) provided by this method.
References
1. Shah M, Cabrera-Ghayouri S, Christie LA, et al. Translational Preclinical Pharmacologic Disease Models for Ophthalmic Drug Development. Pharm Res 2019;36:58.
2. Maurice D. The effect of the low blink rate in rabbits on topical drug penetration. J Ocul Pharmacol Ther 1995;11:297-304.
3. Takahashi K, Saishin Y, Mori K, et al. Topical nepafenac inhibits ocular neovascularization. Invest Ophthalmol Vis Sci 2003;44:409-415.
4. Hay M, Thomas DW, Craighead JL, et al. Clinical development success rates for investigational drugs. Nature Biotechnology 2014;32:40-51.
5. Harrison RK. Phase II and phase III failures: 2013–2015. Nature Reviews Drug Discovery 2016;15:817-818.
6. Kol A, Arzi B, Athanasiou KA, et al. Companion animals: Translational scientist's new best friends. Sci Transl Med 2015;7:308ps321.
7. Garden OA, Volk SW, Mason NJ, et al. Companion animals in comparative oncology: One Medicine in action. Vet J 2018;240:6-13.
167
8. Partridge B, Rossmeisl JH. Companion animal models of neurological disease. J Neurosci Methods 2020;331:108484.
9. Shell JW. Pharmacokinetics of topically applied ophthalmic drugs. Surv Ophthalmol 1982;26:207-218.
10. Sakai T. Major ocular glands (harderian gland and lacrimal gland) of the musk shrew (Suncus murinus) with a review on the comparative anatomy and histology of the mammalian lacrimal glands. J Morphol 1989;201:39-57.
11. Gilger BC, Abarca E, Salmon JH. Selection of Appropriate Animal Models in Ocular Research: Ocular Anatomy and Physiology of Common Animal Models In: Gilger BC, ed. Ocular Pharmacology and Toxicology. Totowa, NJ: Humana Press, 2014;7-32.
12. Sullivan DA, Hann LE, Yee L, et al. Age- and gender-related influence on the lacrimal gland and tears. Acta Ophthalmol (Copenh) 1990;68:188-194.
13. Saito A, Izumisawa Y, Yamashita K, et al. The effect of third eyelid gland removal on the ocular surface of dogs. Vet Ophthalmol 2001;4:13-18.
14. Park SA, Taylor KT, Zwingenberger AL, et al. Gross anatomy and morphometric evaluation of the canine lacrimal and third eyelid glands. Vet Ophthalmol 2016;19:230-236.
15. El-naseery N, El-behery E, El-Ghazali H, et al. The structural characterization of the lacrimal gland in the adult dog (Canis familiaris). Benha Veterinary Medical Journal 2016;31:106-116.
16. Murphy CJ, Samuelson DA, Pollock RVH. Chapter 21: The eye. Miller’s anatomy of the dog 2013:746-785.
17. Zwingenberger AL, Park SA, Murphy CJ. Computed tomographic imaging characteristics of the normal canine lacrimal glands. BMC Vet Res 2014;10:116.
18. Bulbul E, Yazici A, Yanik B, et al. Evaluation of Lacrimal Gland Dimensions and Volume in Turkish Population with Computed Tomography. J Clin Diagn Res 2016;10:TC06-08.
19. Lamagna B, Peruccio C, Guardascione A, et al. Conjunctival dacryops in two golden retrievers. Vet Ophthalmol 2012;15:194-199.
20. Conrady CD, Joos ZP, Patel BC. Review: The Lacrimal Gland and Its Role in Dry Eye. J Ophthalmol 2016;2016:7542929.
21. Kim EC, Doh SH, Chung SY, et al. Direct visualization of aqueous tear secretion from lacrimal gland. Acta Ophthalmol 2017;95:e314-e322.
168
22. Schechter JE, Warren DW, Mircheff AK. A lacrimal gland is a lacrimal gland, but rodent's and rabbit's are not human. Ocul Surf 2010;8:111-134.
23. Bergmanson JP, Doughty MJ, Blocker Y. The acinar and ductal organisation of the tarsal accessory lacrimal gland of Wolfring in rabbit eyelid. Exp Eye Res 1999;68:411-421.
24. Hirt R, Tektas OY, Carrington SD, et al. Comparative anatomy of the human and canine efferent tear duct system--impact of mucin MUC5AC on lacrimal drainage. Curr Eye Res 2012;37:961-970.
25. Ali MJ, Rehorek SJ, Paulsen F. A major review on disorders of the animal lacrimal drainage systems: Evolutionary perspectives and comparisons with humans. Ann Anat 2019;224:102-112.
26. Burling K, Murphy CJ, Curiel JS, et al. Anatomy of the rabbit nasolacrimal duct and its clinical implications. Progress in veterinary & comparative ophthalmology 1991;1:33-40.
27. Rehorek SJ, Holland JR, Johnson JL, et al. Development of the Lacrimal Apparatus in the Rabbit (Oryctolagus cuniculus) and Its Potential Role as an Animal Model for Humans. Anat Res Int 2011;2011:623186.
28. Paulsen FP, Föge M, Thale AB, et al. Animal model for the absorption of lipophilic substances from tear fluid by the epithelium of the nasolacrimal ducts. Invest Ophthalmol Vis Sci 2002;43:3137-3143.
29. Frame NJ, Burkat CN. Identifying an appropriate animal model for the nasolacrimal drainage system. Ophthalmic Plast Reconstr Surg 2009;25:354-358.
30. Arends G, Schramm U. The structure of the human semilunar plica at different stages of its development--a morphological and morphometric study. Ann Anat 2004;186:195-207.
31. Chae JJ, Shin YJ, Lee JD, et al. Nictitating membrane fixation improves stability of the contact lens on the animal corneal surface. PLoS One 2018;13:e0194795.
32. Stevenson W, Chen Y, Lee SM, et al. Extraorbital lacrimal gland excision: a reproducible model of severe aqueous tear-deficient dry eye disease. Cornea 2014;33:1336-1341.
33. Petznick A, Evans MD, Madigan MC, et al. A comparison of basal and eye-flush tears for the analysis of cat tear proteins. Acta Ophthalmol 2011;89:e75-81.
34. Saito A, Watanabe Y, Kotani T. Morphologic changes of the anterior corneal epithelium caused by third eyelid removal in dogs. Vet Ophthalmol 2004;7:113-119.
35. Leiva M, Pena Gimenez T. Diseases of the lacrimal system. Slatter’s fundamentals of veterinary ophthalmology 6th ed Missouri: Elsevier 2017:186-212.
169
36. Paranjpe V, Phung L, Galor A. The Tear Film: Anatomy and Physiology In: Guidoboni G, Harris A,Sacco R, eds. Ocular Fluid Dynamics: Anatomy, Physiology, Imaging Techniques, and Mathematical Modeling. Cham: Springer International Publishing, 2019;329-345.
37. Milz S, Neufang J, Higashiyama I, et al. An immunohistochemical study of the extracellular matrix of the tarsal plate in the upper eyelid in human beings. J Anat 2005;206:37-45.
38. Prince JH, Diesem CD, Eglitis I, et al. Anatomy and histology of the eye and orbit in domestic animals: Springfield, Illinois: Charles C. Thomas. Oxford: Blackwell Scientific Publications., 1960. 39. Greiner JV, Glonek T, Korb DR, et al. Volume of the human and rabbit meibomian gland system. Adv Exp Med Biol 1998;438:339-343.
40. Sebbag L, Kirner NS, Allbaugh RA, et al. Kinetics of Fluorescein in Tear Film After Eye Drop Instillation in Beagle Dogs: Does Size Really Matter? Frontiers in Veterinary Science 2019;6:457.
41. Peiffer RL, Pohm-Thorsen L, Corcoran K. CHAPTER 19 - Models in Ophthalmology and Vision Research**Supported in part by a Grant from Research to Prevent Blindness and the North Carolina Lions Foundation In: Manning PJ, Ringler DH,Newcomer CE, eds. The Biology of the Laboratory Rabbit (Second Edition). San Diego: Academic Press, 1994;409-433.
42. Rajaei SM, Rafiee SM, Ghaffari MS, et al. Measurement of Tear Production in English Angora and Dutch Rabbits. J Am Assoc Lab Anim Sci 2016;55:221-223.
43. Qiu Y, Yang H, Lei B. Effects of three commonly used anesthetics on intraocular pressure in mouse. Curr Eye Res 2014;39:365-369.
44. Finlayson K, Lampe JF, Hintze S, et al. Facial Indicators of Positive Emotions in Rats. PLOS ONE 2016;11:e0166446.
45. Odigie AE, Ekeolu KO, Asemota DO, et al. Comparative non-metric and morphometric analyses of rats at residential halls of the University of Benin campus, Nigeria. J Infect Public Health 2018;11:412-417.
46. Lee HK, Woo IH, Kang B, et al. Meibography and Immunohistochemistric Structures in Animal Models. Journal of the Korean Ophthalmological Society 2018;59:9-16.
47. Moore CP, McHugh JB, Thorne JG, et al. Effect of cyclosporine on conjunctival mucin in a canine keratoconjunctivitis sicca model. Invest Ophthalmol Vis Sci 2001;42:653-659.
48. Kang Y, Li S, Liu C, et al. A rabbit model for assessing symblepharon after alkali burn of the superior conjunctival sac. Sci Rep 2019;9:13857.
170
49. Jutley G, Carpenter D, Hau S, et al. Upper and lower conjunctival fornix depth in healthy white caucasian eyes: a method of objective assessment. Eye (Lond) 2016;30:1351-1358.
50. Watsky MA, Jablonski MM, Edelhauser HF. Comparison of conjunctival and corneal surface areas in rabbit and human. Curr Eye Res 1988;7:483-486.
51. Gipson IK, Tisdale AS. Visualization of conjunctival goblet cell actin cytoskeleton and mucin content in tissue whole mounts. Exp Eye Res 1997;65:407-415.
52. Moore CP, Wilsman NJ, Nordheim EV, et al. Density and distribution of canine conjunctival goblet cells. Invest Ophthalmol Vis Sci 1987;28:1925-1932. 53. Kessing SV. Mucous gland system of the conjunctiva. A quantitative normal anatomical study. Acta Ophthalmol (Copenh) 1968:Suppl 95:91+.
54. Vujković V, Mikac G, Kozomara R. Distribution and density of conjunctival goblet cells. Med Pregl 2002;55:195-200.
55. Umeda Y, Nakamura S, Fujiki K, et al. Distribution of goblet cells and MUC5AC mRNA in the canine nictitating membrane. Exp Eye Res 2010;91:721-726.
56. Doughty MJ. On the in vivo assessment of goblet cells of the human bulbar conjunctiva by confocal microscopy - A review. Cont Lens Anterior Eye 2020.
57. Kishishita H, Nakayasu K. Distribution of conjunctival goblet cells and observation of goblet cells after conjunctival autotransplantation in rabbits. Nippon Ganka Gakkai Zasshi 1996;100:433-442.
58. Doughty MJ. Scanning electron microscopy study of the tarsal and orbital conjunctival surfaces compared to peripheral corneal epithelium in pigmented rabbits. Doc Ophthalmol 1997;93:345-371.
59. Doughty MJ. A systematic assessment of goblet cell sampling of the bulbar conjunctiva by impression cytology. Exp Eye Res 2015;136:16-28.
60. Steven P, Gebert A. Conjunctiva-associated lymphoid tissue - current knowledge, animal models and experimental prospects. Ophthalmic Res 2009;42:2-8.
61. Chodosh J, Nordquist RE, Kennedy RC. Comparative anatomy of mammalian conjunctival lymphoid tissue: a putative mucosal immune site. Dev Comp Immunol 1998;22:621-630.
62. Sakimoto T, Shoji J, Inada N, et al. Histological study of conjunctiva-associated lymphoid tissue in mouse. Jpn J Ophthalmol 2002;46:364-369.
63. Giuliano EA, Moore CP, Phillips TE. Morphological evidence of M cells in healthy canine conjunctiva-associated lymphoid tissue. Graefes Arch Clin Exp Ophthalmol 2002;240:220-226. 171
64. Knop N, Knop E. Ultrastructural anatomy of CALT follicles in the rabbit reveals characteristics of M-cells, germinal centres and high endothelial venules. J Anat 2005;207:409- 426.
65. Zhong X, Liu H, Pu A, et al. M cells are involved in pathogenesis of human contact lens- associated giant papillary conjunctivitis. Arch Immunol Ther Exp (Warsz) 2007;55:173-177.
66. Vézina M. Comparative Ocular Anatomy in Commonly Used Laboratory Animals In: Weir AB,Collins M, eds. Assessing Ocular Toxicology in Laboratory Animals. Totowa, NJ: Humana Press, 2013;1-21. 67. Marfurt C, Anokwute MC, Fetcko K, et al. Comparative Anatomy of the Mammalian Corneal Subbasal Nerve Plexus. Invest Ophthalmol Vis Sci 2019;60:4972-4984.
68. Reichard M, Hovakimyan M, Wree A, et al. Comparative in vivo confocal microscopical study of the cornea anatomy of different laboratory animals. Curr Eye Res 2010;35:1072-1080.
69. Babrauskienė V, Žymantienė J, Aniulienė A, et al. Comparative morphological evaluation of animal corneal parameters. Medycyna Weterynaryjna 2018;74:452-455.
70. Nautscher N, Bauer A, Steffl M, et al. Comparative morphological evaluation of domestic animal cornea. Vet Ophthalmol 2016;19:297-304.
71. Holloway CL. Changes with age in the eye of the dog and hog from birth to senility. 1969.
72. Heywood R. Some clinical observations on the eyes of Sprague-Dawley rats. Lab Anim 1973;7:19-27.
73. Hughes A. A schematic eye for the rat. Vision Res 1979;19:569-588.
74. Henriksson JT, McDermott AM, Bergmanson JP. Dimensions and morphology of the cornea in three strains of mice. Invest Ophthalmol Vis Sci 2009;50:3648-3654.
75. Augusteyn RC, Nankivil D, Mohamed A, et al. Human ocular biometry. Exp Eye Res 2012;102:70-75.
76. Wang X, Dong J, Wu Q. Mean central corneal thickness and corneal power measurements in pigmented and white rabbits using Visante optical coherence tomography and ATLAS corneal topography. Vet Ophthalmol 2014;17:87-90.
77. Thomasy SM, Eaton JS, Timberlake MJ, et al. Species Differences in the Geometry of the Anterior Segment Differentially Affect Anterior Chamber Cell Scoring Systems in Laboratory Animals. J Ocul Pharmacol Ther 2016;32:28-37.
172
78. Strom AR, Cortés DE, Rasmussen CA, et al. In vivo evaluation of the cornea and conjunctiva of the normal laboratory beagle using time- and Fourier-domain optical coherence tomography and ultrasound pachymetry. Vet Ophthalmol 2016;19:50-56.
79. Li HF, Petroll WM, Møller-Pedersen T, et al. Epithelial and corneal thickness measurements by in vivo confocal microscopy through focusing (CMTF). Curr Eye Res 1997;16:214-221.
80. Schulz D, Iliev ME, Frueh BE, et al. In vivo pachymetry in normal eyes of rats, mice and rabbits with the optical low coherence reflectometer. Vision Res 2003;43:723-728.
81. Spoerl E, Mrochen M, Sliney D, et al. Safety of UVA-riboflavin cross-linking of the cornea. Cornea 2007;26:385-389. 82. MD M, J C, M C, et al. A comparative study of Bowman's layer in some mammals: Relationships with other constituent corneal structures. Eur J Anat 2002;6:133-139.
83. Ehlers N. Morphology and histochemistry of the corneal epithelium of mammals. Acta Anat (Basel) 1970;75:161-198.
84. Molon-Noblot SM, Duprat P. Anatomy of the Ocular Surfaces, Cornea, and Conjunctiva, Rat and Mouse In: Jones TC, Mohr U,Hunt RD, eds. Eye and Ear. Berlin, Heidelberg: Springer Berlin Heidelberg, 1991;3-16.
85. Filizay MC, Gökçınar NB, Şahinturk V, et al. Evaluation of Retinol Palmitate Treatment of Photokeratitis in Rat Eyes Exposed to Ultraviolet B Radiation. Evaluation 2019;4:55-61.
86. Thomasy SM, Raghunathan VK, Winkler M, et al. Elastic modulus and collagen organization of the rabbit cornea: epithelium to endothelium. Acta Biomater 2014;10:785-791.
87. Leonard BC, Cosert K, Winkler M, et al. Stromal Collagen Arrangement Correlates with Stiffness of the Canine Cornea. Bioengineering (Basel) 2020;7.
88. Huang J, Camras LJ, Yuan F. Mechanical analysis of rat trabecular meshwork. Soft Matter 2015;11:2857-2865.
89. Xu P, Londregan A, Rich C, et al. Changes in Epithelial and Stromal Corneal Stiffness Occur with Age and Obesity. Bioengineering 2020;7:14.
90. Duman R, Tok Çevik M, Görkem Çevik S, et al. Corneal endothelial cell density in healthy Caucasian population. Saudi J Ophthalmol 2016;30:236-239.
91. Zagon IS, Campbell AM, Sassani JW, et al. Spontaneous episodic decreased tear secretion in rats is related to opioidergic signaling pathways. Invest Ophthalmol Vis Sci 2012;53:3234-3240.
173
92. Wieser B, Tichy A, Nell B. Correlation between corneal sensitivity and quantity of reflex tearing in cows, horses, goats, sheep, dogs, cats, rabbits, and guinea pigs. Vet Ophthalmol 2013;16:251-262.
93. Reins RY, Courson J, Lema C, et al. MyD88 contribution to ocular surface homeostasis. PLoS One 2017;12:e0182153.
94. McLaughlin PJ, Sassani JW, Titunick MB, et al. Efficacy and safety of a novel naltrexone treatment for dry eye in type 1 diabetes. BMC Ophthalmol 2019;19:35.
95. Bolzanni H, Oriá AP, Raposo ACS, et al. Aqueous tear assessment in dogs: Impact of cephalic conformation, inter-test correlations, and test-retest repeatability. Vet Ophthalmol 2020.
96. He J, Bazan HE. Neuroanatomy and Neurochemistry of Mouse Cornea. Invest Ophthalmol Vis Sci 2016;57:664-674.
97. Good KL, Maggs DJ, Hollingsworth SR, et al. Corneal sensitivity in dogs with diabetes mellitus. Am J Vet Res 2003;64:7-11.
98. Ledbetter EC, Marfurt CF, Dubielzig RR. Metaherpetic corneal disease in a dog associated with partial limbal stem cell deficiency and neurotrophic keratitis. Vet Ophthalmol 2013;16:282- 288.
99. Murphy CJ, Marfurt CF, McDermott A, et al. Spontaneous chronic corneal epithelial defects (SCCED) in dogs: clinical features, innervation, and effect of topical SP, with or without IGF-1. Invest Ophthalmol Vis Sci 2001;42:2252-2261.
100. Woo HM, Bentley E, Campbell SF, et al. Nerve growth factor and corneal wound healing in dogs. Exp Eye Res 2005;80:633-642.
101. Gilger BC, Reeves KA, Salmon JH. Ocular parameters related to drug delivery in the canine and equine eye: aqueous and vitreous humor volume and scleral surface area and thickness. Vet Ophthalmol 2005;8:265-269.
102. Vurgese S, Panda-Jonas S, Jonas JB. Scleral thickness in human eyes. PLoS One 2012;7:e29692.
103. Trier K, Olsen EB, Kobayashi T, et al. Biochemical and ultrastructural changes in rabbit sclera after treatment with 7-methylxanthine, theobromine, acetazolamide, or L-ornithine. Br J Ophthalmol 1999;83:1370-1375.
104. Pazos M, Yang H, Gardiner SK, et al. Rat optic nerve head anatomy within 3D histomorphometric reconstructions of normal control eyes. Exp Eye Res 2015;139:1-12.
174
105. Myers KM, Cone FE, Quigley HA, et al. The in vitro inflation response of mouse sclera. Exp Eye Res 2010;91:866-875.
106. Stern ME, Gao J, Siemasko KF, et al. The role of the lacrimal functional unit in the pathophysiology of dry eye. Exp Eye Res 2004;78:409-416.
107. Tomlinson A, Khanal S. Assessment of tear film dynamics: quantification approach. Ocul Surf 2005;3:81-95.
108. Garaszczuk IK, Montes Mico R, Iskander DR, et al. The tear turnover and tear clearance tests - a review. Expert Rev Med Devices 2018;15:219-229.
109. Mishima S, Gasset A, Klyce SD, et al. Determination of tear volume and tear flow. Invest Ophthalmol 1966;5:264-276. 110. van Best JA, Benitez del Castillo JM, Coulangeon LM. Measurement of basal tear turnover using a standardized protocol. European concerted action on ocular fluorometry. Graefes Arch Clin Exp Ophthalmol 1995;233:1-7.
111. Sebbag L, Allbaugh RA, Wehrman RF, et al. Fluorophotometric Assessment of Tear Volume and Turnover Rate in Healthy Dogs and Cats. J Ocul Pharmacol Ther 2019;35:497-502.
112. Chrai SS, Patton TF, Mehta A, et al. Lacrimal and instilled fluid dynamics in rabbit eyes. J Pharm Sci 1973;62:1112-1121.
113. Yoon KC, De Paiva CS, Qi H, et al. Desiccating environmental stress exacerbates autoimmune lacrimal keratoconjunctivitis in non-obese diabetic mice. J Autoimmun 2008;30:212-221.
114. Prabhasawat P, Tseng SC. Frequent association of delayed tear clearance in ocular irritation. Br J Ophthalmol 1998;82:666-675.
115. Shimizu A, Yokoi N, Nishida K, et al. [Fluorophotometric measurement of tear volume and tear turnover rate in human eyes]. Nippon Ganka Gakkai Zasshi 1993;97:1047-1052.
116. Whittaker AL, Williams DL. Evaluation of Lacrimation Characteristics in Clinically Normal New Zealand White Rabbits by Using the Schirmer Tear Test I. J Am Assoc Lab Anim Sci 2015;54:783-787.
117. Sullivan DA, Allansmith MR. Hormonal modulation of tear volume in the rat. Experimental Eye Research 1986;42:131-139.
118. McClellan AJ, Volpe EA, Zhang X, et al. Ocular surface disease and dacryoadenitis in aging C57BL/6 mice. Am J Pathol 2014;184:631-643.
175
119. King-Smith PE, Fink BA, Hill RM, et al. The thickness of the tear film. Curr Eye Res 2004;29:357-368.
120. Purslow C, Wolffsohn JS. Ocular surface temperature: a review. Eye Contact Lens 2005;31:117-123.
121. Carrington SD, Bedford PGC, Guillon JP, et al. Polarized light biomicroscopic observations on the pre-corneal tear film. 1. The normal tear film of the dog. Journal of Small Animal Practice 1987;28:605-622.
122. Bentivoglio AR, Bressman SB, Cassetta E, et al. Analysis of blink rate patterns in normal subjects. Mov Disord 1997;12:1028-1034.
123. Korb DR, Greiner JV, Glonek T, et al. Human and Rabbit Lipid Layer and Interference Pattern Observations In: Sullivan DA, Dartt DA,Meneray MA, eds. Lacrimal Gland, Tear Film, and Dry Eye Syndromes 2: Basic Science and Clinical Relevance. Boston, MA: Springer US, 1998;305-308.
124. Kaminer J, Powers AS, Horn KG, et al. Characterizing the spontaneous blink generator: an animal model. J Neurosci 2011;31:11256-11267.
125. Kilic S, Kulualp K. Efficacy of Several Therapeutic Agents in a Murine Model of Dry Eye Syndrome. Comp Med 2016;66:112-118.
126. Sassa T, Tadaki M, Kiyonari H, et al. Very long-chain tear film lipids produced by fatty acid elongase ELOVL1 prevent dry eye disease in mice. The FASEB Journal 2018;32:2966- 2978.
127. Mishima S, Maurice DM. The oily layer of the tear film and evaporation from the corneal surface. Exp Eye Res 1961;1:39-45.
128. Dartt DA. Neural regulation of lacrimal gland secretory processes: relevance in dry eye diseases. Prog Retin Eye Res 2009;28:155-177.
129. Butovich IA, Lu H, McMahon A, et al. Toward an animal model of the human tear film: biochemical comparison of the mouse, canine, rabbit, and human meibomian lipidomes. Invest Ophthalmol Vis Sci 2012;53:6881-6896.
130. Inomata T, Iwagami M, Hiratsuka Y, et al. Maximum blink interval is associated with tear film breakup time: A new simple, screening test for dry eye disease. Scientific Reports 2018;8:13443.
131. Wei XE, Markoulli M, Zhao Z, et al. Tear film break-up time in rabbits. Clin Exp Optom 2013;96:70-75.
176
132. Van Ooteghem MM. Factors Influencing the Retention of Ophthalmic Solutions on the Eye Surface. Ophthalmic Drug Delivery 1987;7-17.
133. Zaki I, Fitzgerald P, Hardy JG, et al. A comparison of the effect of viscosity on the precorneal residence of solutions in rabbit and man. J Pharm Pharmacol 1986;38:463-466.
134. Aihara M, Lindsey JD, Weinreb RN. Reduction of intraocular pressure in mouse eyes treated with latanoprost. Invest Ophthalmol Vis Sci 2002;43:146-150.
135. Linn CL, Webster SE, Webster MK. Eye Drops for Delivery of Bioactive Compounds and BrdU to Stimulate Proliferation and Label Mitotically Active Cells in the Adult Rodent Retina. Bio Protoc 2018;8.
136. Davies NM. Biopharmaceutical considerations in topical ocular drug delivery. Clin Exp Pharmacol Physiol 2000;27:558-562. 137. Oriá AP, Rebouças MF, Martins Filho E, et al. Photography-based method for assessing fluorescein clearance test in dogs. BMC Vet Res 2018;14:269.
138. Chrai SS, Makoid MC, Eriksen SP, et al. Drop size and initial dosing frequency problems of topically applied ophthalmic drugs. J Pharm Sci 1974;63:333-338.
139. Zeng M, Shen J, Liu Y, et al. The HIF-1 antagonist acriflavine: visualization in retina and suppression of ocular neovascularization. J Mol Med (Berl) 2017;95:417-429.
140. You IC, Li Y, Jin R, et al. Comparison of 0.1%, 0.18%, and 0.3% Hyaluronic Acid Eye Drops in the Treatment of Experimental Dry Eye. J Ocul Pharmacol Ther 2018;34:557-564.
141. Pillion DJ, Bartlett JD, Meezan E, et al. Systemic absorption of insulin delivered topically to the rat eye. Invest Ophthalmol Vis Sci 1991;32:3021-3027.
142. Dartt DA, Willcox MD. Complexity of the tear film: importance in homeostasis and dysfunction during disease. Exp Eye Res 2013;117:1-3.
143. Sebbag L, McDowell EM, Hepner PM, et al. Effect of tear collection on lacrimal total protein content in dogs and cats: a comparison between Schirmer strips and ophthalmic sponges. BMC Vet Res 2018;14:61.
144. Yu V, Bhattacharya D, Webster A, et al. Clusterin from human clinical tear samples: Positive correlation between tear concentration and Schirmer strip test results. Ocul Surf 2018;16:478-486.
145. Roberts S, Erickson O. Dog tear secretion and tear proteins. Journal of Small Animal Practice 1968;3:1-5.
177
146. Kaswan RL, Fullard RJ. Components in normal dog tears and tears from dogs with KCS treated with cyclosporin. Fourth International Symposium 1994;265.
147. Hemsley S, Cole N, Canfield P, et al. Protein microanalysis of animal tears. Research in Veterinary Science 2000;68:207-209.
148. Redl B. Human tear lipocalin. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 2000;1482:241-248.
149. Winiarczyk M, Winiarczyk D, Banach T, et al. Dog Tear Film Proteome In-Depth Analysis. PLoS One 2015;10:e0144242.
150. de Freitas Campos C, Cole N, Van Dyk D, et al. Proteomic analysis of dog tears for potential cancer markers. Res Vet Sci 2008;85:349-352.
151. Wei XE. Biochemical studies of the tear film in humans and rabbits. School of Optometry and Vision Science: The University of New South Wales, Sydney, Australia, 2012.
152. Mantelli F, Argüeso P. Functions of ocular surface mucins in health and disease. Curr Opin Allergy Clin Immunol 2008;8:477-483.
153. Leonard BC, Yañez-Soto B, Raghunathan VK, et al. Species variation and spatial differences in mucin expression from corneal epithelial cells. Exp Eye Res 2016;152:43-48.
154. Royle L, Matthews E, Corfield A, et al. Glycan structures of ocular surface mucins in man, rabbit and dog display species differences. Glycoconj J 2008;25:763-773.
155. Butovich IA, Borowiak AM, Eule JC. Comparative HPLC-MS analysis of canine and human meibomian lipidomes: many similarities, a few differences. Sci Rep 2011;1:24.
156. Hendrix DV, Cox SK. Pharmacokinetics of topically applied ciprofloxacin in tears of mesocephalic and brachycephalic dogs. Vet Ophthalmol 2008;11:7-10.
157. Grumetto L, Cennamo G, Del Prete A, et al. Pharmacokinetics of cetirizine in tear fluid after a single oral dose. Clin Pharmacokinet 2002;41:525-531.
158. Sebbag L, Thomasy SM, Woodward AP, et al. Pharmacokinetic modeling of penciclovir and BRL42359 in the plasma and tears of healthy cats to optimize dosage recommendations for oral administration of famciclovir. Am J Vet Res 2016;77:833-845.
159. Sebbag L, Showman L, McDowell EM, et al. Impact of Flow Rate, Collection Devices, and Extraction Methods on Tear Concentrations Following Oral Administration of Doxycycline in Dogs and Cats. J Ocul Pharmacol Ther 2018;34:452-459.
178
160. Sebbag L, Yan Y, Smith JS, et al. Tear Fluid Pharmacokinetics Following Oral Prednisone Administration in Dogs With and Without Conjunctivitis. J Ocul Pharmacol Ther 2019;35:341- 349.
161. Green-Church KB, Nichols KK, Kleinholz NM, et al. Investigation of the human tear film proteome using multiple proteomic approaches. Mol Vis 2008;14:456-470.
162. Zhou L, Zhao SZ, Koh SK, et al. In-depth analysis of the human tear proteome. J Proteomics 2012;75:3877-3885.
163. Li B, Sheng M, Li J, et al. Tear proteomic analysis of Sjögren syndrome patients with dry eye syndrome by two-dimensional-nano-liquid chromatography coupled with tandem mass spectrometry. Sci Rep 2014;4:5772.
164. Jung JH, Ji YW, Hwang HS, et al. Proteomic analysis of human lacrimal and tear fluid in dry eye disease. Sci Rep 2017;7:13363. 165. Huang Z, Du CX, Pan XD. The use of in-strip digestion for fast proteomic analysis on tear fluid from dry eye patients. PLoS One 2018;13:e0200702.
166. Nättinen J, Aapola U, Jylhä A, et al. Comparison of capillary and Schirmer strip tear fluid sampling methods using SWATH-MS proteomics approach. Translational Vision Science & Technology 2020;9:16-16.
167. Lam SM, Tong L, Duan X, et al. Extensive characterization of human tear fluid collected using different techniques unravels the presence of novel lipid amphiphiles. J Lipid Res 2014;55:289-298.
168. Cicalini I, Rossi C, Pieragostino D, et al. Integrated Lipidomics and Metabolomics Analysis of Tears in Multiple Sclerosis: An Insight into Diagnostic Potential of Lacrimal Fluid. Int J Mol Sci 2019;20.
169. von Thun Und Hohenstein-Blaul N, Funke S, Grus FH. Tears as a source of biomarkers for ocular and systemic diseases. Exp Eye Res 2013;117:126-137.
170. Pieragostino D, D'Alessandro M, di Ioia M, et al. Unraveling the molecular repertoire of tears as a source of biomarkers: beyond ocular diseases. Proteomics Clin Appl 2015;9:169-186.
171. Hagan S, Martin E, Enríquez-de-Salamanca A. Tear fluid biomarkers in ocular and systemic disease: potential use for predictive, preventive and personalised medicine. EPMA J 2016;7:15.
172. Ngo W, Chen J, Panthi S, et al. Comparison of Collection Methods for the Measure of Human Meibum and Tear Film-Derived Lipids Using Mass Spectrometry. Curr Eye Res 2018;43:1244-1252.
179
173. Lee CH, Yeo A, Tae Im Kim MD, et al. Comparison of the Effectiveness between Sampling Methods for Protein Analysis of Tear Fluids. J Korean Ophthalmol Soc 2015;56:1677-1683.
174. Guyette N, Williams L, Tran MT, et al. Comparison of low-abundance biomarker levels in capillary-collected nonstimulated tears and washout tears of aqueous-deficient and normal patients. Invest Ophthalmol Vis Sci 2013;54:3729-3737.
175. Sitaramamma T, Shivaji S, Rao GN. HPLC analysis of closed, open, and reflex eye tear proteins. Indian J Ophthalmol 1998;46:239-245.
176. Posa A, Bräuer L, Schicht M, et al. Schirmer strip vs. capillary tube method: non-invasive methods of obtaining proteins from tear fluid. Ann Anat 2013;195:137-142.
177. Hanstock HG, Walsh NP, Edwards JP, et al. Tear Fluid SIgA as a Noninvasive Biomarker of Mucosal Immunity and Common Cold Risk. Med Sci Sports Exerc 2016;48:569-577.
178. Hanstock HG, Edwards JP, Walsh NP. Tear Lactoferrin and Lysozyme as Clinically Relevant Biomarkers of Mucosal Immune Competence. Front Immunol 2019;10:1178.
179. Esmaeelpour M, Cai J, Watts P, et al. Tear sample collection using cellulose acetate absorbent filters. Ophthalmic Physiol Opt 2008;28:577-583.
180. Markoulli M, Papas E, Petznick A, et al. Validation of the flush method as an alternative to basal or reflex tear collection. Curr Eye Res 2011;36:198-207.
181. López-Cisternas J, Castillo-Díaz J, Traipe-Castro L, et al. Use of polyurethane minisponges to collect human tear fluid. Cornea 2006;25:312-318.
182. Runström G, Mann A, Tighe B. The fall and rise of tear albumin levels: a multifactorial phenomenon. Ocul Surf 2013;11:165-180.
183. Van Agtmaal EJ, ThÖRig L, Van Haeringen NJ. Comparative Protein Patterns in Tears of Several Species In: Peeters H, ed. Protides of the Biological Fluids: Elsevier, 1985;395-397.
184. Wei XE, Markoulli M, Millar TJ, et al. Divalent cations in tears, and their influence on tear film stability in humans and rabbits. Invest Ophthalmol Vis Sci 2012;53:3280-3285.
185. Jones DT, Monroy D, Pflugfelder SC. A novel method of tear collection: comparison of glass capillary micropipettes with porous polyester rods. Cornea 1997;16:450-458.
186. Farias E, Yasunaga KL, Peixoto RVR, et al. Comparison of two methods of tear sampling for protein quantification by Bradford method. Pesquisa Veterinária Brasileira 2013;33:261- 264.
180
187. Davidson HJ, Blanchard GL, Montgomery PC. Comparisons of tear proteins in the cow, horse, dog and rabbit. Adv Exp Med Biol 1994;350:331-334.
188. Brantman KR. Tear film VEGF in dogs with vascularizing corneal disease. Biomedical and Veterinary Sciences. Blacksburg, VA: Virginia Polytechnic Institute and State University, 2013;79.
189. Martinez PS, Storey ES, Pucheu-Haston CM. Survey of cytokines in normal canine tears by multiplex analysis: A pilot study. Vet Immunol Immunopathol 2018;201:38-42.
190. Thörig L, van Agtmaal EJ, Glasius E, et al. Comparison of tears and lacrimal gland fluid in the rabbit and guinea pig. Curr Eye Res 1985;4:913-920.
191. Small D, Hevy J, Tang-Liu D. Comparison of tear sampling techniques for pharmacokinetics analysis: ofloxacin concentrations in rabbit tears after sampling with schirmer tear strips, capillary tubes, or surgical sponges. J Ocul Pharmacol Ther 2000;16:439-446. 192. Coursey TG, Henriksson JT, Marcano DC, et al. Dexamethasone nanowafer as an effective therapy for dry eye disease. J Control Release 2015;213:168-174.
193. Shah M, Edman MC, Reddy Janga S, et al. Rapamycin Eye Drops Suppress Lacrimal Gland Inflammation In a Murine Model of Sjögren's Syndrome. Invest Ophthalmol Vis Sci 2017;58:372-385.
194. Thörig L, van Haeringen NJ, Wijngaards G. Comparison of enzymes of tears, lacrimal gland fluid and lacrimal gland tissue in the rat. Experimental Eye Research 1984;38:605-609.
195. Ahn S, Eom Y, Kang B, et al. Effects of Menthol-Containing Artificial Tears on Tear Stimulation and Ocular Surface Integrity in Normal and Dry Eye Rat Models. Curr Eye Res 2018;43:580-587.
196. Oriá AP, Raposo ACS, Araújo NLLC, et al. Tear ferning test in healthy dogs. Vet Ophthalmol 2018;21:391-398.
197. Dumortier G, Chaumeil JC. Lachrymal determinations: methods and updates on biopharmaceutical and clinical applications. Ophthalmic Res 2004;36:183-194.
198. Karn RC, Laukaitis CM. Comparative Proteomics of Mouse Tears and Saliva: Evidence from Large Protein Families for Functional Adaptation. Proteomes 2015;3:283-297.
199. Rentka A, Koroskenyi K, Harsfalvi J, et al. Evaluation of commonly used tear sampling methods and their relevance in subsequent biochemical analysis. Ann Clin Biochem 2017;54:521-529.
200. Berta A. Collection of tear samples with or without stimulation. Am J Ophthalmol 1983;96:115-116. 181
201. Kaswan RL, Salisbury MA, Ward DA. Spontaneous canine keratoconjunctivitis sicca. A useful model for human keratoconjunctivitis sicca: treatment with cyclosporine eye drops. Arch Ophthalmol 1989;107:1210-1216.
202. Lemp MA, Bron AJ, Baudouin C, et al. Tear osmolarity in the diagnosis and management of dry eye disease. Am J Ophthalmol 2011;151:792-798.e791.
203. Sebbag L, Pesavento PA, Carrasco SE, et al. Feline dry eye syndrome of presumed neurogenic origin: a case report. JFMS Open Rep 2018;4:2055116917746786.
204. Sebbag L, Uhl LK, Schneider B, et al. Investigation of Schirmer tear test-1 for measurement of tear production in cats in various environmental settings and with different test durations. J Am Vet Med Assoc 2020;256:681-686. 205. Leonard BC, Stewart KA, Shaw GC, et al. Comprehensive Clinical, Diagnostic, and Advanced Imaging Characterization of the Ocular Surface in Spontaneous Aqueous Deficient Dry Eye Disease in Dogs. Cornea 2019.
206. Uhl LK, Saito A, Iwashita H, et al. Clinical features of cats with aqueous tear deficiency: a retrospective case series of 10 patients (17 eyes). J Feline Med Surg 2019;21:944-950.
207. Denisin AK, Karns K, Herr AE. Post-collection processing of Schirmer strip-collected human tear fluid impacts protein content. Analyst 2012;137:5088-5096.
208. VanDerMeid KR, Su SP, Krenzer KL, et al. A method to extract cytokines and matrix metalloproteinases from Schirmer strips and analyze using Luminex. Mol Vis 2011;17:1056- 1063.
209. Sebbag L, Moody LM, Allbaugh RA, et al. Nerve growth factor in dogs: Assessment of two immunoassays and selected ocular parameters following a nicergoline challenge per os. Vet Ophthalmol 2019.
210. Balafas E, Katsila T, Melissa P, et al. A Noninvasive Ocular (Tear) Sampling Method for Genetic Ascertainment of Transgenic Mice and Research Ethics Innovation. OMICS 2019;23:312-317.
211. Gaal V, Mark L, Kiss P, et al. Investigation of the Effects of PACAP on the Composition of Tear and Endolymph Proteins. Journal of Molecular Neuroscience 2008;36:321-329.
212. Sebbag L, Allbaugh RA, Weaver A, et al. Histamine-Induced Conjunctivitis and Breakdown of Blood-Tear Barrier in Dogs: A Model for Ocular Pharmacology and Therapeutics. Front Pharmacol 2019;10:752.
213. Collins SP, Labelle AL, Dirikolu L, et al. Tear film concentrations of doxycycline following oral administration in ophthalmologically normal dogs. J Am Vet Med Assoc 2016;249:508-514. 182
214. Sebbag L, Harrington DM, Mochel JP. Tear fluid collection in dogs and cats using ophthalmic sponges. Vet Ophthalmol 2018;21:249-254.
215. Beckwith-Cohen B, Elad D, Bdolah-Abram T, et al. Comparison of tear pH in dogs, horses, and cattle. Am J Vet Res 2014;75:494-499.
216. Inic-Kanada A, Nussbaumer A, Montanaro J, et al. Comparison of ophthalmic sponges and extraction buffers for quantifying cytokine profiles in tears using Luminex technology. Mol Vis 2012;18:2717-2725.
217. Sia RK, Ryan DS, Howard RS, et al. Non-stimulated tear sample collection using polyvinyl alcohol (PVA) foam and polyester wick. Int J Ophthalmol Clin Res 2016;3:048. 218. Quah JH, Tong L, Barbier S. Patient acceptability of tear collection in the primary healthcare setting. Optom Vis Sci 2014;91:452-458.
219. Li S, Sack R, Vijmasi T, et al. Antibody protein array analysis of the tear film cytokines. Optom Vis Sci 2008;85:653-660.
220. Stuchell RN, Feldman JJ, Farris RL, et al. The effect of collection technique on tear composition. Invest Ophthalmol Vis Sci 1984;25:374-377.
221. Bertram M, Mochel J, Allbaugh R, et al. Influence of Schirmer strip wetness on volume absorbed, volume recovered, and total protein content in canine tears. ISU CVM Research Day. Ames, IA, 2019.
222. Monk CS, Jeong SY, Gibson DJ, et al. The presence of minocycline in the tear film of normal horses following oral administration and its anticollagenase activity. Vet Ophthalmol 2018;21:58-65.
223. Lerch M, Allbaugh RA, Sebbag L, et al. Paper spray high-resolution accurate mass spectrometry for quantitation of voriconazole in equine tears. Anal Bioanal Chem 2019;411:5187-5196.
224. Page L, Mochel J, Allbaugh R, et al. Impact of diurnal variability and tear collection method on total protein content and albumin levels in canine tears. ISU CVM Research Day 2019.
225. Hawkins EC, Murphy CJ. Inconsistencies in the absorptive capacities of Schirmer tear test strips. J Am Vet Med Assoc 1986;188:511-513.
226. García-Porta N, Mann A, Sáez-Martínez V, et al. The potential influence of Schirmer strip variables on dry eye disease characterisation, and on tear collection and analysis. Cont Lens Anterior Eye 2018;41:47-53.
183
227. Dionne K, Redfern RL, Nichols JJ, et al. Analysis of tear inflammatory mediators: A comparison between the microarray and Luminex methods. Mol Vis 2016;22:177-188.
228. Sapan CV, Lundblad RL, Price NC. Colorimetric protein assay techniques. Biotechnol Appl Biochem 1999;29 ( Pt 2):99-108.
229. Sebbag L, Moody LM, Mochel JP. Albumin Levels in Tear Film Modulate the Bioavailability of Medically-Relevant Topical Drugs. Front Pharmacol 2019;10:1560.
230. Hirosawa M, Sambe T, Uchida N, et al. Determination of nonsteroidal anti-inflammatory drugs in human tear and plasma samples using ultra-fast liquid chromatography-tandem mass spectrometry. Jpn J Ophthalmol 2015;59:364-371. 231. Maggio F. Ocular surface disease in dogs part 1: aetiopathogenesis and clinical signs. Companion Animal 2019;24:240-245.
232. Holve DL, Mundwiler KE, Pritt SL. Incidence of spontaneous ocular lesions in laboratory rabbits. Comp Med 2011;61:436-440.
233. Bedard KM. Ocular Surface Disease of Rabbits. Vet Clin North Am Exot Anim Pract 2019;22:1-14.
234. Hubert MF, Gerin G, Durand-Cavagna G. Spontaneous ophthalmic lesions in young Swiss mice. Lab Anim Sci 1999;49:232-240.
235. Moore BA, Roux MJ, Sebbag L, et al. A Population Study of Common Ocular Abnormalities in C57BL/6N rd8 Mice. Invest Ophthalmol Vis Sci 2018;59:2252-2261.
236. Monk C. Ocular Surface Disease in Rodents (Guinea Pigs, Mice, Rats, Chinchillas). Vet Clin North Am Exot Anim Pract 2019;22:15-26.
237. Craig JP, Nelson JD, Azar DT, et al. TFOS DEWS II Report Executive Summary. Ocul Surf 2017;15:802-812.
238. Barabino S, Dana MR. Animal models of dry eye: a critical assessment of opportunities and limitations. Invest Ophthalmol Vis Sci 2004;45:1641-1646.
239. Barabino S, Chen W, Dana MR. Tear film and ocular surface tests in animal models of dry eye: uses and limitations. Exp Eye Res 2004;79:613-621.
240. Stern ME, Pflugfelder SC. What We Have Learned From Animal Models of Dry Eye. Int Ophthalmol Clin 2017;57:109-118.
241. Dong ZY, Ying M, Zheng J, et al. Evaluation of a rat meibomian gland dysfunction model induced by closure of meibomian gland orifices. Int J Ophthalmol 2018;11:1077-1083.
184
242. Burgalassi S, Panichi L, Chetoni P, et al. Development of a simple dry eye model in the albino rabbit and evaluation of some tear substitutes. Ophthalmic Res 1999;31:229-235.
243. Li N, Deng X, Gao Y, et al. Establishment of the mild, moderate and severe dry eye models using three methods in rabbits. BMC Ophthalmology 2013;13:50.
244. Dursun D, Wang M, Monroy D, et al. A mouse model of keratoconjunctivitis sicca. Invest Ophthalmol Vis Sci 2002;43:632-638.
245. Viñas M, Maggio F, D'Anna N, et al. Meibomian gland dysfunction (MGD), as diagnosed by non-contact infrared Meibography, in dogs with ocular surface disorders (OSD): a retrospective study. BMC Vet Res 2019;15:443. 246. Murphy CJ, Bentley E, Miller PE, et al. The pharmacologic assessment of a novel lymphocyte function-associated antigen-1 antagonist (SAR 1118) for the treatment of keratoconjunctivitis sicca in dogs. Invest Ophthalmol Vis Sci 2011;52:3174-3180.
247. Dupuis P, Prokopich CL, Hynes A, et al. A contemporary look at allergic conjunctivitis. Allergy Asthma Clin Immunol 2020;16:5.
248. Bundoc VG, Keane-Myers A. Animal models of ocular allergy. Curr Opin Allergy Clin Immunol 2003;3:375-379.
249. Groneberg DA, Bielory L, Fischer A, et al. Animal models of allergic and inflammatory conjunctivitis. Allergy 2003;58:1101-1113.
250. Lourenço-Martins AM, Delgado E, Neto I, et al. Allergic conjunctivitis and conjunctival provocation tests in atopic dogs. Vet Ophthalmol 2011;14:248-256.
251. Bean AG, Baker ML, Stewart CR, et al. Studying immunity to zoonotic diseases in the natural host - keeping it real. Nat Rev Immunol 2013;13:851-861.
252. Marquart ME. Animal models of bacterial keratitis. J Biomed Biotechnol 2011;2011:680642.
253. Pennington MR, Ledbetter EC, Van de Walle GR. New Paradigms for the Study of Ocular Alphaherpesvirus Infections: Insights into the Use of Non-Traditional Host Model Systems. Viruses 2017;9.
254. Ledbetter EC, Dubovi EJ, Kim SG, et al. Experimental primary ocular canine herpesvirus-1 infection in adult dogs. Am J Vet Res 2009;70:513-521.
255. Ollivier FJ. Bacterial corneal diseases in dogs and cats. Clin Tech Small Anim Pract 2003;18:193-198.
185
256. Bartimote C, Foster J, Watson S. The spectrum of Microbial Keratitis: An updated review. The Open Ophthalmology Journal 2019;13.
257. Somayaji R, Priyantha MA, Rubin JE, et al. Human infections due to Staphylococcus pseudintermedius, an emerging zoonosis of canine origin: report of 24 cases. Diagn Microbiol Infect Dis 2016;85:471-476.
258. Thomasy SM, Cortes DE, Hoehn AL, et al. In Vivo Imaging of Corneal Endothelial Dystrophy in Boston Terriers: A Spontaneous, Canine Model for Fuchs' Endothelial Corneal Dystrophy. Invest Ophthalmol Vis Sci 2016;57:OCT495-503.
259. Sanchez RF, Daniels JT. Mini-Review: Limbal Stem Cells Deficiency in Companion Animals: Time to Give Something Back? Curr Eye Res 2016;41:425-432. 260. Dreyfus J, Schobert CS, Dubielzig RR. Superficial corneal squamous cell carcinoma occurring in dogs with chronic keratitis. Vet Ophthalmol 2011;14:161-168.
261. Sebbag L, Crabtree EE, Sapienza JS, et al. Corneal hypoesthesia, aqueous tear deficiency, and neurotrophic keratopathy following micropulse transscleral cyclophotocoagulation in dogs. Vet Ophthalmol 2019.
262. Azari AA, Barney NP. Conjunctivitis: a systematic review of diagnosis and treatment. JAMA 2013;310:1721-1729.
263. Maggs DJ. Diseases of the Conjunctiva In: Maggs DJ, Miller PE,Ofri R, eds. Slatter's fundamentals of veterinary ophthalmology. 6th ed. St Louis, Missouri: Saunders, 2018.
264. Soler E, Mochel J, Allbaugh R, et al. Impact of acute conjunctivitis on tear film dynamics, quantity and quality in healthy Beagle dogs. The 50th Annual Scientific Meeting of the American College of Veterinary Ophthalmologists,. Maui, Hawaii, 2019;E28-E80.
265. Zavaro A, Samra Z, Baryishak R, et al. Proteins in tears from healthy and diseased eyes. Doc Ophthalmol 1980;50:185-199.
266. Janssen PT, Van Bijsterveld OP. Blood-tear barrier and tear fluid composition. The Precorneal Tear Film in Health, Disease and Contact Lens Wear, Holly FJ, editor Lubbock, TX, Dry Eye Institute 1986:471-475.
267. Li K, Liu X, Chen Z, et al. Quantification of tear proteins and sPLA2-IIa alteration in patients with allergic conjunctivitis. Mol Vis 2010;16:2084-2091.
268. Mikkelson TJ, Chrai SS, Robinson JR. Altered bioavailability of drugs in the eye due to drug-protein interaction. J Pharm Sci 1973;62:1648-1653.
269. Takahashi A, Sumi T, Tada K, et al. Evaluation of histamine-induced conjunctival oedema in guinea pigs by means of image analysis. Br J Ophthalmol 2010;94:1657-1661. 186
270. Terhaar M, Allbaugh R, Mochel J, et al. Serum albumin and total protein concentration in the tear film of horses with healthy or diseased eyes, 2019.
271. van Bijsterveld OP, Janssen PT. The effect of calcium dobesilate on albumin leakage of the conjunctival vessels. Curr Eye Res 1981;1:425-430.
272. Guo Y, Ramachandran C, Satpathy M, et al. Histamine-induced myosin light chain phosphorylation breaks down the barrier integrity of cultured corneal epithelial cells. Pharm Res 2007;24:1824-1833.
273. de Wolf FA, Brett GM. Ligand-binding proteins: their potential for application in systems for controlled delivery and uptake of ligands. Pharmacol Rev 2000;52:207-236. 274. Dalhoff A. Seventy-Five Years of Research on Protein Binding. Antimicrob Agents Chemother 2018;62.
275. Prausnitz MR, Noonan JS. Permeability of cornea, sclera, and conjunctiva: a literature analysis for drug delivery to the eye. J Pharm Sci 1998;87:1479-1488.
276. Ramsay E, Ruponen M, Picardat T, et al. Impact of Chemical Structure on Conjunctival Drug Permeability: Adopting Porcine Conjunctiva and Cassette Dosing for Construction of In Silico Model. J Pharm Sci 2017;106:2463-2471.
277. Gronkiewicz KM, Giuliano EA, Kuroki K, et al. Development of a novel in vivo corneal fibrosis model in the dog. Exp Eye Res 2016;143:75-88.
278. Robaei D, Watson S. Corneal blindness: a global problem. Clin Exp Ophthalmol 2014;42:213-214.
279. Stepp MA, Zieske JD, Trinkaus-Randall V, et al. Wounding the cornea to learn how it heals. Exp Eye Res 2014;121:178-193.
280. Proietto LR, Whitley RD, Brooks DE, et al. Development and Assessment of a Novel Canine Ex Vivo Corneal Model. Curr Eye Res 2017;42:813-821.
281. Berkowski WM, Gibson DJ, Craft SL, et al. Development and assessment of a novel ex vivo corneal culture technique involving an agarose-based dome scaffold for use as a model of in vivo corneal wound healing in dogs and rabbits. Am J Vet Res 2020;81:47-57.
282. Berkowski WM, Gibson DJ, Seo S, et al. Assessment of Topical Therapies for Improving the Optical Clarity Following Stromal Wounding in a Novel Ex Vivo Canine Cornea Model. Invest Ophthalmol Vis Sci 2018;59:5509-5521.
187
283. Harman RM, Bussche L, Ledbetter EC, et al. Establishment and Characterization of an Air- Liquid Canine Corneal Organ Culture Model To Study Acute Herpes Keratitis. Journal of Virology 2014;88:13669.
284. Chrai SS, Robinson JR. Ocular evaluation of methylcellulose vehicle in albino rabbits. J Pharm Sci 1974;63:1218-1223.
285. Beauchamp G. Half-Blind to the Risk of Predation. Frontiers in Ecology and Evolution 2017;5.
286. Owen GR, Brooks AC, James O, et al. A novel in vivo rabbit model that mimics human dosing to determine the distribution of antibiotics in ocular tissues. J Ocul Pharmacol Ther 2007;23:335-342.
287. Seo J, Byun WY, Alisafaei F, et al. Multiscale reverse engineering of the human ocular surface. Nat Med 2019;25:1310-1318.
288. Schneider B, Balbas-Martinez V, Jergens AE, et al. Model-Based Reverse Translation Between Veterinary and Human Medicine: The One Health Initiative. CPT Pharmacometrics Syst Pharmacol 2018;7:65-68.
289. Kühnel W. Morphology of the Harderian Gland in the Rabbit. A Short Review In: Webb SM, Hoffman RA, Puig-Domingo ML, et al., eds. Harderian Glands: Porphyrin Metabolism, Behavioral and Endocrine Effects. Berlin, Heidelberg: Springer Berlin Heidelberg, 1992;109- 125.
290. Ma H, Chen Y, Cai X, et al. Effect of aging in periocular appearances by comparison of anthropometry between early and middle adulthoods in Chinese Han population. J Plast Reconstr Aesthet Surg 2019;72:2002-2008.
291. Packer RM, Hendricks A, Burn CC. Impact of facial conformation on canine health: corneal ulceration. PLoS One 2015;10:e0123827.
292. Bron AJ, Benjamin L, Snibson GR. Meibomian gland disease. Classification and grading of lid changes. Eye (Lond) 1991;5 ( Pt 4):395-411.
293. Wang J, Call M, Mongan M, et al. Meibomian gland morphogenesis requires developmental eyelid closure and lid fusion. Ocul Surf 2017;15:704-712.
294. Last JA, Thomasy SM, Croasdale CR, et al. Compliance profile of the human cornea as measured by atomic force microscopy. Micron 2012;43:1293-1298.
295. Silver DM, Csutak A. Human Eye Dimensions for Pressure-Volume Relations. Investigative Ophthalmology & Visual Science 2010;51:5019-5019.
188
296. Atsumi I, Kurata M, Sakaki H. Comparative study on ocular anatomical features among rabbits, beagle dogs and cynomolgus monkeys. Animal Eye Research 2013;32:35-41.
297. Zhou X, Xie J, Shen M, et al. Biometric measurement of the mouse eye using optical coherence tomography with focal plane advancement. Vision Res 2008;48:1137-1143.
298. Vogel B, Wagner H, Gmoser J, et al. Touch-free measurement of body temperature using close-up thermography of the ocular surface. MethodsX 2016;3:407-416. 299. Efron N, Young G, Brennan NA. Ocular surface temperature. Curr Eye Res 1989;8:901- 906.
300. Biondi F, Dornbusch PT, Sampaio M, et al. Infrared ocular thermography in dogs with and without keratoconjunctivitis sicca. Vet Ophthalmol 2015;18:28-34. 301. Tran CH, Routledge C, Miller J, et al. Examination of murine tear film. Invest Ophthalmol Vis Sci 2003;44:3520-3525.
302. Chen HB, Yamabayashi S, Ou B, et al. Structure and composition of rat precorneal tear film. A study by an in vivo cryofixation. Invest Ophthalmol Vis Sci 1997;38:381-387.
303. Fukuoka S, Arita R. Increase in tear film lipid layer thickness after instillation of 3% diquafosol ophthalmic solution in healthy human eyes. Ocul Surf 2017;15:730-735.
304. Kimball SH, King-Smith PE, Nichols JJ. Evidence for the Major Contribution of Evaporation to Tear Film Thinning between Blinks. Investigative Ophthalmology & Visual Science 2010;51:6294-6297.
305. Paschides CA, Kitsios G, Karakostas KX, et al. Evaluation of tear break-up time, Schirmer's-I test and rose bengal staining as confirmatory tests for keratoconjunctivitis sicca. Clin Exp Rheumatol 1989;7:155-157.
306. Kurtul BE, Özer PA, Aydinli MS. The association of vitamin D deficiency with tear break- up time and Schirmer testing in non-Sjögren dry eye. Eye (Lond) 2015;29:1081-1084.
307. Saito A, Kotani T. Estimation of lacrimal level and testing methods on normal beagles. Vet Ophthalmol 2001;4:7-11.
308. Miyasaka K, Kazama Y, Iwashita H, et al. A novel strip meniscometry method for measuring aqueous tear volume in dogs: Clinical correlations with the Schirmer tear and phenol red thread tests. Vet Ophthalmol 2019.
309. Biricik HS, Oğuz H, Sindak N, et al. Evaluation of the Schirmer and phenol red thread tests for measuring tear secretion in rabbits. Vet Rec 2005;156:485-487.
310. Senchyna M, Wax MB. Quantitative assessment of tear production: A review of methods and utility in dry eye drug discovery. J Ocul Biol Dis Infor 2008;1:1-6. 189
311. De Silva MEH, Hill LJ, Downie LE, et al. The Effects of Aging on Corneal and Ocular Surface Homeostasis in Mice. Invest Ophthalmol Vis Sci 2019;60:2705-2715.
312. Marques DL, Alves M, Modulo CM, et al. Lacrimal osmolarity and ocular surface in experimental model of dry eye caused by toxicity. Revista Brasileira de Oftalmologia 2015;74:68.
313. Doughty MJ. Tear Film Stability and Tear Break Up Time (TBUT) in Laboratory Rabbits— A Systematic Review. Current Eye Research 2018;43:961-964.
314. Sebbag L, Park SA, Kass PH, et al. Assessment of tear film osmolarity using the TearLab(™) osmometer in normal dogs and dogs with keratoconjunctivitis sicca. Vet Ophthalmol 2017;20:357-364.
315. Honkanen R, Huang W, Huang L, et al. A New Rabbit Model of Chronic Dry Eye Disease Induced by Complete Surgical Dacryoadenectomy. Curr Eye Res 2019;44:863-872.
316. Chen FS, Maurice DM. The pH in the precorneal tear film and under a contact lens measured with a fluorescent probe. Experimental Eye Research 1990;50:251-259.
317. Ruiz-Ederra J, Levin MH, Verkman AS. In Situ Fluorescence Measurement of Tear Film [Na+], [K+], [Cl−], and pH in Mice Shows Marked Hypertonicity in Aquaporin-5 Deficiency. Investigative Ophthalmology & Visual Science 2009;50:2132-2138.
318. Prydal JI, Muir MG, Dilly PN. Comparison of tear film thickness in three species determined by the glass fibre method and confocal microscopy. Eye (Lond) 1993;7 ( Pt 3):472- 475.
319. Boonstra A, Kijlstra A. The identification of transferrin, an iron-binding protein in rabbit tears. Exp Eye Res 1984;38:561-567.
320. Zhou L, Beuerman RW, Barathi A, et al. Analysis of rabbit tear proteins by high-pressure liquid chromatography/electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom 2003;17:401-412.
321. Seamon V, Vellala K, Zylberberg C, et al. Sex hormone regulation of tear lipocalin in the rabbit lacrimal gland. Exp Eye Res 2008;87:184-190.
322. Kelly KE. Clinical study of canine tear lacritin as a treatment for dry eye. 2016.
323. Zhang FD, Hao ZQ, Gao W, et al. Effect of topical 0.05% cyclosporine A on the tear protein lacritin in a rat model of dry eye. Int J Ophthalmol 2019;12:189-193.
190
324. Ginel PJ, Novales M, García M, et al. Immunoglobulins in stimulated tears of dogs. Am J Vet Res 1993;54:1060-1063.
325. Fullard RJ, Kaswan RM, Bounous DI, et al. Tear protein profiles vs. clinical characteristics of untreated and cyclosporine-treated canine KCS. J Am Optom Assoc 1995;66:397-404.
326. Sullivan DA, Allansmith MR. Source of IgA in tears of rats. Immunology 1984;53:791-799.
327. Bodelier VMW, van Haeringen NJ, Klaver PSY. Species differences in tears; Comparative investigation in the chimpanzee (Pan troglodytes). Primates 1993;34:77-84.
328. Tei M, Spurr-Michaud SJ, Tisdale AS, et al. Vitamin A deficiency alters the expression of mucin genes by the rat ocular surface epithelium. Invest Ophthalmol Vis Sci 2000;41:82-88.
329. Shirai K, Saika S. Ocular surface mucins and local inflammation--studies in genetically modified mouse lines. BMC Ophthalmol 2015;15 Suppl 1:154.
330. Harvey DJ, Tiffany JM, Duerden JM, et al. Identification by combined gas chromatography-mass spectrometry of constituent long-chain fatty acids and alcohols from the meibomian glands of the rat and a comparison with human meibomian lipids. J Chromatogr 1987;414:253-263.
Tables and Figures
Table 1. Comparative anatomy of the ocular surface and globe between humans, dogs and common laboratory species used in preclinical ophthalmic research.
Human Dog Rabbit Mouse Rat Lacrimal gland, Lacrimal gland Third eyelid gland, Accessory glands Lacrimal gland, Infraorbital Lacrimal glands Infraorbital Infraorbital of Wolfring and Third eyelid gland (intraorbital), (extraorbital) 10,11,22 (extraorbital) 10,11,22 Krause 11,14 Accessory glands of 20 Wolfring 10,11,22,23 Lacrimal 191 glands and Lacrimal glands 0.59-0.61 0.14 / 0.1 – / 1.5 – – nasolacrimal volume (cm3) † 18 17 289 apparatus Absent Absent Present Present Present Harderian gland 22 11,16 11,22 11,22 11,22 Single Two Two punctum/canaliculus, Two Two Nasolacrimal drainage puncta/canaliculi, puncta/canaliculi, 1 two pronounced puncta/canaliculi puncta/canaliculi apparatus no flexure dorsal flexure flexures 25 25 25,27 16,25,27 27,38,41 Absent Present Present Present Present Third eyelid 66 16,66 66 66 66 Palpebral fissure 21.3-34.5 18.9-34.1 10-16 3.7-5 ‡ 6-9 width (mm) 290 40,291 38,41,42 43 45 Eyelids Interpalpebral fissure 2.2 § 1.4 0.13 ‡ 0.5 ¶ 1.8 39 area (cm2) 290 39 43 45 Meibomian glands 20-40 20-40 30-50 20 20-30 # (/eyelid) 39,292 16 38,39,41 293 241
Table 1 Continued Human Dog Rabbit Mouse Rat Conjunctival fornix 15 20.36 – – – depth (mm) †† 49 48 Conjunctival surface 17.65 13.3-18.48 ‡‡ – – – (cm2) 50 50 Conjunctiva/Cornea 17.17 8.62-8.94 – – – surface ratio 50 50 Goblet cell spatial Individual Individual Individual Clusters Clusters configuration 51 52 51 51 51 Highest in plica Highest in third Conjunctiva Highest in palpebral semilunaris and eyelid and lower Highest in fornix conjunctiva Goblet cell lower nasal fornix nasal fornix Low in bulbar Relatively dense in – distribution Low in bulbar Low in bulbar conjunctiva bulbar conjunctiva Kim 2019 conjunctiva conjunctiva 192 57-59 30,53,54,56 52,55
Only present in Conjunctiva- Present Present Present nictitating Absent associated lymphoid 61 16,61 61 membrane 61 tissue 61,62 Corneal diameter, 11.8 / 11.2 13-17 / 12-16 13.4-15 / 13-14 2.3-2.6 / – 2.0-6.8 / 2.0-6.7 horizontal/vertical 75,77 71,77 41,76,77 74 44,72,73 (mm) 1.04-1.3 1.2-2.1 1.55-2.03 0.04-0.05 h 0.27-0.36 §§ Corneal surface (cm2) Cornea 50 71,77 50 74 72,73 and Corneal thickness 505-563 497-594 354-407 90-137 159-170 Sclera (µm) 79 68,78 41,76,79,80 11,74,80 68,80 Corneal epithelial 44-55 52-64 45-49 37-46 26-33 thickness (µm) 79 71,78 79 74 85 Endothelial cell 2732 2818 3233 2875 2242 density (cells/mm2) 90 68 68 68 68
Table 1 Continued
Human Dog Rabbit Mouse Rat Subbasal nerve plexus Whorl-like Whorl-like Horizontal Whorl-like Whorl-like pattern 67 67 67 67 67 Corneal sensitivity 0.2-1.0 2.16-2.9 6.21-10 0.59 0.42-0.47 (g/mm2) 2 92,95 2,92 93 91,94
Stiffness/elastic 16.2-33.1 1.3 1.1 25-40 6.2 modulus (kPa) ¶¶ 87,294 87 86 89 88 Scleral thickness at 0.50 0.80 0.29 0.05-0.06 < 0.1 the limbus (mm) 102 101 103 105 104 5.7-6.0 5.0-5.8 2.3-2.9 0.014 j 0.13 ## Globe volume (mL) 295 296 296 297 72 Anterior chamber 0.17-0.31 0.77 0.28-0.30 0.0044-0.007 0.0136-0.015 Globe (mL) 66,77 101 41,66,77 11,66,77 11,66,77 193
Vitreous chamber 3.5-5.4 1.7-3.0 1.1-1.8 0.0053 0.013-0.054 (mL) 65 101,296 66,296 11,65 11 ,65
– Information not available or not found † Lacrimal gland (human), Lacrimal gland / TEL gland (dog), Lacrimal gland / Harderian gland (rabbit) ‡ Estimated based on clinical images43 § Estimated based on clinical images40 ¶ Calculated based on average palpebral fissure length45 # Estimated based on clinical images241 †† Central upper conjunctival sac (from fornix to lid margin) ‡‡ Does not account for the nictitating membrane, surgically removed prior to the experiment §§ Estimated from corneal radius, assuming a circular shape for the cornea in rodents72 ¶¶ Whole cornea in rats, epithelium and anterior stroma in other species ## Estimated from axial length, assuming a spherical shape for the globe
Table 2. Comparative physiology and characteristics of the ocular surface and tear film between humans, dogs and common laboratory species used in preclinical ophthalmic research.
Human Dog Rabbit Mouse Rat Blink rate (blinks/min) 8.5-17.6 14.2 0.05-0.19 0-4 2-5.3 122-124 121 2,123,127 125,126 91,124 Volumetric capacity of Ocular surface 25-30 31.3 10-25 † ≤ 5 ≤ 20 the palpebral fissure physiology 109,136 40 112,138 139,140 141 (µL) Ocular surface 32.8-37.1 35.2 39.1 37.2 36.5 temperature (°C) 298,299 300 298 298 298 Tear film thickness 2.3-11.5 ‡ 15.1 6.5-18.4 7.4-21.1 2-12.6 (µm) 119 111 119 119,301 119,302 194
Lipid layer thickness 62-78 13-581 > 180 12 – (nm) 123,303 121 123 301 1.9-7.5 Tear volume 7-12.4 65.3 112,116 0.06-0.2 4.6 109,115 111 113,118 117 Tear film (µL) characteristics Basal tear turnover rate 13.1-17.5 12.1 6.2-7.1 5.2 – (%/min) 109,110 111 112 113 Reflex tear turnover rate 31.5-100 50 6.1-6.9 § – – (%/min) 109,115 111 112 Evaporative rate 3.22 0.47 – – – (µm/min) 304 127 Schirmer tear test-1 10.0-18.6 18.1-24.3 4.6-7.6 5.6-9.4 – Tear film (mm) # 305,306 13,52,95,205,307,308 42,116,309 91,241 diagnostics ¶ Phenol red thread test 9-20 17.5-39.2 20.9-25.0 2.8-11.2 7.6 (mm/15s) 310 95,205,307,308 42,309 125,244,311 312
Table 2 Continued Human Dog Rabbit Mouse Rat Tear film breakup time 7.4-13.0 13.9-24.0 2-1788 †† 5-25 5.2-6.0 (sec) 305,306 13,52,205,307 313 125,129 241 Tear osmolarity ‡‡ 300.8 337.4-339.0 291.3 346.3-366.8 284.8
(mOsm/L) 202 205,314 315 310 312 7.83 8.05 8.2 7.59 Tear pH – 316 215 316 317
– Information not available or not found † Based on greater percent of drug lost at 1 min in rabbit eyes receiving 25 or 50 µL eyedrop vs. 5 or 10 µL eyedrop,138 despite no changes in tear turnover rate for instilled volumes up to 50 µL112 ‡ Excludes an outlier measurement of 41-46 µm318 195 § Estimated from rabbit eyes receiving a large volume instilled eyedrop (25-50 µL) ¶ Reported values prioritized studies that did not use sedation or general anesthesia # Values reported in mm/5min (humans) or mm/min (all other species) †† Large variability in studies’ methodology, most using topical and/or general anesthesia prior to testing, resulting in non-physiologic and highly variable measurements for tear film break up time ‡‡ Measurements obtained with the same device (TearLabTM, OcuSense Inc., San Diego, CA)
Table 3. Comparative composition of the major components in tear film between humans, dogs and common laboratory species used in preclinical ophthalmic research.
Human Dog Rabbit Mouse Rat
† Abundant Low Low Low Low Lactoferrin 144,147 146,147 319,320 147 147 Abundant Low Low Low Low Lysozyme 144,147 145,146 190,319 147 147 Low ‡ Abundant Low Low Absent Lipocalin 144,147,148 to moderate 147,148,319,321 147,198 147,148 146,147,149,150 Moderate Moderate Absent Present Proteins Lacritin – 144 322 198 323 196 Moderate Moderate Present Low Moderate
Secretory IgA 144 146,324,325 22 11 326 Low Low Low Present Serum albumin – 182 146,150,212 190,319 198 Low Low Absent Abundant Peroxidase – 327 146,325 190 194 Low Absent Low Amylase – – 327 190 194 Present Present Present Present Present MUC5AC 11 11,55 11 11 11
MUC16 >> MUC1 MUC16 >> MUC1 > MUC1 ≈ MUC4 ≈ Mucins MUC1, MUC4, Present Present > MUC4 MUC4 MUC16 MUC16 328 329 153 153 153 Sialylated glycans Fucosylated glycans Fucosylated glycans Major O-glycans 154 154 154 – –
Table 3 Continued
Human Dog Rabbit Mouse Rat Abundant Abundant Low Abundant Present Wax esters 129,155 129,155 129 129 330 Low Low Low Low Abundant Cholesterol 129,155 129,155 129 129 330 Abundant Abundant Low Abundant Cholesteryl esters – 129,155 129,155 129 129 Low Low Low Low DiHL – 129,155 129,155 129 129 Lipids Low Low Abundant Low DiHL esters – 129,155 129,155 129 129 Low Low Abundant Low DiAD – 129,155 129,155 129 129 197
Abundant Abundant Abundant Abundant OAHFA – 129,155 129,155 129 129 Moderate Moderate Low Moderate Chl-OAHFA – 129,155 129,155 129 129 Low Low Low Low Others § – 129,155 129,155 129 129
– Information not available or not found † Homologous iron-binding protein called transferrin is reported in dogs146 and rabbits319 ‡ Homologous proteins are reported in dogs (major canine allergen),148-150 rabbits (lipophilin)151 and rats (VEGr1)148 § Triacylglycerol, squalene, ceramides, phospholipids and sphingomyelins
198
Figure 1. Graphical representation of the canine ocular surface and lacrimal functional unit.
199
Figure 2. Diagram depicting the complexity of tear film dynamics and ocular surface physiology. Secretion of tear components, distribution of tears through blinking, and elimination through nasolacrimal drainage and evaporation must be precisely regulated to maintain homeostasis. Drug kinetics following topical eyedrop administration are impacted by key parameters highlighted in yellow, each being unique in different species. Adapted with permission from “Tsubota K, Tseng SCG, Nordlund ML. Anatomy and Physiology of the Ocular Surface In: Holland EJ,Mannis MJ, eds. Ocular Surface Disease Medical and Surgical Management. New York, NY: Springer New York, 2002;3-15”. 200
Figure 3. Tear collection in dogs using microcapillary glass tubes (a), Schirmer tear strips (b) or absorbent sponges (c).
Figure 4. Step-by-step protocol to exact tear fluid from Schirmer strips for analytical purposes, using a combination of centrifugation and solvent wash. Centrifugation of wetted Schirmer strip (containing tear sample and internal standard) retrieves tear fluid in a large tube, while subsequent solvent elution washes residual content from the absorbent filter paper. 201
Figure 5. Representative clinical pictures of mild conjunctivitis (a), moderate conjunctivitis (b), and severe conjunctivitis (c) in dogs following topical administration of histamine at concentrations of 1 mg/mL, 10 mg/mL and 375 mg/mL, respectively. Reprinted from “Sebbag L, Allbaugh RA, Weaver A, et al. Histamine-Induced Conjunctivitis and Breakdown of Blood-Tear Barrier in Dogs: A Model for Ocular Pharmacology and Therapeutics. Front Pharmacol 2019;10:752”.
202
Figure 6. Graphical representation of the blood-tear barrier in the canine eye. The barrier is intact in healthy eyes (left) but is disrupted in diseased eyes (right), enhancing the flow of compounds (eg, albumin, xenobiotics) between the tear film and the blood compartments. Breakdown of the blood-tear barrier can have important clinical implications such as enhanced systemic absorption from greater conjunctival vascular permeability, or reduction in ocular drug bioavailability due to drug-albumin interactions in the tear film. 203
APPENDIX A. MATHEMATICAL MODELING USING NONLINEAR MIXED- EFFECTS
Parameters estimation was performed using the stochastic approximation expectation
maximization (SAEM) algorithm for nonlinear mixed effects-models as implemented in the
Monolix Suite. Competing models were evaluated numerically using Bayesian information
criteria (BIC) and precision of parameter estimates – defined as relative standard error of the
estimate (RSE). Standard goodness-of-fit diagnostics, including observed vs. predicted
fluorescein concentrations, individual fits and weighted residuals time-course were used to
graphically assist comparison. SAEM convergence and final model parameterization were
assessed graphically by inspection of search stability, distribution of the individual parameters,
distribution of the random effects, individual prediction vs. observation, individual fits, and
distributions of the weighted residuals. The numerical precision of parameter estimates was
assessed using RSE. The numerical normality of individual parameters was assessed using a
Shapiro-Wilk test for normality. The normality of the distribution of residuals was assessed using
a Shapiro-Wilk test and the centering of the distribution of residuals (i.e. 0) was assessed using a
Van Der Waerden test. P values < 0.05 were considered as statistically significant.
A suitable mathematical model has the following features (see Figures below): (i) the line of
identity is aligned with the regression line while (ii) the residues (differences between
observations and predictions) are centered on a mean value of 0, with (iii) a homogeneous
dispersion around the mean.
204
Figure A1. Comparison of predicted tear fluorescence over time (purple curve) to observed data (blue points) for a random sample of dogs (A) and cats (B). Censored data are shown as vertical red bars. The ID of each individual is listed above the individual fit, followed by #1 for the right eye, and #2 for the left eye.
Figure A2. Standard Goodness-Of-Fit diagnostics: Individual predictions vs. Observations (log10) for the fluorophotometry data in dogs (A) and cats (B). The solid black line represents the identity line; the regression line is portrayed in light green color; censored data points (<10%) are represented with red dots. 205
Figure A3. Individual weighted residuals (IWRES) vs. fluorescein concentrations in tears of dogs (A) and cats (B). Censored data points (<10%) are represented with red dots. The turquoise line represents the spline (loess regression).
Figure A4. Simulations of fluorescein vs. time disposition from 500 Monte Carlo simulations using final parameter estimates from the NLME model. Predictions derived from the 5th to the 95th percentile of the model simulations were able to reproduce the variability in the observed data from the original population of dogs/cats. 206
APPENDIX B. TEAR FILM THICKNESS AND THEORETICAL TEAR VOLUME
Tear thickness was measured with spectral-domain optical coherence tomography (SD-
OCT, Optovue iVue, Fremont, CA) in six beagle dogs, selected based on their homogenous subject characteristics (e.g. similar breed, age, skull type, body weight, etc.). Dogs were manually restrained with their eyelids held open by an assistant. Tear thickness was measured with ImageJ software (National Institutes of Health,
Bethesda, MD) from images captured with the SD-OCT. The average tear thickness from all eyes (d = 0.01512 mm) was used to calculate the theoretical canine tear volume (TV), assuming a radius of the canine globe (r) of 10.45 mm.
1 4 1 4 TV (µL) = ( + ) 2 3 2 3 3 3 � 𝜋𝜋 𝑟𝑟 𝑑𝑑 � − � 𝜋𝜋𝑟𝑟 �
The theoretical TV value calculated with this method (10.4 µL) was lower than the one calculated with fluorophotometry (59 µL for a median beagle body weight of 9 kg), a finding likely explained by the unevenness of tear film thickness on the ocular surface. The mathematical equation assumes the tear film thickness to be homogenous when in reality tears are pooling in conjunctival fornices and tear film is much thicker at the lower and upper tear menisci. However, the tear menisci could not be readily imaged in our dogs as we did not use sedation or general anesthesia.
207
APPENDIX C. MATHEMATICAL MODELING USING NONLINEAR MIXED- EFFECTS
Figure C1. Individual predictions versus observations (log10) for the fluorophotometry data in 8 dogs (16 eyes) receiving 10-100 µL of 0.1% fluorescein solution. The solid black line represents the identity line; the regression line is portrayed in light green color.
Figure C2. IWRES plotted against the tear fluorescein concentrations in 8 dogs (16 eyes) receiving 10-100 µL of 0.1% fluorescein solution. The orange line represents the spline (loess regression). IWRES, individual weighted residuals. 208
Figure C3. Simulations of fluorescein vs. time disposition from 500 Monte Carlo simulations using final parameter estimates from the NLME model. Predictions derived from the 5th to the 95th percentile of the model simulations were able to reproduce the variability in the observed data from the original population dogs. NLME, nonlinear mixed effects.
APPENDIX D. HISTAMINE-INDUCED CONJUNCTIVITIS 209
Figure D1: Diagram of the study design showing the balanced crossover trial (top) and the timing of the procedures prior to and following topical histamine/placebo administration (bottom). BP= Blood pressure; CS = Conjunctivitis scoring; IOP = Intraocular pressure.
Table D1: A semi-quantitative conjunctivitis scoring system.
Score Description
Palpebral conjunctiva 0 (none) No swelling of the palpebral conjunctival tissue. 1 (mild) Diffuse thin swelling with no eversion of the eyelid(s) or change in eyelid margin contour. 2 (moderate) Diffuse definite swelling with misalignment of the normal approximation of the lower and upper eyelids. Chemosis Diffuse marked swelling with partial eversion of the eyelid(s). The eyelid margin(s) may have an irregular, ‘undulating’ contour, but can still be closed 3 (severe) completely. 4 (very Extremely severe swelling with pronounced eversion of both eyelids. Eyelid closure is incomplete with exposed swollen conjunctiva protruding between the severe) eyelid margins and masking the corneal surface. 0 (none) Small individual vessels are noted, blanched to pale pink in color. It is normal to observe a few prominent vessels on the palpebral surface of the third eyelid. 1 (mild) Dilatation of only a few vessels with minimal branching and/or tortuosity. Pink to light red in color. Hyperemia Dilatation of the majority of vessels with pronounced branching and/or tortuosity. Bright red to crimson red in color. The conjunctiva between large vessels 2 (moderate) may have a flushed pink-to-red appearance. 3 (severe) Diffuse beefy red appearance to the conjunctiva, difficult to distinguish individual blood vessels.
0 (none) No manifestations 210 1 (mild) 1-9 follicles Follicles 2 (moderate) 10-19 follicles 3 (severe) 20 or more follicles Bulbar conjunctiva 0 (none) No swelling of the bulbar conjunctival tissue. 1 (mild) Focal perilimbal and/or diffuse thin swelling. Underlying episcleral tissue is easily observed through the conjunctiva. Chemosis 2 (moderate) Diffuse definite swelling. Underlying episcleral tissue is difficult to observe. 3 (severe) Diffuse marked swelling, masking the corneoscleral limbal region. 0 (none) Small individual vessels are noted, blanched to pale pink in color. 1 (mild) Dilatation of only a few vessels with minimal branching and/or tortuosity. Pink-to-reddish in color. Hyperemia Dilatation of the majority of vessels with pronounced branching and/or tortuosity. Bright red to crimson red in color. The conjunctiva between large vessels 2 (moderate) may have a flushed pink-to-red appearance. 3 (severe) Diffuse beefy red appearance to the conjunctiva, difficult to distinguish individual blood vessels. 0 (none) No manifestations 1 (mild) 1-9 follicles Follicles 2 (moderate) 10-19 follicles 3 (severe) 20 or more follicles
Table D1 Continued
Score Description
Conjunctival discharge 0 (none) No discharge, or small amount of clear/mucoid material found in the medial canthus 1 (mild) Discharge is above normal and present on the surface of the eye or in the medial canthus, but not on the lids or hairs of the eyelids
2 (moderate) Discharge is abundant, easily observed, and has collected on the lids and around the hairs of the eyelids. 3 (severe) Discharge has been flowing over the eyelids so as to wet the hairs substantially on the skin around the eyes, past the orbital rim Ocular pruritus 0 (none) No ocular itching 1 (mild) Subtle, rapidly resolving itch 2 (moderate) Mild persistent itch, resolving within 30 seconds
3 (severe) Pronounced itch, not resolving within 30 seconds 4 (extremely Incapacitating itch severe)
211
Table D2: Summary table detailing each subsection of the conjunctivitis score for each histamine dose. The score selected in each individual was the maximal score documented between 0 and 420 min following histamine administration. Tmax describes the time (in min) to reach this maximal score in each dog. Results are described as mean ± standard deviation.
Palpebral Palpebral Palpebral Bulbar Bulbar Bulbar Conjunctival Ocular chemosis hyperemia follicles chemosis hyperemia follicles discharge pruritus Dogs 0/6 6/6 0/6 1/6 3/6 0/6 0/6 0/6 H1 affected (0.005 Score 0 1.2 ± 0.4 0 1 1.0 ± 0 0 0 0 mg/mL) Tmax (min) 0 4.7 ± 1.5 0 7 15 ± 10 0 0 0 Dogs 0/6 6/6 0/6 2/6 6/6 0/6 0/6 0/6 affected H2 (0.1 mg/mL) Score 0 1.8 ± 0.4 0 1 ± 0 1.5 ± 0.5 0 0 0 212 Tmax (min) 0 4.5 ± 3.1 0 6.0 ± 1.4 4.3 ± 2.1 0 0 0
Dogs 2/6 6/6 0/6 1/6 6/6 0/6 0/6 2/6 affected H3 (1.0 mg/mL) Score 1.0 ± 0 2.0 ± 0 0 1 2.0 ± 0 0 0 1.0 ± 0
Tmax (min) 5.0 ± 0 4.0 ± 2.1 0 25 6.8 ± 3.5 0 0 1.0 ± 0 Dogs 6/6 6/6 0/6 6/6 5/6 0/6 0/6 4/6 affected H4 1.3 ± Score 1.0 ± 0 2.0 ± 0 0 1.5 ± 0.5 2.0 ± 0 0 0 (10 mg/mL) 0.5
Tmax (min) 5.2 ± 2.6 2.0 ± 1.1 0 6.5 ± 2.0 3.2 ± 3.9 0 0 1.0 ± 0 Dogs 6/6 6/6 0/6 6/6 6/6 0/6 1/6 6/6 H5 affected 2.5 ± (375 Score 2.2 ± 0.8 2.2 ± 0.4 0 2.5 ± 0.5 2.0 ± 0 0 1 mg/mL) 0.8 Tmax (min) 6.3 ± 4.5 2.0 ± 2.4 0 10.7 ± 5.8 1.0 ± 0 0 15 1.0 ± 0
Table D2 Continued Palpebral Palpebral Palpebral Bulbar Bulbar Bulbar Conjunctival Ocular
chemosis hyperemia follicles chemosis hyperemia follicles discharge pruritus Dogs 6/6 6/6 0/6 6/6 6/6 0/6 0/6 6/6 H6 affected 2.8 ± (500 Score 2.7 ± 1.0 2.0 ± 0 0 2.3 ± 0.8 2.0 ± 0 0 0 mg/mL) 0.4 Tmax (min) 9.2 ± 5.4 1.0 ± 0 0 11.2 ± 15.0 1.0 ± 0 0 0 1.0 ± 0
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APPENDIX E. PREPARATION OF SCHIRMER STRIPS FOR LC-MS ANALYSIS
1) Transfer the Schirmer strip (wetted until 20-mm mark) to a 2-mL tubea using single-use tweezersb 2) Spike 5 µL of 10 ng/µL prednisone internal standardc (10 ng/µL prepared in 1:1 acetonitrile:water) on the dry portion of the strip (i.e. 25 to 35 mm strip end) 3) Place the Schirmer strip into a 0.2-mL tubed that was pre-punctured at its bottom with a 18- gauge needle 4) Secure the 0.2-mL tube into a 2-mL tube using adhesive tape 5) Centrifugee the combination for 2 minutes at 3884 x g to extract tear fluid out of the Schirmer strip into the 2-mL tube 6) Transfer the centrifuged strip to another 2-mL tube and cut it into multiple <5mm pieces 7) Add 600 µL of methyl tert-butyl ether (MTBE) into the tube containing the cut Schirmer strip 8) Grind for 1 min with a handheld pestlef 9) Store in +4℃ fridge for 2 hours 10) Ultrasonic agitationg for 30 min 11) Centrifugeh for 1 min at 3824 g 12) Transfer the fluid to a new 2-mL tube, leaving the cut/shredded Schirmer strip behind 13) Dry solvent with nitrogeni for 6-8 min at 5-8 psi 14) Add 75 µL of 25% acetonitrile 15) Vortexj for 1 min 16) Centrifugeh for 1 min at 3824 g 17) Transfer the fluid to the 2-mL tube containing the centrifuged tear sample (step #5) 18) Vortexj for 30 sec 19) Centrifugeh for 30 sec at 3824 g 20) Transfer into LC-MS vialk containing a glass insert and a snap cap 21) Centrifugel at 1074 x g for 10 min 22) Transfer the sample from the glass insert to a 2-mL tube 23) Add 320 µL of iced cold 100% acetonitrile 24) Centrifugeh for 10 min at 10621 g 215
25) Transfer to a new 2-mL tube, leaving the precipitated proteins behind 26) Dry solvent with nitrogeni for 10-15 min 27) Add 100 µL of 25% acetonitrile 28) Vortexj for 5 seconds 29) Transfer the sample to a new glass insert 30) Centrifugel at 998 g for 15 min
*** Steps 23-31 were added after the initial samples were deemed inappropriate for LC-MS analysis. The proteins contained in the centrifuged tear sample were clogging the LC columns, so further precipitation in acetonitrile was required. *** a. 2 mL cryogenic vial, Fisherbrand, Fisher Scientific, Pittsburgh, PA, USA b. Evident® disposable plastic tweezer, Evident, LLC, Union Hall, VA, USA c. Prednisone-d7, Toronto Research Chemicals Inc., North York, Canada d. Eppendorf® safe-lock micro test tubes, Eppendorf North America Inc., Westbury, NY, USA e. VWR® mini centrifuge, VWR International, Mississauga, Ontario, Canada f. Argos® pestle mixer and pestle, Argos Technologies Inc., Elgin, IL, USA g. Bransonic® ultrasonic cleaner, Branson Ultrasonics Corporation, Danbury, CT, USA h. Eppendorf® Centrifuge 5417C, Eppendorf North America, Inc., Hamburg, Germany i. Biotage® nitrogen evaporator, Biotage, LLC, Charlotte, NC, USA j. LP vortex mixer, Thermo scientific Inc., Waltham, MA, USA k. Xpertek® 2-mL12mm*32mm snap seal vial, P.J. Cobert Associates, Inc., St. Louis, MI, USA l. Sorvall ST40 centrifuge, Thermo scientific Inc., Waltham, MA, USA