VALIDATION OF TONOMETERS AND EVALUATION OF AQUEOUS HUMOR FLOW IN THE NONHUMAN

PRIMATE

By

Faith M. McAllister

THESIS

In partial satisfaction of the requirements for the degree of

MASTERS of SCIENCE

in

PHYSIOLOGICAL OPTICS

Presented to the Graduate Faculty of the

College of Optometry

University of Houston

May 2019

Approved:

Nimesh B. Patel O.D. Ph.D.

Lisa Ostrin O.D. Ph.D.

VijayKrishna Raghunathan Ph.D.

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Dedication

To my dearest family, thank you for your unconditional love and support throughout these many years of my education. Your unwavering support since day one has meant the world to me.

To my cherished friends, thank you for your friendship, for your constant encouragement, and for inspiring me to surmount the challenges of graduate school and of life. I could not have done any of this without Lauren Roverse, Maranda McGonigle, Liz Bell, Anna Khoja, Lauren Tatlock, Kayci Perez, and

Lauren Davis.

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Acknowledgments

First and foremost, I must express my profound gratitude for my advisor, Dr. Nimesh Patel. For his continued patience, wisdom, and support in my graduate studies, I am thankful. It was Dr. Patel who inspired me to pursue a Master’s degree my very first year in Optometry School. His patience, brilliance, and care for his patients continued to inspire me and renewed my passion for this research every day.

He has taught me so much more than how to just do research, I became a better scientist, clinician and doctor because of his guidance. I couldn’t have asked for a better mentor than Dr. Patel.

Thank you to Dr. Harwerth. The opportunities I’ve had, and things I’ve been able to accomplish are merely by standing on the shoulders of Giants. Dr. Harwerth, he is truly one of those giants in this field.

I’m so thankful for the wisdom and mentorship that he’s shared with me.

Thank you to my fellow lab members, including Laura Pardon and Kwame Antwi-Boasiako for their valuable assistance and advice in my research studies.

I would like to extend my thanks to my thesis committee members, Dr. VijayKrishna Raghunathan and

Dr. Lisa Ostrin for their astute comments and sharing their experience and expertise in this area of research.

I would like to thank my editor, Zy Mazza, for his work and guidance. His patient feedback was instrumental in articulating many of the more complex concepts I engaged with over the course of this study.

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Finally, I would like to express my gratitude to the faculty and staff at the University of Houston College of Optometry for giving me the opportunity to pursue both my Doctorate and Masters degrees at this established institution. I will forever be grateful for the exceptional education I have received here.

Funding: T35-EY07088, K23-EY21761, P30-EY007551, R01-EY001139

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Abstract

PURPOSE: Intraocular pressure (IOP) is a major risk factor for glaucoma, and the only therapeutic intervention for clinical care. While glaucoma has an elusive cause, much about disease pathophysiology has been established using animal models of ocular hypertension. There is a gap in the literature on validation of IOP measuring tools and aqueous dynamics in experimental models. The main emphasis of this thesis is on; 1) validating non-invasive clinical measures of IOP, and 2) determining the role of homeostasis for maintaining IOP in healthy Non-Human Primate eyes.

METHODS: For these experiments, each eye was cannulated with a 27G butterfly needle, which was attached to a pressure transducer and syringe pump system. In the first experiment, 17 eyes, 11 normal and 6 with experimental intervention, of 12 rhesus macaque monkeys were used. IOP was adjusted between 10 and 60 mmHg in 5 mmHg steps, and tonometry measures obtained using the Tono-Pen and

TonoVet systems at pressures 10-50 mmHg. For the second experiment, 5 healthy monkeys were used.

To assess response to transient, but sustained elevated pressure, the fluid needed to maintain a pressure of 25 mmHg and 40 mmHg was established for a duration of 2hrs.

RESULTS: Experiment 1: TonoVet measures overestimated IOP at all pressure settings (mean difference of 3.17 mmHg, 95% CI 12.53 to −4.74 normal, 3.90 mmHg, 95% CI 12.90 to −6.53 experimental glaucoma). In contrast, Tono-Pen measures overestimated IOP at lower IOPs and underestimated at higher IOP (slope = −0.26 normal, −0.21 experimental glaucoma). Experiment 2: The total inflow, rate of inflow, and variability was greater at 40 mmHg (total fluid median=2.49 mL, range = 0.80- 5.8mL), (rate of fluid inflow median=13.2µl/min, range 4.26 – 42.88 µl/min) compared to 25 mmHg (total fluid

vi median=1.5mL, range = 0.44- 1.85mL), (rate of increase of fluid inflow median=5.4 µl/min, range 1.8 –

9.6 µl/min).

CONCLUSIONS: In the rhesus macaque, the Tono-Pen and TonoVet generally reflect IOP, but for accurate measures, both instruments require individualized calibration. Outflow of the healthy NHP eye responds rapidly to changes in IOP, but with significant inter-individual variability. These findings are important for future work in understanding the impact of IOP and topical medications in experimental models.

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Table of Contents

Dedication ...... iii Acknowledgments...... iv Abstract ...... vi List of Figures ...... x List of Tables ...... xi 1Chapter 1. Introduction ...... 1 1.1 Introduction to glaucoma...... 1 1.1.1 Prevalence ...... 1 1.1.2 Risk factors ...... 1 1.2 Aqueous Humor ...... 2 1.2.1 Aqueous role ...... 2 1.2.2 Anatomy ...... 2 1.2.3 Aqueous formation ...... 3 1.2.4 Aqueous outflow ...... 3 1.2.5 Outflow resistance ...... 7 1.3 IOP ...... 8 1.3.1 Normal distribution ...... 8 1.3.2 IOP calculations ...... 8 1.4 Tonometry ...... 9 1.4.1 History of tonometry ...... 9 1.4.2 Applanation tonometry ...... 10 1.4.3 Indentation tonometry ...... 11 1.4.4 Rebound Tonometry ...... 11 1.4.5 Non-contact tonometry ...... 12 1.4.6 Digital Palpation ...... 12 1.4.7 Dynamic Contour Tonometry and Pneumatonometry...... 13 1.5 Glaucoma Models ...... 14 1.6 Use of NHP as an experimental model for glaucoma ...... 14

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1.7 General scope and question ...... 15 2 Chapter 2. Assessing the True Intraocular Pressure in the Non-human Primate ...... 17 2.1 Abstract ...... 18 2.2 Introduction...... 19 2.3 Methods ...... 20 2.4 Results ...... 24 2.5 Discussion ...... 34 3 Chapter 3 Outflow Response to Elevated IOP in the Non-human primate ...... 37 3.1 Abstract ...... 38 3.2 Introduction...... 39 3.3 Methods ...... 40 3.4 Results ...... 45 3.5 Discussion ...... 54 3.6 Conclusions...... 57 4 General discussion ...... 58 4.1 Main apparatus ...... 58 4.2 Summary of chapters ...... 59 4.3 Tonometry Validation ...... 59 4.4 Aqueous humor evaluation ...... 60 4.5 Future Directions ...... 61 4.6 Conclusions...... 61 References ...... 63

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List of Figures

Figure 1. Trabecular meshwok illustration...... 6 Figure 2. (A) Experimental apparatus calibration ...... 23 Figure 3. (A&B) TonoVet vs Intracameral pressure, (C&D) Modified Bland Altman of TonoVet vs Intracameral pressure...... 29 Figure 4. (A&B) Tono-Pet vs Intracameral pressure, (C&D) Modified Bland Altman of Tono-Pet vs Intracameral pressure...... 31 Figure 5. (A&B) Tono-Pen vs TonoVet IOP, (C&D) Bland-Altman for Tono-Pen vs TonoVet IOP . 33 Figure 6. Experimental Apparatus ...... 43 Figure 7. Anterior canulation of Primate eye ...... 44 Figure 8. (A) Rate of BSS inflow for each eye (B) Mean and standard eror of BSS inflow...... 47 Figure 9. Total fluid inflow for maximum (NHP13) and minimum (NHP14) response to sustained moderate pressure challenge...... 50 Figure 10. Total fluid inflow for maximum (NHP13) and minimum (NHP14) response to sustained mild pressure challenge...... 51 Figure 11. Rate of inflow fluid for maximum (NHP13) and minimum (NHP14) response to sustained moderate pressure challenge...... 52 Figure 12. Rate of inflow fluid for maximum (NHP13) and minimum (NHP14) response to sustained mild pressure challenge...... 53

x

List of Tables

Table 1. Baseline Optical biometry ...... 25 Table 2. Average and repeatability of Tono-Pen and TonoVet ...... 27 Table 3. Rate of inflow fluid to maintain IOP ...... 46 Table 4. Total Normalized fluid and rate of increase in fluid flow for moderate and mild pressure challenge...... 49

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1Chapter 1. Introduction

1.1 Introduction to glaucoma

1.1.1 Prevalence

Glaucoma is a group of progressive optic neuropathies that results in retinal ganglion cell death and optic nerve head (ONH) damage. It is the number one cause of irreversible blindness and the second leading cause of all blindness, following cataracts.1 In general, glaucoma can be categorized as either open or closed angle, based on the iris-trabecular meshwork configuration. Of the various forms, primary open angle (POAG) is the most prevalent. Based on meta-analysis, the prevalence of POAG in the United States is estimated at 1.86% in individuals over 40. Hence, it is estimated that over 2.2 million

Americans have POAG, and around 200,000 of these patients are legally blind from the condition. With an aging population, the prevalence of glaucoma is expected to increase, and by the year 2020, 3.4 million individuals in the United States are expected to have the disease. Because vision loss in glaucoma is irreversible, efforts to reduce the magnitude of vision loss in glaucoma has focused on early diagnosis and treatment to slow ganglion cell loss. 1

1.1.2 Risk factors

Because the etiology of glaucoma is not well understood, risk factors are important for disease determination and detection in the early and mild stages of glaucoma. 2 Risk factors for glaucoma include, advancing age,3, 4 Intraocular pressure (IOP),5 thin central corneas,6 racial background,7 family history,8 and cup to disc ratio at the ONH. Of these, age and IOP are considered major factors, with IOP being the only modifiable risk factor.9 In fact, pharmacological or surgical reduction of IOP is known to decrease the rate of progression in all forms of glaucoma. 10 Hence, IOP measures serve as an excellent

1 indicator of the therapeutic effect of medications which aids in clinical decision of therapeutic treatment.

To better understand disease etiology, and structural and functional changes in glaucoma, animal models are often used. The main objective of this thesis was to validate intraocular pressure measurements in the NHP, and to assess how the healthy eye responds to changes in pressure. The pertinent aspects of aqueous humor, intraocular pressure, and animal models are discussed. As a clinician, I have also included a brief overview of the extant non-surgical clinical IOP-reducing therapies.

1.2 Aqueous Humor

1.2.1 Aqueous role

Aqueous humor itself plays a key role in the structure and function of the eye. IOP from aqueous humor supports structural integrity of the globe, without which hypotony occurs. Aqueous is an optically clear liquid and functions to supply nutrients and remove metabolic wastes from to the lens and cornea that must remain avascular to promote optical clarity.11 POAG is often associated with a decrease in outflow facility causing elevated pressure.12

1.2.2 Anatomy

The ciliary process is considered the functional unit of aqueous humor production. These processes extend from the pars plicata region of the ciliary body. They are lined internally with a bilayer of epithelium joined by tight junctions laterally and gap junctions apically. Rich vasculature of the ciliary body extends into these processes surrounded by stroma and the double epithelial layer. The inner layer of epithelium is non-pigmented with adjacent tight junctions creating one limit of the blood aqueous border. The outer layer is pigmented and is connected via gap junctions to the non-pigmented ciliary epithelium (NPCE). A plentiful number of mitochondria and Na, K-ATPase, ion channels reside in both

2 layers of the epithelium.13 This high level of metabolic organelles supports that these cells are an active site of aqueous humor production. The large number of ciliary processes along the ciliary body provide a large surface area for production and secretion of aqueous humor.11, 14

1.2.3 Aqueous formation

There are three mechanisms by which aqueous humor is formed: simple diffusion, ultrafiltration, and active secretion. Diffusion occurs as a passive movement of ions through a membrane down the concentration gradient. Ultrafiltration results as a pressure-dependent movement of fluid based on the difference of hydrostatic pressure in capillaries and IOP. Active secretion occurs in the ciliary epithelium bilayer and accounts for most of the aqueous humor production that does not fluctuate in response to IOP. Mitochondria-produced energy drives ion pumps in the NPCE moving solutes including sodium against concentration gradients into the posterior chamber followed by water.

Movement of water is facilitated by aquaporins in the NPCE cells.11, 15 Sodium potassium activated adenosine triphosphatase, carbonic anhydrase, and solutes including sodium, chloride, and bicarbonate all play a large role in active secretion, however, there are also other factors involved that are less understood.11 In humans, cumulative aqueous production is 2.5-3.0 µl/min measured with fluorophotometry and aqueous turnover in the anterior chamber is about 1- 1.5% per minute.11,

15 Aqueous humor formation is typically lower at night, and aqueous humor production decreases slightly with age.16

1.2.4 Aqueous outflow

The majority of aqueous outflow occurs through two pathways: the trabecular pathway and the alternate uveoscleral pathway. The classic or trabecular pathway is pressure dependent, while the uveoscleral pathway, is pressure independent.17 While most studies suggest that the trabecular pathway

3 is the major route for outflow a recent studies shows that uveoscleral outflow may account for up to

55% of drainage in some individuals.18

Aqueous humor follows the trabecular outflow pathway through the porous, triangular shaped trabecular meshwork in the angle of the anterior chamber. The trabecular meshwork consists of three regions. The uveal part extends in bands from the iris root and ciliary body to the peripheral cornea to form the lateral border of the anterior chamber. The middle and most extensive layer of the TM consists of the corneal scleral portions; these portions consist of perforated sheets of trabecula reaching from the scleral spur to the lateral wall of the scleral sulcus and become progressively smaller as they near

Schlemm’s canal. The innermost layer of the TM, the juxtacanalicular tissue, lies adjacent to the inner wall of Schlemm's canal. This region is composed of cells embedded in a dense extracellular matrix. (Fig

1.) The major site of outflow resistance is thought to be found in the outermost juxtacanalicular region of the TM. The lumen of Schlemm’s canal drains into an array of collector channels and then communicates with episcleral venous plexus to drain aqueous humor.11, 14 Aqueous humor flows into the episcleral venous system down the pressure gradient created by the difference in IOP and episcleral venous pressure (Pv). Episcleral venous pressure, thus plays a key role in IOP along with aqueous formation rate (F), and aqueous outflow facility (C); this is represented in the modified Goldmann equation (Po=F/C+Pv) where Po = IOP. Posture changes may cause short term changes in IOP due to increase in EVP; chronic alterations in blood pressure may also have an impact on IOP.19, 20

Uveal scleral flow occurs independent of pressure and is thought to account for up to 5-15% of drainage, which decreases with age. 18 Aqueous exits the anterior chamber and enters into the ciliary muscle through the interstitial spaces between the longitudinal ciliary muscle bundles then moves into the supraciliary and suprachoroidal spaces.21 Theories for the movement of aqueous after the supraciliary and suprachoroidal spaces vary and likely include multiple routes. These theories include the 4 uveoscleral pathway, in which aqueous is absorbed by the orbital vascular after passing through the sclera and episcleral,22-24 and the uveovortex pathway in which aqueous is osmotically absorbed by the choroid and passed into vortex veins. 25, 26

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Figure 1. Trabecular meshwok illustration.

This image illustrates the three regions of the TM

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1.2.5 Outflow resistance

Outflow resistance (R) is defined as ratio of change in pressure, IOP (Po) – episcleral venous pressure (Pv), to flow rate (F) i.e. R = (Po-Pv)/F. Outflow facility (C) then, is inverse of resistance. i.e. C =

F/(Po-Pv). This value is assessed by measuring the rate at which IOP declines in response to a mechanical force. 15, 18, 27 In order to measure outflow facility a Schiotz tonometer with a known weight is placed on the cornea. This provides both a mechanical increase in pressure and means to measure IOP as it returns to homeostatic levels. This pseudofacility measurement is based on the decline in IOP. This measurement can then, over time be used to calculate outflow facility. There are several concerns with the accuracy of this method of determining outflow facility including needed assumptions for calculations and multiple sources of measurement error. Assumptions include elastic properties of the eye and stability of aqueous formation and ocular blood flow volume. Measurement errors may arise from factors including calibration and eye movements. Eye movements and forced blinking are shown to create IOP spikes up to 14 mmHg which can confound IOP measurement. 28, 29

Because of this a better method to more directly measure outflow facility is needed.

Pressure dependent outflow occurs through Schlemm’s canal, and variation in resistance provided by size and pressure can impact IOP. Other factors associated with increase outflow resistance and aging include: increased pigment granules, decrease in phagocytic activity of trabecular cells, and an increase in extracellular matrix are all sources of increased resistance that are common in patients with POAG.

Episcleral venous pressure indirectly impacts aqueous outflow as shown in the Goldmann equation.

Episcleral venous pressure can be altered by blood pressure, postural changes, and vessel changes.

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1.3 IOP

1.3.1 Normal distribution

IOP is generated and maintained by the balance of aqueous production and outflow resistance.

IOP data from large epidemiological studies are demonstrate non-Gaussian data, with its distribution skewed toward higher pressures. Despite this, average (15.5 ± 2.6 mmHg) and standard deviation (SD) values are used to determine clinical standards for ‘normal’ and ‘elevated’ IOP. 95% confidence intervals include IOP from 12 to 22 mmHg forming the clinically recognized ‘normal range’.6, 30 However, this statistical average of IOP does not always correlate to nonpathological IOP levels and pathological IOP levels are different for each person. ‘Normal’ IOP is much better defined as IOP levels that do not cause ganglion cell loss. Many factors play a role in generating and maintaining IOP; These are summarized by the Goldmann equation: PO=F/C+Pv

1.3.2 IOP calculations

The Goldmann equation demonstrates the relationship of aqueous formation rate (F), aqueous outflow facility (C), and episcleral venous pressures (Pv). Hence, the three major factors that play a role in IOP are aqueous humor formation, aqueous humor outflow, and episcleral venous pressure, all of which are complex dynamic aspects. The Goldmann equation also illustrates methods by which IOP can be altered. Specifically, most therapeutic glaucoma drugs (beta blockers, carbonic anhydrase inhibitors, and alpha agonists) reduce aqueous formation. Some drugs, such as the prostaglandin analogs, increase uveoscleral outflow.10, 31 The most recent advance in therapeutics are drugs which increase aqueous outflow from the conventional pathway, such as Vyzulta™ and Rhopressa® .32, 33

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1.4 Tonometry

1.4.1 History of tonometry

Increased ocular rigidity and its association with blindness has been noted for many centuries.

At-Tabari, an Arabian surgeon in the 10th century, and Sams-ad-Din of Cairo from the 14th century noted rigidness of globe in individuals whose vision was not improved with cataract surgery. Richard

Bannister noted in his book in 1622 that he observed chronic glaucoma in association with elevated

IOP.34 Platner of Leipzig also noted this association in the mid-1700s. The early 19th century association between increased tension in the eye and blindness was frequently noted. 35, 36

Tonometry is the primary method by which assessments of intraocular pressure can be made in a clinical setting. Today there exists a variety of tonometry methods, and each have their own merits and shortcomings. Depending on patient factors, a clinician may have good reason to prefer one tonometry method over another. While all tonometry methods aim to assess IOP, current tonometers do not measure IOP directly. Direct measures can only be obtained through manometry which involves invasive techniques not suitable for clinical measurements or for research testing. Being able to accurately measure IOP is critical for management of patient care as well as developing further research investigating the role of IOP in glaucoma.

In 1863, von Graefe developed the first instrument to measure IOP using a weight-loaded plunger that measured indentation of the sclera.37 In the late 19th century Maklakoff developed an applanation tonometer showing relatively reliable and accurate measurements, especially for the time.

This tonometer was used across most of Eastern Europe. In the beginning of the 20th century, Schiötz developed an indentation tonometer used throughout the world.38 Additionally, Schiötz tonometers were used to develop “normal” ranges of IOP for statistical purposes. In 1888 Fick designed a novel tonometer with a unique approach to measuring pressure; he used a constant area of flattening varied

9 by the force applied. Baillart, Giroux, and Müller built upon this idea to create tonometers. The Imbert-

Fick principle would later play a key role as the principal that the Goldmann tonometer was based on.

This tonometer remains the “gold standard” for tonometry and serves as a comparison for new methods of tonometry developed. 36

1.4.2 Applanation tonometry

Applanation tonometry is based on the Imbert-Fick principle, which states that the pressure inside an ideal dry, thin-walled sphere equals the force necessary to flatten its surface divided by the area of flattening (P = F/A, where P = pressure, F = force and A = area). In order to compensate for the fact that the cornea is neither dry, thin-walled, nor a perfect sphere, Goldmann tonometry probes have an external diameter of 3.06mm to equal a surface area of 7.35mm2 At this diameter, the resistance of the cornea to flattening is counterbalanced by the capillary attraction of the tear film meniscus for the tonometer head. The IOP (in mmHg) equals the flattening force (in grams) multiplied by 10. 39 While variations in corneal biomechanics may influence readings, Goldmann tonometry remains the “gold standard” and is widely used in glaucoma care for both identification of ocular hypertension and monitoring pharmaceutical efficacy.40, 41 While it has a high degree of accuracy, Goldmann tonometry presents certain disadvantages, including the need for anesthetic and fluorescence dye. Patient cooperation and ability to hold eyelids opens are other potentially disadvantageous factors of this method. This presents a need for new tonometers that minimize these disadvantages. A tonometer that could quickly, easily, and accurately assess IOP without need for anesthetic or other drop instillation that is not affected by corneal biomechanics is ideal and what many modern tonometers aim for today.

To provide more background to support the need of this work, four of the most common tonometry methods have been surveyed here. They are: (1) indentation tonometry, (2) rebound tonometry, (3) non-contact tonometry, and (4) ocular palpitation, also known as digital tonometry. The

10 relative advantages, disadvantages, and clinical applications of these methods are examined below, beginning with indentation tonometry.

1.4.3 Indentation tonometry

Indentation tonometry determines IOP by evaluating the force needed to indent the cornea to a desired amount. The Schiotz tonometer applies varying weights to the cornea evaluating the degree of corneal indentation. This tonometer was also used for determining average ranges of IOP which are still used today.6, 42 However, this tonometer is time consuming, difficult to maneuver and uncomfortable for patients. Newer innovation of indentation tonometry includes the Tono-Pen which uses the Mackay-

Marg principal to determine IOP. The Mackay-Marg principal assumes that applanation tonometry measures both IOP and force needed to indent the cornea. In order to isolate IOP a central plunger surrounded by a footplate measures rising record of force by a strain gauge as it is applied to the cornea.

At applanation, the force is shared by the foot plate and the plunger resulting in a momentary small decrease from the steadily increasing force. This is the point of applanation which is read electronically.

43, 44 Total corneal contact area of Tono-Pen footplate is about the same diameter as a Goldmann tonometry probe. Tono-Pen is commonly employed in animal models and veterinary use as it is very portable and does not require a specific orientation to be used. 45

1.4.4 Rebound Tonometry

In contrast to indentation tonometry, rebound tonometry does not use applanation techniques and provides better patient comfort during measurement as seen in the TonoVet.39 The TonoVet is an induction/impact tonometer in which a small probe interacting with a solenoid coil rebounds off the cornea. A 1.8 mm diameter plastic ball on a stainless-steel wire is held in place by an electromagnetic field. The deceleration rate of the probe caused by corneal contact is recorded and used to determine

IOP. Advantages of rebound tonometry include ease, convenience, portability. TonoVet and its clinical

11 counterpart Icare require no anesthetic, have a much smaller contact point, and do not require a skilled clinician to use. However, instruments must be held appropriately to insure the probe lies perpendicular to the corneal surface and patients must be able to sit upright. These types of tonometers show great potential for tonometry but are relatively new. Limitations of these include potential for corneal thickness impacting measurements and overall accuracy of this tonometer. 46-48

1.4.5 Non-contact tonometry

While rebound tonometry aims to minimize corneal contact, non-contact tonometry, clinically referred to as the “air puff test,” eliminates corneal contact by using air as an application force. Non- contact tonometry, developed in the 1970s allows IOP measurements to be taken without out corneal contact thus eliminating the need for anesthetic to be instilled along with decreasing the risk of infection.39, 47 These tonometers applanate the corneal with a rapid pulse of air. Resistance to this air pulse is detected via an electro-optical system that determines the force at moment of applanation, then used to calculate IOP. Similar to rebound tonometers, non-contact tonometry poses great potential for convenience, however reliability and accuracy are lower compared to other tonometers. For this reason, it is generally only used as a screening tool. Other limitations include needed space for the instrument and lack of portability.

1.4.6 Digital Palpation

All previously mentioned tonometers require equipment of some kind and a certain degree of patient compliance for measurement. When these requirements cannot be met (for instance, when examining a young child), simple ocular palpation can be used. Ocular palpation, or digital tonometry, is the most basic form of assessing IOP in which a clinician uses their fingertips to palpate the globe over the eyelid. While this method first illuminated the connection between elevated pressure and vision loss, it is only used in highly uncooperative patients, such as small children, to rule out extremely

12 elevated IOP. This method’s benefits are that it can be done without any equipment; it is supremely portable; and it is easy. However, even with extremely experienced clinician’s accuracy, it is insufficient to appropriately evaluate and monitor IOP.

1.4.7 Dynamic Contour Tonometry and Pneumatonometry.

Other, less commonly used tonometry instruments include dynamic contour tonometry and pneumotonometry. Dynamic contour tonometry uses contour matching to evaluate IOP. The curved piezoresistive pressure sensor measures IOP. Because this method does not alter the shape of the cornea, it minimizes the effect of variation of corneal thickness and other corneal biomechanical properties on measurement; however, it can be influenced by corneal curvature.36, 49 Pneumatonometry determines IOP by balancing the flow of air, pumped via a piston through a small fenestrated membrane against the cornea, and the resistance to that flow from IOP and the cornea. This pressure affects the movement of the piston and is used to calculate the intraocular pressure. Both tonometers measurements require corneal contact for 5 to 10 seconds. This decreases comfort and ease of use, but provides important data on the pulsatile fluctuation of IOP. While both tonometers provide specific benefits, neither of these methods provide improved accuracy or ease of use compared to other tonometers, and thus they are not commonly used in clinical practice or for research models.

Having examined these methods of tonometry, a clear gap emerges in the literature.

Specifically, while these instruments have been developed primarily for human use, their validation in animal models is not fully investigated. Furthermore, some instruments such as the Goldmann tonometer, which is ‘gold standard’ in the clinical setting, cannot be used in research because the assumptions are violated for eyes with different corneal thickness and curvature measures. In this work, we sought to validate critical clinical tonometry assumptions against true IOP as measured by manometric cannulation system developed for this study.

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1.5 Glaucoma Models

While a lot is learnt through cross sectional and longitudinal studies in patients with glaucoma, these are usually limited to non-invasive diagnostic procedures, with only a small group of studies investigating aqueous and tissue properties. Much on the pathophysiology of glaucoma has been established using in vitro and animal glaucoma models. Glaucoma animal models include a variety of animal species, which are for the majority all dependent on elevated IOP. The methods for inducing ocular hypertension include scaring of the Schlemm’s canal, lasering of the trabecular meshwork, use of microbeads to clog the trabecular meshwork, and disruption of the episcleral vessels. 50-54

The NHP is an important glaucoma model because it provides critical information not available in vitro. It provides invaluable experimental data with minimized variables due to its anatomy, which is similar to that of human beings with respect to the underlying processes of glaucoma pathology.

Posterior changes observed with OCT of the ONH follow similar patters seen in human glaucoma in ocular hypertensive NHP models, serving as excellent models for glaucoma. Furthermore, it helps to identify new risk factors in addition to serving as a test model for new pharmacotherapies.

1.6 Use of NHP as an experimental model for glaucoma

Experimental evaluation of both IOP and aqueous humor dynamics require good experimental models to accurately assess instrumentation and pathophysiology. Monkeys serve as an excellent model for studies of ocular pathology due to similarities in ocular anatomy and visual function.50, 55, 56

NHP are good models for glaucoma as progression of the disease is similar between NHPs and humans.

Experimental glaucoma in the NHP uses artificially elevated IOP models to create glaucomatous damage to the optic nerve head. Elevated IOP must be induced in NHP glaucoma models as NHPs rarely demonstrate spontaneous glaucoma, and observations of neuropathy at normal levels, as seen with

14 normal tension glaucoma in humans, is not observed. NHPs can only be genetically manipulated through tissue-specific, viral vectors, thus make poor genetic models.

Methods of increasing IOP by decreasing outflow through the TM include intracameral injection of microspheres, gels, and enzymes. The most common form of inducing elevated pressure is by scarring of the TM by contiguous argon laser burns. Photocoagulation remains the most predictable form of elevating IOP but repeat treatment of the TM is often required. This is an effective way of increasing

IOP, but limitations exist in concerns with stability of elevated IOP as well as inability to allow investigation of the TM as a dynamic organ that can self-regulate IOP through changes in outflow; thus, evaluation of normal homeostatic response before laser application is important. 57

1.7 General scope and question

IOP plays a crucial role in the diagnosis and management of clinical glaucoma and in research models to further the understanding of IOP and ganglion cell death. Because of this, it is important to have precise and accurate measurements with easily accessible instruments. To better understand the factors that play a role in mediating IOP, it is important to understand how the healthy eye responds to elevated IOPs.

Currently, the relationship between tonometers and intraocular pressure in the in vivo NHP model is unknown, especially at pressures outside normal limits. Additionally, there exists no framework to define the relationship between rate of outflow and pressure increases in this model 58. After identifying these gaps in the relevant literature, we hypothesized that tonometry and pressure have a positive relationship, but may have variability based upon differences in current literature. 46, 58, 59 The first experiment indicated this hypothesis was most likely true because we observed a positive correlation between tonometers with increasing variability at higher IOPs. We further hypothesized a positive correlation between rate of outflow and pressure increase in the NHP model based off of

15 current literature supporting pressure induced outflow mechanisms in the trabecular meshwork.57, 60 In the second set of experiments, we show that the rate of aqueous outflow increased exponentially with

IOP while showing significant inter animal variability, with these differences highlighted at higher pressures and at longer duration of pressure.

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2 Chapter 2. Assessing the True Intraocular Pressure in the Non-human Primate

Contributing Authors

McAllister, Faith, BA; Harwerth, Ronald, OD, PhD, FAAO; Patel, Nimesh, OD, PhD, FAAO

University of Houston College of Optometry, 4901 Calhoun Rd, Houston, TX, USA

* This chapter was published in Optometry and Vision Science

Permission for use of this paper has been granted for purposes of this Thesis

February 2018 - Volume 95 - Issue 2 - p 113–119

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2.1 Abstract

INTRODUCTION: For glaucoma patients, high intraocular pressure (IOP) is a risk factor for progressive neuropathy. Similarly, animal models used to study the disease are based on an experimental elevation of IOP. Thus, accurate IOP measurements are important in characterizing experimental models and resulting effects.

PURPOSE: The purpose of the present study was to investigate IOP measurements in a NHP model of experimental glaucoma by comparing clinical tonometry (Tono-Pen and TonoVet) to the true IOP from intracameral manometry.

METHODS: A total of 17 rhesus macaque eyes from 12 animals were used for this study. Eleven eyes had no previous experimental intervention, whereas six eyes were at varying stages of laser-induced experimental glaucoma. IOPs were adjusted by inserting a needle in the anterior chamber that was attached to a pressure transducer and syringe pump system. The anterior chamber IOP was adjusted to values between 10 and 50 mmHg and corresponding measures with Tono-Pen and TonoVet were taken.

RESULTS: The IOPs by TonoVet and Tono-Pen were linearly related over the range of pressures tested

(slope = 0.68 normal/healthy and 0.72 experimental glaucoma). For the most part, TonoVet measures overestimated IOP at all anterior chamber pressure settings (mean difference of 3.17 mmHg, 95% CI

12.53 to −4.74 normal and 3.90 mmHg, 95% CI 12.90 to −6.53 experimental glaucoma). In contrast,

Tono-Pen measures overestimated IOP at lower IOPs and underestimated at higher IOP (slope = −0.26 normal and −0.21 experimental glaucoma). CONCLUSIONS: The TonoVet and Tono-Pen tonometers that are often used to assess IOP in both clinical and experimental settings generally reflect the status of IOP, but the results from this study suggest that the instruments need calibration with true anterior chamber pressure for accurate measures in experimental models of glaucoma.

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2.2 Introduction

Glaucoma is a group of optic neuropathies that can lead to irreversible blindness. Although the pathophysiology of retinal ganglion cell loss is not fully understood, intraocular pressure (IOP) is a major risk factor and clinical trials have demonstrated that lowering the IOP is an effective treatment.61-63 This mechanistic relationship is also the basis for most animal models of experimental glaucoma, which are generally created by procedures that decrease aqueous outflow and subsequently elevate IOP.

Because the ocular anatomy and visual function of monkeys are similar to those of humans, the NHP is an excellent model for studies of ocular pathology. Experimental glaucoma in the NHP is often induced by scarring of the trabecular meshwork by contiguous argon laser burns.56, 64, 65 Subsequently, the status of experimental glaucoma (i.e. structure and function) is typically assessed as a function of cumulative

IOP over post-treatment time to quantify pressure insult.55, 66 It is, therefore, important to have accurate assessments of IOP, which typically involves tonometry, often using standard clinical/veterinary instruments.

The clinical standard for IOP quantification is Goldmann tonometry, which builds on the Imbert–Fick principal. In human eyes with normal corneal thickness (500–570 μm), a 3.06-mm-diameter applanation zone is shown to be ideal as a 0.1 gm force corresponds to 1 mmHg. However, IOP quantified using

Goldmann tonometry is dependent on corneal biomechanics,41, 67, 68 corneal thickness,69, 70 and corneal curvature.71, 72 Because the NHP cornea is significantly steeper and thinner than that of humans,

Goldmann-type tonometers are not ideal. Hence, most laboratories investigating experimental glaucoma use either a Tono-Pen XL (Reichert, Inc., Depew, NY, USA) or TonoVet (Icare, Vantaa, Finland) to monitor IOP.

The Tono-Pen XL is an applanation/indentation tonometer that works on the Mackay–Marg principal. While the sleeve of the tonometer is similar in width to that of the Goldmann applanation

19 zone (~3.0 mm), the central plunger that is connected to the strain gauge is significantly smaller. The

TonoVet is an induction/impact tonometer in which a small probe rebounds off the cornea. The deceleration rate of the probe caused by corneal contact is recorded and used to determine IOP.73, 74 In theory, IOP measures from both instruments should be repeatable, accurate, and precise in both healthy and disease eyes. The purpose of this study was to investigate IOP measurements in a NHP model of experimental glaucoma by comparing standard tonometry to intracameral manometry. Some of the results of these studies have been presented in abstract form (McAllister F, et al. OVS 2016;93:E- abstract 160077).

2.3 Methods

Animal Subjects: Data were collected from a total of 17 eyes of 12 rhesus macaques. Eleven eyes had no previous experimental intervention, whereas six eyes had laser-induced experimental glaucoma. Of the normal control eyes, six were used to determine intra-session test–retest variability.

Experimental and animal care procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Houston. The use of animals for this experiment adhered to the

National Institutes of Health guidelines for the care and use of laboratory animals, and to the

Association for Research in Vision and Ophthalmology statement for the use of animals in ophthalmic and vision research.

Animal Preparation: Animals were anesthetized with an intramuscular injection of ketamine (20–25 mg/kg) and xylazine (0.8–0.9 mg/kg) and treated with a subcutaneous injection of atropine sulfate (0.04 mg/kg). Throughout the experiment, body temperature was monitored and maintained using a thermal blanket (TC1000 temperature controller; CWE, Ardmore), while heart rate and pulse was monitored with a pulse oximeter (model 9847; Nonin, Plymouth, MN).

20

Laser-Induced Ocular Hypertension: Application of argon laser energy to the trabecular meshwork to induce experimental glaucoma has been described previously.56, 64, 65 Briefly, for the initial laser procedure, performed through a one-mirror gonioscopy lens (Ocular Kaufman; Ocular Instruments,

Bellevue, WA, USA), contiguous laser burns (1.0 W laser power, 0.5 s duration, 50 μm spot size) were applied over 270 degrees of the drainage angle. If a sustained increase in IOP was not achieved by 4 weeks after the initial laser session, additional laser treatments over 180 degrees of the drainage angle were applied until sustained elevated IOPs were achieved. For the six experimental glaucoma eyes used in this study, the mean IOP from the first sustained elevated pressure measured using Tono-Pen was

32.6 mmHg (range 25.3–36.5 mmHg). Cannulation studies on these eyes were done on average at day

576 (range 454–693 days) from the first sustained elevated pressure.

Ocular Biometry: An ocular biometer (Lenstar LS900) was used to measure axial length, anterior chamber depth, lens thickness, anterior corneal curvature, and central corneal thickness in all eyes. The biometry data were used to assess the associated relationship with tonometry error.

Anterior Chamber IOP Control: To prevent infection, 5% ophthalmic betadine (Alcon

Laboratories, Fort Worth, TX) was applied to the eyelids, instilled on the ocular surface, and subsequently washed off with sterile balanced salt solution (BSS; Alcon Laboratories) after a period of 2 minutes. The head of the animal was stabilized using mouth and occipital bars, and a sterile speculum was inserted to keep the eyelids open. The anterior chamber was then accessed using a 27G butterfly needle, inserted approximately 1 mm from the temporal limbus and extending up to 2 mm into the anterior chamber. The needle was connected with sterile microtube filled with balanced salt solution, to a pressure control system that included a capacitive pressure transmitter (Keller PR-41X; Keller America,

Newport News, VA) coupled with a syringe pump (Cole-Parmer, Vernon Hills, IL) that was controlled through a MATLAB (The Mathworks, Natick, MA) program. For each of the cannulation experiments, the 21 pressure in the anterior chamber was adjusted from 10 to 50 mmHg. After changing the pressure setting, eyes were allowed to equilibrate for at least 5 minutes before IOP was measured. Throughout the experiment, the ocular surface was kept hydrated with sterile balanced salt solution. To assess repeatability, the IOP in six normal control eyes was increased from 10 to 50 mmHg in 10 mmHg steps, and at each pressure measure, three measures from both Tono-Pen and TonoVet were acquired.

Because TonoVet does not significantly indent the cornea, measures from this instrument were obtained before Tono-Pen with an interval of at least 2 minutes between measures. For the six eyes with repeated measures, all TonoVet measures were acquired prior to Tono-Pen, allowing for at least a

2-minute interval between all measures. For five eyes of normal controls, and six of the experimental glaucoma eyes, anterior chamber pressure was adjusted from 10 to 50 mmHg in 5 mmHg steps. For these eyes, only one Tono-Pen and TonoVet IOP was obtained at the highest reliability for each instrument (i.e. 5% std dev for Tono-Pen and 1.0 mmHg std dev for TonoVet). Following anterior chamber cannulation and completion of data collection, the needle was removed, and topical antibiotics

(polymyxin B/trimethoprim and moxifloxacin) were instilled on the eye.

IOP Control System: Calibration of the pressure control system was assessed before the start of experiments and after all data collection. The system was calibrated by setting up the pressure transducer, syringe pump, tubing, and needle as would be done during a cannulation experiment. The tubing connected to the needle was taped to a vertical meter ruler that was aligned with the pressure transducer. The pressure setting was adjusted from 10 to 60 mmHg, and the rise of fluid in the tubing was measured in centimeters of water, and subsequently converted to millimeter of mercury (1.0 cmH2O = 0.736 mmHg). Overall, there was excellent agreement between the rise of the water column and the pressure setting (Fig. 2A), with 95% confidence limits of about 0.3 mmHg for the manometer– transducer relationship (Fig. 2B). 22

Figure 2. (A) Experimental apparatus calibration

Correspondence between pressure setting and rise of water column converted to mmHg and comparison to the 1:1 line (B) Bland-Altman plot for the agreement between pressure setting and water column rise, illustrating the bias and 95% limits of agreement.

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Statistical Analysis: All of the tonometry measurements were acquired by one investigator (NBP).

Measurement error and repeatability was assessed for six healthy eyes. Linear regression was used to determine the relationship IOP measures from the two tonometers. To determine the accuracy of the

Tono-Pen and TonoVet, the differences between IOP and pressure transducer were plot against the pressure setting/pressure transducer reading for each animal. To evaluate if there were differences in calibration with disease, data were separated for normal control and experimental glaucoma eyes.

2.4 Results

Data were collected from 11 normal control and 6 experimental glaucoma eyes. Of the 11 normal controls, repeatability was assessed in 6 eyes. All animals maintained good systemic health and no ocular complications from cannulation and IOP quantification were noted during the experiment or at follow-up evaluation of each animal.

Table 1 illustrates the baseline biometry data for normal and experimental NHP glaucoma eyes. The only significant difference between the two groups was the longer axial lengths in experimental glaucoma eyes (P = .02).

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Table 1. Baseline Optical biometry Optical biometry measures for normal (n=11) and experimental glaucoma (n=6) eyes. Statistically significant differences (p<0.05) between the two groups are noted by an*

Normal control Experimental Glaucoma

Axial Length (mm) 18.9±0.67 19.9±0.75 *

Corneal Curvature (D) 53.8±2.31 52.8±2.24

Central Corneal 461.7±22.85 490.5±34.34 Thickness (μm)

Anterior Chamber 3.33±0.28 3.07±0.23 Depth (mm)

Lens Thickness (mm) 3.39±0.07 3.42±0.20

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Table 2. illustrates the repeatability (2.77 Sw) from these six eyes, for pressure settings from 10 to 50 mmHg. In general, there is an increase in test–retest error with increasing IOP for both tonometers. The measurement errors of both tonometers were assessed using the estimated within- subject standard deviation (Sw) from repeated measures in only six normal eyes.

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Table 2. Average and repeatability of Tono-Pen and TonoVet

Average and repeatability of IOP measures for Tono-Pen and TonoVet tonometers at increasing pressure levels (n=6 eyes).

Pressure TONO-PEN TONOVET Setting

(mmHg) Average IOP Repeatability Average IOP Repeatability

(mmHg) (2.77 × Sw, mmHg) (mmHg) (2.77 × Sw, mmHg)

10 10.8 1.67 12.8 1.13 20 18.2 2.83 22.1 1.56 30 25.8 3.48 33.3 2.66 40 33.6 2.51 44.3 3.75 50 40.7 4.82 54.4 2.69

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When compared to the IOP’s as set by the transducer system, the IOPs measured by the

TonoVet were on average 3–4 mmHg higher across all pressure settings (Fig. 3A&B), but the slopes of the linear regressions were near unity (difference from slope of 1, P = .45 normal, and P = .86 glaucoma) with high correlation coefficients. Consequently, the analyses of agreement between the transducer pressure and the TonoVet measurement illustrated small biases, but relatively large limits of agreement

(Fig. 3C&D). In addition, there was no difference in the mean bias and 95% confidence interval for healthy normal (mean difference = 3.17 mmHg, 95% confidence interval 12.53 to −4.74 mmHg) or eyes with experimental glaucoma (mean difference = 3.90 mmHg, 95% confidence interval 12.90 to −6.53 mmHg). It is important to note that the standard deviation of errors was greater at higher pressure settings (2.3 vs. 4.1 mmHg in normal, P = .3 and 1.7 vs. 4.1 mmHg, P = .13 in experimental glaucoma eyes at 10 and 50 mmHg, respectively), but did not reach statistical significance. These findings are consistent with the increase in measurement error with increase in IOP (Table 2).

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Figure 3. (A&B) TonoVet vs Intracameral pressure, (C&D) Modified Bland Altman of TonoVet vs Intracameral pressure. (A & B) The relationship between TonoVet IOP and intracameral pressure in normal healthy and experimental glaucoma eyes. (C & D) Difference in tonometer IOP measures from that of the pressure transducer. Healthy eyes are represented by circles and experimental glaucoma with triangles. Each animal is represented by a unique color.

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In contrast, when IOPs from the Tono-Pen were compared to the pressures set by the transducer system, there were systematic errors as a function of pressure. For lower pressure settings, the Tono-Pen overestimated IOPs while underestimating IOPs at higher pressures (Fig. 4A, B). The IOP relationship was linear (R F3 2 > 0.9) and although the slopes from linear regression were slightly different (−0.26 for normal eyes and −.21 in eyes with experimental glaucoma), the difference was not significant (P = .45). These relationships are also illustrated by the analyses of agreement between the transducer pressure and the Tono-Pen measurements (Fig. 4C, D), which also demonstrated relatively large limits of agreement. Similar to measures from the TonoVet, the standard deviation of error of

Tono-Pen measures increased at higher pressure settings (2.6 vs. 2.8 mmHg in normal, P = .86 and 1.8 vs. 4.6 mmHg, P = .04 in experimental glaucoma eyes at 10 and 50 mmHg, respectively).

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Figure 4. (A&B) Tono-Pet vs Intracameral pressure, (C&D) Modified Bland Altman of Tono-Pet vs Intracameral pressure. (A & B) The relationship between Tono-Pen IOP and intracameral pressure in normal healthy and experimental glaucoma eyes. (C & D) Difference in tonometer IOP measures from that of the pressure transducer. Healthy eyes are represented by circles and experimental glaucoma with triangles. Each animal is represented by a unique color.

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IOP measures from the Tono-Pen and TonoVet were linearly related for both normal and experimental glaucoma eyes (Fig. 5). Although there was good correspondence between Tono-Pen and

TonoVet IOP measures, for both normal eyes (slope = 0.68, R2 = 0.90) and eyes with glaucoma (slope =

0.72, R2 = 0.88), the slope of the best fit linear functions for IOP measures from both instruments were significantly different from unity (P < .01). Although the function was slightly steeper for eyes with glaucoma, the slopes were not statistically different (P = .46). However, the limits of agreement between the two tonometers were as relatively large and, although the width of the 95% confidence interval band was greater for experimental glaucoma (16.3 vs. 13.8 mmHg), the difference was within measurement error.

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Figure 5. (A&B) Tono-Pen vs TonoVet IOP, (C&D) Bland-Altman for Tono-Pen vs TonoVet IOP

Scatter plots illustrating the comparison of Tono-Pen and TonoVet IOP measures in normal (A) and experimental glaucoma (B) eyes for all anterior chamber pressure settings. Bland-Altman plot for the agreement between the two tonometers in normal(C) and experimental glaucoma (D) eyes, illustrating the bias and 95% limits of agreement. Each animal is represented by a unique color.

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Multiple regression analysis was used to test if the IOP error was related to the IOP setting and ocular biometry measures. The analysis showed that the difference of measured versus anterior chamber IOP was significantly (P < .05) related to the IOP setting and central corneal thickness for Tono-

Pen (F = 25.63, P < .01) and central corneal thickness for TonoVet (F = 10.93, P < .01). However, independently, the relationship between central corneal thickness and the mean difference for TonoVet measures was not statistically significant (P = .08). However, the two animals with the largest difference seen on Fig. 2A (pink circle) and 2B (green square) also had the thickest corneas. As Tono-Pen error was related to anterior chamber IOP, the slopes for error measures for individual animals was compared to central corneal thickness, and no significant relationship was found (P = .99).

2.5 Discussion

The main finding from this study was that in rhesus macaques, although IOP measures from Tono-

Pen and TonoVet instruments typically were different, they were highly correlated. However, neither instrument had good accuracy when compared to the anterior chamber pressure settings. The precision of both tools for repeated measures on a single subject was better for lower IOPs and decreased with higher IOPs, but more remarkable are the larger inter-subject limits of agreement for both tonometers.

Overall, the findings from this study support animal-specific instrument calibration for experimental glaucoma studies.

For average anterior chamber IOP levels in healthy eyes (<20 mmHg), the error in Tono-Pen measures was smaller, However, IOP was overestimated for pressures less than ~15 mmHg and underestimated for higher pressure settings. This finding is similar to that reported in the cynomolgus monkey where the pressure crossing was between 17 and 25 mmHg. 59 The data in Fig. 4 also illustrate the variability in true versus measured IOP. For example, the error in IOP measured at an intracameral pressure of 30 mmHg can range from 2 to −6 mmHg. While these differences might seem small as a ratio

34 or percentage of the true pressure, they exponentially add when computing the pressure–time insult to the nerve.

In contrast to the Tono-Pen, IOP error from rebound tonometry was not related to the anterior chamber IOP setting. However, there was greater variability in the measured error between subjects.

Specifically, although the TonoVet accurately measured IOP for several animals, there was one subject for which the error exceeded 10 mmHg for all pressure settings. In addition, the data show that the variability in error increases with pressure (Fig. 3). The results in this study are in contrast to a previous publication where excellent correspondence was reported, but was only tested in two animals. In a different species of NHP (Macaca fascicularis, n = 3), the TonoVet was shown to have good correspondence with manometry, but underestimated IOP by 1.72 ± 5.5 mmHg.

Central corneal thickness has been shown to be a factor influencing IOP measures using both the Tono-Pen and rebound tonometer in clinical populations.75, 76 In the present study, although central corneal thickness was identified on multiple regression analysis, it was not significantly related to IOP error on its own. However, it is noteworthy that the two animals with higher rebound tonometry error also were the animals with the thickest corneas. Overall, the lack of relationship might be a reflection on the number of subjects included in the study, and the strength of the relationship itself in the NHP.

The similarity in results from healthy and experimental glaucoma eyes would suggest that repeat calibration of tonometers on the same eye might not be necessary. It is possible that multiple repeat calibrations on the same eye might result in short-term IOP damage to the optic nerve head that might confound longitudinal studies. However, because there are connective tissue biomechanical changes of the cornea, it is important that longitudinal calibration studies are done.

There are several limitations to this study. While central corneal thickness was measured before inserting the needle, change in corneal thickness with IOP was not investigated. Inserting a needle at the 35 limbus has the potential of changing corneal biomechanics and, subsequently, the IOPs quantified by the instruments. Ideally, pressure would be controlled through cannulation of the vitreous chamber; however, this method carries added risk of complications that can confound findings in experimental glaucoma. In addition, all tonometry measurements were made by one user, and intra-user variability could not be assessed. Only one time point for all eyes was quantified, including the eyes with experimental glaucoma, and intersession variability was not investigated.

Based on the results of this study, tonometers used to monitor IOP in NHP experimental glaucoma should be calibrated to the individual eye. Although these clinical tools provide important methods to assess IOP, IOP is known to fluctuate during the day, and it is important that tools are developed to continuously and accurately monitor IOP in experimental models.29 The findings from this study can also be extended to clinical practice. While IOP measures from clinical tonometers relate well to intracameral pressure, there are significant inter-individual differences that prevent from using a single, simple formula to convert measured IOP to real IOP. However, monitoring change in IOP over time with or without treatment should follow similar trends in eyes with high or low error using Tono-

Pen or rebound tonometry.

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3 Chapter 3 Outflow Response to Elevated IOP in the Non-human primate

Contributing Authors

McAllister F1, Pardon L1, Harwerth R1, Patel N1.

1 University of Houston College of Optometry, 4901 Calhoun Rd, Houston, TX, USA

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3.1 Abstract

Introduction: Homeostatic regulation of Intraocular pressure (IOP) is essential for maintaining normal

IOP; lack of which can lead to elevated IOP. Hence, it is important to understand how the eye responds to changes in intraocular pressure. The purpose of this study was to determine the rate of fluid inflow needed to maintain short term IOP elevations in healthy NHP.

Methods: Anterior chamber pressure was adjusted through cannulation using a 27 gauge needle connected to a pressure control system. Experiment 1: In 10 animals with no previous intervention, IOPs were adjusted between 10 and 60 mmHg in 10 mmHg increments for a period of 10 minutes to determine acute changes in flow. Experiment 2: In 5 healthy animals without experimental intervention, anterior chamber pressure was maintained at 40 mmHg and 25 mmHg, each for 2 hours at separate sessions, to determine dynamics of fluid flow change,

Results: Experiment 1: Inflow rate needed to maintain IOP increased with anterior chamber pressure, and was best fit with an exponential fit = 6.2285e0.0279x µl/min/mmHg. Experiment 2: total inflow fluid, rate of fluid inflow, and variability was greater at 40 mmHg (median=2.49 mL, range = 0.80- 5.8mL),

(rate of fluid inflow median=13.2µl/min, range 4.26 – 42.88 µl/min) compared to 25 mmHg (total fluid median=1.5mL, range = 0.44- 1.85mL), (rate of increase of fluid inflow median=5.4 µl/min, range 1.8 –

9.6 µl/min).

Conclusion: This study shows that the healthy NHP eye responds rapidly to changes in IOP but with significant variability. This variability is highlighted at higher pressures and longer duration of elevated pressures, demonstrating the potential compounding impact of IOP homeostatic dysregulation.

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3.2 Introduction

IOP is the primary, and only modifiable risk factor for glaucoma, a disease which results in irreversible damage to retinal ganglion cells. While the pathophysiology of ganglion cell loss is not well understood, intraocular pressure (IOP) is the only modifiable risk factor known. 61-63 IOP is a function of aqueous production and outflow resistance, and the eye’s ability to maintain homeostasis. Aqueous is produced by the ciliary body and passes into the anterior chamber through the pupil, finally flowing into the episcleral venous system simultaneously through two pathways: the pressure-independent uveoscleral pathway, and pressure-dependent corneal scleral pathway through the trabecular meshwork (TM). Hence, steady-state IOP occurs at the point where there is sufficient aqueous to overcome outflow resistance, driving aqueous into Schlemm’s canal and the episcleral venous system.

Homeostatic regulation occurs through a feedback pathway in the pressure-sensitive TM that increases outflow in response to increasing IOP. Imbalances, such as an increase in production, decrease in outflow, or loss of homeostatic control, can result in elevated IOP.

While an increase in production can result in increased IOP, the most common cause of elevated

IOP is due to increased outflow resistance. In the healthy eye, outflow resistance is greatest in the juxtacanalicular region of the TM. Endothelial cells embedded in a ground substance produce a wide variety of macromolecules that help to regulate resistance.77, 78

There are several groups that have studied aqueous pathway flow in an enucleated eye over time. While these models have a lot of benefit, all aspects of homeostatic mechanisms cannot be evaluated due to enucleation. 79-81 Additionally, normal function of the eye, including blood flow, EOM contribution, and normal external forces do not apply. These models inform about changes that may occur to regain homeostasis including potential changes in blood flow and outflow resistance. In vivo models demonstrate potential for changes in areas of the TM in response to decreased outflow induced 39 from trabecular laser application.57, 82 Normal ability and response to elevated pressure is poorly understood. This study aims to investigate homeostatic variability in the in vivo NHP. These differences have the potential to identify higher risk eyes.

NHPs serve as an excellent model for studies of normal vision and ocular pathology due to similarities in ocular anatomy and visual function. This model has provided important insights into glaucoma pathophysiology and structure function relationships. While significant work has been done to evaluate the neural visual system and optic nerve physiology, there are significantly less studies which investigate the eye’s response to changes in anterior chamber pressure, in vivo. The purpose of this study was to investigate normal variability of acute, graded and sustained response to a non- physiological increase in anterior chamber pressure in the NHP.

3.3 Methods

Animal Subjects. For the first experiment, determining the fluid needed to maintain graded changes in pressure, data were collected from single eyes from 10 animals, with no previous experimental intervention. For the second experiment, determining the fluid needed to maintain a sustained pressure, data were collected from five animals with no previous experimental intervention. In conjunction with anterior segment pressure manipulation, OCT imaging was obtained at each pressure step to evaluate optic nerve head changes in response to IOP. OCT results will not be discussed in this chapter, and have been previously published. 83

Experimental and animal care procedures were reviewed and approved by the Institutional Animal

Care and Use Committee of the University of Houston. The use of animals for these experiments adhered to the National Institutes of Health guidelines for the care and use of laboratory animals, and to the Association for Research in Vision and Ophthalmology statement for the use of animals in ophthalmic and vision research. 40

Animal Preparation. Animals were anesthetized with an intramuscular injection of ketamine (20-

25 mg/kg) and xylazine (0.8-0.9 mg/kg) and treated with a subcutaneous injection of atropine sulfate

(0.04 mg/kg). Throughout the experiment, body temperature was monitored and maintained using a thermal blanket (TC1000 temperature controller, CWE, Ardmore), while heart rate and pulse was monitored with a pulse oximeter (model 9847; Nonin, Plymouth, MN).

Anterior Chamber IOP Control. To prevent infection, 5% ophthalmic betadine (Alcon

Laboratories, Fort Worth, TX) was applied to the eyelids, instilled on the ocular surface, and subsequently washed off with sterile balanced salt solution (BSS, Alcon Laboratories, Fort Worth, TX) after a period of two minutes. The head of the animal was stabilized using mouth and occipital bars, and a sterile speculum was inserted to keep the eyelids open. The anterior chamber was then accessed using a 27G butterfly needle, inserted approximately 1 mm from the temporal limbus and extending up to 2 mm into the anterior chamber. The needle was connected with sterile microtube filled with balanced salt solution, to a pressure control system that included a capacitive pressure transmitter

(Keller PR-41X, Keller America, Newport News, VA) coupled with a syringe pump (Cole-Parmer, Vernon

Hills, IL) that was controlled through a MATLAB program (The Mathworks, Natick, MA). Figure (6). For the first experiment, pressure in the anterior chamber was adjusted from 10 to 60 mmHg in 10 mmHg steps and maintained at each pressure for 10 minutes. For the second experiment, anterior chamber was cannulated at two different sessions, separated by at least two weeks. At one session anterior chamber pressure was set at 25 mmHg, while at the second set to 40 mmHg, for a period of 2 hours. Following the 2 hour period, to evaluate response to reduced pressure, pressure in the anterior chamber was adjusted to 10 mmHg for 2 hours. For both experiments, the total fluid needed to maintain anterior chamber pressure, was used to estimate fluid outflow. Throughout the experiment, the ocular surface was kept hydrated with a rigid gas permeable lens with diameter of 10.0mm, which 41 covered the entire corneal surface. Following anterior chamber cannulation and completion of data collection, the needle was removed, and topical antibiotics (polymyxin B/trimethoprim and moxifloxacin) were instilled on the eye.

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Figure 6. Experimental Apparatus

Experimental set up including cannulated eye, pressure sensor and syringe, all connected in a closed loop system filled with Sterile Balanced salt solution.

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Figure 7. Anterior canulation of Primate eye

This image illustrates anterior cannulation of a primate eye with 27 gauge butterfly needle. Following cannulation, a rigid gas permeable lens, as seen in this image, is placed on the eye to maintain corneal clarity and hydration.

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3.4 Results

All animals maintained good systemic health, and no ocular complications from cannulation were noted during the experiment or at follow up evaluation. In the first experiment outflow was estimated as being equivalent to the fluid inflow needed to maintain pressure. All animals demonstrated fluid displacement with an increase in total inflow fluid. In the first experiment, inflow rate needed to maintain IOP increased exponentially with anterior chamber pressure. In experiment 2 total inflow fluid, rate of fluid inflow, and variability was greater at 40 mmHg than at 25 mmHg. While there is variability between animals, data illustrated that there was no substantial leakage from the cannulation site, which was also visually monitored throughout the experiment.

In the first experiment, pressure was increased from 10 mmHg to 60 mmHg in 10-minute intervals.

Fluid total inflow at each pressure was used to determine rate of inflow at each pressure level. With graded increase in anterior chamber pressure, there was an increase in the rate of fluid inflow for all eyes (Table 3). The fluid needed to maintain anterior chamber pressure increased, without reaching asymptote, even after 10 minutes at very high pressures of 60 mmHg for all eyes. The total fluid displacement for the graded pressure experiments was 1.41 ± 0.284 ml (median = 1.46, range 1.064-

1.963ml). For each eye, the rate of increase was best fit with an exponential function (inflow = y + a x exp (b x IOP), Fig. 8A. Fig. 8A, B illustrates the mean and standard deviation of response; demonstrating significant variability in how animals respond to graded increases in anterior chamber pressure. Overall, the average increase in inflow rate was 4.8 + 1.2 x exp (0.05 x IOP) µl /min/mmHg. We noted a large variability in rate of fluid flow at 10 mmHg, with some animals having greater flow rates at this pressure than at higher pressure settings. This finding is likely an error on our experimental design, not allowing enough time for the eye to equilibrate following anterior chamber cannulation.

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Table 3. Rate of inflow fluid to maintain IOP

Rate of outflow required to maintain designated IOP for each animal and exponential fit of inflow across pressures for each animal.

Rate of fluid inflow (µl /min) Exponenti 10 mmHg 20 mmHg 30 mmHg 40 mmHg 50 mmHg 60 mmHg al fit, (b)

NHP 1 9.42 14.94 23.28 30.9 48.36 70.38 2280 NHP 2 6.9 6.18 11.64 17.82 27.66 42.54 2880 NHP 3 11.64 9.72 14.16 21 29.76 43.26 3000 NHP 4 15.6 7.74 11.94 16.32 20.64 27.48 3960 NHP 5 8.76 7.56 10.86 16.44 20.34 28.86 2520 NHP 6 31.62 6.06 7.8 9.9 14.94 28.2 5760 NHP 7 6.42 8.46 11.82 16.02 22.32 37.74 3420 NHP 8 19.26 7.92 11.7 15.9 22.26 33.42 2580 NHP 9 5.736 8.76 13.32 19.38 29.64 46.62 2760 NHP 10 17.04 6.18 8.34 12.36 18.3 27.42 4440

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A B

Figure 8. (A) Rate of BSS inflow for each eye (B) Mean and standard eror of BSS inflow

(A) shows increasing rate of BSS inflow (µl /min) for each eye represented by different markers at each pressure measurement. (B) shows increasing Mean and standard error for rate of BSS inflow (µl /min) at each pressure measure.

47

In the second experiment, IOP was sustained at 25 and 40 mmHg for a period of 2 hours followed by IOP reduction to 10 mmHg for an additional 2 hours. Homeostatic response was evaluated by total inflow fluid over time and by rate of inflow over time. For each animal, the fluid replacement volume increased exponentially as a function of time for both pressure settings (Fig 9, 10). For demonstrating the range, we have plotted two animals, the largest fluid inflow (NHP 13), and the lowest

(NHP 14), for both 25 mmHg and 40 mmHg pressures. Overall, less total fluid was replaced for all animals with pressure maintained at 25 mmHg (median=1.5 mL, range = 0.44- 1.85 mL) compared to 40 mmHg (median=2.49 mL, range = 0.80- 5.8 mL, Table 5A).

To quantify the rate of inflow increase, we calculated the inflow rate for 5 min intervals. For each animal, the rate of increase with higher pressure was linear in nature (Fig 11, 12). The rate of change shows less variety among homeostatic response to 25 mmHg (median=5.4 µl/min, range 1.8 –

9.6 µl/min) compared to 40 mmHg (median=13.2 µl/min, range 4.26 – 42.876 µl/min). The rate of flow as a function of time dropped and remained relatively stable when pressure was returned to 10 mmHg, returning to similar values as initial normalized flow rates for NHP 14, but remaining elevated for NHP

13. Overall, there was variability in homeostatic response to both pressures, and similar to experiment

1, was larger at the higher pressure setting. In general, the eyes ability to regulate pressure quickly observed in both short-term and sustained IOP.

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Table 4. Total Normalized fluid and rate of increase in fluid flow for moderate and mild pressure challenge. 4A and B shows Total fluid in ml displaced for all five animals and rate of outflow for pressure sustained at 40 mmHg (A) and 25 mmHg (B) then reduced to 10 mmHg. NHP 13 demonstrated the greatest total fluid displaced and the highest rate of fluid increase for both pressure settings of 40 and 25 mmHg. NHP14 demonstrated the lowest total fluid displaced and the lowest rate of fluid increase for both pressure settings of 40 and 25 mmHg.

Table 4A. Normalized total fluid (ml) Rate of increase in fluid flow

displaced at 40 mmHg (µl/min) during 40 mmHg 40 mmHg 10 mmHg 40 mmHg 10 mmHg NHP 11 1.723 0.508 11.322 -2.07 NHP 12 2.486 0.851 15.192 -1.578 NHP 13 5.896 1.995 42.876 -3.048 NHP 14 0.797 0.222 4.35 -1.776 NHP 15 5.65 1.658 49.842 -4.728

Table 4B. Normalized total fluid (ml) Rate of increase in fluid flow

displaced at 25 mmHg (µl/min) during 25 mmHg 25 mmHg 10 mmHg 25 mmHg 10 mmHg NHP 11 0.717 0.58 3.57 -1.24 NHP 12 1.609 0.785 7.596 -0.09 NHP 13 1.846 1.302 9.882 0.246 NHP 14 0.445 0.206 1.83 -0.198 NHP 15 1.517 0.979 9.402 0.114

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Figure 9. Total fluid inflow for maximum (NHP13) and minimum (NHP14) response to sustained moderate pressure challenge.

Total normalized outflow over time for the maximum (NHP13) and minimum (NHP14) response to sustained 40 mmHg followed by a 10 mmHg return. The animals with the highest (NHP13) and lowest (NHP14) change in outflow rate were the same for both experiments. This demonstrates a relative similarity in response to a mild or moderate pressure increase for a given individual animal.

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Figure 10. Total fluid inflow for maximum (NHP13) and minimum (NHP14) response to sustained mild pressure challenge.

Total normalized outflow over time for the maximum (NHP13) and minimum (NHP14) response to sustained 25 mmHg followed by a 10 mmHg return. The total normalized outflow over time for the maximum (NHP13) and minimum (NHP14) response to sustained 40 mmHg (A) and 25 mmHg (B) followed by a 10 mmHg return.

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Figure 11. Rate of inflow fluid for maximum (NHP13) and minimum (NHP14) response to sustained moderate pressure challenge.

Rate of normalized fluid displacement in five-minute intervals for maximum (NHP 13) and minimum (NHP 14) response with pressure maintained at 40 mmHg and a subsequent pressure setting of 10 mmHg.

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Figure 122. Rate of inflow fluid for maximum (NHP13) and minimum (NHP14) response to sustained mild pressure challenge.

Rate of normalized fluid displacement in five-minute intervals for maximum (NHP 13) and minimum (NHP 14) response with pressure maintained at 25 mmHg and subsequent pressure setting of 10 mmHg. These values are comparable for both which suggests a relative similarity in response to a mild and moderate pressure increase for any individual animal.

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3.5 Discussion

The main finding from this study was that in rhesus macaques, total inflow fluid and rate of fluid inflow increased exponentially as a function of anterior chamber pressure. This suggests that the TM responds rapidly, increasing outflow, to maintain homeostasis. Specifically, in the first experiment, involving acute changes in IOP, there was a corresponding acute increase in fluid flow. Further, in the second experiment, when elevated IOP was sustained, the TM responded to this non-physiologically high IOP by increasing outflow at an exponential rate, which did not asymptote over the 2 hour time period studied.

Both of these experiments show pliability and accommodation of outflow pathways in the normal eye, but with variability between subjects. In the first experiment, a continuous increase in flow occurred through the full duration past 1 hour and at 60 mmHg, well above homeostatic IOP for rhesus macaques. In the second experiment, an increase in both flow and rate of flow continued at 25 and 40 mmHg after 2 hours without reaching asymptote. Neither of these experiments were able to demonstrate the maximal outflow rate for the experimental animals suggesting a large accommodative capacity in pressure dependent outflow pathways.

In both experiments there was a wide variability between the animal’s responses; this illustrates a natural range of response to elevated IOP as well as a range in ability for maintaining homeostasis.

Differences between subjects were greater at higher pressures, suggesting that some animals are better able to accommodate pressure changes than others. Animals included in both studies showed a similar relative relationship; animals with a larger change in outflow in response to increasing elevated pressures also showed a larger response to 2-hour pressures of both 25 and 40 mmHg compared to other animals.

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As this experiment was performed within a short time frame and an asymptote was not reached, we can only make conclusions on short term IOP changes, and it is difficult to conclude what long term adaptations individual animals may make. Additionally, animals were not evaluated for test- retest variability in response to IOP insult. What this does suggest is that some animals are much better at regaining homeostatic IOP in response to short term IOP fluctuations. Because short term increases in IOP occur secondary to blinking, eye movements, and eye rubbing;84-88 the role of short term IOP response may play a larger role in long term elevations of IOP. Inability to lower IOP in response to short term elevations may lead to a compounding effect of an overall higher IOP. This is supported by observations of higher variations in circadian IOP fluctuations in individuals with POAG.89

Anatomical and physiological changes of the TM have been observed in both short-term and sustained IOP. Previous studies have shown significant changes in outflow pathway morphology in response to changing IOP.82, 90, 91 Changes include distention of juxtacanalicular tissue and endothelial cells, up to 50% have been observed in primate eyes in vivo with induced pressures of up to 30 mmHg. 92

Elevations in IOP also caused increases in the size and number of vacuoles.

Long term changes in unlasered portions of TM in response to elevated IOP in experimental primate models include softening and thinning of unlasered TM and collapse of Schlemm’s canal.

93 Pliability and softening of TM, along with this study’s observation of increasing accommodation and increase of inflow fluid with increasing IOP in healthy animals, informs on differences seen by thickening of trabecular lamellae and increases in ECM beneath the inner wall of Schlemm’s canal found in human patients with POAG and in normal aging changes with Rhesus Macaques 16. Thickening of trabecular lamellae and increased in ECM may be directly related to inability to accommodate elevated pressures and decease of homeostatic regulation in disease eyes.

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These studies suggest that in response to elevated IOP, both mechanical and cellular changes occur in normal eyes as a complex system of compensating mechanisms in both short term and long- term elevations. Our findings support that there is a wide range of variability among responses to elevated IOP in the normal eye, which results from a variety of changes occurring in pressure dependent outflow tissues.

These observations suggest homeostatic regulatory mechanisms present in outflow pathway tissues are capable of an adaptive response. Alterations in outflow resistance accommodate changes in

IOP in order to maintain physiologic IOP. The sources of these differences are poorly understood. This veritably may be influenced by other variations in TM anatomy and physiology.

This study has several limitations. Collection of data included quantification of necessary inflow fluid into the system to maintain IOP, not outflow itself. The assumption that the amount of inflow equals outflow is based upon the maintenance of a closed loop system through the anterior cannulation and observation for leaks in the system. Inflow fluid measured only accounts balanced salt solution from the experimental apparatus syringe. We cannot account for normal aqueous production and changes in production from xylazine, which is an alpha agonist, and is known to decrease production. Other changes from animal sedation including blood pressure and autonomic nervous system changes cannot be accounted for. However; changes in needed inflow and total fluid were much higher than normal aqueous production (2.6 2.5-3.0 µl/min.11, 15) and it is unlikely that other changes played a significant role in inflow fluid. Though all experiments a closed loop system was maintained. Additionally, only one cannulation experiment was performed on each animal with no re-test information.

Because outflow was not directly measured and subsequently outflow through the trabecular meshwork was not directly measured, it is possible that a partial amount of fluid was displaced through the uveoscleral pathway. Regardless of the pathway through which outflow occurred homeostatic 56 regulatory mechanisms were still observed. Pressure changes were administered after IOP returned to pre-cannulation to minimize the effects of corneal puncture during cannulation. All animals used in this study were young healthy animals thus, it would be beneficial to reassess these experiments in older primates as well to assess normal aging changes. It is still important to identify the normal variability in young healthy patients as well as risk factors for the disease as early diagnosis is key is glaucoma due to its irreversible progressive nature.

3.6 Conclusions

This study shows that the eye responds rapidly to changes in IOP, but there are differences in how eyes respond. These differences are highlighted at higher pressures and with longer duration of elevated pressure. Animals with larger outflow rate changes demonstrated a larger response at all pressure levels. Primate eyes with less ability to increase outflow may be more susceptible to increases in IOP. 15

Normal daily activities including blinking, eye movements, and eye rubbing have all been shown to create IOP elevations. 84-88 Animals who have a higher and quicker response to elevations in IOP will regain a homeostasis quicker. This may also decrease the compounding effect of spikes through the day and diurnal variations. It is possible that these differences can foreshadow eyes that are more or less susceptible to glaucoma. Current pharmaceutical treatments are focused on lowering IOP by inhibiting aqueous production and increasing outflow. The efficacy of these medications is highly variable from patient to patient. Through a better understanding of the underlying factors behind the variety of homeostatic mechanisms and outflow resistance mechanisms, future treatments could be targeted at decreasing outflow resistance directly. The results from this study will be important to investigate the influence of glaucoma drugs on IOP homeostatic mechanisms.

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4 General discussion

Glaucoma encompasses a group of optic neuropathies that remain a leading cause of irreversible blindness worldwide. Based on 2002 estimates, glaucoma affects over 2.2 million individuals in the United States and 37 million worldwide. Due in part to an aging population, the disease is estimated to impact twice as many by 2020, making it a significant health problem.1 While the disease is multifactorial, IOP is a major and only modifiable risk factor. In fact, while glaucoma detection and progression are often dependent on optic nerve head structure and visual function, therapeutic goals are always IOP based in all forms of glaucoma.

Much about glaucoma disease pathophysiology has been established using animal models. Of these, the NHP has the most similar ocular and brain anatomy, and visual response to that of humans.

While several veterinary instruments are available to quantify intraocular pressure in animals, there is scant data on the relationship between these non-invasive tools and true anterior chamber pressure.

Determining the precision and accuracy of these instruments is critical for improving quantification of individualized cumulative pressure insult in these models. Similarly, while much is known on the mechanisms on aqueous humor outflow, there is scant in vivo data. The most common technique study glaucoma in the NHP is ocular hypertension induced through laser scarring of the trabecular meshwork.

The two experiments in this thesis investigated the accuracy and precision of two commonly used tonometers and the response in outflow to non-physiological increases in IOP in the NHP.

4.1 Main apparatus

Our experiments were dependent on several important apparatus. The most important was the pressure control system. This closed loop system included a syringe pump, pressure transducer and a

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MATLAB algorithm. Calibration of this system included comparison with height of water column converted into mmHg and showed excellent correlation (Fig 2). Using this system, we could maintain anterior chamber pressure, and quantify the amount of fluid needed for stability. Second, our first experiment was dependent on clinical tonometers used in veterinary practice. We studied both the

Tono-Pen and TonoVet instruments. Through this process, I learnt how tonometers work, the biology of aqueous production and drainage, and how to apply statistics to the data collected. I will build on these skills as I venture into the next phase of my research career.

4.2 Summary of chapters

4.3 Tonometry Validation

The NHP experimental glaucoma model is a pressure dependent model. Hence, accurate measures of IOP are essential for determining the cumulative pressure insult to the eye. As in clinical practice, the most common methods used to quantify pressure include non-invasive measures using either the Tono-Pen or TonoVet. However, data on the accuracy and precision of these instruments were not available.

To determine the accuracy and precision of two commonly used tonometers, we used a closed loop pressure system to maintain anterior chamber pressure while tonometry was performed. In 17 animals IOP was raised in 10 mmHg steps from 10 to 50 mmHg with measurements taken at each step using the closed-loop anterior chamber cannulation system. Both Tono-Pen and Tonovet showed good precision at normal pressures under 30 mmHg but had increasing test-retest variability with increasing

IOP into pathologic levels. Tono-Pen and Tonovet measurements showed good correlation to each other but, when compared to the pressure sensor, Tonovet consistently overestimated IOP by an average of

3.17 mmHg in normal eyes and 3.90 in laser induce ocular hypertensive eyes across all IOPs. Tono-Pen

59 overestimated IOP at lower IOPs and underestimated IOP at higher IOPs. Overall, based on the variability seen, these findings indicate that instruments should be calibrated for each individual subject.

This work also suggests that there is room for improvement for the development of new tonometry instrumentation, or algorithms used to quantify pressure using existing systems.

4.4 Aqueous humor evaluation

Homeostatic regulation of outflow resistance is essential in maintaining a healthy IOP for the eye. For the most, the trabecular meshwork is the most active in this regulation. Using our pressure control system, the total inflow of fluid is recorded, and can be used to estimate the rate at which outflow mechanism adapt to pressure exposure. In chapter 2, two different series of experiments were performed to assess the impact of increases in IOP on outflow of the NHP eye. In the first experiment, acute changes were investigated, while the goal of the second experiment was to assess the rate at which outflow changes with sustained pressure.

In the first series of experiments outflow response increased with increasing IOP in all animals, but with large variability in response across animals. These differences between inter-animal responses are highlighted at higher pressures. This suggests that some eyes may be more prone to variation in pressure. In the second series of experiments, at two different sessions, separated by at least 2 weeks,

IOP was raised to and maintained at either 25 mmHg or 40 mmHg for 120 minutes; it was then lowered and maintained at 10 mmHg for 120 minutes. The total replacement fluid needed to maintain the experimental IOP was quantified. Replacement fluid flow rates were calculated in 5-minute intervals and used as an estimate of the outflow rate. As with the first arm of this study, the rate of fluid inflow had significant variability between animals. However, in all animals, the rate of fluid inflow increased with time suggesting that to maintain homeostasis there is rapid change in outflow. It was also notable that

60 animals with larger changes in outflow response with high IOPs, quickly dropped following IOP lowering to 10 mmHg but did not return to normalized baseline levels.

4.5 Future Directions

IOP in the NHP is typically measured when animals are sedated. However, it is well established that diurnal variation and daily variation of IOP are important risk factors for disease. Using calibrated tools, the future goals are to behaviorally train animals to obtain such measures. This would significantly improve how pressure insult to the eye is quantified, which is important for distinguishing animals that show greater or slower progression. The data from the experiments in chapter 2 also illustrate that there is a need to improve tonometry technology. With current technology, there is significant inter and intra subject variability. While our goal is not to develop new technologies, we will continue to test tonometers, invasive and non-invasive, as they become available.

Data from chapter 2 show there is significant variability between animals in how they accommodate to changes in anterior chamber pressure. Our next studies will be directed at repeatability of these results within and across animals. In addition, it will be important to establish histological and mechanobiological differences between animals. Furthermore, this model and apparatus can also be used to assess how the eye responds to new therapeutic interventions.

4.6 Conclusions

Intraocular pressure plays an important role in the diagnosis and management of glaucoma as the only modifiable risk factor of this blinding disease.94 It is important to have instruments that accurately and precisely measure IOP both clinically and in research. Animal models serve an important role in our understanding of glaucoma. To further our understanding, it is essential that these animal

61 models accurately represent clinical disease progression and that measurements taken are reliable and accurate. The series of studies presented in this dissertation illustrate the importance of proper calibration of two commonly used tonometers and the wide range of outflow response to non- physiological increases in intraocular pressure. The future directions are aimed at developing a better understanding of the role that IOP plays in glaucomatous optic neuropathy and improving glaucomatous animal models and wider understanding of normal and abnormal response to IOP and homeostatic regulation of IOP.

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