Physiology of the Tear Film and Adnexa

Special Thanks Physiology of the Tear Film and Acknowledgements and Adnexa (content and photos) 1. Dr. Mike Davidson 2016 William Magrane Basic Science Course 2. Contributions by in Veterinary & Comparative Ophthalmology ACVO/ECVO colleagues Elizabeth A. Giuliano, DVM, MS 3. Current/former members Diplomate, ACVO of the MU-CVM Comparative Ophthalmology service

Today’s Objectives Today’s Objectives 3) Summary charts – helpful for boards 1) Discuss the “lacrimal functional unit” with emphasis on innervation, normal 4) “The brain can only absorb what the #%$@ physiologic function, and a review of can withstand” pathologic mechanisms in dry eye Clinical cases – our passion What does this lecturer “bring to the table”? 2) Review mammalian eyelid anatomy CALT and PDT Encourage your creative juices

“Lacrimal Functional Unit (L.F.U.)” A complex functional unit which modulates the homeostasis of the ocular surface Lacrimal gland Tear film Ocular surface epith. Cornea, conjunctiva, meibomian glands Eyelids Interconnecting sensory and motor nerves

1 “Lacrimal Functional Unit (L.F.U.)” Control of Tear Secretion – New Concepts Traditional Normal tears: the result of intrinsic lacrimal gland activity; neural participation in reflex tears only New concept: tears under constant neural regulation On-going homeostatic regulation of the ocular surface Suggests a relatively constant level of neural signals that precisely meter tear production; may mediate lipid & mucin secretion also

Stern, Gao, Siemasko et al Experimental Eye Research, 2004

Control of Tear Secretion – New Concepts Lacrimal Gland Innervation: Afferent Pathway of Trigeminal Ganglion-Mediated Reflex Control mechanism includes Irritation of cornea/conjunctiva stimulates afferent afferent nerves from the cornea nerves: Impulses are carried along lacrimal nerve & other ocular surface tissues  the ophthalmic division of the trigeminal nerve central nervous system relay  sensory nuclei in trigeminal ganglion (TG) nuclei  efferent nerves Lacrimal nerve - smallest branch of the ophthalmic n. comprise the autonomic innervation to secretory tissues Courses laterally within the orbital cavity above and along the upper border of the lateral rectus muscle whose products contribute to Relevance? the tear film

Lacrimal Gland Innervation: Lacrimal Gland Innervation: Afferent Pathway of Trigeminal Ganglion-Mediated Reflex Afferent Pathway of Trigeminal Ganglion- Trigeminal ganglion is Mediated Reflex Cornea connected to lacrimal conjunctiva Lacrimal nerve Ophthalmic nerve stimulus nucleus of facial nerve in Trigeminal pons by internuncial ganglion neurons Lacrimal gland Maxillary nerve Impulses are directed, by Mandibular nerve Pons way of the trigeminal ganglion, to the lacrimal nucleus Lacrimal nucleus of the facial nerve in the pons Slide courtesy of Dr. Ota

2 Lacrimal Gland Lacrimal Gland – Innervation Additional Sensory Innervation Recommend Gia Klauss’s chapter on “non-hypotensive autonomic agents in The afferent innervation of the lacrimal gland is also veterinary ophthalmology” VCNA, Ocular therapeutics, 2004 provided by the ipsilateral superior vagal ganglion (SVG) and superior glossopharyngeal ganglion Autonomic innervation - sympathetic: (SGG) The sympathetic postganglionic fibers arise from the There may be SVG and SGG-mediated reflexes in cranial cervical sympathetic ganglion which is the addition to the TG-mediated reflex uppermost ganglion of the sympathetic trunk and travel in S.B Cheng et al. Three novel neural pathways to the the plexus of nerves around the internal carotid artery lacrimal glands of the cat. Brain Research 873 (2000) They join the maxillary nerve, the zygomatic nerve, the In humans, there is a connection between zygomaticotemporal nerve and finally the lacrimal nerve hypothalamus and lacrimal nucleus (emotional (a branch of the ophthalmic nerve) tears), and between olfactory system and lacrimal nucleus (“wasabe tears”)

Slide courtesy of Dr. Ota Lacrimal Gland – Innervation Recommend Gia Klauss’s chapter on “non-hypotensive autonomic agents in Sympathetic innervation veterinary ophthalmology” VCNA, Ocular therapeutics, 2004

Autonomic innervation - sympathetic: Lacrimal gland Lacrimal nerve Post-ganglion fibers join the maxillary nerve, the zygomatic nerve, vascular smooth Zygomatic Ophthalmic nerve the zygomaticotemporal nerve and finally the lacrimal nerve (a muscle branch of the ophthalmic nerve) nerve Acinar cells, Maxillary nerve T1 Distributed to interstitium surrounding acini and to vascular ductules and tubules smooth muscle fibers of glands Plexus of nerves around the internal Sympathetic nerves contain norepinephrine and neuropeptide Y carotid artery (NPY) Sympathetic stimulation increases tear secretion by affecting Cranial cervical ↑ protein secretion sympathetic ganglion vascular supply to lacrimal gland as well as activating a G ↑ blood flow protein pathway In some species, the sympathetic nervous system may influence tear secretion, not only by modulating blood flow to the gland and its distribution within it, but also by direct effects on the secretory acini.

Lacrimal Gland – Innervation Lacrimal Gland – Innervation Autonomic innervation - Autonomic innervation - parasympathetic parasympathetic The efferent postganglionic parasympathetic impulses Parasympathetic secromotor are then transmitted via sphenopalatine nerve to the nerve supply to lacrimal gland zygomatic nerve (a branch of the maxillary division of is derived from the lacrimal the trigeminal nerve). They then pass into zygomatic nucleus of the facial nerve temporal nerve which reaches the lacrimal gland The pre-ganglionic fibers reach The zygomatic temporal nerve also gives off a recurrent branch the pterygopalatine to the lacrimal nerve, from which the efferent fibers terminate in the lacrimal gland (sphenopalatine) ganglion via the great petrosal nerve and vidian nerves as they pass through the pterygoid canal

3 Lacrimal Gland – Innervation Lacrimal Gland – Innervation Autonomic innervation – parasympathetic Autonomic innervation – parasympathetic Postganglionic parasympathetic fibers innervate: Lacrimal gland secretion inhibited by Leu-Enkephaline (L-Enk) Acinar cells, duct cells, and blood vessels A neuropeptide that interacts with inhibitory G proteins Interferes with activation adenylate cyclase by G stimulatory Exert principal neural control of electrolyte, water, and proteins protein secretion Postganglionic parasympathetic fibers also innervate the Stimulatory effect mediated via acetylcholine and nasal glands via the caudal nasal nerve (from the vasoactive intestinal peptide (VIP) maxillary nerve of the trigeminal nerve) Increase in tear secretion through a G protein pathway and perhaps a calcium/calmodulin pathway

Lacrimal Gland – Innervation Parasympathetic innervation Autonomic innervation – parasympathetic Lacrimal nerve Ophthalmic nerve Postganglionic Zygomatic Trigeminal Lacrimal gland nerve ganglion parasympathetic fibers acinar cells, tubules Zygomatico Maxillary nerve ductules and temporal also innervate the nasal vascular wall nerve Mandibular nerve glands via the caudal Pons

nasal nerve (from the Electrolyte and maxillary nerve of the water secretion Pterygoid canal trigeminal nerve) Sphenopalatine Vidian n. Greater petrosal n. nerve Lacrimal nucleus Pterygopalatine CN VII (Sphenopalatine) ganglion

Post-ganglionic parasympathetic fibers Pre-ganglionic parasympathetic fibers

Slide courtesy of Dr. Ota

Additional References Parasympathetic innervation Lacrimal Gland Innervation Ding C, Walcott B, Keyser KT. Sympathetic neural control of the More than one parasympathetic ganglion is mouse lacrimal gland. Invest Ophthalmol vis Sci. 2003; 44: 1513- involved in the neural regulation of lacrimal gland 1520 secretion. Cheng S, Kuchiiwa S, Kuchiiwa T et al. Three novel pathways to the Ciliary ganglion (CG) lacrimal glands of the cat: an investigation with cholera toxin B Otic ganglion (OG) subunit as a retrograde tracer. Brain Res. 2003; 873:160-164

J. Anat. 199 (1996), Brain Res 873 (2000) Powell CC, Martin CL. Distribution of cholinergic and adrenergic nerve fibers in the lacrimal glands of dogs. Am JVet Res 1989; 50: Some anatomical studies suggest that small 2084-2088 neurons mediate the vasodilation of the lacrimal Text Books gland, while the large neurons mediate the Milder B. The lacrimal system lacrimal secretion Snell RS, Lemp MA. Clinical Anatomy of the eye Kaufman PL, Alm A. Adler’s Physiology of the eye Brain Res 873(2000), Brain Res 522 (1990) Krachmer JH, Mannis MJ, Holland EJ. Cornea

4 “Veterinary Neurology” by Oliver, Hoerlein, and Mayhew

“Lacrimal Functional Unit (L.F.U.)” Tear Film – Anatomy & Physiology the “traditional” teaching Normal tears essential: Lipid Most superficial layer 1. To prevent surface infection Stabilize & prevent evaporation of 2. Provide a pure optical surface for light refraction aqueous layer Produced by the meibomian glands 3. Maintenance of surface “homeostatic” environment Aqueous Intermediate layer Provides corneal nutrition; removes waste products Concept of L.F.U. – first introduced by Stern Produced by orbital gland AND et al Cornea, 1998 gland of the 3rd eyelid Describe the relationship between ocular surface Mucus and the lacrimal glands in normal tear secretion Interface of tear film with and during inflammation hydrophobic cornea Secretory IgA Composed of tear film, ocular surface epithelium, Produced by conjunctival goblet eyelids, interconnecting sensory and motor nerves cells

Tear Film – Functional Anatomy Continually Emerging Newer Techniques Traditionally, tear film has been described as having 3 layers with a total thickness of 7 -10 µm (Adlers, older, 10th edition – note, typo in this edition, µm not mm) In the last 15-20 years, evidence has called this earlier estimate into question (newer techniques, mathematical models); hundreds of papers! Prydal et al, IOVS, 1992 estimated tear film thickness of 35-40 µm, composed mainly of a gel containing mucins Danjo et al, Jpn J Ophthal, 1994 - 11 µm Ewen King-Smith et al, IOVS, 2000 disputes Prydal’s and Danjo’s earlier work and states a value of 3 µm for the thickness of human precorneal tear film Another review: 6-20 µm, J Cataract Refract Surg 2007

5 Average central T.F. thickness: 4.78 +/- 0.88 µm

6 Tear Film – Functional Anatomy Adler’s Physiology of the Eye, 11th ed (2011) thickness of the precorneal tear film in humans: 3.4 +/- 2.6 µm and composed of 4 layers (glycocalyx on corneal & conjunctival epithelia, mucous, aqueous, and lipid layers)

Whatever the true thickness of tear film, the structural rigidity of 3 discernible layers has changed with time Tear layers are considered to be more of a continuum with the lipid layer most anterior to the aqueous and mucin components

Tear Film Content and Thickness Analytical methods Why the discrepancy? Tears – attractive for sampling (accessibility, rich in content, largely acellular) Volume of minimally stimulated tears (i.e. environmental stimulation) ~7 µL, collection of > 2 µL at any time point results in reflex tearing  alters both volume and composition of tears Qualitative and quantitative techniques 1 and 2-dimentional polyacrylamide gel electrophoresis (PAGE) Isoelectric focusing (IEF) Crossed immunoelectrophoresis ELISA Size-exclusion high-pressure liquid chromatography (HPLC) Reversed phase and ion-exchange HPLC Matrix-assisted laser absorption/ionization (MALDI) mass spectrometry Surface-enhanced laser desorption-time of flight (SELDI-TOF) ProteinChip technology OCT – latest and greatest versions (e.g. ultrahigh resolution OCT) Hoang-Xuan et al, Inflammatory Diseases of the Conjunctiva, 2001

7 Tear Film Discrepancies in Vet. Med.? We are NOT immune to same issues/problems Non-invasive meibometry can be used in conscious dogs and has been described as a means to quantify meibomian gland secretions in this species, however results have been variable Ofri R, Orgad K, Kass PH, Dikstein S. 2007. Canine meibometry: establishing baseline values for meibomian gland secretions in dogs. Veterinary Journal, 174, 536-40. Benz P, Tichy A, Nell B. 2008. Review of the measuring precision of the new Meibometer MB550 through repeated measurements in dogs. Veterinary Ophthalmology, 11, 368- 74. “Repeated measurement results obtained by two examiners, with the new device Meibometer MB550 linked to a computer, showed a wide range of values. The measuring precision of the new Meibometer MB550 is therefore questionable.”

Tear Film – Functions

1) Protect cornea from desiccation and lubricate eyelids 2) Maintain refractive power of cornea by smoothing its surface for refraction of incoming rays 3) Protect against infections via specific and nonspecific antibacterial substances 4) Supply oxygen/nutrients to cornea and transport metabolic by-products from corneal surface 5) Avoid corneal dehydration due to hyperosmolarity 6) Remove foreign materials from the cornea and conjunctiva 7) Provide WBCs/other immune cells with access to cornea and conjunctiva

Tear Film – Lipid Layer Tear Film – Lipid Layer Meibomian glands Molecular weight of meibomian lipids (i.e., Meibomian glands meibum) is higher, and the polarity is lower, than Holocrine, modified sebaceous glands arranged that of sebum, thus meibomian lipids are fluid at lid linearly within the dense connective tissues (i.e., tarsal temperature plate) of the eyelid margin A recent model proposed that a combination of PTF proteins and lipids could interact and behave similarly to Secretions consist of wax monoesters, sterol esters, lung surfactant to provide a non-collapsible viscoelastic hydrocarbons, triglycerides, diglycerides, free sterols gel that would allow for proteins to remain in their lowest (i.e., cholesterol), free fatty acids, and polar lipids free energy states while in contact with lipids (Rantamaki (including phospholipids) (Levin et al., 2011) et al., 2011, Butovich, 2011)

8 Tear Film – Meibomian Glands Tear Film – Meibomian Glands These acini open into central ductules arranged at right Highly developed in the dog, angles to the eyelid margin, and they deliver lipid to the with 20 to 40 glands per eyelid surface of the eyelid through small openings just external typically being present (i.e., anterior) to the mucocutaneous junction Glands - located within the i “Gray line”- an important surgical landmark in a variety of tarsal plate, in which they blepharoplastic procedures (photo courtesy Dr. Heinrich) form linear aggregates of secretory acini that are usually visible through the semitransparent palpebral conjunctiva

Tear Film – Lipid Layer Tear Film – Lipid Layer Thickness varies throughout the Secretion influenced by several factors: day (maximum upon Mechanical (blinking reflex) awakening) and composition Nervous (as shown after trigeminal nerve sectioning) may differ between individuals, Hormonal (stimulatory action of androgens, estrogens inhibitory) age (children higher than adults) Physical (feedback regulation according to surface tension – S.T. Compression of the eyelids decreases when lipid spreads over surface) during normal blinking contribute to release of meibomian secretions, but precise neural and hormonal mechanisms regulating secretion of meibomian lipid are not well understood

Tear Film Break Up Time Tear Film – Aqueous Layer Video courtesy Shelby L. Reinstein, DVM, MS, DACVO Secreted by lacrimal glands of the orbit and nictitating membrane Aqueous tear component provides most of the avascular cornea’s metabolic needs by supplying glucose, electrolytes, oxygen, and water to superficial cornea Lubricates the cornea, conjunctiva, and nictitating membrane Removes metabolites such as carbon dioxide and lactic acid Flushes away particulate debris and bacteria from the ocular surface

9 Tear Film – Aqueous Layer Tear Film – Aqueous Layer The aqueous portion of the PTF is 98.2% water PTF contains proteinase inhibitors as well as and 1.8% solids (i.e., mostly proteins) proteinases - important in both ocular immunity and Consists of water, electrolytes, glucose, urea, surface- in the prevention of excessive degradation of normal active polymers, glycoproteins, and tear proteins healthy ocular tissues (de Souza et al., 2006) Examples of primary tear proteins include globulins (i.e., Total proteolytic activity in tears has been found to be secretory IgA, IgG, IgM), albumin, lysozyme, lactoferrin, significantly increased after corneal wounding lipocalin, epidermal growth factor, transforming growth Ulcerative keratitis in animals has been associated with initially factors, lacritin, and interleukens high levels of tear film proteolytic activity which decrease as Antibodies, immunoglobulins, lysozyme, lactoferrin, ulcers heal and proteinase levels in melting ulcers remain transferrin, ceruloplasmin, and glycoproteins all contribute to elevated leading to rapid progression of the ulcers (Ollivier et the antibacterial properties of tears al., 2007) Certain topical medications (e.g. EDTA) may reduce the gelatinase activity present in tears of normal dogs (Couture et al., 2006)

Tear Film – Aqueous Layer Tear Film – Aqueous Layer The lacrimal glands of the orbit and the nictitating “I’ve never seen one of these ductules from the nictitans….” membrane are tubuloacinar and histologically similar Ductules from these glands deliver aqueous tear secretions into the conjunctival fornices In dogs 3-5 ductules from the orbital lacrimal gland open into the dorsolateral conjunctival fornix, whereas the nictitans gland delivers aqueous tears onto the corneal surface through multiple ducts opening between lymphoid follicles on the posterocentral third eyelid In humans, pH varies 7.14-7.82, osmotic pressure of 305 mOsm/kg, refractive index of 1.357 pH in our patients?

Tear Film pH – What’s new in Vet Med? Tear Film – Aqueous Layer The relative contributions by each of the main lacrimal glands to reflex tear secretion have been investigated in the dog by surgical removal of either one or both glands and measurement of the resulting tear production (Helper, 1970, Helper, 1976, Saito et al., 2001, Helper et al., 1974) Tear volume produced by each gland varied considerably among animals The orbital lacrimal gland was the main source of aqueous tears in some dogs, whereas the nictitating membrane gland was the main source in others When either gland was removed singly, a compensatory increase in tear production appeared to occur in the remaining gland Removal of both glands resulted in near-total absence of secretions Suggests that accessory conjunctival glands may not be present in the dog, or that they play an inconsequential role in aqueous secretions

10 Tear Film – Aqueous Layer Tear Film – Aqueous Layer The role of each gland (i.e., orbital or nictitans glands) in the production of basal secretions versus reflex tear secretions has not Chemical mediators of lacrimal gland secretion are been determined cholinergic agonists, released from parasympathetic nerves, Destruction of lacrimal gland results in an estimated decrease and norepinephrine, released from sympathetic nerves, of 23-46% and nictitans gland results in 12-26% decrease located in both the cornea and conjunctiva (Dartt, 2004, Dartt, 2009, Tiffany, 2008) These neurotransmitters activate signal transduction pathways affecting the myoepithelial, acinar, and duct cells, and blood vessels of the lacrimal gland leading to secretion Other stimuli of lacrimal gland secretion include various proteins (i.e. EGF growth factor, neuropeptide Y, substance P, calcitonin gene-related peptide) and hormones (Dartt, 2004, Davidson and Kuonen, 2004, Lemp, 2008)

Harderian Gland Specialized lacrimal gland found in amphibians, reptiles, birds, and mammals 5 types recognized: serous, mucous, seromucoid, mixed, and lipid glands Typically, located on nasal side of orbit and its single duct empties on to bulbar surface of nictitans Contiguous with gland of 3rd eyelid except: Rabbits – below and medial to lacrimal gland Pigs – separate from 3rd eyelid gland Rodents (rat, hamster, gerbil) – posterior to globe and produces porphyrin  imparts a reddish brown color to tears and will fluoresce under ultraviolet light

Tear Film – Mucus Layer

Deepest tear film layer Adheres firmly to underlying epithelial cells Thickness ranges from 0.8 µm over cornea to 1.4 µm over conjunctiva Facilitates adherence of aqueous layer to surface of conjunctival and corneal epithelial cells

11 Tear Film – Mucus Layer Tear Film – Role in Ocular Immunity Mucin – composed of a heterogeneous group of Rich in lysozyme, betalysin, lactoferrin, and antibody hydrated O-linked oligosaccharides linked to protein (low levels of lysozyme in cattle tears - Gionfriddo et. al. Proteins synthesized in endoplasmic reticulum of AJVR, 2000) goblet cells Saccharide branches added in Golgi apparatus IgA & IgG secreted by lacrimal glands (rich in Glycoproteins are condensed and stored in plasma cells and lymphocytes) membrane-bound secretory granules at apical side Serum proteins of goblet cells Derived from vascular compartment by filtration Various compounds (secretagogues, serotonin, epinephrine, phenylephrine, dopamine) stimulate Represent 1% of total tear proteins in the absence of goblet cells to release mucin, as well as antigen, infection immune complexes, mechanical action, other Albumin, haptoglobin, IgG, IgA, IgM, IgE, α2- factors macroglobulins, complement-derived proteins, transferrin, Adler’s physiology of the eye (2011) – regulation of α1-antitrypsin, and β2-microglobulin goblet cell and mucin production Davidson HJ and Kuonen VJ. The tear film and ocular mucins. Vet Opthal 2004; 7 (2) 71-77

Immunopathogenesis of KCS in the dog Clinical Significance Further David L. Williams, VCNA, 2008 Keratoconjunctivitis Sicca Reading Diagnosis and treatment of cKCS – familiar “Abnormality in either the quantity or Mechanism by which inflammatory changes lead quality of any primary tear component may to reduced tear production? compromise tear function.” (C Moore, 1. Lymphocyte-associated cytotoxicity 1999) 2. Apoptosis of glandular epithelial cells 3. Cytokine release from inflammatory cells “Dry eye is a disorder of the tear film due to 4. Inflammatory cells/associated cytokines or tear deficiency or excessive tear autoantibodies may influence neurotransmitter evaporation.” (National Eye Institute, NIH). function in lacrimal gland  inhibits neurologic stimulation of tear secretion

Immunopathogenesis of KCS in the dog Want to learn about more about tear film Further David L. Williams, VCNA, 2008 Reading and practice your Italian? Federica Maggio DVM, DACVO One or more of the aforementioned proposed mechanisms may be involved in pathogenesis of Maggio F, Pizzirani S. Patologie del film lacrimale e disease delle superfici oculari nel cane e nel gatto. Parte I. Cenni Important murine models – significant body of di fisiopatologia. Veterinaria 2009 23:5;35-51 literature on the immunologic aspects of dry eye & Maggio F. Pizzirani S. Patologie del film lacrimale e SjÖgrens Syndrome delle superfici oculari nel cane e nel gatto. Parte II. Segni MRL/lpr mouse – defect in the Fas receptor clinici, diagnosi e terapia. Veterinaria 2009 23:5;55-70 NOD mouse – CD4+ T cell infiltrate in submandibular, lacrimal, and pancreas glands

12 Dry Eye Therapy Precorneal Tear Film (requested in last BSC reviews) Lipid – Meibomian glands Aqueous – Lacrimal glands Mucin – Conjunctival goblet cells

Lacrimal Gland

Gland of 3rd Eyelid

G. Constantinesqu

Lacrimogenic agents Lacrimostimulation

For the treatment of dry eye, Pilocarpine or keratoconjunctivitis sicca Parasympathetic innervation to lacrimal glands Lacrostimulants Topical solution is given orally Systemic increase in parasympathetic activitiy cholinergic agonists immunosuppressive agent (CsA) Cyclosporine A Topical Lacrimomimetics (tear substitutes) aqueous replacement mucinomimetics lipid replacement

Cyclosporine A CsA regulation of T lymphocytes Inhibits T cell activation CsA binds Cyclophilin (CpN) Decreases IL-2 release and IL-2  Prevents Calcineurin (CaN) activity receptor expression Nuclear Factor of Activated T cells (NF-AT-p) 2+ Stimulates tear production – Dephosphorylated by CaN Ca Ag Available as – Translocated to nucleus – Promotes IL-2 transcription 0.2% ointment (Optimmune® Schering Plough) CD3 TCR CsA decreases Compounded as a 1 or 2 % solution at a licensed Ca2+ + NF-AT p pharmacy IL-2 production + p Apply topically 2-3 times daily CsA CsA CaN NF-AT Systemic absorption may occur at higher dosing CpN frequency and smaller dogs

Non-inhibitory to corneal healing NF-AT IL-2 promoter IL-2 Gene

13 Tacrolimus Conjunctiva - overview Vascularized mucous membrane – covers Tacrolimus belongs to a group of drugs called the anterior surface of eyeball, posterior surface of macrolide lactones or calcineurin inhibitors eyelids, and ant. & post. surface of 3rd eyelid. Similar immunosuppressant activity to cyclosporin Secretes mucus – required for tear film stability & Binds to a receptor within the cell called the FK corneal transparency binding protein This resulting drug-protein complex inhibits Mucosal defense – immunocompetent cells calcineurin (a calcium-dependent phosphatase Initiate and mediate inflammatory reactions transmitting chemical) that in turn reduces the activity Synthesize immunoglobulin of T-lymphocytes in the immune system Morphologic characteristics (microvilli) and biochemical properties (enzyme activity) allow phagocytosis of foreign Wedgewood 1-800-331-8272 particles such as viruses Veterinary Pharmaceuticals 1-800-682-4664 Stokes Pharmacy 1-800-754-5222

Conjunctiva - anatomy Conjunctiva - histology 1) Epithelium Palpebral conjunctiva Between 2 and 8-10 layers thick, depending on location Mucocutaneous junction: transition zone behind the meibomian gland gland Single layer of basal cells openings where stratified Variable # of layers of keratinized squamous epi of lid margin  stratified intermediate cells nonkeratinized squamous epi of conjunctiva Superficial cells of variable shape Tarsal conjunctiva Flattening of superficial cells believed to be an adaptation Orbital conjunctiva – from tarsal plate into fornix to mechanical pressure Conjunctival Cul-de-sac, or Fornix Melanocytes, located among basal cells Immunocompetent cells (esp. Langerhans cells) Bulbar conjunctiva 2) Basement Membrane Zone (BMZ) Scleral division: extends from fornix to limbus Conj, sclera, and tenon’s capsule are firmly attached ~ 3mm from the Separates the epithelium from the limbus and conj is more difficult to mobilize in this area conjunctival stroma or chorion Limbal division: ~ 3mm wide ring at junction of conj and 3) Chorion corneal epithelia

Conjunctiva - histology Conjunctival Glands Chorion (conjunctival stroma) 1) Serous: 1) Scleral division: extends from fornix to limbus 1) Krause’s glands Deep in the conj tissue of fornix (~ 40 in superior & 6-8 in inferior Conj, sclera, and tenon’s capsule are firmly attached ~ fornix in humans) 3mm from the limbus and conj is more difficult to mobilize in this area Histologically, similar to lacrimal glands 2) Wolfring’s glands 2) Limbal division: ~ 3mm wide ring at junction of 2-5 in upper lid (along the upper edge of tarsus) and fewer present conjunctival and corneal epithelia along lower edge of inferior tarsus 3) Rich collagen framework 1) Mucous: 4) Abundant vessels and immunocompetent cells 1) Henle’s glands or crypts Accounts for rapid and Epithelial invaginations within chorion and composed of goblet cells sometimes “violent” Situated along upper edge of superior tarsus inflammatory reactions 2) Manz’s glands At limbus: reported in pigs, cattle, & dogs; absent in humans

 Other: Goblet cells in the conjunctival epithelium

14 Goblet Cells Mucus production per eye per day: 2-3 mL (humans) = one thousandth of total tear production Mucins: High molecular weight glycoproteins (2000-4000 kDa) with subunits of 0.5-2x106Da, which form a gel when their concentration reaches 0.5-1% Peroxidases Contribute to the anti-infectious defense of the ocular surface by the tear film Some goblet cells synthesize hyaluronic acid Helps stabilize the tear film

Mucus – Functions: Glycocalyx: Anchor the aqueous layer of the tear film Glycoproteins and glycolipids that cover the Tear film is organized into increasingly dense filaments as microvilli and microplicae of corneal and conjunctival one approaches the cell layers epithelium Trap desquamated epithelial cells and acellular surface debris (microorganisms) Extends ~ 300 nm from microvilli and microplicae Transported to medial canthus during blinking  evacuated Angular and branching and often extends laterally Immunological barrier: between microvilli Immobilize more than 30% of the secretory IgA contained Filaments branch distally and are associated with cell in tear film membrane Mucus layer of tear film attaches to the carbohydrate- rich glycocalyx Protects epithelium by causing shear forces of blinking to break up mucus layer further away from cell surface Mucus attachment to glycocalyx allows aqueous layer to spread evenly over corneal epithelium

Eyelid Functions Eyelid Functions Comparative approach: where are the exceptions? 1) Screening and Fish lack eyelids (constantly bathed sensing – cilia and in aqueous environment) Land creatures need a way to vibrissae “bring the ocean with them” st 2) Mechanical wiping Amphibians: 1 creatures to have true eyelids and nictitans Tadpoles have no eyelids  frogs have eyelids, action lacrimal glands and a NL system

3) Secretions and Specialized eyelids spreading of Crocodilians – bony tarsus of upper lid glandular tissue Chameleons – tight-fitting around globe and move with globe Snakes, geckos, skinks – spectacle which has a vascular 4) Screening of light to network allow sleep

15 Eyelids - composition Eyelids - Skin, collagen, muscle, glandular tissue, palpebral conjunctiva composition 1. Levator palpebrae Eyelid superioris Musculofibrous Skin Palpebral Conjunctiva layer 2. Orbital septum & 2’ = tarsal plate Cilia & assoc. sebaceous glands Orbicularis oculi Tactile hairs 3. Orbicularis oculi 4. Puncta lacrimalie Orbital septum: • These 3 components Arises from orbit margin intermingle inseparably 5. Cilium w/ associated sebaceous glands • Components are succeeded Aponeurosis levator muscle: by “tarsus” or “tarsal plate” Originate from orbit 6. Tarsal or meibomian glands toward free margin – poorly defined Smooth tarsal muscle: Veterinary Anatomy, 1987, Originate from orbit Dyce Sack & Wensing

Eyelid Skin Cilia Epidermis Species Location on Eyelids Strata corneum & granular, spinous and basal layers People Upper and lower Dermis Dogs Upper Dense, irregular C.T. Pigs Upper Most species, dermis devoid of fat exception in some dogs: Shar Pei Horses Upper and few on lower Ruminants Upper and lower Hair follicles extend deep into dermis Palpebral margin Cats None per se (normal hair can appear as cilia) Skin changes keratinized, stratified squamous EPI  non- keratinized, stratified squamous EPI Birds Some species (i.e. budgerigar) Eyelashes / CILIA located on eyelid leading edge have filoplumes: rudimentary Normal turnover time: 3-5 months; regrow in 2 months feathers without barbs

Eyelid Musculature

Prominent orbito-palpebral sulcus (skin fold) of the superior eyelid (arrows) delineating the orbital portion (above the skin fold) and tarsal portion (below the skin fold) of the eyelid Textbook of Sm An Surg, 1993, Slatter

16 Eyelid Musculature – Orbicularis oculi Major eyelid muscle Arranged in concentric rings around the palpebral opening Fibers originate and terminate on the medial palpebral ligament Innervation: CN VII (Facial) Function: eyelid closure Specialized devisions: Horner’s muscle: Branch that runs under lacirmal sac and inserts on medial orbital wall Negative pressure within lacrimal sac so as to pull tears into sac Muscles of Riolan: Travel along eyelid margin, surrounding the eyelash bulbs May rotate eyelashes toward eye & propel glandular contents during blink

Musculature – Levator palpebrae Musculature – Levator anguli oculi medialis superioris & Müller’s muscle and Frontalis Levator palpebrae superioris Both eyelid elevators Originates deep within orbit, dorsal to optic canal between Both muscles innervated by CN VII – palpebral branch origins of dorsal rectus and dorsal oblique Functions to elevate upper eyelid LAOM - also known as the corrugator supercilia Innervated by CN III (oculomotor) Small muscle that arises caudodorsal Müller’s muscle to the medial commissure Portion of the levator palpebrae Contraction raises the medial superioris that extends deeper into dermis portion of upper eyelid Composed of smooth muscle fibers In the horse, gives rise to Innervated by sympathetic nervous system (carried by a prominent lid notch infratrochlear n, a branch of nasociliary n., branch of ophthalmic devision of CN V) Functions to widen/elevate palpebral fissure Well described in cat, poorly described in horse, even less described in other species

Musculature – Musculature – Retractor anguli oculi lateralis Pars palpebralis of the m. sphincter colli profundus (or the Malaris muscle) Located parallel and superficial to lateral palpebral Consists of several delicate straps of muscle which ligament originate near the ventral midline and course dorsally to Innervated by zygomatic branch of CN VII insert on the lower eyelid Functions to draw the lateral canthus posteriorly Ventral portion lies deep to the platysma Dorsal portion is subcutaneous and close to eyelid skin and laterally when eyelids close Innervated by buccal branches of CN VII Functions to depress the lower eyelid

17 Eyelid Musculature Eyelid Glands MUSCLE INNERVATION FUNCTION Glands of Zeis and Moll Orbicularis Oculi CN VII Eyelid closure Located in anterior lamella of eyelid (Horner’s and Riolan) (palpebral branch) Aids lacrimal pump mechanism Associated with eyelash cilia Secrete their contents around lash follicle shaft Levator palpebrae superioris CN III Maintain open pal. fissure Zeis: Chief elevator of upper eyelid Modified sebaceous glands Müller’s muscle Sympathetic N.S. Maintain open pal. fissure Surround base of hair follicles Elevates upper eyelid Moll: Frontalis (upper eyelid) CN VII Maintain open pal. fissure Eccrine, or modified sweat glands (palpebral branch) Elevates upper eyelid Located just deep to the hair follicles Levator anguli oculi medialis CN VII Raises medial portion of superior Meibomian glands (corrugator supercilia) (auriculopalpebral) eyelid Holocrine, sebaceous glands not associated with lash cilia Retractor anguli oculi lateralis CN VII Contraction during eyelid closure Produce the lipid layer of the tear film (zygomatic branch of pulls lateral canthus posteriorly & Secretion may be partially under neural or homonal control auriculopalpebral) laterally Meibum contains waxy esters, sterols, triacylglycerols, cholesterols, polar lipids, free fatty acids Malaris (lower eyelid) CN VII Maintain open pal. fissure Lower melting temp than seibum, thus liquid on ocular surface (dorsal buccal branch) Depresses lower lid

Eyelid Vasculature Eyelid Movement Well described in dog and horse Most domestic species: superior lid is most mobile In all species, variation exists between different animals Innervation to levator palpebrae superioris m follows Hering’s – not all reports identical, but all similar law: Synergistic muscles receive simultaneous and equal innervation References: Motor neurons for levator m. arise from a single unpaired Anatomy of the Equine Eye and Orbit: Histologic Structure and central caudal nucleus of the oculomotor complex, and a Blood Supply of the Eyelids. BG Anderson, M Wyman. J Equine single motor neuron may innervate the levator m. bilaterally Med Surg 3, 4-9, 1979 Hence, any supranuclear input into motor neuron influences Miller’s Anatomy of the Dog. Ed. HE Evans, GC Christensen. BOTH levator muscles Saunders 1979 Clinical significance: When the levator on one side is weak, Fundamentals of Veterinary Ophthalmology, 3rd edition. DH the lid on opposite side may be retracted in an unconscious Slatter. Saunders 2001 attempt to elevate the ptotic lid

Eyelid Movement Birds, many reptiles: inferior lid raises to meet superior Humans have ability to move eyebrows: Elevation: frontalis muscle Depression: orbicularis muscle in forced lid closure Drawn together: corrugator supercili

WHERE DO YOU PLACE YOUR EQUINE LAVAGE TUBES??

18 Eyelid Movement - Blinking Types of eyelid closure: Inferomedial Placement of a Single-Entry 1. Spontaneous blinking Most common (15 / min humans) Subpalpebral Lavage Tube for Treatment of Lateral  medial Equine Eye Disease (part of lacrimal pump mechanism) # blinks / min % bilateral blinks Veterinary Ophthalmology Dog: 3-5 b/min 85% Volume 3 (2-3), pp 153-156, September 2000 Cat: 1-5 b/min 70% Giuliano EA, Maggs D, Moore CP, et al Horse: 5-25 b/min 30% Pig: 10 b/min 90%

Eyelid Movement - Blinking Third Eyelid Nictitating Membrane, Palpebral Tertia, Semilunar Fold of the Types of eyelid closure: Conjunctiva, Plica Semilunaris Conjunctivae 1. Spontaneous blinking Topographical distribution: Originates in the anterior ventromedial orbit 2. Reflex blinking Triangular in shape; covered Elicited by sensory stimuli (cutaneous touch, auditory with conjunctiva signals, bright visual stimuli, ocular irritation) “T-shaped” hyaline cartilage Gland of the third eyelid 3. Controlled winking – learned response in humans Function: 4. Blepharospasm Protects the globe Forcible contraction of the orbicularis and muscles of the Secretion and distribution of brow  IOP is raised tears Causes: idiopathic, ocular disease, neurodegenerative Aid in removal of particulate disorders (i.e. Parkinson dz) matter from the eye

Nictitans (3rd Eyelid) – Anatomy & Nictitans (3rd Eyelid) – Anatomy & Physiology Physiology Gland of the 3rd Eyelid Movement - Passive Orbital tone Encompasses base of cartilage Orbital fat Seromucous secretions in dog (serous Hydration status in horses) exit through ducts open in the Exception - CATS posterior aspect of the TE between Believed to have some smooth muscle and lymphoid follicles sympathetic innervation to their 3rd eyelid movement Important contributor to basal tear production

19 Mucosa-Associated Lymphoid Tissue (MALT) Morphological evidence of M cells in healthy canine conjunctiva-associated A distinct network of diffuse aggregates of lymphoid tissue lymphoid tissue located in various mucosal surfaces EA Giuliano, CP Moore, TE Phillips Gut (GALT) Graefe’s Arch Clin Exp Ophthalmol (2002) 240:220-226 Bronchus (BALT) Conjunctiva (CALT) Characterization of membranous (M) cells in Nasal mucosa (NALT) normal feline conjunctiva-associated lymphoid tissue (CALT) Steven P, Gebert A. Conjunctiva-associated lymphoid EA Giuliano, K Finn tissue – current knowledge, animal models and Veterinary Ophthalmology (2011) 14, Supplement 1, 60–66 experimental prospects Ophthalmol Res 2009; 42: 2-8

BACKGROUND INFORMATION

Mammalian mucosae are routinely in contact with large numbers of different antigens Mucosa-Associated Lymphoid Tissue (MALT): Afferent branch Efferent branch Effector functions may occur at some distance from original stimulus Roitt, I., et al; Immunology, 5th ed, 1998

MICROFOLD (M) CELLS SIGNIFICANCE of M-CELLS Morphologic characteristics: Gut-Associated Lymphoid Tissue (GALT) A less elaborate apical cell surface with small Exploited by infectious agents microvilli and microfolds An invaginated basolateral membrane forming a Preferential binding and translocation of cytoplasmic pocket containing lymphocytes, antigens across the mucosal barrier with macrophages, & dendritic cells subsequent delivery to underlying antigen A diminished distance between the apical and pocket membrane to enable more efficient presenting cells transcytosis Shigella, Salmonella, Yersinia, Located in the Follicle Associated Epithelium Campylobacter, Vibrio, E. coli, Polio, HIV (FAE) overlying organized immune cells (Neutra et al., 1996; Sansonetti & Phalipon, 1999)

20 SIGNIFICANCE of M-CELLS

Intense research efforts aimed at the development of mucosal vaccines by targeting M-cells (reviewed by Killeen et al., 1999)

New drug delivery strategies may be possible

Roitt, I., et al; Immunology, 5th ed, 1998

OVERVIEW of Graduate Work RESULTS Characterize the Follicle-Associated Epithelium Morphologic studies: (FAE) of Canine Conjunctiva-Associated Lymphoid rd Tissue (CALT) Canine 3 eyelid lymphoid follicles compared with surrounding conjunctival epithelium – light Hypothesis: microscopy, transmission & scanning electron microscopy The FAE of CALT in healthy dogs contains microfold (M) cells morphologically and functionally analogous to Healthy canine CALT contains cells those described in intestinal Mucosa-Associated structurally analogous to M-cells: Lymphoid Tissue (MALT) Diminished cytoplasmic thickness Project design (3 phases): Attenuated apical cell surface structures Morphologic examination using LM, TEM & SEM Invaginated basolateral membranes forming Immunohistochemistry cytoplasmic pockets with subtending lymphocytes Functional studies using Staphylococcus aureus organisms and plasma cells

MORPHOLOGY Light Microscopy SEM

Scanning Electron Transmission Electron Microscopy Microscopy

21 RESULTS IMMUNOHISTOCHEMISTRY Immunohistochemistry: Lymphocytes have an organized distribution in CD 79 - B cells CD 3 - T cells MALT: B-cells in germinal centers T-cells comprise apical cap Canine CALT lymphocytes (Nictitans from 5 dogs) CD 79 staining for B-cells (monoclonal mouse anti-human B Cell, DAKO Corp., Carpinteria, CA) CD 3 staining for T-cells (rabbit anti-human T-cell, DAKO Corp., Carpinteria, CA) Findings consistent with other descriptions of organized MALT

FUNCTIONAL STUDIES MATERIALS & METHODS Functional Uptake Studies

Hypothesis: Nictitating membranes obtained from 10 healthy, 2 year old, M/C, tricolored research hounds. Canine conjunctival M-cells exhibit Health status based on: selective binding and preferential Physical Examination uptake of antigens compared with Blood work (CBC, Serum Chemistry Panel, surrounding epithelial cells Urinalysis) Complete Ophthalmic Examination (Slit-lamp Biomicroscopy, Fluorescein Stain, and Schirmer Tear Test)

MATERIALS & METHODS MATERIALS & METHODS Functional Uptake Studies Functional Uptake Studies

10 dogs treated with 30 l of a heat- Primary fixation: killed, 1 x 1010 Staphylococcus aureus 2.5% glutaraldehyde-2% paraformaldehyde (BioParticles, Molecular Probes, Eugene, OR) per 1 ml buffered solution for 12 hours sterile eyewash (Dacriose, CIBA Vision Ophthalmics, Atlanta, GA) Tissues subsectioned and processed using 5 dogs: 1 gtt OU q 5 minutes for 30 minutes standard techniques for: immediately prior to euthanasia Scanning Electron Microscopy (SEM) 5 dogs: 1 gtt OU q 5 minutes for 30 minutes Transmission Electron Microscopy (TEM) 12 hours prior to euthanasia

22 SEM FAE M-cell surface Non-FAE cell surface

60,000 X magnification 30,000 X magnification

Treatment Immediately Prior to Tissue Collection

SEM

Treatment Immediately Prior to Tissue Collection

TEM

23 Treatment 12 Treatment 12 Hours Prior to Hours Prior to Tissue Collection Tissue Collection

*

TEM

TEM CONCLUSIONS Staph. aureus 1) There are distinct differences in epithelial cell morphology overlying the canine conjunctival lymphoid follicles compared to the surrounding conjunctival epithelium. 2) The FAE overlying lymphoid follicles in canine CALT, as well as the distribution of T and B lymphocytes subtending this region, contain morphologic features analogous to MALT described in other regions.

CONCLUSIONS CLINICAL RELEVANCE 3) Canine CALT contains M-cells that 1) Conjunctival M-cells capable of antigen demonstrate selective binding and sampling will link ocular immunology to a preferential uptake of heat-killed Staph. growing body of research concerned with aureus organisms. targeted mucosal vaccines and potentially serve as a means of developing new 4) The transport of antigens across M-cells strategies for drug delivery. to the lymphoid cells in organized CALT 2) Recognition of conjunctival M-cells opens appears to be time dependent. the door to new research aimed at determining if any ocular bacterial, parasitic, or viral pathogens exploit this entry site.

24 Future Directions

NATIONAL EYE INSTITUTE: Project Title: M-cells in Mammalian Conjunctiva Grant Number – 1 R03 EY13779-01

Meagher, CK, Hongshan L, Moore CP, Phillips TE. Conjunctival M cells selectively bind and translocate Maackia amurensis leukoagglutinin. Exp Characterization of membranous (M) cells in Eye Research. 2005; 80: 545-553 normal feline conjunctiva-associated lymphoid Liu H. Meagher CK. Moore CP. Phillips TE. M cells in the follicle-associated epithelium of the rabbit conjunctiva preferentially bind and translocate latex tissue (CALT) beads. Investigative Ophthalmology & Visual Science. 2005; 46(11):4217-23 EA Giuliano, K Finn Knop N. Knop E. Ultrastructural anatomy of CALT follicles in the rabbit reveals Veterinary Ophthalmology (2011) 14, Supplement 1, 60–66 characteristics of M-cells, germinal centers and high endothelial venules. Journal of Anatomy. 2005; 207(4):409-26 Knop E. Knop N. The role of eye-associated lymphoid tissue in corneal immune protection. [Review] Journal of Anatomy. 2005; 206(3):271-85

PHOTODYNAMIC THERAPY FOR EQUINE PERIOCULAR TUMORS

Elizabeth A. Giuliano, DVM, MS Diplomate ACVO

IEOC, 2013

25 OUR PROBLEM: EQUINE OPHTHALMIC NEOPLASIA Approximately 10% of equine neoplasms affect Most common ocular neoplasms: the eye or periocular structures SCC, sarcoid, papilloma, lymphosarcoma, melanoma Neoplastic adnexal disease represents the most Identical treatments administered to similarly- common & often the most frustrating, of all appearing tumor types can have different clinical equine eyelid diseases outcomes Reasons for which one horse responds well to therapy and another does not … ??

Medical Treatment for Periocular Sarcoids in Horses EQUINE OPHTHALMIC NEOPLASIA Type of Therapy Drug Dose Number of % of non-recurrence cases Topical Therapy AW4-LUDES ointment135 Once daily for 5 146 35% Variability in clinical response  a myriad of days different treatment options exist for any given 5% 5-fluorouracil135 Bid x 5 days, then 9 67% qd for 5 days, the equine neoplasm QOD for 5 application Immunotherapy BCG141 1 ml per cm2 of 26 100% tumor surface every 2-4 weeks BCG135 Ocult, verrucose, mixed 1.0 ml per cm2 52 0% every 2-4 weeks Nodular or fibroblastic “ 300 69% Chemotherapy Cisplatin177 1 mg / cm3 every 2 19 95% weeks for 4 treatments Cisplatin135 “ 18 33% Equine Ocular Adnexal and Nasolacrimal Disease Chapter 4 for the second edition of Equine Ophthalmology, Ed by Brian C. Gilger

Surgical Treatment for Periocular Sarcoids in Horses TREATMENT QUANDARIES… Type of Therapy Description Number of cases % of non-recurrence Surgical excision 138 Excision -- 50% Definitive conclusions regarding biologic behavior or Surgical excision 135 Excision 28 18% response to treatment from reviewing the veterinary Cryotherapy 186 Double or triple freeze – literature difficult thaw to –25oC Cryotherapy 135 “239% Study design varies widely / limited to isolated case reports Hyperthermia135 Tissue temperatures 20% Very few reports exist with >100 horses included and between 41oC and 45oC containing at least a year follow-up time after specific Brachytherapy for Periocular Sarcoids in Horses therapy for a particular tumor-type has been used % of non- In stark contrast to studies routinely published in physician Radioisotope Dose Range Number of cases recurrence ophthalmology 222Radon seeds195 6000 cGy 19 92% 198Gold seeds197 7000 cGy 19 90% 192Iridium seeds142,194 60 Gy 115 87%, 100% 192Iridium pins135 7000-9000 cGy 53 98% Equine Ocular Adnexal and Nasolacrimal Disease Chapter 4 for the second edition of Equine Ophthalmology, Ed by Brian C. Gilger

26 TREATMENT QUANDARIES… TREATMENT QUANDARIES… Methods of data reporting (i.e. recurrence rate Treatment efficacy: challenging to critically assess versus disease-free interval) also vary among Some studies not designed with a control population publications for adequate comparison purposes & are often based on a subset of cases referred to specialty/teaching Point: be aware of these limitations when hospitals reviewing the literature and in planning the most Referral cases are frequently those that are appropriate therapy for your patients refractory to “conventional” treatment modalities May have already undergone treatment prior to being included in the study; therefore, the population of horses examined is skewed

PHOTODYNAMIC THEAPY LECTURE OBJECTIVES, PDT

1) Overview of Photodynamic Therapy 2) My interest in this modality 1) Summary of early work 3) How I perform this procedure 1) Review of treatment protocol and clinical cases Historically, concept of combining light with chemical agent 4) Response to frequently asked questions has ancient beginnings 1) Laser type/source, cost, good vs. bad candidates The success of PDT in early 1960s with subsequent PS for procedure, etc – discussion welcomed approvals generated world-wide interest 1900-1955: 112 publications 1955-2013: > 16,000 publications; > 8000 papers in past 5 years Chem. Rev. 2010 (110) 2795-2838

PHOTODYNAMIC THERAPY (PDT) “PHOTOCHEMISTRY” Mechanism of Action Type I reaction λ Energy transferred to Selective Photosensitizer Photosensitizer other compounds  Tissue Ground state Excited state Superoxide, free radicals Destruction Type II reaction Energy transferred to O2 Photosensitizer Light 1  O2 (singlet oxygen) Cancer therapy using photosensitizing agent and light in 1 Cell Killing - O2 and oxygen free radical production the presence of oxygen to produce a localized 1. Damage of cellular structures - plasma membrane, phototoxic effect mitochondria, lysosomes 2. Inflammation 2-stage process 3. Vascular effects and tumor ischemia Delivery of photosensitizer (IV, PO, topical) Selective tissue destruction Preferential localization of photosensitizer in cancer cells Light irradiation of target tissue Focused light beam (lasers – major advancement in PDT) 1 O2 - short half-life (0.04 µ sec), diffusion distance (<0.1 µm)

27 Modified Jablonksi Diagram, CA CANCER J CLIN 2011; 61: 250-281 PHOTOPHYSICS & PHOTOCHEMISTRY Light exposure takes a photosensitizer

molecule from the ground singlet state (S0) to an excited singlet state (S1) Excited PS – very unstable Emit its excess energy as fluorescence and/or heat OR Undergo “intersystem crossing” to form a more PS in triplet state can either decay without radiation stable triplet state to the ground state or transfer its energy to molecular oxygen (O2) O2 is unique: triplet in its ground state This step leads to the formation of Singlet Oxygen (Type II reaction)

Modified Jablonksi Diagram, CA CANCER J CLIN 2011; 61: 250-281 PHOTOPHYSICS & PHOTOCHEMISTRY Reactive oxygen species (ROS) (predominantly the generation of singlet 1 oxygen or O2) via Type II chemistry is mechanistically much simpler than via Type I Most PSs are believed to predominately exert their cytotoxic effects through Type II reactions

A Type I reaction can also occur: PS reacts directly with organic substrates, acquiring a hydrogen atom or electron to form a radical Superoxide anions, hydroxyl radicals, peroxides

MECHANISMS OF PDT-MEDIATED APOPTOSIS CYTOTOXICITY Characterized by chromatin condensation, DNA cleavage, cell shrinkage, membrane blebbing, with release of signals required Apoptosis ** for phagocytic cell activity (e.g. Damage Necrosis Associated Molecular Patterns) Autophagy-associated cell death At biochemical level: mitochondria outer membrane permeabilization (MOMP) after PDT  activation of caspases (highly conserved family of cysteine- dependent, aspartate-specific proteases)

Yoo JO, Ha KS. New Insights into the Mechanisms for Photodynamic Therapy-Induced Cancer Cell Death. In: International Review of Cell & Molecular Biology. 2012; 139-174

28 NECROSIS AUTOPHAGY Characterized by cytoplasm Characterized by formation of a double vacuolization & breakdown of plasma membrane structure called the membrane  release of cellular autophagosome, which sequesters contents and proinflammatory cytoplasmic organelles and traffics them molecules to lysosomes Results from pathological insults or a Autophagosome-lysosome fusion results bioenergetic catastrophe and ATP in degradation of cytoplasmic depletion components by lysosomal hydrolases At biochemical level: cytochrome c In adult organisms, functions as a self- release and DNA oligonucleaosomal digestion pathway promoting cell fragmentation survival in an adverse environment & as a quality control mechanism by Yoo JO, Ha KS. New Insights into the Mechanisms for Photodynamic Therapy-Induced Cancer Cell Death. In: removing damaged organelles, toxic International Review of Cell & Molecular Biology. 2012; 139-174 metabolites, or intracellular pathogens

PDT AND THE IMMUNE RESPONSE PDT AND THE IMMUNE RESPONSE Inflammation elicited by PDT is a tumor antigen Phagocytized tumor cells migrate to local lymph nonspecific process orchestrated by the innate nodes immune system Differentiate into antigen-presenting cells ROS within tumor cells after PDT leads to cell death (apoptosis & necrosis) Tumor antigen presentation within lymph nodes is Tumor cell death potentiated by damage to followed by clonal expansion of tumor-sensitized microvasculature, restricting oxygen and nutrient lymphocytes that home to the tumor and eliminate supply residual tumor cells Cell death activates the complement cascade, Korbelik M. PDT-associated host response and its role in the therapy outcome. secretion of proinflammatory cytokines, and rapid Lasers Surg Med. 2006; 38: 500-508 recruitment of neutrophils, macrophages, and Korbelik M, Dougherty GJ. Photodynamic therapy-mediated immune response dendritic cells against subcutaneous mouse tumors. Cancer Res. 1999; 59: 1941-1946

PHOTOSENSITIZERS

29 PHOTOSENSITIZERS (PSs) PSs – IDEAL PROPERTIES Most PSs used in cancer therapy based on a Single, pure compound (quality control analysis, low tetrapyrrole structure manufacturing costs, stable when stored) Similar to the protoporphyrin present in hemoglobin High absorption peak between 600-800 nm (red – deep red) Absorption of photons with λ longer than 800 nm does not provide enough energy to excite oxygen to its singlet state No dark toxicity Relatively rapid clearance from normal tissues, minimizing phototoxic side effects

Allison RR, Sibata CH. Oncologic photodynamic therapy photosensitizers: a clinical review. Photodiagnosis Photodyn Ther. 2010; 7: 61-75.

PSs – IDEAL PROPERTIES PSs – DEVELOPMENT Penetration of light into tissue increases with its λ 1st PS used in cancer therapy: HPD Agents with strong absorbance in deep red most effective at (hematoporphyrin derivative) tumor control (e.g. chlorins, bacteriochlorins, phthalocyanines) A water-soluble mixture of porphyrins Later, a purified form, porfimer sodium (Photofrin®) developed Still widely used Disadvantages: long-lasting skin photosensitivity, relatively low absorbance at 630 nm Major effort aimed at producing second generation PSs Increased absorbance at longer λ, less toxicity

Agostinis P, et al. Photodynamic therapy of cancer: an update. CA Cancer J Clin. 2011; 61: 250-281

30 PSs – TUMOR-LOCALIZING PROPERTIES

“Enhanced permeability and retention effect” LIGHT SOURCES IN PDT Leaky tumor blood vessels due to neovascularization Absence of lymphatic drainage Some PSs preferentially bind to low-density lipoprotein (LDL) LDL receptors up-regulated on tumor cells

Hamblin MR, Newman EL. On the mechanism of the tumour-localising effect in photodynamic therapy. J Photochem Photobiol B. 1994; 23: 3-8

CHOOSING A LIGHT SOURCE LIGHT SOURCES in PDT No single light source is ideal for all PDT indications, even using the same PS “Any light source, either laser or nonlaser, with Choice based on: suitable spectral characteristics and a high output at PS absorption an absorption maximum of the photosensitizer can Disease (location, lesion size, accessibility, and tissue be used for PDT” characteristics http://emedicine.medscape.com/article/1121517-overview#aw2aab6b5 Cost Size GOAL: match output λ to spectral absorption peak Clinical efficacy of PDT is dependent on complex of PS dosimetry: Total light dose, light exposure time, light delivery mode (single vs fractionated), etc

LIGHT SOURCES LASERS in PDT ADV of Lasers: First successful treatment of tumors in animals reported in Monochromatic: provides maximum effectiveness if λ 1975 corresponds with the peak absorption of PS Slow to gain acceptance Coherent, high irradiance: minimize therapeutic exposure time Lasers – major advancement Can be readily coupled to fiberoptics, enabling light delivery Monochromatic to any organ (e.g. bladder, GI tract, lungs) Coherent DIS of Lasers: Intense Expensive Fiberoptic cables Require special maintenance (automated dosimetry & calibration) When coupled with fiberoptics, may only be used on small skin lesions

31 NONCOHERENT LIGHT in PDT LIGHT EMITTING DIODES/DEVICES ADV: (LEDs) in PDT Large illumination field compared to laser Relatively narrow spectral bandwidths Low cost, smaller size, and readily available High fluence rates Polychromatic light sources allow the use of different photosensitizers with different absorption maxima Large illumination field compared to laser DIS: Low cost Recall: blue light penetrates least efficiently through tissue; red & infrared more deeply 600-1200 nm = “Optical Window” of tissue 1 Do not exceed 800 nm (cannot generate O2)

LIGHT DOSIMETRY LIGHT DOSE Efficacy of PDT affected by power EXAMPLE: Definition: dose of light delivered from optical fiber density and fluence Area is r2, where  = 3.1415927 Fiber output and distance to surface Energy: Joules / cm2 3 cm spot  1.5 cm radius2 x  = 7.07 cm2 Typical dose is 100 J / cm2 Dose Rate = Power Density = mW/cm2 = 700*/7 = 100 mW/cm2 = 0.1 W/cm2 : # photons to given area – J/cm2 Typical treatment “spot” is 16 – 20 Fluence minutes Dose rate x time (1009s) = 0.1 W/cm2 x 1009 sec = 100 (W/sec)/cm2 = 100 J/cm2 Dose = amount of light delivered Some researchers advocate “pulsing” 2 the laser energy so that the Where * is mW from fiber tip Power density: # photons per unit time – W/cm photosensitizer can return to ground Dose rate = rate of light delivery state and then be “re-excited” to achieve better tumor necrosis MW Berns et al., Exposure (Dose) Tables High – tissue hyperthermia at >125 mW/cm2 for hematoporphyrin derivative *********************************************** photoradiation therapy. Lasers in Surgery Low and Medicine, 1984, 4: 107-131 Power density Fewer microvascular effects # of photons / unit time delivered to a Slower O2 depletion, more efficient tumor killing given area My special thanks to our collaborator: Ian Higher local control rate 2 J. MacDonald, Ph.D. Biophysics. PDT mWatts / cm Center, Roswell Park Cancer Institute, Treatment time = Fluence/Power density Fluence (light dose from laser) Buffalo, NY Expressed as Joules / cm2 where a Joule Longer exposure times preferred in some cases is a Watt-Second

PILOT STUDY: Photochlor® (HPPH) CLINICAL EXPERIENCE 2-[1-hexyloxy]-2-devinylpyropheophorbide-a Investigational cancer therapy at MU-VMTH McCaw, DL et al. Treatment of canine oral SCC with photodynamic therapy. British Journal of Cancer (2000) 82: 1297- 1299 McCaw, DL et al. Treatment of canine hemangiopericytomas with photodynamic therapy. Lasers in Surgery and Medicine (2001) 29: 23-26 Giuliano EA, MacDonald PJ, McCaw DL, et al. Veterinary Ophthalmology 2008; 11(1):27-34 Drug not commercially available

32 http://www.epgonline.org/page.cfm/pageId/789/type/print/

REFERENCES REFERENCES Peer Reviewed Publications Peer Reviewed Publications Ota J, Giuliano EA, Mullen SF, Turk JR, Lewis MR, Cohn LA, Moore CP, Critser J. Giuliano EA, MacDonald I, McCaw DL, Dougherty TJ, Klauss G, Ota J, Pearce Xenotransplantation of cryopreserved equine squamous cell carcinoma to JW, Johnson PJ. Photodynamic therapy for the treatment of periocular athymic nude mice and SCID mice. Research in Veterinary Science squamous cell carcinoma in horses: a pilot study. Veterinary Ophthalmology 2007;83(3):355-359 2008;11(Suppl 1):27-34

Morgan EJ, Whetstine JL, Giuliano EA, Tucker SA. Application of a fluorescence Barnes LD, Giuliano EA, Ota J, Cohn LA, Moore, CP. The effect of assay for the quantification of the photodynamic agent Photofrin® in horses. photodynamic therapy on squamous cell carcinoma in a murine model: Applied Spectroscopy 2007;61(4):450-454 evaluation of time between intralesional injection to laser irradiation. The Veterinary Journal 2009;180(1):60-65 Giuliano EA, Ota J, Tucker SA. Photodynamic therapy: basic principles and potential uses for the veterinary ophthalmologist. Veterinary Ophthalmology Barnes LD, Giuliano EA, Ota J. Cellular localization of Visudyne® as a function 2007;10(6):337-343 of time after local injection in an in-vivo model of squamous cell carcinoma: an investigation into tumor cell death. Veterinary Ophthalmology Ota J, Giuliano EA, Cohn LA, Lewis MR, Moore CP. Local photodynamic therapy 2010;13(3):158-165 for equine squamous cell carcinoma: evaluation of a novel treatment method in a murine model. Veterinary Journal 2008;176(2): 170-176

REFERENCES REFERENCES Peer Reviewed Publications Abstracts (not listed in publications) Giuliano EA. Equine periocular neoplasia: Current concepts in aetiopathogenesis and emerging treatment modalities. Equine Veterinary Morgan EJ, Giuliano EA, Johnson PJ, Tucker SA. Quantitative determination of Journal 2010; Suppl (37):9-18 Photofrin® following periocular subcutaneous injection in horses. Pittcon National Conference, Pittsburg, Pennsylvania, 2005 Giuliano EA, Johnson PJ, Delgado C, Pearce JW, Moore CP. Local photodynamic therapy delays recurrence of equine periocular squamous cell Tucker SA, Whetstine JL, Morgan EJ, Giuliano EA, Johnson PJ. Developing a carcinoma compared to cryotherapy. Veterinary Ophthalmology 2013, 1-9 spectroscopic method to determine the efficacy of a novel photodynamic doi:10.1111/vop.12099 therapy modality in the treatment of equine squamous cell carcinoma. 32nd Annual FACSS Meeting, Quebec City, Canada, October 9-13, 2005 Abstracts (not listed in publications) Giuliano EA, Ota J, Klauss G, Johnson PJ. Equine eyelid melanoma treated with Giuliano EA, Morgan EJ, Johnson PJ, Tucker SA. Spectroscopic determination surgical resection and local photodynamic therapy. Proc. European College of Photofrin® following periocular subcutaneous injection in horses. Proc. of Veterinary Ophthalmologists/ European Society of Veterinary American College of Veterinary Ophthalmologists, 35th Annual Meeting, Ophthalmologists, Oporto, Portugal, June 17th, 2005. Veterinary Washington, DC, October 20-23, 2004, page 46. Veterinary Ophthalmology Ophthalmology 2005; 8(6): 429 [abstract 12] 2004; 7(6): 443 [abstract 35]

33 REFERENCES Abstracts (not listed in publications) Thank you! Giuliano EA, Morgan EJ, Whetstine JL, Johnson PJ, Tucker SA. Determination of Photofrin® concentration and dissipation rate after periocular subcutaneous Enjoy Eye Camp, 2016 injection of horses. Proc. American College of Veterinary Ophthalmologists, 36th Annual Meeting, Nashville, Tennessee, October 12-15, 2005, page 45. Veterinary Ophthalmology 2005; 8(6): 442 [abstract 32]

Giuliano EA, Whestine JL, Tucker SA. Application of a fluorescence assay to determine the stability of verteporfin in Domoso® and sterile water over time. Proc. American College of Veterinary Ophthalmologists, 39th Annual Meeting, Boston, MA, October 15-18, 2008. Veterinary Ophthalmology 2008; 11(6): 427 [abstract 92]

Giuliano EA, Johnson PJ, Pearce JW, Moore CP. Treatment of periocular sarcoid with surgical resection and photodynamic therapy: 7 cases. Proc. American College of Veterinary Ophthalmologists, 43st Annual Meeting, Portland, OR, October 17-20, 2012. Veterinary Ophthalmology 2013; 16(1): E8 [abstract 47]

34