Reduced retinal transduction and enhanced transgene-directed immunogenicity with intravitreal delivery of rAAV following posterior vitrectomy in dogs.

Adeno-associated virus (AAV) vector-based gene therapy is a promising treatment strategy for delivery of neurotrophic transgenes to retinal ganglion cells (RGCs) in glaucoma patients. Retinal distribution of transgene expression following intravitreal injection (IVT) of AAV is variable in animal models and the vitreous humor may represent a barrier to initial vector penetration. The primary goal of our study was to investigate the effect of prior core vitrectomy with posterior hyaloid membrane peeling on pattern and efficiency of transduction of a capsid amino acid substituted AAV2 vector, carrying the green fluorescent protein (GFP) reporter transgene following IVT in dogs. When progressive intraocular inflammation developed starting 4 weeks post IVT, the study plan was modified to allow detailed characterization of the etiology as a secondary goal. Unexpectedly, surgical vitrectomy was found to significantly limit transduction, whereas in non-vitrectomized eyes transduction efficiency reached upwards to 37.3% of RGC layer cells. The developing retinitis was characterized by mononuclear cell infiltrates resulting from a delayed-type hypersensitivity reaction, which we suspect was directed at the GFP transgene. Our results, in a canine large animal model, support caution when considering surgical vitrectomy before IVT for retinal gene therapy in patients, as prior vitrectomy appears to significantly reduce transduction efficiency and may predispose the patient to development of vector-induced immune reactions.

Photoreceptor-targeted gene delivery using intravitreally administered AAV vectors in dogs.

Delivery of therapeutic transgenes to retinal photoreceptors using adeno-associated virus (AAV) vectors has traditionally required subretinal injection. Recently, photoreceptor transduction efficiency following intravitreal injection (IVT) has improved in rodent models through use of capsid-mutant AAV vectors; but remains limited in large animal models. Thickness of the inner limiting membrane (ILM) in large animals is thought to impair retinal penetration by AAV. Our study compared two newly developed AAV vectors containing multiple capsid amino acid substitutions following IVT in dogs. The ability of two promoter constructs to restrict reporter transgene expression to photoreceptors was also evaluated. AAV vectors containing the interphotoreceptor-binding protein (IRBP) promoter drove expression exclusively in rod and cone photoreceptors, with transduction efficiencies of ~4% of cones and 2% of rods. Notably, in the central region containing the cone-rich visual streak, 15.6% of cones were transduced. Significant regional variation existed, with lower transduction efficiencies in the temporal regions of all eyes. This variation did not correlate with ILM thickness. Vectors carrying a cone-specific promoter failed to transduce a quantifiable percentage of cone photoreceptors. The newly developed AAV vectors containing the IRBP promoter were capable of producing photoreceptor-specific transgene expression following IVT in the dog.

Targeting gene expression to cones with human cone opsin promoters in recombinant AAV.

Specific cone-directed therapy is of high priority in the treatment of human hereditary retinal diseases. However, not much information exists about the specific targeting of photoreceptor subclasses. Three versions of the human red cone opsin promoter (PR0.5, 3LCR-PR0.5 and PR2.1), and the human blue cone opsin promoter HB569, were evaluated for their specificity and robustness in targeting green fluorescent protein (GFP) gene expression to subclasses of cones in the canine retina when used in recombinant adeno-associated viral vectors of serotype 5. The vectors were administered by subretinal injection. The promoter PR2.1 led to most effective and specific expression of GFP in the long- and medium-wavelength-absorbing cones (L/M cones) of normal and diseased retinas. The PR0.5 promoter was not effective. Adding three copies of the 35-bp LCR in front of PR0.5 lead to weak GFP expression in L/M cones. The HB569 promoter was not specific, and GFP was expressed in a few L/M cones, some rods and the retinal pigment epithelium. These results suggest that L/M cones, the predominant class of cone photoreceptors in the retinas of dogs and most mammalian species can be successfully targeted using the human red cone opsin promoter.

Optic nerve head neuroretinal rim blood flow differences in monkeys with laser-induced glaucoma.

INTRODUCTION: The blood flow of the neuroretinal rim (NRR) of the optic nerve head (ONH) of the rhesus monkey with laser-induced glaucoma was examined. METHODS: Argon laser photocoagulation of the trabecular meshwork to induce elevated intraocular pressure (IOP) was performed in one eye of nine normal male rhesus monkeys. The nasal and temporal NRR of the monkey ONH were examined by the Heidelberg retina tomograph/flowmeter (HRT/HRF) under neuromuscular blockade. A mixed effect analysis of variance was used to determine significant differences between eyes and between locations in the eyes. RESULTS: The average IOP in the hypertensive glaucoma and normal eyes was 34.8 +/- 7.2 and 16.0 +/- 1.9 mmHg, respectively. The HRT determined average overall cup to disc (C/D) area ratio in the glaucoma and normal eyes, which was 0.49 +/- 0.28 and 0.22 +/- 0.16, respectively. The mean temporal NRR HRF flow in the hypertensive eyes was significantly greater than in the normotensive eyes (P < 0.0001), than in the nasal NRR of the hypertensive eyes (P < 0.0001) and than in the nasal NRR of the normotensive eyes (P < 0.01). The mean nasal NRR HRF flow in the hypertensive eyes was significantly less than in the nasal NRR of the normotensive eyes (P < 0.01). There was no statistical difference between the mean HRF flow of the temporal and nasal NRR of the normotensive eyes. The elevated IOP positively influenced the flow values in the hypertensive eye (r = 0.724). CONCLUSIONS: The capillary microcirculation of the temporal NRR of the rhesus monkey ONH with laser-induced glaucoma has significantly increased blood flow, and the nasal NRR significantly reduced blood flow compared to blood flow in the NRR of normal normotensive monkey eyes.

Profiles of matrix metalloproteinase activity in equine tear fluid during corneal healing in 10 horses with ulcerative keratitis.

OBJECTIVE: Levels of tear film matrix metalloproteinases (MMPs) activity are significantly elevated in horses with ulcerative keratitis and contribute to the excessive breakdown of stromal collagen. Changes in the amount of proteolytic activity in horse tear film during corneal healing and stromal remodeling have not yet been reported, but we hypothesize they should decrease. In the present study we analyzed serial tear fluid from horses with ulcerative keratitis to identify any changes in MMP activity during corneal healing and stromal remodeling. PROCEDURES: Samples of tear fluid were obtained from both eyes of 10 horses with ulcerative keratitis on the day of admission (day 1) at the hospital and then at various time points until complete healing of the cornea. Tear film MMP2 and MMP9 activity was determined by quantitative gelatin zymography. In all cases medical treatment included topical applications of equine serum, antibiotics, atropine and systemic administration of anti-inflammatory drugs. Surgical procedures were performed in several cases on day 2 in addition to the medical treatment. RESULTS: The mean total MMP activity (+/- SD) measured in relative standard units (RSU) in the tear fluid of the ulcerated eye (2.44 +/- 1.44) of the 10 horses was significantly higher than the mean in the contralateral eye (0.81 +/- 0.68) (P = 0.006), on the day of admission at the VMTH. The mean MMP activity in these ulcerated eyes significantly decreased (-82.4%) between the first day of admission and the day when the ulcer had completely healed (P = 0.0002). The activity level in the healed eye (0.43 +/- 0.17) was not significantly different to the one in the contralateral eye (0.36 +/- 0.18) on the day of complete corneal healing (P = 0.374). The level of MMP activity in the contralateral eye also decreased from 0.81 +/- 0.68-0.36 +/- 0.18 but this decrease (56%) was not significant (P = 0.069). CONCLUSIONS: Ulcerative keratitis in horses is associated with initially high levels of tear film proteolytic activity that decrease as the ulcers heal. The success of medical and surgical treatment of the corneal ulcers is reflected by the enzyme activity in tears. In horses successful treatment does lead to a rapid reduction in tear film proteolytic activity that corresponded with the improvement in the clinical signs of corneal ulceration. Measurement of MMP activity in the tear film might represent a way to monitor the progression of corneal healing in horses with ulcerative keratitis.

Time-specific intraocular pressure curves in Rhesus macaques (Macaca mulatta) with laser-induced ocular hypertension.

OBJECTIVE: To detect and categorize time-specific variations in daytime intraocular pressure (IOP) found in Rhesus monkeys with laser-induced ocular hypertension. PROCEDURES: Ten male monkeys with argon laser-induced ocular hypertension in one eye were anesthetized with ketamine hydrochloride, and the IOP measured in both eyes at 7 a.m., 7.30 a.m., and then hourly until 1 p.m. with a Tonopen trade mark XL applanation tonometer. Intraocular pressure time profiles for both eyes in each animal were developed. The means +/- SD of the IOPs for both eyes were calculated for the whole 6-h study period, and the values compared statistically. The difference between the lasered eye mean IOP standard deviation and the normal eye mean IOP standard deviation for each animal during the 6-h follow-up was also calculated and compared. RESULTS: Mean IOP (+/- SD) in the glaucoma and normal eyes for the 10 animals during the 6-h study was 32.6 +/- 2.5 and 14.9 +/- 2.5 mmHg, respectively. The IOP was significantly higher in the experimental eye than in the normal eye (P = 0.0008). The mean IOP in the lasered eye did not significantly change during the study period, whereas a slight but significant increase in IOP of the normal eye over the study period was recorded (P = 0.003). The variance in IOP in the hypertensive eyes was considerably greater than that in the untreated control eyes. From 7 a.m. to 1 p.m. the IOP declined in five eyes and increased in the other five eyes with laser-induced ocular hypertension. CONCLUSIONS: The time-specific IOP variation pattern in the daytime in the laser treated eyes is significantly greater than the variation in the normotensive eyes. This shows that in order to detect statistical differences between IOP variations induced by an IOP-reducing drug, and the exaggerated spontaneous IOP variations present in the laser-induced hypertensive eye, sufficient animals should be included in any study. Understanding the time-specific IOP variation present in a group of monkeys with laser-induced ocular hypertension is essential prior to using the model for the evaluation of IOP-reducing drugs.

Comparative morphology of the tapetum lucidum (among selected species).

OBJECTIVES: The phenomenon of 'eye-shine' is seen in a variety of animal species, and is generally thought to be related to the presence of an intraocular reflecting structure, the tapetum lucidum. The tapetum lucidum is a biologic reflector system that is a common feature in the eyes of vertebrates. It normally functions to provide the light-sensitive retinal cells with a second opportunity for photon-photoreceptor stimulation, thereby enhancing visual sensitivity at low light levels. The tapetum lucidum is presented here according to a classification based on the location, as well as the composition, of this reflective layer. Finally, the physical and chemical properties, as well as the origins of the different tapeta lucida, are discussed and compared. METHODS: The anatomic and biochemical aspects of the tapetum lucidum in various vertebrates are examined. Morphologic observations were made from paraffin and plastic embedded specimens. Specimens were treated with traditional stains and observed by light and transmission electron microscopy. RESULTS: Some species (primates, squirrels, birds, red kangaroo and pig) do not have this structure and they usually are diurnal animals. In vertebrates, the tapetum lucidum exhibits diverse structure, organization and composition. Therefore, the retinal tapetum (teleosts, crocodilians, marsupials, fruit bat), the choroidal guanine tapetum (elasmobranchs), the choroidal tapetum cellulosum (carnivores, rodents, cetacea), and the choroidal tapetum fibrosum (cow, sheep, goat, horse) are described. CONCLUSIONS: The tapetum lucidum represents a remarkable example of neural cell and tissue specialization as an adaptation to a dim light environment and, despite these differences, all tapetal variants act to increase retinal sensitivity by reflecting light back through the photoreceptor layer. These variations regarding both its location and structure, as well as the choice of reflective material, may represent selective visual adaptations associated with their feeding behavior, in response to the use of specific wavelengths and amount of reflectance required.

Connective tissue growth factor in tear film of the horse: detection, identification and origin.

BACKGROUND: Healing of corneal ulcers in horses is often associated with profound corneal stromal fibrosis and scar formation resulting in visual impairment. Connective tissue growth factor (CTGF) is a fibrogenic cytokine involved in wound healing and scarring. The purpose of this study was to determine whether CTGF was present in the tear fluid of normal horse eyes and the eyes of horses with corneal ulcers in order to evaluate the role of CTGF in corneal wound healing and corneal scar formation. METHODS: Tear fluid samples were collected from 65 eyes of 44 horses; 32 samples from normal eyes, 21 samples from eyes with corneal ulceration, and 12 samples from the unaffected contralateral eyes of horses with ulcers. CTGF levels in the tears were determined by enzyme immunoassay using goat IgG against human CTGF. Antigenetic similarity of human and horse CTGF was established in a bio-equivalence assay. The identity of horse CTGF was confirmed by western blot. Lacrimal and nictitating membrane glands were investigated by immunohistochemistry in the attempt to clarify the origin of tear fluid CTGF. RESULTS: CTGF was detected in tear film of 23 normal unaffected eyes (72%) and 8 normal contralateral eyes (67%), with the mean CTGF levels (+/- SEM) being 51.5+/-19.2 and 13.4+/-3.9 ng/ml respectively. CTGF was found in 8 eyes with corneal ulcers (38%) with the mean CTGF concentration of 26.3+/-14.8 ng/ml. Western blot identified the protein detected as CTGF. The identification of CTGF in lacrimal glands suggests a major role of these glands in the presence of CTGF in tears. CONCLUSIONS: CTGF is present in horse tear fluid and derives, at least partly, from the lacrimal gland. Equine CTGF has strong antigenic similarity with human CTGF. Corneal disease leads to a decrease of CTGF concentrations in tears. The possible role of CTGF in the healing process of ocular surface requires further investigation.

Corneal thickness and endothelial cell density measured by non-contact specular microscopy and pachymetry in Rhesus macaques (Macaca mulatta) with laser-induced ocular hypertension.

PURPOSE: Sustained increase in intraocular pressure (IOP) in humans results in a loss of corneal endothelial cells and an increase of corneal thickness. The effects of chronically elevated IOP on the corneal endothelium of monkeys with laser-induced ocular hypertension, a commonly used animal model of human glaucoma have not been documented. This study examined the central corneal thickness (CCT), the corneal endothelial cell density (ECD), and the corneal endothelial cell size (ACS) in Rhesus monkeys with experimental ocular hypertension. Materials and methods. Ten male monkeys with argon laser-induced ocular hypertension in one eye for an average duration of 2.4+/-0.7 years, were sedated with ketamine hydrochloride, and the CCT, ECD, and ACS measured at the center of the cornea of both eyes with a Topcon SP-2000P non-contact specular microscope (Topcon America Corporation((R)), Paramus, NJ, USA). CCT was also measured using a DHG-500 Pachette ultrasonic pachymeter (DHG Technology Inc., Exton, PA, USA). Mean and standard deviation (S.D.) of CCT, ECD and ACS for each eye was calculated and statistically compared.Results. Mean CCT in the hypertensive and normal eyes measured by specular microscopy was 0.477+/-0.023mm and 0.468+/-0.020 mm, respectively. Mean ECD in the hypertensive and normal eyes was 2601.7+/-631.8 and 3990.2+/-402.9 cells mm(-2), respectively. The mean size of the endothelial cells was 252.4+/-23.9 micro m(2) in the normal eye and 408.7+/-115.0 microm m(2) in the hypertensive eye. No significant difference in the measurement of CCT was observed between the specular microscope and the pachymeter (p=0.46). No significant difference in the mean CCT was observed between the two eyes (p=0.4820), whereas the mean ECD was significantly lower in the hypertensive eye than in the normal eye (p<0.001). The ECD was inversely related to the length of IOP elevation (p<0.001). CONCLUSIONS: No difference in the corneal thickness measurement was observed between the specular microscopy and the pachymetry techniques. Chronic ocular hypertension did not significantly affect the CCT, but caused a significant loss of endothelial cells in the center of the cornea of the laser treated eyes compared to the normotensive eyes. The duration of elevated IOP was the most important factor affecting the ECD.

Disease of the equine posterior segment.

The appearance of the equine fundus is reviewed from the perspective of differentiating normal variations from disease, and the descriptions have been updated to include recently published ocular fundic abnormalities. Most pathological lesions are identified near the optic nerve head, and typically involve depigmentation or hyperpigmentation. Depending upon configuration and appearance, linear pigmented bands may reflect the course of the vortex veins, the transition from tapetal to nontapetal fundus, or indicate chorioretinitis or equine motor neuron disease. Choroidal vasculature is readily apparent in color-dilute (subalbinotic) horses and must be differentiated from hemorrhage. Retinal hemorrhages in foals are common and may occur independently to hypoxic ischemic encephalopathy. Retinal cysts may signal more significant disease in the eye such as anterior segment dysgenesis. Prominence of gray or tan-colored material on or near the optic nerve head may represent traumatic optic neuropathy, benign optic neuropathy, proliferative optic neuropathy or actual neoplasia.

Immunohistochemistry of the extracellular matrix of the normal equine lamina cribrosa.

Purpose To use immunohistochemical techniques to identify and localize the structural macromolecules of the extracellular matrix (ECM) of the normal adult equine lamina cribrosa in order to make comparisons to the extracellular matrix of the lamina cribrosa of horses with glaucoma. METHODS: Normal eyes of five adult horses between 5 and 10 years of age were fixed in 10% neutral buffered formalin and embedded in paraffin. Polyclonal rabbit-derived antibodies against human elastin, laminin, fibrillin-1, and collagen types I, III and IV, and polyclonal goat-derived antibodies against collagen type VI were used as primary antibodies. Transverse and longitudinal histologic sections of the optic nerve head and lamina cribrosa were stained using several dilutions of the primary antibodies, biotinylated link antibody, horseradish peroxidase-labeled streptavidin, and 3,3'-diaminobenzidine as a chromogen. The immunohistochemical staining patterns were qualitatively interpreted. RESULTS: The normal adult horse lamina cribrosa labeled positively for collagen types I, III and VI, laminin, elastin and fibrillin. Collagen type VI staining of the laminar ECM was most intense, followed by labeling for collagen types III and I, respectively. Laminar blood vessels were weakly positive for laminin and slightly positive for type IV collagen. The scleral ECM of the laminar insertion zone had more intense labeling for collagen types I and VI than did the laminar plates. CONCLUSIONS: The extracellular matrix of the laminar plates of the adult equine lamina cribrosa is similar to the dog as it consists of elastic and collagen fibers (with collagen types VI, III and I). Both the normal dog and horse lamina display more intense staining of collagen type VI than is found in the ECM of the normal human lamina cribrosa. The macromolecular structure of the equine lamina cribrosa suggests that it is a very resilient structure that may provide some protection to the optic nerve axons during episodes of elevated intraocular pressure.

Comparative retinal ganglion cell and optic nerve morphology.

The optic nerve is divided in four regions: intraocular, intraorbital, intracanalicular, and intracranial. The vertebrate retinal ganglion cells are classified by morphology, physiology and soma size. Species differences and similarities occur with retinal ganglion cells. Alpha retinal ganglion cells have large somata, large dendritic fields, large-diameter axons, and are most dense in the peripheral retina. Beta retinal ganglion cells have smaller diameter somata, smaller dendritic fields, small diameter axons, and predominate in the central retina. Gamma retinal ganglion cells are a heterogenous class of cells and have small diameter axons, and slow axon conduction velocities. The spatial distribution and organization of the retinal ganglion cells extends retinotopically through the nerve fiber layer, optic nerve, optic chiasm, optic tract, lateral geniculate nucleus, and visual cortex. The retinal nerve fiber layer thickness decreases from the optic disk toward the periphery of the retina. The retrobulbar optic nerve axon counts and axon density vary by species, with larger nerves having higher axon counts. Decussation of the optic nerve axons at the optic chiasm varies with 100% decussation in most birds and fish, 65% in cats, 75% in dogs, 80-90% in large animals, and 50% in primates. Centrifugal axons also occur in the optic nerve and may represent a method by which the brain can influence retinal activity.

Comparative optic nerve physiology: implications for glaucoma, neuroprotection, and neuroregeneration.

The axoplasm of optic nerve axons moves bidirectionally at various speeds along an intra-axonal pressure gradient from the retinal ganglion cell (RGC) somata toward its synapse, and from the synapse towards the RGC somata. The axoplasmic flow of optic nerve axons is precarious even at normal intraocular pressures (IOP) as it moves from the intraocular optic nerve through the scleral lamina cribrosa to the intraorbital optic nerve. The scleral lamina cribrosa is not simply a porous region of the sclera but a specialized extracellular matrix of the central nervous system whose movement during fluctuations in IOP can affect optic nerve axoplasmic flow. The abundant optic nerve blood supply maintains adequate optic nerve head perfusion through a process of vascular autoregulation. Glaucoma is associated with reduced optic nerve axoplasmic flow and compromised optic nerve circulation such that RGC death due to glutamate excitotoxicity and neurotrophin deprivation result.

Diurnal intraocular pressure curves in healthy rhesus macaques (Macaca mulatta) and rhesus macaques with normotensive and hypertensive primary open-angle glaucoma.

PURPOSE: The authors identify any diurnal intraocular pressure (IOP) variation in healthy Cayo Santiago macaques, as well as Cayo Santiago macaques with normotensive and hypertensive primary open-angle glaucoma (POAG), to further evaluate their potential value as a model for human POAG. METHODS: Twenty-four monkeys (eight animals each of the healthy control, normotensive glaucoma, and hypertensive glaucoma groups) were sedated with ketamine hydrochloride and the IOP measured hourly from 8:00 AM until 3:00 PM with a Tonopen XL applanation tonometer (Mentor, Norwell, MA, U.S.A.). Mean IOP time profiles, mean IOPs at each observation time, and linear trends in mean IOP over time were compared among groups. Intraocular pressure rate-of-change over time and residual variation about the regression line for each individual eye were analyzed. Cup/disc ratio (C/D) was compared with baseline IOP, IOP profile mean and slope, and IOP residual variation. RESULTS: A significant group and time effect on mean IOP (p = 0.0001 and 0.011, respectively), with highest values at 2:00 PM or 3:00 PM, and a significant increasing linear trend in mean IOP over time in the hypertensive group were observed (p = 0.012). Intermediate readings between control and hypertensive mean IOPs were identified for the normotensive glaucoma group. The biggest range of IOP variation was found in the hypertensive glaucoma monkeys. Higher variations in IOP did not lead to an increase in C/D. CONCLUSIONS: This study provides evidence that the Cayo Santiago macaques are a valuable model for human normotensive and hypertensive POAG.

Histomorphometry of the porcine scleral lamina cribrosa surface.

The lamina cribrosa surface of the pig was examined using trypsin digestion, scanning electron microscopy, and computerized image analysis. Six normal eyes from healthy pigs were studied. The total intralaminar scan area, total number of laminar pores, median individual laminar pore areas, median individual pore form factors, and mean pore density were determined for the dorsal, ventral, nasal and temporal hemicircles, the dorsal-nasal, dorsal-temporal, ventral-nasal, and ventral-temporal quadrants, and their central and peripheral subdivisions. The mean (+/- SD) total intralaminar scan area was 8.29 +/- 1.54 mm2. The mean (+/- SD) total laminar pore count was 517 +/- 73 pores. The mean pore count was significantly larger in the ventral than the dorsal hemicircle (292 +/- 39 vs. 225 +/- 38 pores, respectively; P = 0.001), and significantly greater in the periphery compared to the center (388 +/- 58 vs. 129 +/- 27 pores, respectively; P = 0.0001). The overall mean (+/- SD) pore density was 67 +/- 7 pores mm-2. Mean pore density was significantly greater in the ventral than the dorsal hemicircle (70 +/- 8 vs. 64 +/- 6 pores mm-2, respectively; P = 0.019), and significantly greater in the center compared to the periphery (75 +/- 9 vs. 60 +/- 8 pores mm-2, respectively; P = 0.020). The mean (+/- SD) median individual pore area was 3752 +/- 572 &mgr;m2. The mean (+/- SD) median pore form factor was 0.680 +/- 0.035. No significant regional differences were found in mean median pore form factor or mean median individual pore areas. The intralaminar optic nerve of pigs is 55.1% non-neural connective and vascular tissue. A pigmented ventral fascial groove in the scleral lamina cribrosa appears unique to the porcine lamina cribrosa, and may be a vestige of the embryonic optic fissure.

Primary adenocarcinoma of the gland of the nictitating membrane in a cat.

An 11-year-old, neutered, male domestic shorthair was presented with a five-month history of recurrent, unilateral, seromucoid discharge from the right eye. A verrucous mass extended from the posterior aspect of the nictitating membrane. Adenocarcinoma of the gland of the nictitating membrane (GNM) was diagnosed upon biopsy. The cat subsequently developed metastases to the lungs, pleura, mediastinum, liver, and kidneys and died six months after clinical signs first were observed. Little is known about the biological behavior of adenocarcinoma of the GNM in cats. This is the first report that describes the natural progression of this disease.