Endoscopic diagnostic and surgical orbital approach in canines.

The aim of this study is to describe new diagnostic and surgical orbital approaches using video endoscopy in canines. Four different endoscopic approaches were investigated in this study of video endoscopy in cadavers: dorsal transorbital ligament approach via incision of the orbital ligament (DTOLA), dorsal subpalpebral transconjunctival approach (DSTA), ventral subpalpebral transconjunctival approach (VSTA), and transoral orbital approach (TOA). Two additional approaches, the ventral transpalpebral approach (VTA) and dorsal caudal transmuscular approach (DCTA) along with the DTOLA and DSTA were used in clinical patients. The most technically demanding approach was DTOLA; however, it provided the best visualisation of different anterior and posterior orbital structures. Visualisation of primarily the dorsal orbital wall, dorsal portion of the eye globe, and dorsal extraconal space also was achieved by DSTA. The VSTA enabled good visualisation of the ventral orbital floor and the ventral extraconal and intraconal space. In contrast, the TOA provided relatively poor visualisation of orbital structures, limited to the ventral orbital quadrant. Meanwhile, the VTA provided visualisation similar to the VSTA, while DCTA visualisation was limited to the dorsal and caudal orbital space. Orbital endoscopy is an effective and minimally invasive procedure that can be used for diagnostic and surgical orbital procedures.

Presumed cancer-associated retinopathy (CAR) mimicking Sudden Acquired Retinal Degeneration Syndrome (SARDS) in canines.

OBJECTIVE: To describe functional and structural features of presumed cancer-associated retinopathy (CAR) mimicking sudden acquired retinal degeneration syndrome (SARDS) in dogs and describe treatment outcomes. ANIMALS: Subjects were 17 dogs from 8 eight US states and Canada diagnosed with SARDS or immune-mediated retinitis (IMR) by 12 ophthalmologists. Nine eyes from seven deceased patients were used for microarray (MA), histology, or immunohistochemical (IHC) analysis. PROCEDURES: Dogs underwent complete ophthalmic examination, including retinal photography, optical coherence tomography (OCT), chromatic pupil light reflex testing (cPLR), and electroretinography (ERG), in addition to complete systemic examination. Histology, microarray, and IHC analysis were performed in CAR retinas to evaluate histological and molecular changes in retinal tissue. RESULTS: None of the patients evaluated satisfied previously established criteria for diagnosis of SARDS (flat ERG+ no red - good blue PLR), and all were diagnosed with IMR. All patients were diagnosed with a cancer: meningioma (24%), sarcoma (18%), pituitary tumor (12%), and squamous cell carcinoma (12%), other (34%). Median survival time was 6 months from diagnosis (range 1-36 months). Most frequent systemic abnormalities were as follows: proteinuria (78%); elevated liver enzymes (47%); and metabolic changes (PU/PD, polyphagia - 24%). Immunosuppressive therapy resulted in the reversal of blindness in 44% of treated patients, with 61% of all treated patients recovering and/or maintaining vision. Median time for preservation of vision was 5 months (range 1-35 months). CONCLUSIONS: Observed changes are highly suggestive of immune-mediated damage in IMR-CAR eyes. A relatively high percentage of patients with CAR responded positively to immunosuppressive therapy.

The future of canine glaucoma therapy.

Canine glaucoma is a group of disorders that are generally associated with increased intraocular pressure (IOP) resulting in a characteristic optic neuropathy. Glaucoma is a leading cause of irreversible vision loss in dogs and may be either primary or secondary. Despite the growing spectrum of medical and surgical therapies, there is no cure, and many affected dogs go blind. Often eyes are enucleated because of painfully high, uncontrollable IOP. While progressive vision loss due to primary glaucoma is considered preventable in some humans, this is mostly not true for dogs. There is an urgent need for more effective, affordable treatment options. Because newly developed glaucoma medications are emerging at a very slow rate and may not be effective in dogs, work toward improving surgical options may be the most rewarding approach in the near term. This Viewpoint Article summarizes the discussions and recommended research strategies of both a Think Tank and a Consortium focused on the development of more effective therapies for canine glaucoma; both were organized and funded by the American College of Veterinary Ophthalmologists Vision for Animals Foundation (ACVO-VAF). The recommendations consist of (a) better understanding of disease mechanisms, (b) early glaucoma diagnosis and disease staging, (c) optimization of IOP-lowering medical treatment, (d) new surgical therapies to control IOP, and (e) novel treatment strategies, such as gene and stem cell therapies, neuroprotection, and neuroregeneration. In order to address these needs, increases in research funding specifically focused on canine glaucoma are necessary.

Optical coherence tomography and molecular analysis of sudden acquired retinal degeneration syndrome (SARDS) eyes suggests the immune-mediated nature of retinal damage.

OBJECTIVE: To perform detailed analysis of retinal changes in dogs with SARDS using optical coherence tomography (OCT), funduscopy, and molecular analysis. ANIMALS: Subjects were 29 dogs from 12 US states and Canada diagnosed with SARDS by 8 ophthalmologists. An additional 7 eyes from 5 deceased SARDS dogs were used for molecular and histological analysis. PROCEDURES: Dogs were evaluated using chromatic pupil light reflex testing (cPLR), and electroretinography (ERG); subjects underwent complete ophthalmic examination, including funduscopy, retinal photography, and OCT, in addition to complete laboratory analysis, blood pressure evaluation, abdominal and thoracic radiographs, and computerized tomography (CT) imaging to assess possible systemic abnormalities. Histology and immunohistochemistry analysis was performed in 2 SARDS eyes. Microarray analysis was performed in 5 SARDS retinas. RESULTS: Thirty-eight percent of patients had <1-mm wide retinal detachments (RD) on OCT analysis, which could not be detected by funduscopy or retinal photographs. Systemic hypertension did not seem to be a contributing factor (RD 22.2%; ND 20%, Odds ratio = 1.1). No dogs showed neoplastic changes by thoracic or abdominal radiography, or CT imaging. There was no statistically significant difference in age (RD 7.9 ± 1.9 years (mean ± SD); ND 7.6 ± 1.7 years, p = 0.69) or duration of blindness prior to presentation (RD 18 ± 7 days (mean±SD); ND 21 ± 12 days, p = 0.28). Microarray and histology analysis of SARDS eyes revealed molecular changes suggestive of immune-mediated damage. CONCLUSIONS: Observed histological, molecular, and OCT changes are highly suggestive of immune-mediated damage in SARDS eyes.

Variants in Nebulin (NEB) Are Linked to the Development of Familial Primary Angle Closure Glaucoma in Basset Hounds.

Several dog breeds are susceptible to developing primary angle closure glaucoma (PACG), which suggests a genetic basis for the disease. We have identified a four-generation Basset Hound pedigree with characteristic autosomal recessive PACG that closely recapitulates PACG in humans. Our aim is to utilize gene mapping and whole exome sequencing approaches to identify PACG-causing sequence variants in the Basset. Extensive clinical phenotyping of all pedigree members was conducted. SNP-chip genotyping was carried out in 9 affected and 15 unaffected pedigree members. Two-point and multipoint linkage analyses of genome-wide SNP data were performed using Superlink-Online SNP-1.1 and a locus was mapped to chromosome 19q with a maximum LOD score of 3.24. The locus contains 12 Ensemble predicted canine genes and is syntenic to a region on chromosome 2 in the human genome. Using exome-sequencing analysis, a possibly damaging, non-synonymous variant in the gene Nebulin (NEB) was found to segregate with PACG which alters a phylogenetically conserved Lysine residue. The association of this variants with PACG was confirmed in a secondary cohort of unrelated Basset Hounds (p = 3.4 × 10-4, OR = 15.3 for homozygosity). Nebulin, a protein that promotes the contractile function of sarcomeres, was found to be prominently expressed in the ciliary muscles of the anterior segment. Our findings may provide insight into the molecular mechanisms that underlie PACG. The phenotypic similarities of disease presentation in dogs and humans may enable the translation of findings made in this study to patients with PACG.

Identification of genetic loci associated with primary angle-closure glaucoma in the basset hound.

PURPOSE: Primary angle-closure glaucoma (PACG) in dogs is usually caused by the gradual collapse of the iridocorneal angle and cleft, eventually leading to aqueous humor (AH) outflow obstruction. The condition occurs in several breeds of dogs and the prognosis for affected animals is typically poor. We have identified several basset hound (BH) pedigrees, as well as unrelated cases with characteristic PACG that in many aspects recapitulates PACG in human patients. The goal of this study was to utilize the BH PACG model to characterize the genetics of PACG, and potentially discover genetic factors contributing to PACG in humans and animals. METHODS: We conducted a genome-wide logistic regression test for association using 37 PACG cases and 41 unaffected controls. Population stratification and cryptic relatedness were assessed using a multidimensional scaling analysis. The expression of two candidate genes within the target tissues of the BH eye was assessed by immunohistochemistry. RESULTS: We report significant associations at two novel loci, specifically BICF2P31912 in COL1A2 on chromosome 14 with a per-allele odds ratio (OR, 95% confidence interval [CI]) of 3.35 (1.73-6.51), P(genome)=3.6×10⁻4; and BICF2P893476 residing in proximity to RAB22A on chromosome 24 with a per-allele OR (95% CI) of 3.93 (1.78-8.66), P(genome)=4.9×10⁻4. COL1A2 and RAB22A demonstrated widespread expression throughout the eye and were prominently noted in the ciliary body (CB), trabecular meshwork (TM), and iris. CONCLUSIONS: Our finding of two genetic associations supports the potential segregation of PACG risk-conferring variants in the BH. The genetic associations identified may contribute to mechanisms underlying the pathogenesis of PACG, which remain to be elucidated.

Detection and characterization of glaucoma-like canine retinal tissues using Raman spectroscopy.

Early detection of pathological changes and progression in glaucoma and other neuroretinal diseases remains a great challenge and is critical to reduce permanent structural and functional retina and optic nerve damage. Raman spectroscopy is a sensitive technique that provides rapid biochemical characterization of tissues in a nondestructive and noninvasive fashion. In this study, spectroscopic analysis was conducted on the retinal tissues of seven beagles with acute elevation of intraocular pressure (AEIOP), six beagles with compressive optic neuropathy (CON), and five healthy beagles. Spectroscopic markers were identified associated with the different neuropathic conditions. Furthermore, the Raman spectra were subjected to multivariate discriminate analysis to classify independent tissue samples into diseased/healthy categories. The multivariate discriminant model yielded an average optimal classification accuracy of 72.6% for AEIOP and 63.4% for CON with 20 principal components being used that accounted for 87% of the total variance in the data set. A strong correlation (R2>0.92) was observed between pattern electroretinography characteristics of AEIOP dogs and Raman separation distance that measures the separation of spectra of diseased tissues from normal tissues; however, the underlining mechanism of this correlation remains to be understood. Since AEIOP mimics the pathological symptoms of acute/early-stage glaucoma, it was demonstrated that Raman spectroscopic screening has the potential to become a powerful tool for the detection and characterization of early-stage disease.

Rapid diagnosis of retina and optic nerve abnormalities in canine patients with and without cataracts using chromatic pupil light reflex testing.

OBJECTIVE: To develop fast and reliable testing routines for diagnosing retina and optic nerve diseases in canine cataract patients based on chromatic properties of the pupillary light reflex response. PROCEDURES: Seventy-seven canine patients with a history of cataract and decreased vision (43 patients with cataracts and no evidence of retina or optic nerve disease, 21 patients with cataracts and retinal degeneration [RD], 13 patients with cataracts and retinal detachment [RDT]), 11 canine patients with optic neuritis (ON) and 23 healthy dogs were examined using chromatic pupillary light reflex (cPLR) analysis with red and blue light and electroretinography. RESULTS: Electroretinography analysis showed statistically significant deficits in a- and b-wave amplitudes in dogs with cataracts and RD, or cataracts and RDT, when compared to dogs with cataracts without evidence of retinal abnormalities. Evaluation of b-wave amplitudes showed that presence of 78.5-µV (or lower) amplitudes had high sensitivity of 100% (95% CI: 87.2-100%) and high specificity of 96.7% (95% CI: 88.4-100%) in RD and RDT. Evaluation of cPLR responses using red light showed that presence of the pupil end constriction diameter of 5.5 mm (or higher) had moderately high sensitivity of 76.5% (95% CI: 50.1-93.2%) and high specificity of 100% (95% CI: 91.2-100%) in detecting RD and RDT. Optic neuritis patients had absent cPLR responses, regardless of the visual status. CONCLUSIONS AND CLINICAL RELEVANCE: Chromatic evaluation of the pupillary light reflex is a rapid and accurate test for diagnosing retina and optic nerve diseases in canine patients.

Characterization of structure and function of the mouse retina using pattern electroretinography, pupil light reflex, and optical coherence tomography.

OBJECTIVE: To perform in vivo analysis of retinal functional and structural parameters in healthy mouse eyes. ANIMAL STUDIED: Adult C57BL/6 male mice (n = 37). PROCEDURES: Retinal function was evaluated using pattern electroretinography (pERG) and the chromatic pupil light reflex (cPLR). Structural properties of the retina and nerve fiber layer (NFL) were evaluated using spectral-domain optical coherence tomography (SD-OCT). RESULTS: The average pERG amplitudes were found to be 11.2 ± 0.7 µV (P50-N95, mean ± SEM), with an implicit time for P50-N95 interval of 90.4 ± 5.4 ms. Total retinal thickness was 229.5 ± 1.7 µm (mean ± SEM) in the area centralis region. The thickness of the retinal nerve fiber layer (mean ± SEM) using a circular peripapillary retinal scan centered on the optic nerve was 46.7 ± 0.9 µm (temporal), 46.1 ± 0.9 µm (superior), 45.8 ± 0.9 µm (nasal), and 48.4 ± 1 µm (inferior). The baseline pupil diameter was 2.1 ± 0.05 mm in darkness, and 1.1 ± 0.05 and 0.56 ± 0.03 mm after stimulation with red (630 nm, luminance 200 kcd/m(2)) or blue (480 nm, luminance 200 kcd/m(2)) light illumination, respectively. CONCLUSIONS: Pattern electroretinography, cPLR and SD-OCT analysis are reproducible techniques, which can provide important information about retinal and optic nerve function and structure in mice.

Topical ocular sodium 4-phenylbutyrate rescues glaucoma in a myocilin mouse model of primary open-angle glaucoma.

PURPOSE: Mutations in the myocilin gene (MYOC) are the most common known genetic cause of primary open-angle glaucoma (POAG). The purpose of this study was to determine whether topical ocular sodium 4-phenylbutyrate (PBA) treatment rescues glaucoma phenotypes in a mouse model of myocilin-associated glaucoma (Tg-MYOC(Y437H) mice). METHODS: Tg-MYOC(Y437H) mice were treated with PBA eye drops (n = 10) or sterile PBS (n = 8) twice daily for 5 months. Long-term safety and effectiveness of topical PBA (0.2%) on glaucoma phenotypes were examined by measuring intraocular pressure (IOP) and pattern ERG (PERG), performing slit lamp evaluation of the anterior chamber, analyzing histologic sections of the anterior segment, and comparing myocilin levels in the aqueous humor and trabecular meshwork of Tg-MYOC(Y437H) mice. RESULTS: Tg-MYOC(Y437H) mice developed elevated IOP at 3 months of age when compared with wild-type (WT) littermates (n = 24; P < 0.0001). Topical PBA did not alter IOP in WT mice. However, it significantly reduced elevated IOP in Tg-MYOC(Y437H) mice to the level of WT mice. Topical PBA-treated Tg-MYOC(Y437H) mice also preserved PERG amplitudes compared with vehicle-treated Tg-MYOC(Y437H) mice. No structural abnormalities were observed in the anterior chamber of PBA-treated WT and Tg-MYOC(Y437H) mice. Analysis of the myocilin in the aqueous humor and TM revealed that PBA significantly improved the secretion of myocilin and reduced myocilin accumulation as well as endoplasmic reticulum (ER) stress in the TM of Tg-MYOC(Y437H) mice. Furthermore, topical PBA reduced IOP elevated by induction of ER stress via tunicamycin injections in WT mice. CONCLUSIONS: Topical ocular PBA reduces glaucomatous phenotypes in Tg-MYOC(Y437H) mice, most likely by reducing myocilin accumulation and ER stress in the TM. Topical ocular PBA could become a novel treatment for POAG patients with myocilin mutations.

Spectral domain optical coherence tomography (SD-OCT) assessment of the healthy female canine retina and optic nerve.

OBJECTIVE: To provide normative data for canine whole retinal thickness (WRT), nerve fiber layer thickness (NFL), photoreceptor layer thickness (PR), and outer nuclear layer thickness (ONL) using spectral domain optical coherence tomography. ANIMAL STUDIED: Twelve healthy adult intact female beagles. PROCEDURE: Horizontal volume scans through the area dorso-temporal from the optic nerve (superior retina), and the area ventro-temporal from the optic nerve (inferior retina) were used to evaluate the thickness of retinal NFL, PR, ONL, and WRT. Peripapillary circular scans were used to evaluate NFL thickness. Statistical analyses were performed to compare the thickness of the individual layers between the superior and inferior retina (paired t-test). One-way analysis of variance (ANOVA) was used to compare the thickness of peripapillary NFL between the superior, inferior, temporal and nasal quadrants of the circle scan. RESULTS: The WRT, PR, and NFL thickness were greater in the superior than in the inferior retina (198.7 ± 9.6 µm vs. 164.4 ± 6.4 µm, P < 0.0001; 95.5 ± 6.5 µm vs. 78.8 ± 7.4 µm, P < 0.0001; and 26.4 ± 1.6 µm vs. 25.0 ± 1.9 µm, P = 0.0236, respectively). No statistical difference was found between the ONL thickness of the superior and inferior retina (50.1 ± 6.4 µm vs. 44.3 ± 3.6, P = 0.0578). Peripapillary NFL thickness showed a similar tendency as the linear scans, with the superior quadrant having the greatest thickness (91.26 ± 7.0 µm) and the inferior quadrant being the thinnest (76.42 ± 9.2 µm) (P < 0.001). CONCLUSIONS: Results of our in vivo studies showed significant differences between thickness values for the superior (tapetal) and inferior (nontapetal) retinal regions.

Exploring Raman spectroscopy for the evaluation of glaucomatous retinal changes.

Glaucoma is a chronic neurodegenerative disease characterized by apoptosis of retinal ganglion cells and subsequent loss of visual function. Early detection of glaucoma is critical for the prevention of permanent structural damage and irreversible vision loss. Raman spectroscopy is a technique that provides rapid biochemical characterization of tissues in a nondestructive and noninvasive fashion. In this study, we explored the potential of using Raman spectroscopy for detection of glaucomatous changes in vitro. Raman spectroscopic imaging was conducted on retinal tissues of dogs with hereditary glaucoma and healthy control dogs. The Raman spectra were subjected to multivariate discriminant analysis with a support vector machine algorithm, and a classification model was developed to differentiate disease tissues versus healthy tissues. Spectroscopic analysis of 105 retinal ganglion cells (RGCs) from glaucomatous dogs and 267 RGCs from healthy dogs revealed spectroscopic markers that differentiated glaucomatous specimens from healthy controls. Furthermore, the multivariate discriminant model differentiated healthy samples and glaucomatous samples with good accuracy [healthy 89.5% and glaucomatous 97.6% for the same breed (Basset Hounds); and healthy 85.0% and glaucomatous 85.5% for different breeds (Beagles versus Basset Hounds)]. Raman spectroscopic screening can be used for in vitro detection of glaucomatous changes in retinal tissue with a high specificity.

Reduction of ER stress via a chemical chaperone prevents disease phenotypes in a mouse model of primary open angle glaucoma.

Mutations in myocilin (MYOC) are the most common genetic cause of primary open angle glaucoma (POAG), but the mechanisms underlying MYOC-associated glaucoma are not fully understood. Here, we report the development of a transgenic mouse model of POAG caused by the Y437H MYOC mutation; the mice are referred to herein as Tg-MYOC(Y437H) mice. Analysis of adult Tg-MYOC(Y437H) mice, which we showed express human MYOC containing the Y437H mutation within relevant eye tissues, revealed that they display glaucoma phenotypes (i.e., elevated intraocular pressure [IOP], retinal ganglion cell death, and axonal degeneration) closely resembling those seen in patients with POAG caused by the Y437H MYOC mutation. Mutant myocilin was not secreted into the aqueous humor but accumulated in the ER of the trabecular meshwork (TM), thereby inducing ER stress in the TM of Tg-MYOC(Y437H) mice. Furthermore, chronic and persistent ER stress was found to be associated with TM cell death and elevation of IOP in Tg-MYOC(Y437H) mice. Reduction of ER stress with a chemical chaperone, phenylbutyric acid (PBA), prevented glaucoma phenotypes in Tg-MYOC(Y437H) mice by promoting the secretion of mutant myocilin in the aqueous humor and by decreasing intracellular accumulation of myocilin in the ER, thus preventing TM cell death. These results demonstrate that ER stress is linked to the pathogenesis of POAG and may be a target for treatment in human patients.

Transplantation of BDNF-secreting mesenchymal stem cells provides neuroprotection in chronically hypertensive rat eyes.

PURPOSE: To evaluate the ability of mesenchymal stem cells (MSCs) engineered to produce and secrete brain-derived neurotrophic factor (BDNF) to protect retinal function and structure after intravitreal transplantation in a rat model of chronic ocular hypertension (COH). METHODS: COH was induced by laser cauterization of trabecular meshwork and episcleral veins in rat eyes. COH eyes received an intravitreal transplant of MSCs engineered to express BDNF and green fluorescent protein (BDNF-MSCs) or just GFP (GFP-MSCs). Computerized pupillometry and electroretinography (ERG) were performed to assess optic nerve and retinal function. Quantification of optic nerve damage was performed by counting retinal ganglion cells (RGCs) and evaluating optic nerve cross-sections. RESULTS: After transplantation into COH eyes, BDNF-MSCs preserved significantly more retina and optic nerve function than GFP-MSC-treated eyes when pupil light reflex (PLR) and ERG function were evaluated. PLR analysis showed significantly better function (P = 0.03) in BDNF-MSC-treated eyes (operated/control ratio = 63.00% ± 11.39%) than GFP-MSC-treated eyes (operated/control ratio = 31.81% ± 9.63%) at 42 days after surgery. The BDNF-MSC-transplanted eyes also displayed a greater level of RGC preservation than eyes that received the GFP-MSCs only (RGC cell counts: BDNF-MSC-treated COH eyes, 112.2 ± 19.39 cells/section; GFP-MSC-treated COH eyes, 52.21 ± 11.54 cells/section; P = 0.01). CONCLUSIONS: The authors have demonstrated that lentiviral-transduced BDNF-producing MSCs can survive in eyes with chronic hypertension and can provide retina and optic nerve functional and structural protection. Transplantation of BDNF-producing stem cells may be a viable treatment strategy for glaucoma.

Neuroinflammation in advanced canine glaucoma.

PURPOSE: The pathophysiological events that occur in advanced glaucoma are not well characterized. The principal purpose of this study is to characterize the gene expression changes that occur in advanced glaucoma. METHODS: Retinal RNA was obtained from canine eyes with advanced glaucoma as well as from healthy eyes. Global gene expression patterns were determined using oligonucleotide microarrays and confirmed by real-time PCR. The presence of tumor necrosis factor (TNF) and its receptors was evaluated by immunolabeling. Finally, we evaluated the presence of serum autoantibodies directed against retinal epitopes using western blot analyses. RESULTS: We identified over 500 genes with statistically significant changes in expression level in the glaucomatous retina. Decreased expression levels were detected for large number of functional groups, including synapse and synaptic transmission, cell adhesion, and calcium metabolism. Many of the molecules with decreased expression levels have been previously shown to be components of retinal ganglion cells. Genes with elevated expression in glaucoma are largely associated with inflammation, such as antigen presentation, protein degradation, and innate immunity. In contrast, expression of many other pro-inflammatory genes, such as interferons or interleukins, was not detected at abnormal levels. CONCLUSIONS: This study characterizes the molecular events that occur in the canine retina with advanced glaucoma. Our data suggest that in the dog this stage of the disease is accompanied by pronounced retinal neuroinflammation.

Exogenous modulation of intrinsic optic nerve neuroprotective activity.

BACKGROUND: To characterize the molecular and functional status of the rat retina and optic nerve after acute elevation of intraocular pressure (IOP). METHODS: Retinal ischemia was induced in rats by increasing the IOP (110 mmHg/60 minutes). Microarray analysis, quantitative RT-PCR (qRT-PCR) and immunohistochemistry were used to characterize retinal tissue. PLGA microspheres containing neurotrophic factors (BDNF, GDNF, or CNTF) or empty microspheres were injected into the vitreous of operated animals 1 day after elevation of IOP. Pupil light reflex (PLR) parameters and electroretinograms (ERG) were monitored at multiple time points during the 60-day postoperative recovery period. RESULTS: Molecular analysis showed a significant intrinsic up-regulation of CNTF at 10 and 25 days after induction of the acute ocular hypertension (p = 0.0067). Molecular tissue analysis of GDNF and its receptors (GDNFR1, GDNFR2), and BDNF and its receptor (trkB) showed no change in expression. Animals that received CNTF microspheres had no significant functional recovery compared to animals which received blank microspheres (p > 0.05). Animals that received GDNF or BDNF microspheres showed significant PLR recovery (p < 0.05 and p < 0.001 respectively) compared to non-treated animals. CONCLUSIONS: Continuous release of neurotrophic growth factors (NGFs) significantly protects optic nerve function in the experimental model of retinal ischemia observed by PLR analysis.

Functional and structural changes in a canine model of hereditary primary angle-closure glaucoma.

PURPOSE: To characterize functional and structural changes in a canine model of hereditary primary angle-closure glaucoma. METHODS: Intraocular pressure (IOP) was evaluated with tonometry in a colony of glaucomatous dogs at 8, 15, 18, 20, and 30 months of age. Retinal function was evaluated using electroretinography (scotopic, photopic, and pattern). Examination of anterior segment structures was performed using gonioscopy and high-frequency ultrasonography (HFU). RESULTS: A gradual rise in IOP was observed with an increase in age: 8 months, 14 mm Hg (median value); 15 months, 15.5 mm Hg; 18 months, 17.5 mm Hg; 20 months, 24 mm Hg; 30 months, 36 mm Hg. Provocative testing with mydriatic agents (tropicamide and atropine 1%) caused significant increases in IOP (35% and 50%, respectively). HFU analysis showed complete collapse of iridocorneal angles by 20 months of age. Scotopic and photopic ERG analysis did not reveal significant deficits, but pattern ERG analysis showed significantly reduced amplitudes in glaucomatous dogs (glaucoma, 3.5 +/- 0.4 muV; control, 6.2 +/- 0.3 muV; P = 0.004; Student's t-test). Histologic analysis revealed collapse of the iridocorneal angle, posterior bowing of the lamina cribrosa, swelling and loss of large retinal ganglion cells, increased glial reactivity, and increased thickening of the lamina cribrosa. CONCLUSIONS: Canine hereditary angle-closure glaucoma is characterized by a progressive increase in intraocular pressure, loss of optic nerve function, and retinal ganglion cell loss.

Brain-derived neurotrophic factor released from engineered mesenchymal stem cells attenuates glutamate- and hydrogen peroxide-mediated death of staurosporine-differentiated RGC-5 cells.

The purpose of this study was to determine the viability of cell-based delivery of brain-derived neurotrophic factor (BDNF) from genetically modified mesenchymal stem cells (MSCs) for neuroprotection of RGC-5 cells. RGC-5 cells were differentiated with the protein kinase inhibitor staurosporine (SS) and exposed to the cellular stressors glutamate or H2O2. As a neuroprotective strategy, these cells were then co-cultured across a membrane insert with mesenchymal stem cells (MSCs) engineered with a lentiviral vector for production of BDNF (BDNF-MSCs). As a positive control, recombinant human BDNF (rhBDNF) was added to stressed RGC-5 cells. After SS-differentiation RGC-5s developed neuronal-like morphologies, and a significant increase in the proportion of RGC-5s immunoreactive for TuJ-1 and Brn3a was observed. Differentiated RGC-5s also had prominent TrkB staining, demonstrating expression of the high-affinity BDNF receptor. Treatment of SS-differentiated RGC-5s with glutamate or H2O2, produced significant cell death (56.0 +/- 7.02 and 48.90 +/- 4.58% of control cells, respectively) compared to carrier-solution treated cells. BDNF-delivery from MSCs preserved more RGC-5 cells after treatment with glutamate (80.0 +/- 5.40% cells remaining) than control GFP expressing MSCs (GFP-MSCs, 57.29 +/- 1.89%, p < 0.01). BDNF-MSCs also protected more RGC-5s after treatment with H2O2 (65.6 +/- 3.47%) than GFP-MSCs (46.0 +/- 4.20%, p < 0.01). We have shown survival of differentiated RGC-5s is reduced by the cellular stressors glutamate and H2O2. Additionally, our results demonstrate that genetically modified BDNF-producing MSCs can enhance survival of stressed RGC-5 cells and therefore, may be effective vehicles to deliver BDNF to retinal ganglion cells affected by disease.

Intestinal cytokine mRNA expression in canine inflammatory bowel disease: a meta-analysis with critical appraisal.

Data implicating mucosal cytokines in the pathogenesis of canine inflammatory bowel disease (IBD) are limited. The aims of the present study were to report new findings of intestinal cytokine expression in dogs with IBD and to compare these data with previous studies through meta-analysis. Cytokine mRNA abundance in intestinal biopsies collected prospectively was evaluated by using a semiquantitative RT-PCR technique. For meta-analysis, an electronic database search revealed 3 clinical trials, all of which were nonrandomized (type III) case series. Prospective analysis showed that the intestines of healthy dogs and those with IBD express numerous cytokines and that a proinflammatory expression profile is not a feature of small or large-intestinal IBD. The meta-analysis data included 158 dogs characterized as healthy (n = 45), diarrheic nonIBD dogs (n = 6), nonresponders (n = 2), small-intestinal IBD (n = 41), colonic IBD (n = 25), and chronic enteropathy (n = 39). German shepherd dogs were overrepresented in 3 of the 4 studies. Healthy dogs showed mRNA expression for most cytokines including IL2, IL4, IL5, IL10, IL12, IFNgamma, TNFalpha, and TGFbeta. Only IL12 mRNA expression was increased consistently in small-intestinal IBD, whereas IBD colitis lacked consistent patterns of expression. In summary, dogs with IBD fail to express a predominant Th1- or Th2 cytokine bias in inflamed mucosa. Heterogeneity of results among these studies might be explained by numerous factors including the method of mRNA quantification, stage of disease, and demographic differences in study populations.