A Spontaneous Nonhuman Primate Model of Myopic Foveoschisis.

Purpose: Foveoschisis involves the pathologic splitting of retinal layers at the fovea, which may occur congenitally in X-linked retinoschisis (XLRS) or as an acquired complication of myopia. XLRS is attributed to functional loss of the retinal adhesion protein retinoschisin 1 (RS1), but the pathophysiology of myopic foveoschisis is unclear due to the lack of animal models. Here, we characterized a novel nonhuman primate model of myopic foveoschisis through clinical examination and multimodal imaging followed by morphologic, cellular, and transcriptional profiling of retinal tissues and genetic analysis. Methods: We identified a rhesus macaque with behavioral and anatomic features of myopic foveoschisis, and monitored disease progression over 14 months by fundus photography, fluorescein angiography, and optical coherence tomography (OCT). After necropsy, we evaluated anatomic and cellular changes by immunohistochemistry and transcriptomic changes using single-nuclei RNA-sequencing (snRNA-seq). Finally, we performed Sanger and whole exome sequencing with focus on the RS1 gene. Results: Affected eyes demonstrated posterior hyaloid traction and progressive splitting of the outer plexiform layer on OCT. Immunohistochemistry showed increased GFAP expression in Müller glia and loss of ramified Iba-1+ microglia, suggesting macro- and microglial activation with minimal photoreceptor alterations. SnRNA-seq revealed gene expression changes predominantly in cones and retinal ganglion cells involving chromatin modification, suggestive of cellular stress at the fovea. No defects in the RS1 gene or its expression were detected. Conclusions: This nonhuman primate model of foveoschisis reveals insights into how acquired myopic traction leads to phenotypically similar morphologic and cellular changes as congenital XLRS without alterations in RS1.

CRISPR-based VEGF suppression using paired guide RNAs for treatment of choroidal neovascularization.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based genomic disruption of vascular endothelial growth factor A (Vegfa) with a single gRNA suppresses choroidal neovascularization (CNV) in preclinical studies, offering the prospect of long-term anti-angiogenesis therapy for neovascular age-related macular degeneration (AMD). Genome editing using CRISPR-CRISPR-associated endonucleases (Cas9) with multiple guide RNAs (gRNAs) can enhance gene-ablation efficacy by augmenting insertion-deletion (indel) mutations with gene truncations but may also increase the risk of off-target effects. In this study, we compare the effectiveness of adeno-associated virus (AAV)-mediated CRISPR-Cas9 systems using single versus paired gRNAs to target two different loci in the Vegfa gene that are conserved in human, rhesus macaque, and mouse. Paired gRNAs increased Vegfa gene-ablation rates in human cells in vitro but did not enhance VEGF suppression in mouse eyes in vivo. Genome editing using paired gRNAs also showed a similar degree of CNV suppression compared with single-gRNA systems. Unbiased genome-wide analysis using genome-wide unbiased identification of double-stranded breaks (DSBs) enabled by sequencing (GUIDE-seq) revealed weak off-target activity arising from the second gRNA. These findings suggest that in vivo CRISPR-Cas9 genome editing using two gRNAs may increase gene ablation but also the potential risk of off-target mutations, while the functional benefit of targeting an additional locus in the Vegfa gene as treatment for neovascular retinal conditions is unclear.

Host Immune Responses after Suprachoroidal Delivery of AAV8 in Nonhuman Primate Eyes.

The suprachoroid is a potential space located between the sclera and choroid of the eye, which provides a novel route for ocular drug or viral vector delivery. Suprachoroidal injection of adeno-associated virus (AAV)8 using transscleral microneedles enables widespread transgene expression in eyes of nonhuman primates, but may cause intraocular inflammation. We characterized the host humoral and cellular immune responses after suprachoroidal delivery of AAV8 expressing green fluorescent protein (GFP) in rhesus macaques, and found that it can induce mild chorioretinitis that resolves after systemic corticosteroid administration, with recovery of photoreceptor morphology, but persistent immune cell infiltration after 3 months, corresponding to a loss of GFP expression from retinal pigment epithelial cells, but persistent expression in scleral fibroblasts. Suprachoroidal AAV8 triggered B cell and T cell responses against GFP, but only mild antibody responses to the viral capsid compared to intravitreal injections of the same vector and dose. Systemic biodistribution studies showed lower AAV8 levels in liver and spleen after suprachoroidal injection compared with intravitreal delivery. Our findings suggest that suprachoroidal AAV8 primarily triggers host immune responses to GFP, likely due to sustained transgene expression in scleral fibroblasts outside the blood-retinal barrier, but elicits less humoral immune reactivity to the viral capsid than intravitreal delivery due to lower egress into systemic circulation. As GFP is not native to primates and not a clinically relevant transgene, suprachoroidal AAV delivery of human transgenes may have significant translational potential for retinal gene therapy.

Retinal vascular changes after glial disruption in rats.

Glial dysfunction is found in a number of retinal vascular diseases but its link with blood-retinal barrier (BRB) breakdown remains poorly understood. The present study tested the hypothesis that glial dysfunction is a major contributor to the BRB breakdown that is a hallmark of retinal vascular diseases. We investigated the specificity of the purportedly selective glial toxin, DL-alpha-aminoadipic acid (DL-alpha-AAA) on different types of ocular cells in vitro and then tested the effect of glial disruption on retinal vasculature after intraocular injection of DL-alpha-AAA or siRNA targeting glutamine synthetase (GS) in rats. DL-alpha-AAA was toxic to astrocytes and Müller cells but not to other types of BRB-related cells in vitro. Subretinal injection of DL-alpha-AAA disrupted retinal glial cells, induced vascular telangiectasis and increased vascular permeability from 4 days to over 2 months post-injection. Vascular changes induced by DL-alpha-AAA were observed predominantly in regions of glial disruption, as reflected by reduced expression of GS and increased expression of glial fibrillary acidic protein and vimentin. Confocal microscopy showed changes in all three layers of the retinal vasculature, which co-localised with areas of Müller cell disruption. Double labeling immunohistochemistry revealed that retinal glial disruption after DL-alpha-AAA injection was accompanied by increased expression of vascular endothelial growth factor and reduced expression of the tight junction protein claudin-5. Intravitreal injection of GS siRNA induced similar changes in Müller cells and BRB breakdown. Our data are consistent with the hypothesis that glial dysfunction is a primary contributor to the BRB breakdown in retinal vascular diseases.

CRISPR Technology for Ocular Angiogenesis.

Among genome engineering tools, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based approaches have been widely adopted for translational studies due to their robustness, precision, and ease of use. When delivered to diseased tissues with a viral vector such as adeno-associated virus, direct genome editing can be efficiently achieved in vivo to treat different ophthalmic conditions. While CRISPR has been actively explored as a strategy for treating inherited retinal diseases, with the first human trial recently initiated, its applications for complex, multifactorial conditions such as ocular angiogenesis has been relatively limited. Currently, neovascular retinal diseases such as retinopathy of prematurity, proliferative diabetic retinopathy, and neovascular age-related macular degeneration, which together constitute the majority of blindness in developed countries, are managed with frequent and costly injections of anti-vascular endothelial growth factor (anti-VEGF) agents that are short-lived and burdensome for patients. By contrast, CRISPR technology has the potential to suppress angiogenesis permanently, with the added benefit of targeting intracellular signals or regulatory elements, cell-specific delivery, and multiplexing to disrupt different pro-angiogenic factors simultaneously. However, the prospect of permanently suppressing physiologic pathways, the unpredictability of gene editing efficacy, and concerns for off-target effects have limited enthusiasm for these approaches. Here, we review the evolution of gene therapy and advances in adapting CRISPR platforms to suppress retinal angiogenesis. We discuss different Cas9 orthologs, delivery strategies, and different genomic targets including VEGF, VEGF receptor, and HIF-1α, as well as the advantages and disadvantages of genome editing vs. conventional gene therapies for multifactorial disease processes as compared to inherited monogenic retinal disorders. Lastly, we describe barriers that must be overcome to enable effective adoption of CRISPR-based strategies for the management of ocular angiogenesis.

Suprachoroidal and Subretinal Injections of AAV Using Transscleral Microneedles for Retinal Gene Delivery in Nonhuman Primates.

Retinal gene therapy using adeno-associated viruses (AAVs) is constrained by the mode of viral vector delivery. Intravitreal AAV injections are impeded by the internal limiting membrane barrier, while subretinal injections require invasive surgery and produce a limited region of therapeutic effect. In this study, we introduce a novel mode of ocular gene delivery in rhesus macaques using transscleral microneedles to inject AAV8 into the subretinal or suprachoroidal space, a potential space between the choroid and scleral wall of the eye. Using in vivo imaging, we found that suprachoroidal AAV8 produces diffuse, peripheral expression in retinal pigment epithelial (RPE) cells, but it elicited local infiltration of inflammatory cells. Transscleral subretinal injection of AAV8 using microneedles leads to focal gene expression with transduction of RPE and photoreceptors, and minimal intraocular inflammation. In comparison, intravitreal AAV8 shows minimal transduction of retinal cells, but elicits greater systemic humoral immune responses. Our study introduces a novel mode of transscleral viral delivery that can be performed without vitreoretinal surgery, with focal or diffuse transgene expression patterns suitable for different applications. The decoupling of local and systemic immune responses reveals important insights into the immunological consequences of AAV delivery to different ocular compartments surrounding the blood-retinal barrier.

Factors Impacting Efficacy of AAV-Mediated CRISPR-Based Genome Editing for Treatment of Choroidal Neovascularization.

Frequent injections of anti-vascular endothelial growth factor (anti-VEGF) agents are a clinical burden for patients with neovascular age-related macular degeneration (AMD). Genomic disruption of VEGF-A using adeno-associated viral (AAV) delivery of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 has the potential to permanently suppress aberrant angiogenesis, but the factors that determine the optimal efficacy are unknown. Here, we investigate two widely used Cas9 endonucleases, SpCas9 and SaCas9, and evaluate the relative contribution of AAV-delivery efficiency and genome-editing rates in vivo to determine the mechanisms that drive successful CRISPR-based suppression of VEGF-A, using a mouse model of laser-induced choroidal neovascularization (CNV). We found that SpCas9 demonstrated higher genome-editing rates, greater VEGF reduction, and more effective CNV suppression than SaCas9, despite similar AAV transduction efficiency between a dual-vector approach for SpCas9 and single-vector system for SaCas9 to deliver the Cas9 orthologs and single guide RNAs (gRNAs). Our results suggest that successful VEGF knockdown using AAV-mediated CRISPR systems may be determined more by the efficiency of genome editing rather than viral transduction and that SpCas9 may be more effective than SaCas9 as a potential therapeutic strategy for CRISPR-based treatment of CNV in neovascular AMD.

Long-term Evolution and Remodeling of Soft Drusen in Rhesus Macaques.

Purpose: To characterize the evolution and structure of soft drusen in aged rhesus macaques using in vivo multimodal retinal imaging and ex vivo histologic and ultrastructural analyses as a nonhuman primate model of early age-related macular degeneration (AMD). Methods: Multimodal imaging including fundus photography, spectral domain optical coherence tomography (SD-OCT), and fundus autofluorescence (FAF) were used to characterize and track individual drusen lesions in 20 aged rhesus macaques (mean age 23.3 ± 2.7 years) with drusenoid lesions over 2 years, followed by semithin histologic analysis and transmission electron microscopy (TEM). Results: Although most drusen gradually increased in size, a portion spontaneously regressed or collapsed over 2 years. Histologic analyses showed that soft drusen exhibit hypertrophy and dysmorphia of overlying retinal pigment epithelium (RPE), as seen in early and intermediate AMD, but do not exhibit RPE atrophy, RPE migration, or photoreceptor degeneration characteristic of advanced AMD. Ultrastructure of soft drusen showed abundant lipid particles within Bruch's membrane and AMD-related basal linear deposits (BlinD) resembling those in human drusen. Conclusions: The dynamic remodeling, histologic findings, and ultrastructural features of soft drusen in aged rhesus macaques support nonhuman primates as an animal model of early AMD and reveal important insights into drusen biogenesis and AMD development.

Quantitative Fundus Autofluorescence in Rhesus Macaques in Aging and Age-Related Drusen.

Purpose: To employ quantitative fundus autofluorescence (qAF) imaging in rhesus macaques to noninvasively assess retinal pigment epithelial (RPE) lipofuscin in nonhuman primates (NHPs) as a model of aging and age-related macular degeneration (AMD). Methods: The qAF imaging was performed on eyes of 26 rhesus macaques (mean age 18.8 ± 8.2 years, range 4-27 years) with normal-appearing fundus or with age-related soft drusen using a confocal scanning laser ophthalmoscope with 488 nm excitation and an internal fluorescence reference. Eyes with soft drusen also underwent spectral-domain optical coherence tomography imaging to measure drusen volume and height of individual drusen lesions. The qAF levels were measured from the perifoveal annular ring (quantitative autofluorescence 8 [qAF8]) using the Delori grid, as well as focally over individual drusen lesions in this region. The association between qAF levels and age, sex, and drusen presence and volume were determined using multivariable regression analysis. Results: Mean qAF levels increased with age (P < 0.001) and were higher in females (P = 0.047). Eyes with soft drusen exhibited reduced mean qAF compared with age-matched normal eyes (P = 0.003), with greater drusen volume showing a trend toward decreased qAF levels. However, qAF levels are focally increased over most individual drusen (P < 0.001), with larger drusen appearing more hyperautofluorescent (R2 = 0.391, P < 0.001). Conclusions: In rhesus macaques, qAF levels are increased with age and female sex, but decreased in eyes with soft drusen, similar to human AMD. However, drusen lesions appear hyperautofluorescent unlike those in humans, suggesting similarities and differences in RPE lipofuscin between humans and NHPs that may provide insight into drusen biogenesis and AMD pathogenesis.

Differentiation of Retinal Glial Cells From Human Embryonic Stem Cells by Promoting the Notch Signaling Pathway.

Dysfunction of retinal glial cells, particularly Müller cells, has been implicated in several retinal diseases. Despite their important contribution to retinal homeostasis, a specific way to differentiate retinal glial cells from human pluripotent stem cells has not yet been described. Here, we report a method to differentiate retinal glial cells from human embryonic stem cells (hESCs) through promoting the Notch signaling pathway. We first generated retinal progenitor cells (RPCs) from hESCs then promoted the Notch signaling pathway using Notch ligands, including Delta-like ligand 4 and Jagged-1. We validated glial cell differentiation with qRT-PCR, immunocytochemistry, western blots and fluorescence-activated cell sorting as we promoted Notch signaling in RPCs. We found that promoting Notch signaling in RPCs for 2 weeks led to upregulation of glial cell markers, including glial fibrillary acidic protein (GFAP), glutamine synthetase, vimentin and cellular retinaldehyde-binding protein (CRALBP). Of these markers, we found the greatest increase in expression of the pan glial cell marker, GFAP. Conversely, we also found that inhibition of Notch signaling in RPCs led to upregulation of retinal neuronal markers including cone-rod homeobox (CRX) and orthodenticle homeobox 2 (OTX2) but with little expression of GFAP. This retinal glial differentiation method will help advance the generation of stem cell disease models to study the pathogenesis of retinal diseases associated with glial dysfunction such as macular telangiectasia type 2. This method may also be useful for the development of future therapeutics such as drug screening and gene editing using patient-derived retinal glial cells.

Innervation of the Flexor Digitorum Profundus: A Systematic Review.

PURPOSE: The aim of this study was to review the innervation of the flexor digitorum profundus (FDP). METHODS: In PubMed and Scopus, terms (Flexor digitorum profundus OR FDP) AND (innervation OR nerve) were used, resulting in 233 and 281 papers, respectively. After excluding 142 duplicates, 73 abstracts were reviewed. Forty-seven abstracts were excluded, 26 full papers were reviewed, and 17 papers were analyzed. RESULTS: In most cases (97.6%), the index FDP was innervated by the anterior interosseous nerve (AIN). Dual innervation from the AIN and ulnar nerve (UN) was observed in 2.4% of papers. In majority (76.8%), the middle FDP received dual innervation from the AIN and the UN. The rest was innervated by the AIN only (22.0%) or the UN only (1.2%). In most cases (85.4%), the ring FDP was innervated by the UN only. The rest (14.6%) received dual innervation from the AIN and the UN. In majority of cases (64.6%), the little FDP was innervated by the UN only. The rest (35.4%) received dual innervation from the AIN and the UN. The AIN entered the FDP at 107.63 (8.80) mm from the elbow, corresponding to 26.75% (2.17%) of the forearm length, measured proximally. The average number of AIN branches to the FDP was 2.27 (1.33). The average number of UN branches to the FDP was 1.37 (0.94). In 8.8% of limbs, a communicating branch supplied the FDP. Among the limbs with a communicating branch, 32.3% had branches supplying the FDP. CONCLUSION: The results of this study may be useful in managing nerve injury patients.

OBJECTIF: La présente étude visait à analyser l'innervation du tendon fléchisseur profond (TFP). MÉTHODOLOGIE: Dans PubMed et Scopus, les chercheurs ont utilisé les termes Flexor digitorum profundus OU FDP ET innervation OU nerve et ont obtenu 233 et 281 articles, respectivement. Après avoir exclu 142 articles dédoublés, ils ont examiné 73 résumés et en ont exclu 47. Ils ont parcouru 26 articles complets et en ont analysé 17. RÉSULTATS: Dans la plupart des cas (97.6%), le TFP de l'index était innervé par le nerf interosseux antérieur (NIA). Les chercheurs ont observé la double innervation par le NIA et le nerf ulnaire (NU) dans 2.4% des cas. Le TFP du majeur était doublement innervé par le NIA et le NU dans la majorité des cas (76.8%). Les autres cas étaient innervés seulement par le NIA (22.0%) ou par le NU (1.2%). Dans la plupart des cas (85.4%), le TFP de l'annulaire était seulement innervé par le NU. Les autres (14.6%) étaient doublement innervés par le NIA et le NU. Le TFP de l'auriculaire était seulement innervé par le NU dans la majorité des cas (64.6%). Les autres (35.4%) étaient doublement innervés par le NIA et le NU. Le NIA pénétrait dans le TFP à 107.63 (8.80) mm du coude, ce qui correspond à une mesure proximale de 26.75% (2.17%) de la longueur de l'avant-bras. Il y avait une moyenne de 2.27 (1.33) ramifications du NIA vers le TFP et de 1.37 (0.94) ramification du NU vers le TFP. Dans 8.8% des membres, une ramification communicante alimentait le TFP; 32.3% étaient alors dotés de ramifications alimentant le TFP. CONCLUSION: Les résultats de la présente étude peuvent être utiles pour la prise en charge des patients ayant une lésion nerveuse.

Differential expression of microRNAs in retinal vasculopathy caused by selective Müller cell disruption.

Vascular changes and photoreceptor degeneration are features of age-related macular degeneration, diabetic retinopathy and macular telangiectasis. We have profiled the differential expression of microRNAs and analysed their target genes in transgenic mice in which induced Müller cell disruption results in photoreceptor degeneration, vascular leak and deep retinal neovascularisation. We identified 9 miRNAs which were differentially expressed during the development of retinal neovascularization and chose miR-200b and its target genes for further study. Using qRT-PCR and western blot analysis, we found that downregulation of miR-200b was negatively correlated with its target genes, including zinc finger E-box binding homeobox (ZEB) 1 and 2 and vascular endothelial growth factor receptor 1. Double immunofluorescence labelling revealed that the newly formed vessels in the outer retina were positive for ZEB2. Furthermore, intravitreal injections of a miR-200b-mimic and anti-miR-200b confirmed the negative correlation of miR-200b and its target gene expression. We also found that the miR-200b-mimic inhibited vascular leak in the established mild vascular lesions, whereas anti-miR-200b promoted it. Taken together, these data suggest that miR-200b may play a role in the development of intraretinal neovascularisation.

Genomic analysis using Affymetrix standard microarray genechips (169 format) in degenerate murine retina.

Microarray is one of the most useful tools for gene expression profiling. The growing development of microarray genechip technology has enabled increasingly sophisticated studies on the differences in gene transcription between diseased and non-diseased tissues and provides clues to their contribution to the disease in a single experiment. Thus, microarray is used in not only in clinical diagnostics, but also to understand pathological processes and identify leads for new treatments. Here, we present a detailed protocol for performing genomic analysis of retinal tissue with Affymetrix genechip microarray together with additional guidelines from the authors.

Profiling of microRNAs involved in retinal degeneration caused by selective Müller cell ablation.

Dysfunction of Müller cells has been implicated in the pathogenesis of several retinal diseases. In order to understand the potential contribution of Müller cells to retinal disease better, we have developed a transgenic model in which foci of Müller cell ablation can be selectively induced. MicroRNAs (miRNAs), small non-coding RNAs that are involved in post-transcriptional modulation, have critical functions in various biological processes. The aim of this study was to profile differential expression of miRNAs and to examine changes in their target genes 2 weeks after Müller cell ablation. We identified 20 miRNAs using the miScript HC PCR array. Data analysis using two target gene prediction databases (TargetScan and mirTarBase) revealed 78 overlapping target genes. DAVID and KEGG pathway analysis suggested that the target genes were generally involved in cell apoptosis, p53, neurotrophin, calcium, chemokine and Jak-STAT signalling pathways. Changes in seven target genes including Cyclin D2, Caspase 9, insulin-like growth factor 1, IL-1 receptor-associated kinase (IRAK), calmodulin (CALM) and Janus kinase 2 (Jak2), were validated with qRT-PCR and western blots. The cellular localisation of cleaved-caspase 9, Cyclin D2, Jak2 and CALM was examined by immunofluorescence studies. We found that the transcription of some miRNAs was positively, rather than negatively, correlated with their target genes. After confirming that overexpressed miR-133a-3p was localised to the outer nuclear layer in the damaged retina, we validated the correlation between miR-133a-3p and one of its predicted target genes, cyclin D2, with a luciferase assay in 661 photoreceptor cells. Results revealed by miRNA profiling, target gene analysis and validation were generally consistent with our previous findings that selective Müller cell ablation causes photoreceptor degeneration and neuroinflammation. Our data on alterations of miRNAs and their target gene expression after Müller cell ablation provide further insights into the potential role of Müller cell dysfunction in retinal disease.

Laser capture microdissection-directed profiling of glycolytic and mTOR pathways in areas of selectively ablated Müller cells in the murine retina.

PURPOSE: We have reported previously down-regulation of key metabolic pathways, the glycolytic and mTOR pathways, from a global retinal microarray analysis after selective Müller cell ablation in a novel transgenic model. The purpose of the present study was to examine changes in expression of key molecules of glycolytic and mTOR pathways specifically in patches of Müller cell loss. METHODS: Eyes were enucleated 1 and 3 months after induced Müller cell ablation, directly embedded in optimal cutting temperature medium, and snap frozen in liquid nitrogen. Laser capture microdissection (LCM) was conducted to dissect patches of Müller cell loss for quantitative RT-PCR (qRT-PCR) analysis of key genes of the glycolytic (glyceraldehyde-3-phosphate dehydrogenase, enolase 1 and 2, lactate dehydrogenase A and B) and mTOR pathways (insulin-like growth factor receptor 1, phosphatidylinositide-3-kinase, Akt1, and regulatory-associated protein of mTOR). Protein validations were performed by immunohistochemistry. RESULTS: The LCM-directed qRT-PCR analysis of Müller cell ablated specimens demonstrated reduced transcription of genes involved in the glycolytic and mTOR metabolic pathways. Of the proteins we chose to study, only enolase 1 was expressed by Müller cells. Other glycolytic and mTOR pathway proteins were expressed by photoreceptor inner and outer segments, which were lost in patches of Müller cell ablation. CONCLUSIONS: We found suppression of genes encoding various glycolytic and mTOR pathway-associated enzymes in areas of Müller cell loss. This appeared mainly to be due to loss of photoreceptor inner and outer segments. The consequences of metabolic derangement caused by Müller cell ablation warrant further investigation.

Differential gene expression profiling after conditional Müller-cell ablation in a novel transgenic model.

PURPOSE: Müller cells, the principal glial cells in the mammalian retina, play an important role in the maintenance of retinal homeostasis. Recent reports suggest that Müller-cell dysfunction may contribute to the pathogenesis of retinal diseases such as idiopathic macular telangiectasia type 2. In the present study, we used microarray to compare retinae isolated from transgenic mice in which the Müller cells of adult mice retinae can be selectively ablated with control mice. METHODS: Retinae were isolated 1 week, 1 month, and 3 months after tamoxifen-induced selective Müller-cell ablation and microarray were performed with Affymatrix microarrays. Differentially expressed (DE) genes, temporal trends of DE genes, and pathway analysis were conducted. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to validate the results. RESULTS: Strong upregulation of mRNA of proteins involved in gliosis, apoptosis, and neurotrophism was found 1 week after ablation and their related pathways such as the apoptotic and Jak/Stat pathways were identified. Three months after induced Müller-cell ablation, Müller-cell metabolic pathways and vasculopathy-related pathways such as genes involved in glycolysis and tight junctions were downregulated. qRT-PCR analysis showed consistent expression trends of selected genes. CONCLUSIONS: The results were generally consistent with the previous morphologic findings in this model, in which photoreceptor degeneration soon after Müller-cell ablation, accompanied by blood-retinal barrier breakdown and subsequent retinal neovascularization were reported. These results are consistent with a significant contribution of Müller-cell dysfunction on retinal neuronal injury and vascular pathology at the mRNA level.

Submacular DL-alpha-aminoadipic acid eradicates primate photoreceptors but does not affect luteal pigment or the retinal vasculature.

PURPOSE: Macular telangiectasia type 2 (MT2) is a condition of uncertain etiology characterized by retinal vascular abnormalities, depletion of luteal pigment, and photoreceptor loss. To model this condition, the authors recently used a purportedly glial-selective toxin, DL-α-aminoadipic acid (DL-α-AAA), to test the effect of Müller cell disruption on the blood-retinal barrier in rats. In this study, they investigated macular changes after subretinal injection of DL-α-AAA in monkeys. METHODS: Various doses of DL-α-AAA were injected beneath the macula in eight monkey eyes. Eyes were examined by multifocal electroretinography (mfERG), optical coherence tomography (OCT), fundus autofluorescence, color photography, and fluorescein angiography. Five months after injection, eyes were examined by histology and immunohistochemistry for changes in photoreceptors and the retinal glia. In vitro studies evaluated the effect of DL-α-AAA on 661W cone photoreceptor viability. RESULTS: Subretinal injection of DL-α-AAA resulted in virtually complete ablation of photoreceptors in the injected area, as shown by OCT and histology, and severely impaired mfERG responses. Müller cells, albeit activated, survived the injury. Macular pigment remained unchanged in the central fovea. Subretinal injection of DL-α-AAA did not induce vascular leakage, though it increased the fundus autofluorescence. DL-α-AAA had a dose-dependent toxic effect on 661W photoreceptors. CONCLUSIONS: Submacular injection of DL-α-AAA induced severe damage to photoreceptors but failed to eliminate Müller cells in monkeys. Central macular pigment persisted despite loss of photoreceptors, and the retinal vasculature was unaffected. These observations may have significance in studying the roles of different cellular components in the pathogenesis of MT2.

Tyrosine phosphorylation of VE-cadherin and claudin-5 is associated with TGF-ß1-induced permeability of centrally derived vascular endothelium.

Breakdown of the inner blood-retinal barrier and the blood-brain barrier is associated with changes in tight and adherens junction-associated proteins that link vascular endothelial cells. This study aimed to test the hypothesis that transforming growth factor (TGF)-ß1 increases the paracellular permeability of vascular endothelial monolayers through tyrosine phosphorylation of VE-cadherin and claudin-5. Bovine retinal and human brain capillary endothelial cells were grown as monolayers on coated polycarbonate membranes. Paracellular permeability was studied by measuring the equilibration of (14)C-inulin or fluorescence-labelled dextran. Changes in VE-cadherin and claudin-5 expression were studied by immunocytochemistry (ICC) and quantified by cell-based enzyme linked immunosorbent assays (ELISA). Tyrosine phosphorylation of VE-cadherin and claudin-5 was studied by ICC, immunoprecipitation and Western blotting. We found that exposure of endothelial cells to TGF-ß1 caused a dose-dependent increase in paracellular permeability as reflected by increases in the equilibration of (14)C-inulin. This effect was enhanced by the tyrosine phosphatase inhibitor orthovanadate and attenuated by the tyrosine kinase inhibitor lavendustin A. ICC and cell-based ELISA revealed that TGF-ß1 induced both dose- and time-dependent decreases in VE-cadherin and claudin-5 expression. Assessment of cell viability indicated that changes in these junction-associated proteins were not due to endothelial death or injury. ICC revealed that tyrosine phosphorylation of endothelial monolayers was greatly enhanced by TGF-ß1 treatment, and immunoprecipitation of cell lysates showed increased tyrosine phosphorylation of VE-cadherin and claudin-5. Our results suggest that tyrosine phosphorylation of VE-cadherin and claudin-5 is involved in the increased paracellular permeability of central nervous system-derived vascular endothelium induced by TGF-ß1.