Interleukin-17A modulates retinal inflammation by regulating microglial activation via the p38 MAPK pathway in experimental glaucoma neuropathy.

As a prototypical member of the IL-17 family, interleukin-17A (IL-17A) has received increasing attentions for its potent proinflammatory role as well as potential to be a key therapeutic target in human autoimmune inflammatory diseases; however, its roles in other pathological scenarios like neuroinflammations are not fully elucidated yet but appear essentially correlating and promising. Glaucoma is the leading cause of irreversible blindness with complicated pathogenesis still to be understood, where neuroinflammation was reported to be critically involved in its both initiation and progression. Whether IL-17A takes part in the pathogenesis of glaucoma through interfering neuroinflammation due to its potent proinflammatory effect is still unknown. In the present study, we investigated the role of IL-17A in the pathological process of glaucoma neuropathy as well as its relationship with the predominant immune inflammation mediator microglia in retina, trying to elucidate the underlying mechanisms from the view of inflammation modulation. In our study, RNA sequencing was performed for the retinas of chronic ocular hypertension (COH) and control mice. Western blot, RT-PCR, immunofluorescence, and ELISA were used to evaluate the microglial activation and proinflammatory cytokines release at conditioned levels of IL-17A, along with assessment of optic nerve integrity including retinal ganglion cells (RGCs) counting, axonal neurofilament quantification, and flash visual-evoked potential (F-VEP) examination. And the possibly involved signaling pathways were screened out to go through further validation in scenarios with conditioned IL-17A. Subsequently, IL-17A was found to be significantly upregulated in COH retina. Furthermore, suppression of IL-17A effectively diminished the loss of RGCs, improved axonal quality, and F-VEP performance in COH mice. Mechanistically, IL-17A promoted microglial activation and proinflammatory cytokines release along with enhanced phenotypic conversion of activated microglia to M2-type in early stage and to M1-type in late stage in glaucomatous retinas. Microglia elimination decreased the proinflammatory factors secretion, enhanced the RGCs survival and axonal quality mediated by IL-17A. Furthermore, IL-17A-induced the overactivation of microglia in glaucomatous condition was alleviated after blocking the p38 MAPK pathway. Taken together, IL-17A is involved in the regulation of retinal immune response and RGCs cell death in experimental glaucoma by essentially promoting retinal microglial activation via p38 MAPK signaling pathway. IL-17A dynamically regulates the phenotypic conversion of retinal microglia in experimental glaucoma partly depending on the duration of elevated intraocular pressure. Suppression of IL-17A contributes to alleviate glaucoma neuropathy and exhibits promising potential as an innovative target for therapeutic strategy in glaucoma.

Multifocal electroretinography increases following experimental glaucoma in nonhuman primates with retinal ganglion cell axotomy.

PURPOSE: To determine whether short-latency changes in multifocal electroretinography (mfERG) observed in experimental glaucoma (EG) are secondary solely to retinal ganglion cell (RGC) loss or whether there is a separate contribution from elevated intraocular pressure (IOP). METHODS: Prior to operative procedures, a series of baseline mfERGs were recorded from six rhesus macaques using a 241-element unstretched stimulus. Animals then underwent hemiretinal endodiathermy axotomy (HEA) by placing burns along the inferior 180° of the optic nerve margin in the right eye (OD). mfERG recordings were obtained in each animal at regular intervals following for 3-4 months to allow stabilization of the HEA effects. Laser trabecular meshwork destruction (LTD) to elevate IOP was then performed; first-order kernel (K1) waveform root-mean-square (RMS) amplitudes for the short-latency segment of the mfERG wave (9-35 ms) were computed for two 7-hexagon groupings-the first located within the superior (non-axotomized) macula and the second within the inferior (axotomized) macula. Immunohistochemistry for glial fibrillary acidic protein (GFAP) was done. RESULTS: By 3 months post HEA, there was marked thinning of the inferior nerve fiber layer as measured by optical coherence tomography. Compared with baseline, no statistically significant changes in 9-35 ms K1 RMS amplitudes were evident in either the axotomized or non-axotomized portions of the macula. Following LTD, mean IOP in HEA eyes rose to 46 ± 9 compared with 20 ± 2 mmHg (SD) in the fellow control eyes. In the HEA + EG eyes, statistically significant increases in K1 RMS amplitude were present in both the axotomized inferior and non-axotomized superior portions of the OD retinas. No changes in K1 RMS amplitude were found in the fellow control eyes from baseline to HEA epoch, but there was a smaller increase from baseline to HEA + EG. Upregulation of GFAP in the Müller cells was evident in both non-axotomized and axotomized retina in eyes with elevated IOP. CONCLUSIONS: The RMS amplitudes of the short-latency mfERG K1 waveforms are not altered following axotomy but undergo marked increases following elevated IOP. This suggests that the increase in mfERG amplitude was not solely a result of RGC loss and may reflect photoreceptor and bipolar cell dysfunction and/or changes in Müller cells.

Retinal Changes in Astrocytes and Müller Glia in a Mouse Model of Laser-Induced Glaucoma: A Time-Course Study.

Macroglia (astrocytes and Müller glia) may play an important role in the pathogenesis of glaucoma. In a glaucoma mouse model, we studied the effects of unilateral laser-induced ocular hypertension (OHT) on macroglia in OHT and contralateral eyes at different time points after laser treatment (1, 3, 5, 8 and 15 days) using anti-GFAP and anti-MHC-II, analyzing the morphological changes, GFAP-labelled retinal area (GFAP-PA), and GFAP and MHC-II immunoreactivity intensities ((GFAP-IRI and MHC-II-IRI)). In OHT and contralateral eyes, with respect to naïve eyes, at all the time points, we found the following: (i) astrocytes with thicker somas and more secondary processes, mainly in the intermediate (IR) and peripheral retina (PR); (ii) astrocytes with low GFAP-IRI and only primary processes near the optic disc (OD); (iii) an increase in total GFAP-RA, which was higher at 3 and 5 days, except for at 15 days; (iv) an increase in GFAP-IRI in the IR and especially in the PR; (v) a decrease in GFAP-IRI near the OD, especially at 1 and 5 days; (vi) a significant increase in MHC-II-IRI, which was higher in the IR and PR; and (vii) the Müller glia were GFAP+ and MHC-II+. In conclusion, in this model of glaucoma, there is a bilateral macroglial activation maintained over time involved in the inflammatory glaucoma process.

Age and intraocular pressure in murine experimental glaucoma.

Age and intraocular pressure (IOP) are the two most important risk factors for the development and progression of open-angle glaucoma. While IOP is commonly considered in models of experimental glaucoma (EG), most studies use juvenile or adult animals and seldom older animals which are representative of the human disease. This paper provides a concise review of how retinal ganglion cell (RGC) loss, the hallmark of glaucoma, can be evaluated in EG with a special emphasis on serial in vivo imaging, a parallel approach used in clinical practice. It appraises the suitability of EG models for the purpose of in vivo imaging and argues for the use of models that provide a sustained elevation of IOP, without compromise of the ocular media. In a study with parallel cohorts of adult (3-month-old, equivalent to 20 human years) and old (2-year-old, equivalent to 70 human years) mice, we compare the effects of elevated IOP on serial ganglion cell complex thickness and individual RGC dendritic morphology changes obtained in vivo. We also evaluate how age modulates the impact of elevated IOP on RGC somal and axonal density in histological analysis as well the density of melanopsin RGCs. We discuss the challenges of using old animals and emphasize the potential of single RGC imaging for understanding the pathobiology of RGC loss and evaluating new therapeutic avenues.

Antibody and Protein Profiles in Glaucoma: Screening of Biomarkers and Identification of Signaling Pathways.

Glaucoma represents a group of chronic neurodegenerative diseases, constituting the second leading cause of blindness worldwide. To date, chronically elevated intraocular pressure has been identified as the main risk factor and the only treatable symptom. However, there is increasing evidence in the recent literature that IOP-independent molecular mechanisms also play an important role in the progression of the disease. In recent years, it has become increasingly clear that glaucoma has an autoimmune component. The main focus nowadays is elucidating glaucoma pathogenesis, finding early diagnostic options and new therapeutic approaches. This review article summarizes the impact of different antibodies and proteins associated with glaucoma that can be detected for example by microarray and mass spectrometric analyzes, which (i) provide information about expression profiles and associated molecular signaling pathways, (ii) can possibly be used as a diagnostic tool in future and, (iii) can identify possible targets for therapeutic approaches.

Inhibition of the cysteinyl leukotriene pathways increases survival of RGCs and reduces microglial activation in ocular hypertension.

Glaucoma is the second leading cause of blindness worldwide. This multifactorial, neurodegenerative group of diseases is characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, leading to irreversible visual impairment and blindness. There is a huge unmet and urging need for the development of new and translatable strategies and treatment options to prevent this progressive loss of RGC. Accumulating evidence points towards a critical role of neuroinflammation, in particular microglial cells, in the pathogenesis of glaucoma. Leukotrienes are mediators of neuroinflammation and are involved in many neurodegenerative diseases. Therefore, we tested the leukotriene receptors CysLT1R/GPR17-selective antagonist Montelukast (MTK) for its efficacy to modulate the reactive state of microglia in order to ameliorate RGCs loss in experimental glaucoma. Ocular hypertension (OHT) was induced unilaterally by injection of 8 µm magnetic microbead (MB) into the anterior chamber of female Brown Norway rats. The contralateral, untreated eye served as control. Successful induction of OHT was verified by daily IOP measurement using a TonoLab rebound tonometer. Simultaneously to OHT induction, one group received daily MTK treatment and the control group vehicle solution by oral gavage. Animals were sacrificed 13-15 days after MB injection. Retina and optic nerves (ON) of OHT and contralateral eyes were analyzed by immunofluorescence with specific markers for RGCs (Brn3a), microglial cells/macrophages (Iba1 and CD68), and cysteinyl leukotriene pathway receptors (CysLT1R and GPR17). Protein labeling was documented by confocal microscopy and analyzed with ImageJ plugins. Further, mRNA expression of genes of the inflammatory and leukotriene pathway was analyzed in retinal tissue. MTK treatment resulted in a short-term IOP reduction at day 2, which dissipated by day 5 of OHT induction in MTK treated animals. Furthermore, MTK treatment resulted in a decreased activation of Iba1+ microglial cells in the retina and ON, and in a significantly increased RGC survival in OHT eyes. Within the retina, GPR17 and CysLT1R expression was demonstrated in single RCGs and in microglial cells respectively. Further, increased mRNA expression of pro-inflammatory genes was detected in OHT induced retinas. In the ON, OHT induction increased the number of GPR17+ cells, showing a trend of reduction following MTK treatment. This study shows for the first time a significantly increased RGC survival in an acute OHT model following treatment with the leukotriene receptor antagonist MTK. These results strongly suggest a neuroprotective effect of MTK and a potential new therapeutic strategy for glaucoma treatment.

Targeted delivery of LM22A-4 by cubosomes protects retinal ganglion cells in an experimental glaucoma model.

Glaucoma, a major cause of irreversible blindness worldwide, is associated with elevated intraocular pressure (IOP) and progressive loss of retinal ganglion cells (RGCs) that undergo apoptosis. A mechanism for RGCs injury involves impairment of neurotrophic support and exogenous supply of neurotrophic factors has been shown to be beneficial. However, neurotrophic factors can have widespread effects on neuronal tissues, thus targeting neurotrophic support to injured neurons may be a better neuroprotective strategy. In this study, we have encapsulated LM22A-4, a small neurotrophic factor mimetic, into Annexin V-conjugated cubosomes (L4-ACs) for targeted delivery to injured RGCs in a model of acute IOP elevation, which is induced by acute IOP elevation. We have tested cubosomes formulations that encapsulate from 9% to 33% LM22A-4. Our data indicated that cubosomes encapsulating 9% and 17% LM22A-4 exhibited a mixture of Pn3m/Im3m cubic phase, whereas 23% and 33% showed a pure Im3m cubic phase. We found that 17% L4-ACs with Pn3m/Im3m symmetries showed better in-situ and in-vitro lipid membrane interactions than the 23% and 33% L4-ACs with Im3m symmetry. In vivo experiments showed that 17% L4-ACs targeted the posterior retina and the optic nerve head, which prevented RGCs loss and improved functional outcomes in a mouse model of acute IOP elevation. These results provide evidence that Annexin V-conjugated cubosomes-based LM22A-4 delivery may be a useful targeted approach to prevent the progression of RGCs loss in glaucoma. STATEMENT OF SIGNIFICANCE: Recent studies suggest that the therapy of effectively delivering neurotrophic factors to the injured retinal ganglion cells (RGCs) could promote the survival of RGCs in glaucoma. Our present work has for the first time used cubosomes as an active targeted delivery system and have successfully delivered a neuroprotective drug to the damaged RGCs in vivo. Our new cubosomal formulation can protect apoptotic cell death in vitro and in vivo, showing that cubosomes are a promising drug carrier system for ocular drug delivery and glaucoma treatment. We have further found that by controlling cubosomes in Pn3m phase we can facilitate delivery of neuroprotective drug through apoptotic membranes. This data, we believe, has important implications for future design and formulation of cubosomes for therapeutic applications.

Neuroprotective effect of the somatostatin receptor 5 agonist L-817,818 on retinal ganglion cells in experimental glaucoma.

Somatostatin plays important roles in modulating neuronal functions by activating the five specific G-protein coupled receptors (sst1-sst5). Previous studies have demonstrated that sst5 were expressed in retinal ganglion cells (RGCs) and sst5 agonist attenuated the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid-induced retinal neurotoxicity. In this study, we investigated effects and underlying mechanisms of the sst5 agonist L-817,818 on RGC injury induced by elevated intraocular pressure (COH) in experimental glaucoma. Our results showed that intraperitoneal administration of L-817,818 significantly reduced RGC loss and decreased the number of terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL)-positive RGCs in COH retinas, suggesting that L-817,818 may attenuate RGC apoptosis. Consistently, in COH retinas with L-817,818 administration, both the down-regulated mRNA and protein levels of anti-apoptotic Bcl-2 and the up-regulated mRNA and protein levels of pro-apoptotic Bax were partially reversed. L-817,818 administration downregulated the expression of apoptosis-related proteins caspase-9 and caspase-3 in COH retinas. In addition, L-817,818 administration reduced the concentrations of reactive oxygen species/reactive nitrogen species and malondialdehyde, and ameliorated the functions of mitochondrial respiratory chain complex (MRCC). Our results imply that administration of the sst5 agonist L-817,818 reduces RGC loss in COH rats through decreasing RGC apoptosis, which is mediated by regulating Bcl-2/Bax balance, reducing oxidative stress and rescuing activities of MRCC. Activation of sst5 may provide neuroprotective roles for RGCs in glaucoma.

Experimental models of glaucoma filtration surgery.

Glaucoma filtration surgery plays an important role in achieving intraocular pressure (IOP) reduction in patients who have high IOP despite maximum medical therapy. Preclinical experimental models of glaucoma filtration surgery contribute a great deal to our knowledge of the wound healing processes that predispose to scarring and may lead to poor outcomes. However, this research needs to be interpreted in the light of the specific study design, animal model and methods used. We review the existing literature addressing various models of experimental glaucoma filtration surgery, discuss the considerations in assessing these models and describe future steps in evaluating potential therapeutics and bleb characteristics that could impact translational research in this field.

Neuroprotective and Anti-Inflammatory Effects of a Hydrophilic Saffron Extract in a Model of Glaucoma.

Glaucoma is a neurodegenerative disease characterized by the loss of retinal ganglion cells (RGCs). An increase in the intraocular pressure is the principal risk factor for such loss, but controlling this pressure does not always prevent glaucomatous damage. Activation of immune cells resident in the retina (microglia) may contribute to RGC death. Thus, a substance with anti-inflammatory activity may protect against RGC degeneration. This study investigated the neuroprotective and anti-inflammatory effects of a hydrophilic saffron extract standardized to 3% crocin content in a mouse model of unilateral, laser-induced ocular hypertension (OHT). Treatment with saffron extract decreased microglion numbers and morphological signs of their activation, including soma size and process retraction, both in OHT and in contralateral eyes. Saffron extract treatment also partially reversed OHT-induced down-regulation of P2RY12. In addition, the extract prevented retinal ganglion cell death in OHT eyes. Oral administration of saffron extract was able to decrease the neuroinflammation associated with increased intraocular pressure, preventing retinal ganglion cell death. Our findings indicate that saffron extract may exert a protective effect in glaucomatous pathology.

Review of rodent hypertensive glaucoma models.

Glaucoma is a neurodegenerative disease characterized by the progressive loss of retinal ganglion cells (RGCs). Elevated intraocular pressure (IOP) is a primary risk factor for the development and progression of glaucoma. Rodent models of glaucoma have greatly improved our understanding of the pathophysiology of glaucoma and served as a useful tool to investigate neuroprotective agents. An ideal glaucoma animal model should be easy to induce, reproducible, biologically plausible and predictable. Of the available animal models of glaucoma, rodents are commonly studied because they have a relatively short life span and can be genetically altered. A successful hypertensive glaucoma model should induce structural glaucomatous changes: including loss of retinal nerve fibres, retinal ganglion cells and optic-disc cupping along with IOP elevation. The level and duration of IOP elevation should be titratable depending on the targeted glaucomatous damage. This review summarizes the outcomes of induced rodent hypertensive glaucoma models including intracameral injection of microbeads, laser photocoagulation, episcleral vein cauterization, injection of hypertonic saline and hyaluronic acid. We aim to provide a detailed overview of each of the models with a focus on parameters that defines a successful glaucoma model. The induced IOP elevation and duration of elevation varied among the different models and strain of rodent; nonetheless, they all achieved a sustainable raised IOP with corresponding RGC loss. The limitations of each model are discussed.

[Comparison of two experimental models of glaucoma in rabbits] / Comparación de dos modelos experimentales de glaucoma en conejos.

Objective: to compare two models of experimental glaucoma by induction of ocular hypertension in rabbits. Materials and methods: Sixteen New Zealand female rabbits, 2-3 kg were used. Model A (n=6): cauterization of episcleral and perilimbar veins of the right eye (RE) with surgical electrocautery. Model B (n=10): Injection of ?-chymotrypsin in posterior chamber of RE. Intraocular pressure (IOP) was measured before and after the induction of ocular hypertension (OHT), once a week at the same time of day for 40 days, with a manual tonometer. The animals were euthanized by CO2 inhalation. In both models the control was the IOP of the left eye (LE). The mean and standard error (SE) values of IOP, expressed in mmHg, were compared statistically by applying Student's t-test with a significance level of p<0.05. Results: The IOP in LE (control) of model A: was 12.9±1.05 and in model B: 12.9±1.09. There were no significant differences between the models. Model A: The IOP increase in RE was 14.7% (14.8±1.4) with respect to LE. A significant increase in IOP was observed within the first 24 hours: 23.5±1.9 (p<0.05) compared to the control eye. There were no significant differences with subsequent controls. Model B: The increase in IOP in RE was 129.1% (29.6±3.4) with respect to LE. In all cases an increase was observed from Day 1 (p<0.05). The IOP peak in RE was evidenced on Day 25: 35±3.4 (p<0.05). The increase in IOP induced by model B was significantly higher (p<0.01) than in model A. There was loss of ganglion cells of the retina in both models, but the following anatomo-pathological changes were observed only in model B: buphthalmos, subluxation of the lens and increased excavation of the papilla. Conclusion: This study indicates that model B is the most appropriate method to induce a rapid, controlled increase of IOP in rabbits and, more importantly, that this increase may be sustained over extended periods of time. This model could be useful for evaluating the efficacy of new ocular drug delivery systems and for further studies of the physiopathology of glaucoma.

Objetivo: comparar dos modelos de glaucoma experimental por inducción de hipertensión ocular en conejos y describir cambios anatomo-patológicos. Materiales y métodos: Se utilizaron 16 conejos New Zealand, hembras, de 2-3 kg. Modelo A (n=6): cauterización de venas epiesclerales y perilimbares en ojo derecho (OD) con cauterio eléctrico quirúrgico. Modelo B (n=10): inyección intracamerular en OD de ?-quimotripsina. Se midió la presión intraocular (PIO) antes y después de la inducción de la hipertensión ocular (HTO), una vez por semana, a la misma hora del día, durante 40 días, con tonómetro manual. Los animales fueron sacrificados por inhalación de CO2. En ambos modelos la PIO del ojo izquierdo (OI).fue tomado como valor control. La media y error estándar (EE) de los valores de la PIO, expresada en mmHg, fueron evaluadas y comparadas estadísticamente aplicando Test T de Student considerando un nivel de significación de p < 0.05. Resultados: La PIO en OI (control) del modelo A: fue 12,9±1,05 y en el modelo B: 12,9±1,09. No se observaron diferencias significativas entre ambas. Modelo A: el aumento de la PIO en OD fue 14,7% (14,8±1,4) con respecto a OI. Se observó un incremento significativo de la PIO dentro de las primeras 24 hs: 23,5±1,9 (p<0,05) comparado con el valor del ojo control. No hubo diferencias significativas con los controles posteriores. Modelo B: el aumento de la PIO en OD fue 129,1% (29,6±3,4) con respecto al OI. En todos los casos se observó un incremento desde el día 1 (p<0,05). El pico de PIO en OD se evidenció el día 25: 35±3,4 (p<0,05). El incremento de la PIO inducida en el modelo B fue significativamente mayor (p<0,01) que en el modelo A. En ambos modelos hubo pérdida de células ganglionares de la retina, pero sólo en el modelo B se observaron los siguientes cambios anatomo-patológicos: buftalmus, subluxación del cristalino y aumento de la excavación de la papila. Conclusión: De acuerdo a este estudio, el modelo B aparece como el método más apropiado a los fines de inducir un incremento rápido y controlado de la IOP en conejos y más importante, este incremento sería capaz de mantenerse alto a lo largo de periodos de tiempos extendidos. Este modelo podría ser de gran utilidad para evaluar la eficacia de nuevos sistemas oculares de liberación de fármacos y realizar futuros estudios de la fisiopatología del glaucoma Conclusión: De acuerdo a este estudio, el modelo B aparece como el método más apropiado a los fines de inducir un incremento rápido y controlado de la IOP en conejos y más importante, este incremento sería capaz de mantenerse alto a lo largo de periodos de tiempos extendidos. Este modelo podría ser de gran utilidad para evaluar la eficacia de nuevos sistemas oculares de liberación de fármacos y realizar futuros estudios de la fisiopatología del glaucoma

Correlation of Crystallin Expression and RGC Susceptibility in Experimental Glaucoma Rats of Different Ages.

PURPOSE: Glaucoma is one of the leading causes of blindness worldwide with age being an important risk factor. However, the pathogenesis remains poorly understood. Aim of this study was to focus on age-dependent molecular changes in an experimental animal model of glaucoma. METHODS: Intraocular pressure was elevated in Sprague-Dawley rats aged 3, 14, and 47 weeks for a period of 7 weeks by episcleral vein cauterization. Ganglion cell loss was monitored by an immunohistochemical staining of the Brain-specific homeobox/POU (Pit-1, Oct-2, Unc-86) domain protein 3A positive cells in retinal flat-mounts and spectral-domain optical coherence tomography measuring the retinal nerve fiber layer thickness. Molecular protein alterations were analyzed using a comprehensive mass spectrometric proteomics approach of the retina and vitreous body. RESULTS: While juvenile animals did not show a significant loss of retinal ganglion cells due to intraocular pressure elevation, adolescent animals showed a decrease up to 26% (p < 0.05). A shift of retinal crystallin protein expression levels within all protein-family subclasses (α, ß, γ) could be observed in the youngest animal group (p < 0.05), while the upregulation of crystallin proteins in older animals was less striking. In addition, numerous crystallin proteins were also detected in the vitreous body. CONCLUSION: These results provide insights of a potential correlation of age-related glaucomatous damage and the absence of crystallin proteins in the retina.

Bilateral early activation of retinal microglial cells in a mouse model of unilateral laser-induced experimental ocular hypertension.

The immune system plays an important role in glaucomatous neurodegeneration. Retinal microglial reactivation associated with ganglion cell loss could reportedly contribute to the glaucoma progression. Recently we have described signs of microglia activation both in contralateral and ocular hypertension (OHT) eyes involving all retinal layers 15 days after OHT laser induction in mice. However, no works available have analyzed the microglial activation at earliest time points after OHT induction (24 h) in this experimental model. Thus, we seek to describe and quantify signs of microglia activation and differences depending on the retinal layer, 24 h after unilateral laser-induced OHT. Two groups of adult Swiss mice were used: age-matched control (naïve) and lasered. In the lasered animals, OHT eyes as well as contralateral eyes were analyzed. Retinal whole-mounts were immunostained with antibodies against Iba-1 and MHC-II. We quantified the number of microglial cells in the photoreceptor layer (OS), outer plexiform layer (OPL), and inner plexiform layer (IPL); the number of microglial vertical processes connecting the OPL and OS; the area of the retina occupied by Iba-1+ cells (Iba1-RA) in the nerve fiber layer-ganglion cell layer (NFL-GCL), the total arbor area of microglial cells in the OPL and IPL and; Iba-1+ cell body area in the OPL, IPL and NFL-GCL. In contralateral and OHT eyes the morphological features of Iba-1+ cell activation were: migration, enlargement of the cell body, higher degree of branching and reorientation of the processes, radial disposition of the soma and processes toward adjacent microglial plexuses, and presence of amoeboid cells acting as macrophages. These signs were more pronounced in OHT eyes. Most of Iba-1+ cells did not express MHC-II; rather, only dendritic and rounded cells expressed it. In comparison with naïve eyes, in OHT eyes and contralateral eyes no significant differences were found in the microglial cell number; but there was a significant increase in Iba1-RA. The total arbor area of microglial cells was significantly decreased in: i) OHT eyes with respect contralateral eyes and naïve-eyes in IPL; ii) OHT eyes with respect to naïve eyes in OPL. The number of microglial vertical processes connecting the OPL and OS were significantly increased in contralateral eyes compared with naïve-eyes and OHT eyes. In OPL, IPL and NFL-GCL, the cell body area of Iba-1+ cells was significantly greater in OHT eyes than in naïve and contralateral eyes, and greater in contralateral eyes than in naïve eyes. A non-proliferative microglial reactivation was detected both in contralateral eyes and in OHT eyes in an early time after unilateral laser-induced OHT (24 h). This fast microglial activation, which involves the contralateral eye, could be mediated by the immune system.

Hydrogen sulfide supplement attenuates the apoptosis of retinal ganglion cells in experimental glaucoma.

Glaucoma is a group of neurodegenerative eye diseases characterized by progressive impairment of visual function due to loss of retinal ganglion cells (RGC). As hydrogen sulfide (H2S) was reported to play a role in the process of glaucomatous neuropathy and improve RGC survival in experimental glaucoma, the authors aimed to investigate the anti-apoptosis effect of H2S supplement in a rat glaucoma model, and further tried to explore the involved factors in the neuroprotection. A chronic ocular hypertension (COH) rat model induced by intracameral injection of cross-linking hydrogel was employed to simulate glaucoma and 288 rats were subjected to experimental procedures in the present study. After 4 weeks of sodium hydrosulfide (NaHS) administration following COH induction, the apoptosis of RGC isolated from experimented rats as well as apoptosis of neurons in ganglion cell layer (GCL), intrinsic apoptotic pathway activity, mitochondrial function, glial activation, NF-κB pathway activity, NADPH oxidase activity, autophagy activity and TNF-α level in retina were evaluated. The results showed that H2S supplement effectively attenuated the apoptosis of RGC in experimental glaucoma, and the neuroprotection by H2S might correlate with preservation of mitochondrial function, attenuation of oxidative stress, suppression of glial activation, inhibition of inflammatory pathways and downregulation of autophagy. Our study indicated that H2S supplement resulted in significant neuroprotection through attenuation of RGC apoptosis which might be linked with multiple factors in experimental glaucoma. The new therapeutic strategy might potentially contribute to benefit glaucoma treatment, which is worth further concerns.

The Small Heat Shock Protein α-Crystallin B Shows Neuroprotective Properties in a Glaucoma Animal Model.

Glaucoma is a neurodegenerative disease that leads to irreversible retinal ganglion cell (RGC) loss and is one of the main causes of blindness worldwide. The pathogenesis of glaucoma remains unclear, and novel approaches for neuroprotective treatments are urgently needed. Previous studies have revealed significant down-regulation of α-crystallin B as an initial reaction to elevated intraocular pressure (IOP), followed by a clear but delayed up-regulation, suggesting that this small heat-shock protein plays a pathophysiological role in the disease. This study analyzed the neuroprotective effect of α-crystallin B in an experimental animal model of glaucoma. Significant IOP elevation induced by episcleral vein cauterization resulted in a considerable impairment of the RGCs and the retinal nerve fiber layer. An intravitreal injection of α-crystallin B at the time of the IOP increase was able to rescue the RGCs, as measured in a functional photopic electroretinogram, retinal nerve fiber layer thickness, and RGC counts. Mass-spectrometry-based proteomics and antibody-microarray measurements indicated that a α-crystallin injection distinctly up-regulated all of the subclasses (α, ß, and γ) of the crystallin protein family. The creation of an interactive protein network revealed clear correlations between individual proteins, which showed a regulatory shift resulting from the crystallin injection. The neuroprotective properties of α-crystallin B further demonstrate the potential importance of crystallin proteins in developing therapeutic options for glaucoma.

Movement of retinal vessels toward the optic nerve head after increasing intraocular pressure in monkey eyes with experimental glaucoma.

A shift or displacement of the retinal blood vessels (RBVs) with neuroretinal rim thinning indicates the progression of glaucomatous optic neuropathy. In chronic open angle glaucoma, individuals with RBV positional shifts exhibit more rapid visual field loss than those without RBV shifts. The retinal vessels reportedly move onto the optic nerve head (ONH) in response to glaucoma damage, suggesting that RBVs are pulled toward the ONH in response to increased cupping. Whether this phenomenon only applies to RVBs located in the vicinity or inside the ONH or, more generally, to RBVs also located far from the ONH, however, is unclear. The aim of this study was to evaluate the movement of RBVs located relatively far from the ONH edge after increasing intraocular pressure (IOP) in an experimental monkey model of glaucoma. Fundus photographs were obtained in 17 monkeys. High IOP was induced in the monkeys by laser photocoagulation burns applied uniformly with 360° irradiation around the trabecular meshwork of the left eye. The right eye was left intact and used as a non-treated control. Considering the circadian rhythm of IOP, it was measured in both eyes of each animal at around the same time-points. Then, fundus photographs were obtained. Using Image J image analysis software, an examiner (N.E.) measured the fundus photographs at two time-points, i.e. before laser treatment (time 1) and the last fundus photography after IOP elevation (time 2). The following parameters were measured (in pixels): 1) vertical diameter of the ONH (DD), 2) distance from the ONH edge to the first bifurcation point of the superior branch of the central retinal vein (UV), 3) distance from the ONH edge to the first bifurcation point of the inferior branch of the central retinal vein (LV), 4) ONH area, and 5) surface area of the cup of the ONH. We calculated the ratios of UV to DD (UV/DD), LV to DD (LV/DD), and the cup area to disc area ratio (C/D). The mean UV/DD at time 1 (0.656 ± 0.233) was decreased at time 2 (0.542 ± 0.192) (p < 0.01), and the mean LV/DD at time 1 (0.642 ± 0.151) was decreased at time 2 (0.534 ± 0.171) (p < 0.01). The mean C/D at time 1 (0.303 ± 0.035) was increased at time 2 (0.556 ± 0.110) (p < 0.01). The mean IOP at time 1 was 19.8 ± 2.5 and that at time 2 was 54.2 ± 15.8. The amount and rate of the change in LV/DD and C/D between time 1 and time 2 were significantly correlated (r = -0.654 and -0.536, p = 0.004 and 0.026, respectively). Therefore, in an experimental monkey model of glaucoma, RBVs located relatively far from the ONH were pulled toward the ONH as cupping increased.

Relationship between Elevated Intraocular Pressure and Divided Peripapillary Sector Retinal Nerve Fiber Layer Thickness in a Cynomolgus Monkey Laser-Induced Ocular Hypertension Model.

AIMS: We investigated the relationship between elevated intraocular pressure (IOP) and changes in global and peripapillary sector retinal nerve fiber layer (RNFL) thickness around the optic nerve head (ONH) in the laser-induced ocular hypertension monkey model. METHODS: To induce high IOP, green laser photocoagulation burns were applied around the trabecular meshwork of 1 eye from each of 12 cynomolgus monkeys. The animals had been acclimated to IOP measurement under conscious conditions for more than 2 months, and IOP was chronologically measured. RNFL thickness was measured for 6 peripapillary sectors and global area using spectral-domain optical coherence tomography. RESULTS: After model induction, marked IOP elevation and enlarged optic disk cupping were observed. Thinning of the RNFL associated with elevated IOP was observed around the ONH from 6 until 9 weeks after laser treatment, and the degree of reduction in RNFL thickness varied between the peripapillary sectors. Correlations between cumulative IOP elevation and RNFL thickness reduction were statistically significant for the temporal-superior (p = 0.024), nasal-inferior (p = 0.044), and temporal (p = 0.049) sectors, and global RNFL (p = 0.018). CONCLUSIONS: These results suggest that this model reflected the pathology of clinical glaucoma in terms of the specific pattern of RNFL thinning around the ONH.

Retinal neurodegeneration in experimental glaucoma.

In rats and mice, limbar tissues of the left eye were laser-photocoagulated (LP) and ocular hypertension (OHT) effects were investigated 1 week to 6 months later. To investigate the innermost layers, retinas were examined in wholemounts using tracing from the superior colliculi to identify retinal ganglion cells (RGCs) with intact retrograde axonal transport, melanopsin immunodetection to identify intrinsically photosensitive RGCs (m(+)RGC), Brn3a immunodetection to identify most RGCs but not m(+)RGCs, RECA1 immunodetection to examine the inner retinal vessels, and DAPI staining to detect all nuclei in the GC layer. The outer retinal layers (ORLs) were examined in cross sections analyzed morphometrically or in wholemounts to study S- and L-cones. Innervation of the superior colliculi was examined 10 days to 14 weeks after LP with orthogradely transported cholera toxin subunit B. By 2 weeks, OHT resulted in pie-shaped sectors devoid of FG(+)RGCs or Brn3a(+)RGCs but with large numbers of DAPI(+)nuclei. Brn3a(+)RGCs were significantly greater than FG(+)RGCs, indicating the survival of large numbers of RGCs with their axonal transport impaired. The inner retinal vasculature showed no abnormalities that could account for the sectorial loss of RGCs. m(+)RGCs decreased to approximately 50-51% in a diffuse loss across the retina. Cross sections showed focal areas of degeneration in the ORLs. RGC loss at 1m diminished to 20-25% and did not progress further with time, whereas the S- and L-cone populations diminished progressively up to 6m. The retinotectal projection was reduced by 10 days and did not progress further. LP-induced OHT results in retrograde degeneration of RGCs and m(+)RGCs, severe damage to the ORL, and loss of retinotectal terminals.

Macro- and microglial responses in the fellow eyes contralateral to glaucomatous eyes.

Most studies employing experimental models of unilateral glaucoma have used the normotensive contralateral eye as the normal control. However, some studies have recently reported the activation of the retinal macroglia and microglia in the uninjured eye, suggesting that the eye contralateral to experimental glaucoma should not be used as a control. This review analyzes the studies describing the contralateral findings and discusses some of the routes through which the signals can reach the contralateral eye to initiate the glial reactivation.