Pathological high intraocular pressure induces glial cell reactive proliferation contributing to neuroinflammation of the blood-retinal barrier via the NOX2/ET-1 axis-controlled ERK1/2 pathway.

BACKGROUND: NADPH oxidase (NOX), a primary source of endothelial reactive oxygen species (ROS), is considered a key event in disrupting the integrity of the blood-retinal barrier. Abnormalities in neurovascular-coupled immune signaling herald the loss of ganglion cells in glaucoma. Persistent microglia-driven inflammation and cellular innate immune system dysregulation often lead to deteriorating retinal degeneration. However, the crosstalk between NOX and the retinal immune environment remains unresolved. Here, we investigate the interaction between oxidative stress and neuroinflammation in glaucoma by genetic defects of NOX2 or its regulation via gp91ds-tat. METHODS: Ex vivo cultures of retinal explants from wildtype C57BL/6J and Nox2 -/- mice were subjected to normal and high hydrostatic pressure (Pressure 60 mmHg) for 24 h. In vivo, high intraocular pressure (H-IOP) was induced in C57BL/6J mice for two weeks. Both Pressure 60 mmHg retinas and H-IOP mice were treated with either gp91ds-tat (a NOX2-specific inhibitor). Proteomic analysis was performed on control, H-IOP, and treatment with gp91ds-tat retinas to identify differentially expressed proteins (DEPs). The study also evaluated various glaucoma phenotypes, including IOP, retinal ganglion cell (RGC) functionality, and optic nerve (ON) degeneration. The superoxide (O2-) levels assay, blood-retinal barrier degradation, gliosis, neuroinflammation, enzyme-linked immunosorbent assay (ELISA), western blotting, and quantitative PCR were performed in this study. RESULTS: We found that NOX2-specific deletion or activity inhibition effectively attenuated retinal oxidative stress, immune dysregulation, the internal blood-retinal barrier (iBRB) injury, neurovascular unit (NVU) dysfunction, RGC loss, and ON axonal degeneration following H-IOP. Mechanistically, we unveiled for the first time that NOX2-dependent ROS-driven pro-inflammatory signaling, where NOX2/ROS induces endothelium-derived endothelin-1 (ET-1) overexpression, which activates the ERK1/2 signaling pathway and mediates the shift of microglia activation to a pro-inflammatory M1 phenotype, thereby triggering a neuroinflammatory outburst. CONCLUSIONS: Collectively, we demonstrate for the first time that NOX2 deletion or gp91ds-tat inhibition attenuates iBRB injury and NVU dysfunction to rescue glaucomatous RGC loss and ON axon degeneration, which is associated with inhibition of the ET-1/ERK1/2-transduced shift of microglial cell activation toward a pro-inflammatory M1 phenotype, highlighting NOX2 as a potential target for novel neuroprotective therapies in glaucoma management.

Cilastatin as a Potential Anti-Inflammatory and Neuroprotective Treatment in the Management of Glaucoma.

Glaucoma is a neurodegenerative disease that causes blindness. In this study, we aimed to evaluate the protective role of cilastatin (CIL), generally used in the treatment of nephropathologies associated with inflammation, in an experimental mouse model based on unilateral (left) laser-induced ocular hypertension (OHT). Male Swiss mice were administered CIL daily (300 mg/kg, i.p.) two days before OHT surgery until sacrifice 3 or 7 days later. Intraocular Pressure (IOP), as well as retinal ganglion cell (RGC) survival, was registered, and the inflammatory responses of macroglial and microglial cells were studied via immunohistochemical techniques. Results from OHT eyes were compared to normotensive contralateral (CONTRA) and naïve control eyes considering nine retinal areas and all retinal layers. OHT successfully increased IOP values in OHT eyes but not in CONTRA eyes; CIL did not affect IOP values. Surgery induced a higher loss of RGCs in OHT eyes than in CONTRA eyes, while CIL attenuated this loss. Similarly, surgery increased macroglial and microglial activation in OHT eyes and to a lesser extent in CONTRA eyes; CIL prevented both macroglial and microglial activation in OHT and CONTRA eyes. Therefore, CIL arises as a potential effective strategy to reduce OHT-associated damage in the retina of experimental mice.

TLR4 Inhibition Protects against Retinal Ganglion Cell Damage in Rats with Chronic Ocular Hypertension.

BACKGROUND: This study aims to investigate the protective effect of Toll-like receptor 4 (TLR4) inhibitor Resatorvid (TAK-242) on retinal ganglion cells (RGCs) in a chronic ocular hypertension (COH) rat model, as well as to explore the potential involved mechanisms. METHODS: COH model was built up in rats with a single intracameral administration of cross-linking hydrogel. The expression levels of TLR4, NLR family pyrin domain containing 3 (NLRP3), microglial activation and pro-inflammatory cytokines were evaluated in COH retinas and COH retinas treated with TAK-242 using immunofluorescence staining and Western blot. Additionally, retrograde labeling and neuronal nuclear protein (NeuN) staining were performed to count RGCs. RESULTS: Activated microglia and increased TLR4 expression were observed in the retinas of COH rats. This was accompanied by upregulated expressions of NLRP3, tumor necrosis factor alpha (TNF-α), cytokine interleukin-1ß (IL-1ß) and Interleukin-6 (IL-6). Intravitreal injection of TAK-242 promoted the survival of RGCs by attenuating microglial activation, interfering with the TLR4-NLRP3 pathway and regulating pro-inflammatory cytokines. CONCLUSIONS: Targeting TLR4 inhibition could be a potential therapeutic strategy to protect RGCs from COH damage.

ATP-P2X7R-mediated microglia senescence aggravates retinal ganglion cell injury in chronic ocular hypertension.

BACKGROUND: Dysfunction of microglia during aging affects normal neuronal function and results in the occurrence of neurodegenerative diseases. Retinal microglial senescence attributes to retinal ganglion cell (RGC) death in glaucoma. This study aims to examine the role of ATP-P2X7R in the mediation of microglia senescence and glaucoma progression. METHODS: Forty-eight participants were enrolled, including 24 patients with primary open-angle glaucoma (POAG) and age-related cataract (ARC) and 24 patients with ARC only. We used ARC as the inclusion criteria because of the availability of aqueous humor (AH) before phacoemulsification. AH was collected and the adenosine triphosphate (ATP) concentration was measured by ATP Assay Kit. The chronic ocular hypertension (COH) mouse model was established by microbead occlusion. Microglia were ablated by feeding PLX5622 orally. Mouse bone marrow cells (BMCs) were prepared and infused into mice through the tail vein for the restoration of microglia function. Western blotting, qPCR and ELISA were performed to analyze protein and mRNA expression in the ocular tissue, respectively. Microglial phenotype and RGC survival were assessed by immunofluorescence. The mitochondrial membrane potential was measured using a JC-1 assay kit by flow cytometry. RESULTS: ATP concentrations in the AH were increased in older adults and patients with POAG. The expression of P2X7R was upregulated in the retinal tissues of mice with glaucoma, and functional enrichment analysis showed that P2X7R was closely related to cell aging. Through in vivo and in vitro approaches, we showed that pathological activation of ATP-P2X7R induced accelerated microglial senescence through impairing PTEN-induced kinase 1 (PINK1)-mediated mitophagy, which led to RGC damage. Additionally, we found that replacement of senescent microglia in COH model of old mice with BMCs from young mice reversed RGC damage. CONCLUSION: ATP-P2X7R induces microglia senescence by inhibiting PINK1-mediated mitophagy pathway. Specific inhibition of ATP-P2X7R may be a fundamental approach for targeted therapy of RGC injury in microglial aging-related glaucoma.

The Effect of Latanoprost on Choroidal Vascularity Index in Glaucoma and Ocular Hypertension.

PRCIS: The choroidal vascularity index (CVI) is a new marker for the choroid. The decrease in CVI following latanoprost use can provide a better understanding of the pathogenesis of the posterior segment side effects of latanoprost such as cystoid macular edema and central serous choroidopathy. PURPOSE: The purpose of this paper is to evaluate the changes in the CVI, total choroidal area (TCA), stromal area (SA), luminal area (LA), and choroidal thickness (CT) following latanoprost therapy in patients with primary open angle glaucoma and ocular hypertension. MATERIALS AND METHODS: Patients with newly diagnosed primary open angle glaucoma or ocular hypertension who had never received antiglaucoma therapy were included. Each patient received latanoprost 0.005% once daily. Enhanced depth imaging mode of spectral-domain optical coherence tomography scans was taken before the start of latanoprost therapy and in the first and third months. Subfoveal CT, CVI, TCA, LA, and SA for the submacular area, and 4 quadrants of the peripapillary area were calculated from the scans. RESULTS: A total of 36 eyes of 18 patients were analyzed. Subfoveal CT increased significantly ( P =0.007). Mean TCA ( P =0.008) and SA ( P <0.001) in the first and third months were higher than baseline in the submacular regions. Mean CVI was lower in the first and third months ( P <0.001). There was an increase in the mean TCA and SA in the peripapillary temporal ( P =0.001 and 0.028) and inferior ( P =0.002 and <0.001) quadrants and a decrease in mean CVI in the temporal ( P =0.027) and inferior ( P =0.003) peripapillary quadrants. A negative correlation was found between the rate of decrease in intraocular pressure and the macular region CVI. CONCLUSIONS: Following latanoprost use for several months, the CVI was significantly decreased in newly treated patients with glaucoma or ocular hypertension, among other changes to the choroid. These findings may contribute to a better understanding of the effects of prostaglandins on the posterior segment of the eye.

Urrets-Zavalia syndrome following cataract surgery in dogs: A case series.

Background: In human medicine, Urrets-Zavalia syndrome (UZS) is a well-recognized but uncommon postoperative complication characterized by a fixed dilated pupil, accompanied by iris atrophy and glaucoma. Although it was originally reported in 1963 after penetrating keratoplasty surgery for keratoconus, it has been associated with various ophthalmic procedures such as cataract surgery. The condition has not been previously published in the veterinary literature. Case Description: Three client-owned diabetic dogs that developed UZS´s triad after cataract surgery are described. Despite uneventful phacoemulsification in the six eyes, five developed moderate-to-severe postoperative ocular hypertension. Although intraocular pressure (IOP) spikes were initially controlled, fixed dilated pupils accompanied by iris atrophy and chronic ocular hypertension were seen in the five affected eyes. Aggressive medical and surgical management maintained vision in three of those eyes. In one eye, uncontrolled IOP led to blindness. Conclusion: This is the first published description of UZS in dogs, occurring after phacoemulsification. Although no exact, demonstrable causative element could be determined, we believe that should be considered a triggering condition for this syndrome, as it directly affects the ocular blood flow autoregulation and intrinsic uveal tissue integrity. Until the contrary is proved, diabetes mellitus might be considered as a risk factor for developing this syndrome after cataract surgery in dogs.

Astragaloside IV Attenuates Ocular Hypertension in a Mouse Model of TGFß2 Induced Primary Open Angle Glaucoma.

Elevated intraocular pressure (IOP) is a major risk factor in developing primary open angle glaucoma (POAG), which is the most common form of glaucoma. Transforming growth factor-beta 2 (TGFß2) is a pro-fibrotic cytokine that plays an important role in POAG pathogenesis. TGFß2 induced extracellular matrix (ECM) production, deposition and endoplasmic reticulum (ER) stress in the trabecular meshwork (TM) contribute to increased aqueous humor (AH) outflow resistance and IOP elevation. Drugs which alter the glaucomatous fibrotic changes and ER stress in the TM may be effective in reducing ocular hypertension. Astragaloside IV (AS.IV), a novel saponin isolated from the roots of Astragalus membranaceus, has demonstrated antifibrotic and ER stress lowering effects in various tissues during disease conditions. However, the effect of AS.IV on glaucomatous TM fibrosis, ER stress and ocular hypertension has not been studied. Primary human TM cells treated with AS.IV decreased TGFß2 induced ECM (FN, Col-I) deposition and ER stress (KDEL, ATF4 and CHOP). Moreover, AS.IV treatment reduced TGFß2 induced NF-κB activation and αSMA expression in TM cells. We found that AS.IV treatment significantly increased levels of matrix metalloproteases (MMP9 and MMP2) and MMP2 enzymatic activity, indicating that the antifibrotic effects of AS.IV are mediated via inhibition of NF-κB and activation of MMPs. AS.IV treatment also reduced ER stress in TM3 cells stably expressing mutant myocilin. Interestingly, the topical ocular AS.IV eye drops (1 mM) significantly decreased TGFß2 induced ocular hypertension in mice, and this was associated with a decrease in FN, Col-1 (ECM), KDEL (ER stress) and αSMA in mouse TM tissues. Taken together, the results suggest that AS.IV prevents TGFß2 induced ocular hypertension by modulating ECM deposition and ER stress in the TM.

TRPV4-induced Müller cell gliosis and TNF-α elevation-mediated retinal ganglion cell apoptosis in glaucomatous rats via JAK2/STAT3/NF-κB pathway.

BACKGROUND: Glaucoma, the leading cause of irreversible blindness worldwide, is a type of retinal disease characterized by the selective death of retinal ganglion cells (RGCs). However, the pathogenesis of glaucoma has not been fully elucidated. Transient receptor potential vanilloid 4 (TRPV4) is a pressure-sensitive and calcium-permeable cation channel. TRPV4 is widely distributed in the retina and its sustained activation leads to RGC death; indicating that TRPV4 may be a possible target for glaucoma treatment. Here, we investigated the effects of TRPV4 on RGC apoptosis in a rat model of chronic ocular hypertension (COH), then examined the mechanism underlying these effects. METHODS: The COH model was established by injection of micro-magnetic beads into the anterior chamber of adult male rats. The expression levels of TRPV4, glial fibrillary acidic protein, and inflammatory factors were assessed by immunohistochemistry and immunoblotting. RGC apoptosis and visual dysfunction were evaluated by TUNEL assay and photopic negative response. Functional expression of TRPV4 was examined by electrophysiology and calcium imaging. Real-time polymerase chain reaction and immunoblotting were employed to investigate the molecular mechanism underlying the effects of TRPV4 on tumor necrosis factor-α (TNF-α) release. RESULTS: We found that TRPV4 played an essential role in glaucoma, such that high levels of TRPV4 expression were associated with elevated intraocular pressure. Furthermore, TRPV4 activation was involved in glaucoma-induced RGC apoptosis and RGC-related reductions in visual function. Mechanistic investigation demonstrated that TRPV4 activation led to enhanced Müller cell gliosis and TNF-α release via the JAK2/STAT3/NF-kB pathway, while TRPV4 inhibition could reverse these effects. Finally, TRPV4 activation could lead to elevated expression of TNF receptor 1 in RGCs, while inhibition of TNF-α could reduce TRPV4-mediated RGC apoptosis. CONCLUSIONS: TRPV4 activation induces Müller cell gliosis and TNF-α elevation via the JAK2/STAT3/NF-κB pathway, which may exacerbate RGC apoptosis in glaucoma; these results suggest that TRPV4 can serve as a therapeutic target in glaucoma treatment.

Clinical characteristics and prognostic factors in hypertensive anterior uveitis diagnosed with polymerase chain reaction.

Aim of the study is to report the clinical characteristics and prognostic factors in hypertensive anterior uveitis (AU) diagnosed with multiplex polymerase chain reaction (PCR). Eighty-eight eyes of 88 patients with hypertensive AU were enrolled from 2015 to 2019 in a tertiary center in South Korea. All patients underwent multiplex PCR that was performed using aqueous humor samples collected at first visit to detect the DNA of six herpesviruses. Twenty-eight (31.8%) eyes were PCR positive. Herpes simplex virus was found in 6 (6.8%) eyes, varicella-zoster virus in 7 (8.0%) eyes, cytomegalovirus in 14 (15.9%) eyes, and Epstein-Barr virus in 1 (1.1%) eye. On multivariate regression analysis, PCR positivity was significantly associated with coin-shaped keratic precipitates (odds ratio (OR) = 6.01, P = 0.044). Recurrence and final visual acuity were significantly associated with a presumed diagnosis of viral endotheliitis (OR = 21.69, P = 0.04 and OR = 6.3, P = 0.004, respectively). This study showed the importance of PCR positivity, suggesting that identification of the virus through active PCR testing could affect the course, treatment, and prognosis of hypertensive AU.

Primary cilia and the reciprocal activation of AKT and SMAD2/3 regulate stretch-induced autophagy in trabecular meshwork cells.

Activation of autophagy is one of the responses elicited by high intraocular pressure (IOP) and mechanical stretch in trabecular meshwork (TM) cells. However, the mechanosensor and the molecular mechanisms by which autophagy is induced by mechanical stretch in these or other cell types is largely unknown. Here, we have investigated the mechanosensor and downstream signaling pathway that regulate cyclic mechanical stretch (CMS)-induced autophagy in TM cells. We report that primary cilia act as a mechanosensor for CMS-induced autophagy and identified a cross-regulatory talk between AKT1 and noncanonical SMAD2/3 signaling as critical components of primary cilia-mediated activation of autophagy by mechanical stretch. Furthermore, we demonstrated the physiological significance of our findings in ex vivo perfused eyes. Removal of primary cilia disrupted the homeostatic IOP compensatory response and prevented the increase in LC3-II protein levels in response to elevated pressure challenge, strongly supporting a role of primary cilia-mediated autophagy in regulating IOP homeostasis.

Gonioscopic iridocorneal angle morphology and incidence of postoperative ocular hypertension and glaucoma in dogs following cataract surgery.

PURPOSE: To investigate the relationship between gonioscopic iridocorneal angle (ICA) morphology and the incidence of postoperative ocular hypertension (POH) and postoperative glaucoma in dogs undergoing cataract surgery. ANIMALS STUDIED: Retrospective analysis of 138 eyes of 78 canine patients who underwent phacoemulsification at North Carolina State University from December 1, 2015 through April 30, 2017. METHODS: Medical records of all phacoemulsification patients with preoperative RetCam gonioscopic images were reviewed for preoperative, intraoperative, and postoperative variables. Gonioscopic angle indices were calculated using a novel (ZibWest) angle grading system, and these indices were analyzed for outcome-related significance. RESULTS: Increased surgeon experience was associated with increased probability of POH and vision loss. Higher average ZibWest Angle indices (ie, more open angles with less pectinate ligament dysplasia/ abnormality) were associated with a significantly decreased probability of medically unresponsive glaucoma. Increased patient age was significantly associated with an increased probability of both postoperative glaucoma and vision loss. Female dogs were significantly more likely to experience postoperative glaucoma compared to male dogs. Increased surgery time was significantly associated with increased probability of vision loss. CONCLUSIONS: The ZibWest angle index may predict increased risk for developing medically unresponsive glaucoma with cataract surgery. Female sex, and increased patient age, surgical time, and surgeon experience were associated with increased postoperative morbidity.

Involvement of the NLRC4 inflammasome in promoting retinal ganglion cell death in an acute glaucoma mouse model.

PURPOSE: To explore the role of nucleotide-binding oligomerization domain-like receptors (NLRs) family caspase-activation and the recruitment domain containing 4 (NLRC4) inflammasome in retinal ganglion cell (RGC) injury induced by an acute glaucoma mouse model. METHOD: A mouse model of acute ocular hypertension, which can lead to retinal ischemia-reperfusion (I/R) injury, was established. The expression level of NLRC4 was detected by polymerase chain reaction and western blotting. Localized expression of NLRC4 was detected by examining immunofluorescence in eyeball sections. Intravitreal adeno-associated virus 2(AAV2) administration was used to knockdown retinal Nlrc4. Fluoro-Gold labeled RGCs and TdT-mediated dUTP nick end labeling were used to evaluate the survival and apoptosis of RGCs. Tlr4-/- mice were utilized to explore whether NLRC4 inflammasome is influenced by Toll-like receptor4 (TLR4). RESULTS: NLRC4, expressed in RGCs and microglial cells, was actively involved in mouse retinal I/R injury. Knockdown of Nlrc4 using an AAV2 vector caused an obvious reduction in the generation of IL-1ß led by the rapidly elevated intraocular pressure, and thereby improved the RGC survival. In addition, activation of the NLRC4 inflammasome could influence the phosphorylation of p38 and Jun N-terminal kinase, which was largely dependent on TLR4 signaling. CONCLUSION: Our study demonstrated the role of NLRC4 inflammasome in promoting RGC damage in mouse retinal I/R injury. Inhibition of NLRC4 might be leveraged as a potential therapeutic target in glaucomatous retinopathy.

Ocular hypertension suppresses homeostatic gene expression in optic nerve head microglia of DBA/2 J mice.

Glaucoma is the leading cause of irreversible vision loss. Ocular hypertension is a major risk factor for glaucoma and recent work has demonstrated critical early neuroinflammatory insults occur in the optic nerve head following ocular hypertension. Microglia and infiltrating monocytes are likely candidates to drive these neuroinflammatory insults. However, the exact molecular identity / transcriptomic profile of microglia following ocular hypertensive insults is unknown. To elucidate the molecular identity of microglia after long-term exposure to ocular hypertension, we used a mouse model of glaucoma (DBA/2 J). We performed RNA-sequencing of microglia mRNA from the optic nerve head at a time point following ocular hypertensive insults, but preceding detectable neurodegeneration (with microglia identified as being CD45lo/CD11b+/CD11c-). Furthermore, RNA-sequencing was performed on optic nerve head microglia from mice treated with radiation therapy, a potent therapy preventing neuroinflammatory insults. Transcriptomic profiling of optic nerve head microglia mRNA identifies metabolic priming with marked changes in mitochondrial gene expression, and changes to phagocytosis, inflammatory, and sensome pathways. The data predict that many functions of microglia that help maintain tissue homeostasis are affected. Comparative analysis of these data with data from previously published whole optic nerve head tissue or monocyte-only samples from DBA/2 J mice demonstrate that many of the neuroinflammatory signatures in these data sets arise from infiltrating monocytes and not reactive microglia. Finally, our data demonstrate that prophylactic radiation therapy of DBA/2 J mice potently abolishes these microglia metabolic transcriptomic changes at the same time points. Together, our data provide a unique resource for the community to help drive further hypothesis generation and testing in glaucoma.

Relationships between corneal biomechanics and the structural and functional parameters of glaucoma damage / Relações entre a biomecânica da córnea e os parâmetros estruturais e funcionais do dano do glaucoma

ABSTRACT Purpose: To investigate the relationships between (i) thickness of the retinal nerve fiber layer, optic nerve head topography, and visual field parameters and (ii) corneal biomechanical properties in normal controls and patients with ocular hypertension and primary open-angle glaucoma. Methods: This observational, cross-sectional study included 68 eyes with primary open-angle glaucoma, 99 eyes with ocular hypertension and 133 control eyes. Corneal biomechanical properties, optic nerve head topographic features, retinal nerve fiber layer thickness, and visual fields were assessed in all cases. Corneal biomechanical properties, retinal nerve fiber layer thicknesses, and optic nerve head topographic features were compared among the groups. The associations between structural and functional measures of glaucomatous damage and corneal biomechanical factors were also evaluated. Results: Significantly lower corneal hysteresis and corneal resistance factor values were observed in the primary open-angle glaucoma and ocular hypertension groups as compared with the control group, but there were no significant differences between the primary open-angle glaucoma and ocular hypertension groups. In the ocular hypertension group, no associations were observed between the corneal hysteresis and corneal resistance factor with values and the structural and functional parameters. In the primary open-angle glaucoma group, positive correlations were observed between the corneal hysteresis values and the global retinal nerve fiber layer thickness (p<0.01, r=0.27), mean retinal nerve fiber layer thickness (p<0.01, r=0.33), and mean deviation (p<0.01, r=0.26), and negative correlations were observed between the corneal resistance factor values, and the cup area (p<0.01, r=-0.39), cup-to-disk ratio (p=0.02, r=-0.28), linear cup-to-disk ratio (p=0.02, r=-0.28), and cup shape (p=0.03, r=-0.26). In the control group, weak correlations were detected between the corneal hysteresis and the cup area (p=0.03, r=0.19), cup-to-disk ratio (p=0.01, r=0.21), and linear cup-to-disk ratio (p=0.01, r=0.22). Conclusions: Distinct correlations were identified between the corneal hysteresis and corneal resistance factor values and the functional and structural parameters in the primary open-angle glaucoma and control groups. Corneal hysteresis and corneal resistance factor may have different roles in the pathophysiology of glaucoma.

RESUMO Objetivo: Investigar as relações entre (i) espessura da camada de fibras nervosas da retina, topografia do nervo óptico e parâmetros do campo visual e (ii) propriedades biomecânicas da córnea, em controles normais e pacientes com hiperten são ocular e glaucoma primário de ângulo aberto. Métodos: Este estudo observacional, transversal, incluiu 68 olhos com glaucoma primário de ângulo aberto, 99 olhos com hipertensão ocular e 133 olhos controle. As propriedades biomecânicas da córnea, as características topográficas da cabeça do nervo óptico, a espessura da camada de fibras nervosas da retina e os campos visuais foram avaliados em todos os casos. As propriedades biomecânicas da córnea, a espessura da camada de fibras nervosas da retina e as características topográficas da cabeça do nervo óptico foram comparadas entre os grupos. As associações entre medidas estruturais e funcionais de danos glaucomatosos e fatores biomecânicos da córnea também foram avaliadas. Resultados: Valores de histerese corneana e da resistência corneana foram significativamente menores nos grupos com glaucoma primário de ângulo aberto e hipertensão ocular em com paração ao grupo controle, mas não houve diferenças significativas entre os grupos de glaucoma primário de ângulo aberto e hipertensão ocular. No grupo com hipertensão ocular, não foram observadas associações entre histerese da córnea e o fator de resistência corneana com os valores e os parâmetros estruturais e funcionais. No grupo com glaucoma primário de ângulo aberto foram observadas correlações positivas entre os valores de histerese corneana e a espessura a camada de fibras nervosas da retina (p<0,01, r=0,27), espessura média da camada de fibras nervosas da retina (p<0,01, r=0,33) e desvio médio (p<0,01, r=0,26), e correlações negativas entre o os valores do fator de resistência da córnea e a área de escavação (p<0,01, r=-0,39), a relação escavação/disco (p=0,02, r=-0,28), a relação copo-para-disco linear (p=0,02, r=-0,28) e a forma da escavação (p=0,03, r=-0,26). No grupo controle, correlações foram detectadas entre a histerese da córnea e área de escavação (p=0,03, r=0,19), relação escavação/disco (p=0,01, r=0,21) e relação copo-para-disco linear (p=0,01, r=0,22). Conclusões: Correlações distintas foram identificadas entre histerese da córnea e os valores de resistência da córnea e os parâmetros funcionais e estruturais nos grupos de glaucoma primário de ângulo aberto e controle. A histerese da córnea e o fator de resistência da córnea podem ter diferentes papéis na fisiopatologia do glaucoma.

Effects of age on retinal macrophage responses to acute elevation of intraocular pressure.

There is accumulating evidence that aging shifts the central nervous system milieu towards a proinflammatory state, with increased reactivity of microglia in the aging eye and brain having been implicated in the development of age-related neurodegenerative conditions. Indeed, alterations to microglial morphology and function have been recognized as a part of normal aging. Here, we sought to assess the effects of age on the retinal microglial and macrophage response to acute intraocular pressure (IOP) elevation. Further, we performed experiments whereby bone marrow from young or middle-aged mice was used to reconstitute the bone marrow of whole-body irradiated 12 month old mice. Bone marrow chimeric mice then underwent cannulation and IOP elevation 8 weeks after whole-body irradiation and bone marrow transplantation in order to determine whether the age of bone marrow alters the macrophage response to retinal injury. Our data show retinal macrophage reactivity and microglial morphological changes were enhanced in older mice when compared to younger mice in response to injury. When IOP elevation was performed after whole-body irradiation and bone marrow rescue, we noted subretinal macrophage accumulation and glial reactivity was reduced compared to non-irradiated mice that had also undergone IOP elevation. This effect was evident in both groups of chimeric mice that had received either young or middle-aged bone marrow, suggesting irradiation itself may alter the macrophage and glial response to injury rather than the age of bone marrow.

Protection of the Retinal Ganglion Cells: Intravitreal Injection of Resveratrol in Mouse Model of Ocular Hypertension.

Purpose: To investigate the efficacy of intravitreal administration of resveratrol (RSV) in a microbead-induced high intraocular pressure (IOP) murine model for glaucoma. Methods: Experiments were performed using adult C57BL/6JJcl mice. Polystyrene microbeads were injected into the anterior chamber to induce IOP elevation. Retinal flat-mounts and sections were assessed by immunohistochemistry to detect the expression of reactive oxygen species and acetyl-p53 in retinal ganglion cells (RGCs), brain-derived neurotrophic factor (BDNF) in Müller glial cells (MGCs), and the receptor tropomyosin receptor kinase B (TrkB) in RGCs. Light cycler real-time PCR was also used for confirming gene expression of BDNF in primary cultured MGCs exposed to RSV. Results: Microbeads induced high IOP followed by RGC death and axon loss. Administration of RSV rescued RGCs via decreased reactive oxygen species generation and acetyl-p53 expression in RGCs and upregulated BDNF in MGCs and TrkB expression in RGCs, which exhibited a strong cytoprotective action against cell death through multiple pathways under high IOP. Conclusions: Our data suggest that administration of RSV may delay the progress of visual dysfunction during glaucoma and may therefore have therapeutic potential.

Expression of Melatonin and Dopamine D3 Receptor Heteromers in Eye Ciliary Body Epithelial Cells and Negative Correlation with Ocular Hypertension.

BACKGROUND: Experiments in the late nineties showed an inverse relationship in the eye levels of melatonin and dopamine, thereby constituting an example of eye parameters that are prone to circadian variations. The underlying mechanisms are not known but these relevant molecules act via specific cell surface dopamine and melatonin receptors. This study investigated whether these receptors formed heteromers whose function impact on eye physiology. We performed biophysical assays to identify interactions in heterologous systems. Particular heteromer functionality was detected using Gi coupling, MAPK activation, and label-free assays. The expression of the heteroreceptor complexes was assessed using proximity ligation assays in cells producing the aqueous humor and human eye samples. Dopamine D3 receptors (D3Rs) were identified in eye ciliary body epithelial cells. We discovered heteromers formed by D3R and either MT1 (MT1R) or MT2 (MT2R) melatonin receptors. Heteromerization led to the blockade of D3R-Gi coupling and regulation of signaling to the MAPK pathway. Heteromer expression was negatively correlated with intraocular hypertension. CONCLUSIONS: Heteromers likely mediate melatonin and dopamine actions in structures regulating intraocular pressure. Significant expression of D3R-MT1R and D3R-MT1R was associated with normotensive conditions, whereas expression diminished in a cell model of hypertension. A clear trend of expression reduction was observed in samples from glaucoma cases. The trend was marked but no statistical analysis was possible as the number of available eyes was 2.

Nuclear factor-kappa beta signaling is required for transforming growth factor Beta-2 induced ocular hypertension.

A major risk for the development of primary open-angle glaucoma (POAG) is elevated intraocular pressure (IOP). Elevated IOP is caused by increased outflow resistance due in part to excessive extracellular matrix (ECM) deposition in the trabecular meshwork (TM). The role of transforming growth factor beta 2 (TGFß2) in inducing ECM production is well understood. Recent studies suggest that toll-like receptor 4 (TLR4) plays an important role in fibrogenesis. We have previously described a crosstalk between TGFß2 and TLR4 in the development of ocular hypertension and glaucomatous TM damage. Nuclear factor-kappa beta (NF-κB) is critical for TLR4 signaling. To determine the transactivation of NF-κB, TM cells were stimulated with cellular fibronectin containing the EDA isoform (cFN-EDA), TGFß2, or lipopolysaccharide (LPS) in combination with a selective TLR4 inhibitor. cFN-EDA, TGFß2, and LPS all induced transactivation of NF-κB and inhibition of TLR4 blocked the effect of each treatment paradigm. To evaluate the role of NF-κB in IOP regulation, we utilized our inducible mouse model of ocular hypertension by injection of Ad5.TGFß2 in mice harboring a mutation in NF-κB and wild-type controls. IOP was measured over time and eyes accessed by immunohistochemistry for the ECM protein FN and the specific FN-EDA isoform. Ad5.TGFß2 induced ocular hypertension and expression of FN and FN-EDA in wild-type mice, but mutation in NF-κB blocked the effect. These data suggest that NF-κB is necessary for TGFß2-induced ECM production and ocular hypertension and the transactivation of NF-κB is dependent on both TGFß2 and TLR4.

Piezo channel plays a part in retinal ganglion cell damage.

Piezo channel is one of the mechanosensitive channels that senses pressure and shearing stress. Previous reports show that Piezo channel is expressed in many tissues such as skin and lung and they have many important roles. In addition, the mRNA of Piezo has been detected in astrocytes in the optic nerve head of mice. However, it is not yet clear where Piezo channel localize in eye and what kind of effects it have. Thus, the purpose of this study was to determine the expression sites of Piezo channel in mouse eyes and effect of Piezo channel on retinal ganglion cells. Immunostaining analysis showed that the Piezo 1/2 were expressed in the cornea, trabecular meshwork of the anterior ocular segment, lens epithelial cells, and on the retinal ganglion cell layer. The expression of retinal Piezo 2 was increased in retinal disorder model mouse caused by high IOP. Piezo 1 agonist Yoda 1 suppressed neurite outgrowth in retinal ganglion cells. On the other hand, Piezo antagonist GsMTx4 promoted neurite outgrowth in retinal ganglion cells. These findings indicate that Piezo channel may contribute to diseases relating the IOP such as glaucoma.

A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice.

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing stable IOP elevation in mice that mimics the post-operative complication of using silicone oil (SO) as a tamponade agent in human vitreoretinal surgery. In this protocol, SO is injected into the anterior chamber of the mouse eye to block the pupil and prevent inflow of aqueous humor. The posterior chamber accumulates aqueous humor and this in turn increases the IOP of the posterior segment. A single SO injection produces reliable, sufficient, and stable IOP elevation, which induces significant glaucomatous neurodegeneration. This model is a true replicate of secondary glaucoma in the eye clinic. To further mimic the clinical setting, SO can be removed from the anterior chamber to reopen the drainage pathway and allow inflow of aqueous humor, which is drained through the trabecular meshwork (TM) at the angle of the anterior chamber. Because IOP quickly returns to normal, the model can be used to test the effect of lowering IOP on glaucomatous retinal ganglion cells. This method is straightforward, does not require special equipment or repeat procedures, closely simulates clinical situations, and may be applicable to diverse animal species. However, minor modifications may be required.