Sector-specific Association of Intraocular Pressure Dynamics in Dark-room Prone Testing and Visual Field Defect Progression in Glaucoma.

PURPOSE: To investigate sectoral differences in the relationship between intraocular pressure (IOP) dynamics during dark-room prone testing (DRPT) and visual field (VF) defect progression in primary open-angle glaucoma (POAG) patients. DESIGN: Retrospective, longitudinal study. PARTICIPANTS: This retrospective study included 116 eyes of 84 POAG patients who underwent DRPT and had at least 5 reliable VF tests conducted over a more than 2-year follow-up period. We excluded eyes with mean deviation worse than -20 dB or a history of intraocular surgery or laser treatment. METHODS: Average total deviation (TD) was calculated in the superior, central, and inferior sectors of the Humphrey 24-2 or 30-2 program. During DRPT, IOP was measured in the sitting position, and after 60 minutes in the prone position in a dark room, IOP was measured again. The relationship between IOP change during DRPT, IOP after DRPT, and TD slope in each quadrant was analyzed with a linear mixed-effects model, adjusting for other potential confounding factors. MAIN OUTCOME MEASURES: Total deviation slope in each quadrant, IOP change during DRPT, and IOP after DRPT. RESULTS: Intraocular pressure after DRPT and IOP change during DRPT were 18.16 ± 3.42 mmHg and 4.92 ± 3.12 mmHg, respectively. Superior TD slope was significantly associated with both IOP after DRPT (ß = -0.28, P = 0.003) and IOP change during DRPT (ß = -0.21, P = 0.029), while central (ß = -0.05, P = 0.595; ß = -0.05; P = 0.622) and inferior (ß = 0.05, P = 0.611; ß = 0.01, P = 0.938) TD slopes were not. CONCLUSION: Dark-room prone testing might be a useful test to predict the risk of superior VF defect progression in eyes with POAG. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

The association between intraocular pressure dynamics during dark-room prone testing and intraocular pressure over a relatively long-term follow-up period in primary open-glaucoma patients.

PURPOSE: To investigate the relationship between the dynamics of intraocular pressure (IOP) during dark-room prone testing (DRPT) and IOP over a relatively long-term follow-up period. METHODS: This retrospective study enrolled 84 eyes of 51 primary open-angle glaucoma patients who underwent DRPT for whom at least three IOP measurements made using Goldmann applanation tonometry were available over a maximum follow-up period of two years. We excluded eyes with a history of intraocular surgery or laser treatment and those with changes in topical anti-glaucoma medication during the follow-up period. In DRPT, IOP was measured in the sitting position, and after 60 min in the prone position in a dark room, IOP was measured again. In this study, IOP fluctuation refers to the standard deviation (SD) of IOP, and IOP max indicates the maximum value of IOP during the follow-up. The relationship between these parameters was analyzed with a linear mixed-effects model, adjusting for clinical parameters including age, gender, and axial length. RESULTS: IOP increased after DRPT with a mean of 6.13 ± 3.55 mmHg. IOP max was significantly associated with IOP after DRPT (ß = 0.38; p < 0.001). IOP fluctuation was significantly associated with IOP change in DRPT (ß = 0.29; p = 0.007). CONCLUSION: Our findings suggest that short-term and relatively long-term IOP dynamics are associated. Long-term IOP dynamics can be predicted by DRPT to some extent.

Retina as a potential biomarker in schizophrenia spectrum disorders: a systematic review and meta-analysis of optical coherence tomography and electroretinography.

INTRODUCTION: Abnormal findings on optical coherence tomography (OCT) and electroretinography (ERG) have been reported in participants with schizophrenia spectrum disorders (SSDs). This study aims to reveal the pooled standard mean difference (SMD) in retinal parameters on OCT and ERG among participants with SSDs and healthy controls and their association with demographic characteristics, clinical symptoms, smoking, diabetes mellitus, and hypertension. METHODS: Using PubMed, Scopus, Web of Science, and PSYNDEX, we searched the literature from inception to March 31, 2023, using specific search terms. This study was registered with PROSPERO (CRD4202235795) and conducted according to PRISMA 2020. RESULTS: We included 65 studies in the systematic review and 44 in the meta-analysis. Participants with SSDs showed thinning of the peripapillary retinal nerve fiber layer (pRNFL), macular ganglion cell layer- inner plexiform cell layer, and retinal thickness in all other segments of the macula. A meta-analysis of studies that excluded SSD participants with diabetes and hypertension showed no change in results, except for pRNFL inferior and nasal thickness. Furthermore, a significant difference was found in the pooled SMD of pRNFL temporal thickness between the left and right eyes. Meta-regression analysis revealed an association between retinal thinning and duration of illness, positive and negative symptoms. In OCT angiography, no differences were found in the foveal avascular zone and superficial layer foveal vessel density between SSD participants and controls. In flash ERG, the meta-analysis showed reduced amplitude of both a- and b-waves under photopic and scotopic conditions in SSD participants. Furthermore, the latency of photopic a-wave was significantly shorter in SSD participants in comparison with HCs. DISCUSSION: Considering the prior report of retinal thinning in unaffected first-degree relatives and the results of the meta-analysis, the findings suggest that retinal changes in SSDs have both trait and state aspects. Future longitudinal multimodal retinal imaging studies are needed to clarify the pathophysiological mechanisms of these changes and to clarify their utility in individual patient monitoring efforts.

Retinal layers and associated clinical factors in schizophrenia spectrum disorders: a systematic review and meta-analysis.

INTRODUCTION: The retina shares structural and functional similarities with the brain. Furthermore, structural changes in the retina have been observed in patients with schizophrenia spectrum disorders (SSDs). This systematic review and meta-analysis investigated retinal abnormalities and their association with clinical factors for SSD. METHODS: Studies related to retinal layers in SSD patients were retrieved from PubMed, Scopus, Web of Science, Cochrane Controlled Register of Trials, International Clinical Trials Registry Platform, and PSYNDEX databases from inception to March 31, 2021. We screened and assessed the eligibility of the identified studies. EZR ver.1.54 and the metafor package in R were used for the meta-analysis and a random-effects or fixed-effects model was used to report standardized mean differences (SMDs). RESULTS: Twenty-three studies (2079 eyes of patients and 1571 eyes of controls) were included in the systematic review and meta-analysis. The average peripapillary retinal nerve fiber layer (pRNFL) thickness, average macular thickness (MT), and macular ganglion cell layer-inner plexiform layer (GCL-IPL) thickness were significantly lower in patients than in controls (n = 14, 6, and 3, respectively; SMD = -0.33, -0.49, and -0.43, respectively). Patients also had significantly reduced macular volume (MV) compared to controls (n = 7; SMD = -0.53). The optic cup volume (OCV) was significantly larger in patients than in controls (n = 3; SMD = 0.28). The meta-regression analysis indicated an association between several clinical factors, such as duration of illness and the effect size of the pRNFL, macular GCL-IPL, MT, and MV. CONCLUSION: Thinning of the pRNFL, macular GCL-IPL, MT, and MV and enlargement of the OCV in SSD were observed. Retinal abnormalities may be applicable as state/trait markers in SSDs. The accumulated evidence was mainly cross-sectional and requires verification by longitudinal studies to characterize the relationship between OCT findings and clinical factors.

A Plant-Derived Antioxidant Supplement Prevents the Loss of Retinal Ganglion Cells in the Retinas of NMDA-Injured Mice.

Purpose: To investigate the effect of plant-derived antioxidant compounds, identified with primary culture screening, on retinal ganglion cell (RGC) survival in mice under excitotoxic conditions. Additionally, to determine the effect of these compounds on the involvement of calpain inactivation. Materials and Methods: Plant-derived antioxidant compounds including hesperidin, crocetin, and Tamarindus indica were administrated orally to C57BL/6J mice. The levels of lipid oxidation and calpain activation were assessed with a TBARS assay and western blotting. RGC survival was evaluated with a TUNEL assay and RBPMS immunostaining after intravitreal injection of NMDA. Results: Plant-derived antioxidant compounds significantly ameliorated the increase in the level of MDA in the retinas after NMDA injury. Cleaved α-fodrin fragments were detected in the NMDA-injured retinas, and these fragments were significantly lower in mice that received the plant-derived antioxidant compounds. The plant-derived antioxidants also ameliorated increases in TUNEL-positive cells and RGC death after NMDA injection. Conclusion: These results indicate that oral administration of plant-derived antioxidant compounds such as hesperidin, crocetin, and Tamarindus indica suppressed RGC death. This oral supplementation decreased lipid oxidation and excessive calpain activation in NMDA-injured retinas. Thus, our newly developed antioxidant supplement has a potential role in neuroprotective treatment for retinal diseases, such as glaucoma.

CHOP deletion and anti-neuroinflammation treatment with hesperidin synergistically attenuate NMDA retinal injury in mice.

Glaucoma is a leading cause of blindness worldwide and is characterized by degeneration associated with the death of retinal ganglion cells (RGCs). It is believed that glaucoma is a group of heterogeneous diseases with multifactorial pathomechanisms. Here, we investigate whether anti-inflammation treatment with an ER stress blockade can selectively promote neuroprotection against NMDA injury in the RGCs. Retinal excitotoxicity was induced with an intravitreal NMDA injection. Microglial activation and neuroinflammation were evaluated with Iba1 immunostaining and cytokine gene expression. A stable HT22 cell line transfected with an NF-kB reporter was used to assess NF-kB activity after hesperidin treatment. CHOP-deficient mice were used as a model of ER stress blockade. Retinal cell death was evaluated with a TUNEL assay. As results, in the NMDA injury group, Iba1-positive microglia increased 6 h after NMDA injection. Also at 6 h, pro-inflammatory cytokines and chemokine increased, including TNFα, IL-1b, IL-6 and MCP-1. In addition, the MCP-1 promoter-driven EGFP signal, which we previously identified as a stress signal in injured RGCs, also increased; hesperidin treatment suppressed this inflammatory response and reduced stressed RGCs. In CHOP-deficient mice that received an NMDA injection, the gene expression of pro-inflammatory cytokines, chemokines, markers of active microglia, and inflammatory regulators was greater than in WT mice. In WT mice, hesperidin treatment partially prevented retinal cell death after NMDA injury; this neuroprotective effect was enhanced in CHOP-deficient mice. These findings demonstrate that ER stress blockade is not enough by itself to prevent RGC loss due to neuroinflammation in the retina, but it has a synergistic neuroprotective effect after NMDA injury when combined with an anti-inflammatory treatment based on hesperidin.

GCaMP3 expressing cells in the ganglion cell layer of Thy1-GCaMP3 transgenic mice before and after optic nerve injury.

The genetically encoded green fluorescent protein-based calcium sensor, GCaMP, has been used to detect calcium transients and report neuronal activity. We evaluated the specificity of GCaMP3 expression to retinal ganglion cells (RGCs) of the transgenic Thy1-GCaMP3 mouse line in healthy control animals and in those after optic nerve transection (ONT). Retinas from control mice (n = 4) were isolated and stained for RNA-binding protein with multiple splicing (RBPMS) and choline acetyltransferase (ChAT), specific markers for RGCs and cholinergic amacrine cells, respectively. GCaMP3 expression was enhanced with green fluorescent protein (GFP) immunoreactivity. In one subset of animals, ONT was performed 3, 7, or 14 days before sacrifice (n = 4, 4, 4, respectively). Cells positive for GCaMP3, RBPMS, ChAT, as well as the population of co-labeled cells, were quantified. In another subset of animals (n = 4), in vivo confocal scanning laser ophthalmoscope imaging was performed in the same mice at baseline and at 3, 7 and 14 days after ONT. The mean (SD) densities of GCaMP3, RBPMS, and ChAT expressing cells in control retinas were 2663 (110), 3401 (175), and 1041 (47) cells/mm2, respectively. Of the GCaMP3+ cells, 92 (1)% were co-labeled with RBPMS, while 72 (1)% of RBPMS-labeled cells expressed GCaMP3. ChAT expressing cells were not co-labeled with GCaMP3. The number of GCaMP3+ and RBPMS+ cells decreased dramatically after ONT; 78%, 39%, and 18% of GCaMP3+ and 80%, 40%, and 15% of RBPMS+ cells, relative to control retinas, survived at 3, 7, and 14 days after ONT. However, the number of ChAT+ cells did not change. There was a progressive decrease in GCaMP3 fluorescence after ONT in in vivo images. The majority of RGCs in the ganglion cell layer of Thy1-GCaMP3 mice express GCaMP3. There was an expected progressive and specific loss of GCaMP3 expression after ONT. Thy1-GCaMP3 transgenic mice have potential for longitudinal assessment of RGCs in injury models.

The relationship between glutathione levels in leukocytes and ocular clinical parameters in glaucoma.

PURPOSE: To investigate the effect of mitochondrial dysfunction on the autoregulation of blood flow, by measuring levels of glutathione, an indicator of mitochondrial dysfunction, in glaucoma patients. METHODS: Fifty-six OAG patients and 21 age-matched controls underwent a blood assay. Mitochondrial function was measured according to the levels of total glutathione (t-GSH), reduced GSH (GSH), and oxidized GSH (GSSG, glutathione disulfide) in peripheral blood mononuclear cells. Ocular blood flow in the optic nerve head was assessed with laser speckle flowgraphy parameters, including acceleration time index (ATI). We determined correlations between these measurements and other clinical parameters. Furthermore, we investigated the association between glutathione levels and glaucoma with a logistic regression analysis. Finally, we calculated the area under the receiver operating characteristic (ROC) curve in order to determine the power of redox index (the log GSH/GSSG ratio) to distinguish the groups. RESULTS: OAG patients demonstrated significantly higher GSSG levels and a lower redox index than the controls (p = 0.01, p = 0.01, respectively), but total GSH and reduced GSH levels were similar in the OAG subjects and controls (p = 0.80, p = 0.94, respectively). Additionally, redox index was significantly correlated with mean deviation (MD) of the visual field (r = 0.29, p = 0.03) and ATI (r = -0.30, p = 0.03). Multiple linear regression analysis showed that redox index contributed to MD (p = 0.02) and ATI (p = 0.04). The receiver operating characteristic curve (AUC) analysis suggested that redox index could differentiate between control eyes and eyes with glaucoma (AUC; 0.70: 95% interval; 0.57-0.84). The cutoff point for redox index to maximize its sensitivity and specificity was 2.0 (sensitivity: 91.1%, specificity: 42.9%). CONCLUSIONS: These results suggest that redox index is lower in OAG patients than in controls. Thus, it is possible that mitochondrial dysfunction contributes to glaucoma pathogenesis by causing vascular alterations.

Retinal Characterization of the Thy1-GCaMP3 Transgenic Mouse Line After Optic Nerve Transection.

Purpose: GCaMP3 is a genetically encoded calcium indicator for monitoring intracellular calcium dynamics. We characterized the expression pattern and functional properties of GCaMP3 in the Thy1-GCaMP3 transgenic mouse retina. Methods: To determine the specificity of GCaMP3 expression, Thy1-GCaMP3 (B6; CBA-Tg(Thy1-GCaMP3)6Gfng/J) retinas were processed for immunohistochemistry with anti-green fluorescent protein (anti-GFP, to enhance GCaMP3 fluorescence), anti-RBPMS (retinal ganglion cell [RGC]-specific marker), and antibodies against amacrine cell markers (ChAT, GABA, GAD67, syntaxin). Calcium imaging was used to characterize functional responses of GCaMP3-expressing (GCaMP+) cells by recording calcium transients evoked by superfusion of kainic acid (KA; 10, 50, or 100 µM). In a subset of animals, optic nerve transection (ONT) was performed 3, 5, or 7 days prior to calcium imaging. Results: GFP immunoreactivity colocalized with RBPMS but not amacrine cell markers in both ONT and non-ONT (control) groups. Calcium transients evoked by KA were reduced after ONT (50 µM KA; ΔF/F0 [SD]; control: 1.00 [0.67], day 3: 0.50 [0.35], day 5: 0.31 [0.28], day 7: 0.35 [0.36]; P < 0.05 versus control). There was also a decrease in the number of GCaMP3+ cells after ONT (cells/mm2 [SD]; control: 2198 [453], day 3: 2224 [643], day 5: 1383 [375], day 7: 913 [178]; P < 0.05). Furthermore, the proportion of GCaMP3+ cells that responded to KA decreased after ONT (50 µM KA, 97%, 54%, 47%, and 58%; control, 3, 5, and 7 days, respectively). Conclusions: Following ONT, functional RGC responses are lost prior to the loss of RGC somata, suggesting that anatomical markers of RGCs may underestimate the extent of RGC dysfunction.

Ecel1 Knockdown With an AAV2-Mediated CRISPR/Cas9 System Promotes Optic Nerve Damage-Induced RGC Death in the Mouse Retina.

Purpose: To assess the therapeutic potential of endothelin-converting enzyme-like 1 (Ecel1) in a mouse model of optic nerve crush. Methods: Ecel1 expression was evaluated with real time quantitative (qRT)-PCR, Western blotting, and immunohistochemistry in mouse retinas after optic nerve crush. Vinblastine administration to the optic nerve and the intravitreal injection of N-methyl-d-aspartate (NMDA) were used to assess Ecel1 gene expression. Ecel1 was deleted with an adeno-associated viral (AAV) clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9 system, and retinal ganglion cell (RGC) survival was investigated with retrograde labeling, qRT-PCR, and visual evoked potential. Results: Optic nerve crush induced Ecel1 expression specifically in the RGCs, peaking on day 4 after optic nerve crush. Ecel1 gene expression was induced by the vinblastine-induced inhibition of axonal flow, but not by NMDA-induced excitotoxicity, even though both are triggers of RGC death. Knockdown of Ecel1 promoted the loss of RGCs after optic nerve crush. Conclusions: Our data suggest that Ecel1 induction is part of the retinal neuroprotective response to axonal injury in mice. These findings might provide insight into novel therapeutic targets for the attenuation of RGC damage, such as occurs in traumatic optic neuropathy.

The neuroprotective effect of hesperidin in NMDA-induced retinal injury acts by suppressing oxidative stress and excessive calpain activation.

We found that hesperidin, a plant-derived bioflavonoid, may be a candidate agent for neuroprotective treatment in the retina, after screening 41 materials for anti-oxidative properties in a primary retinal cell culture under oxidative stress. We found that the intravitreal injection of hesperidin in mice prevented reductions in markers of the retinal ganglion cells (RGCs) and RGC death after N-methyl-D-aspartate (NMDA)-induced excitotoxicity. Hesperidin treatment also reduced calpain activation, reactive oxygen species generation and TNF-α gene expression. Finally, hesperidin treatment improved electrophysiological function, measured with visual evoked potential, and visual function, measured with optomotry. Thus, we found that hesperidin suppressed a number of cytotoxic factors associated with NMDA-induced cell death signaling, such as oxidative stress, over-activation of calpain, and inflammation, thereby protecting the RGCs in mice. Therefore, hesperidin may have potential as a therapeutic supplement for protecting the retina against the damage associated with excitotoxic injury, such as occurs in glaucoma and diabetic retinopathy.

Evaluating retinal vessel diameter with optical coherence tomography in normal-tension glaucoma patients.

PURPOSE: To investigate a novel optical coherence tomography (OCT)-derived variable, circumpapillary mean retinal shadow width (cpMRSW), and to elucidate its association with normal-tension glaucoma (NTG). METHODS: For the purpose of validation, we measured retinal vascular calibers in 68 arterioles and 100 venules of 12 NTG patients and 12 healthy subjects and compared the width of the visible retinal shadows in spectral-domain OCT images and the caliber of retinal vessels in retinal photographs. Then we calculated cpMRSW in 78 NTG eyes and 25 age-matched healthy control eyes. Additionally, we divided the patients into early (mean deviation: MD > -6 dB), moderate (MD -6 to -12 dB), and severe (MD < -12 dB) NTG groups, and compared cpMRSW in these groups. Finally, we calculated the area under the receiver operating characteristic (ROC) curve in order to determine the power of mean retinal shadow width to distinguish the groups. RESULTS: OCT retinal shadow width was significantly correlated with photography-measured retinal caliber (r = 0.82, P < 0.001). CpMRSW was significantly different between the control and NTG patients (control: 107.3 ± 7.0 µm, mild: 99.4 ± 8.6 µm, moderate: 99.7 ± 9.5 µm, severe: 90.5 ± 12.0 µm, P < 0.001), despite similar distributions in systemic variables. An ROC analysis revealed that cpMRSW could differentiate NTGs from normal eyes (area under the ROC curve: 0.81). CONCLUSIONS: Our new software for measuring mean retinal shadow width in OCT images may be a valuable tool for detecting NTG and diagnosing its severity.