Changes In Retinal Ganglion Cells Research Articles

Longitudinal In Vivo Changes in Retinal Ganglion Cell Dendritic Morphology After Acute and Chronic Optic Nerve Injury.

PurposeTo characterize in vivo dendritic changes in retinal ganglion cells (RGCs) after acute (optic nerve transection, ONT) and chronic (experimental glaucoma, EG) optic nerve injury.MethodsONT and EG (microbead model) were carried out in Thy1-YFP mice in which the entire RGC dendritic arbor was imaged with confocal fluorescence scanning laser ophthalmoscopy over two weeks in the ONT group and over two and six months, respectively, in two (groups 1 and 2) EG groups. Sholl analysis was used to quantify dendritic structure with the parameters: area under the curve (AUC), radius of the dendritic field, peak number of intersections (PI), and distance to the PI (PD).ResultsDendritic changes were observed after three days post-ONT with significant decreases in all parameters at two weeks. In group 1 EG mice, mean (SD) intraocular pressure (IOP) was 15.2 (1.1) and 9.8 (0.3) mmHg in the EG and untreated contralateral eyes, respectively, with a significant corresponding decrease in AUC, PI, and PD, but not radius. In group 2 mice, the respective IOP was 13.1 (1.0) and 8.8 (0.1) mmHg, peaking at two months before trending towards baseline. Over the first two months, AUC, PI, and PD decreased significantly, with no further subsequent changes. The rates of change of the parameters after ONT was 5 to 10 times faster than in EG.ConclusionsRapid dendritic changes occurred after ONT, while changes in EG were slower and associated with level of IOP increase. The earliest alterations were loss of inner neurites without change in dendritic field.

MiRNA Changes in Retinal Ganglion Cells after Optic Nerve Crush and Glaucomatous Damage.

The purpose of this study was to characterize the miRNA profile of purified retinal ganglion cells (RGC) from healthy and diseased rat retina. Diseased retina includes those after a traumatic optic nerve crush (ONC), and after ocular hypertension/glaucoma. Rats were separated into four groups: healthy/intact, 7 days after laser-induced ocular hypertension, 2 days after traumatic ONC, and 7 days after ONC. RGC were purified from rat retina using microbeads conjugated to CD90.1/Thy1. RNA were sequenced using Next Generation Sequencing. Over 100 miRNA were identified that were significantly different in diseased retina compared to healthy retina. Considerable differences were seen in the miRNA expression of RGC 7 days after ONC, whereas after 2 days, few changes were seen. The miRNA profiles of RGC 7 days after ONC and 7 days after ocular hypertension were similar, but discrete miRNA differences were still seen. Candidate mRNA showing different levels of expression after retinal injury were manipulated in RGC cultures using mimics/AntagomiRs. Of the five candidate miRNA identified and subsequently tested for therapeutic efficacy, miR-194 inhibitor and miR-664-2 inhibitor elicited significant RGC neuroprotection, whereas miR-181a mimic and miR-181d-5p mimic elicited significant RGC neuritogenesis.

Electrophysiological markers of preclinical diagnosis of glaucomatous optic neuropathy

Purpose: to evaluate the changes in electrophysiological indicators reflecting various aspects of the function of retinal ganglion cells (RGC) and their axons in the early diagnosis of glaucomatous optic neuropathy (GON).Material and methods. Two clinical groups, (1) 35 patients (60 eyes) aged 49 to 70 with suspected glaucoma and (2) 16 patients (30 eyes) aged 43–68 with initial primary open-angle glaucoma (POAG), and a comparison group of 38 relatively healthy subjects (45 eyes) aged 42–70 were tested for pattern-reversed visual evoked potentials (PVEP), transient and stationary pattern-ERGs (PERG) according to ISCEV, and photopic negative response (PhNR).Results. The P100 amplitudes in both clinical groups differed significantly from the norm in PVEP on small and large patterns. The elongation of peak latency (T) of P100 compared with norm was significant for the stimulus 1° in group 2. In both groups of patients, increased variability of the temporal parameters of PERG and PVEP for small patterns was found. In groups 1 and 2, a decrease in the amplitude of P50 and N95 peaks of transient PERG for all stimuli was revealed, which was the most significant for the 0.3° pattern. In group 1, the N95 peak was significantly delayed in PERG for large patterns. A statistically significant reduction in the steady-state PERG's amplitude was found in the groups of suspected glaucoma and initial POAG. The sharpest changes were found for small (0.8° and 0.3°) patterns. The elongation of T compared to the norm was most pronounced for PERG at 0.3°, but due to the high variability of temporary indicators within the group, it had no statistical significance. The amplitude of PhNR was significantly different from the norm in the ERG for a flash of 3.0 cd·sec/m2.Conclusion. In patients with suspected glaucoma, a decrease in the P100 VEP amplitude with the simultaneous elongation of T may be considered as a criteria for the plastic stage at the level of lateral geniculate nucleus. Markers of functional changes in RGCs are the decrease in the amplitude of PhNR in response to bright flash, and P50 and N95 of PERG for pattern size 0.3°. The results indicate a greater vulnerability of the parvocellular system to early events in the development of GON.

Relationship between N95 Amplitude of Pattern Electroretinogram and Optical Coherence Tomography Angiography in Open-Angle Glaucoma.

Purpose: The pattern electroretinogram (PERG) is useful to detect retinal ganglion cell (RGC) damage in patients with glaucoma. Optical coherence tomography angiography (OCTA) measures retinal vessel density (VD), which is known to be reduced in glaucoma. There may be correlations between parameters of the PERG and OCTA in open-angle glaucoma (OAG). Methods: In total, 95 eyes of 95 OAG patients and 102 eyes of 102 normal controls were included in this study. N35, P50, and N95 latency along with P50 and N95 amplitude were obtained using the PERG. Retinal VD was measured around the peripapillary and macular area according to the ETDRS grid (concentric circles with diameters of 1, 3, and 6 mm), which is named a center (≤1 mm), an inner (1–3 mm), an outer (3–6 mm), and a full (≤6 mm) area. Pearson correlation analysis was done between parameters, and partial correlation analysis was done after adjusting confounding factors. Results: P50 amplitude, N95 amplitude, and VD of most measured areas were significantly lower in the OAG group compared to the normal group. N95 amplitude showed a statistically significant correlation with parameters of optical coherence tomography and visual field, peripapillary outer and full VD, and macular outer and full VD even after adjusting confounding factors. There was no significant correlation between parameters in the normal group. Conclusions: N95 amplitude was associated with structural and functional change including VD reduction in OAG. Microvascular alterations may be associated with dysfunctional changes of RGC recorded by the PERG in OAG.

Laser Capture Microdissection-Based RNA Microsequencing Reveals Optic Nerve Crush-Related Early mRNA Alterations in Retinal Ganglion Cell Layer.

PurposeTo establish a method of laser capture microdissection (LCM) and RNA microsequencing for exploring optic nerve crush (ONC)–related early mRNA alterations in retinal ganglion cell (RGC) layer.MethodsAn LCM protocol was developed using retinal tissue sections to obtain high-quality RNA for microsequencing. Cells in the RGC layer were collected by laser pressure catapulting (LPC) using a PALM Zeiss UV LCM system. The effect of section thickness and slide type on tissue capture success and RNA yield and the integrity after LCM were evaluated. The optimal LCM protocol was used to explore ONC-related early mRNA alterations in the RGC layer. Candidate genes were validated by real-time polymerase chain reaction of the RGC layer tissue dissected by “cut and LPC” using the same LCM system.ResultsWe successfully established an optimal LCM protocol using 30-µm–thick retinal tissue sections mounted on glass slides and laser pressure catapulting (LPC) to collect cells in the RGC layer and to obtain high-quality RNA for microsequencing. On the basis of our protocol, we identified 8744 differentially expressed genes that were involved in ONC-related early mRNA alterations in the RGC layer. Candidate genes included Atf3, Lgals3, LOC102551701, Plaur, Tmem140, and Maml1.ConclusionsThe LCM-based single-cell RNA sequencing allowed a new sight into the early mRNA changes of RGCs highlighting new molecules associated to ONC.Translational RelevanceThis technique will be helpful for more accurate transcriptome analysis of clinical pathological samples of ophthalmology and provide important reference for the discovery of new pathological diagnosis indicators and drug development targets.

Attenuated Visual Function in Patients with Major Depressive Disorder.

Background: We sought to investigate visual function, primarily, and structural changes in retinal ganglion cells, secondarily, in patients with major depressive disorder. Methods: A total of 50 normal participants and 49 patients with major depressive disorder were included in this cross-sectional study. The participants underwent 24–2 standard automated perimetry and spectral-domain optical coherence tomography. Results: The pattern standard deviation (PSD) in the visual field test was higher in the major depressive disorder patients than in the normal control subjects (p = 0.017). The patients with major depressive disorder showed reduced minimum ganglion cell–inner plexiform layer (GCIPL) thickness relative to the normal control participants (p = 0.015). The average score on the Hamilton Depression Rating scale showed a significant correlation with the PSD, minimum GCIPL thickness, and inferior GCIPL thickness (r = 0.265, p = 0.009; r = −0.239, p = 0.017; and r = −0.204, p = 0.043, respectively). The multivariate analysis of factors associated with PSD showed old age and a high Hamilton Depression Rating score to be relevant (p = 0.002 and 0.028, respectively). Conclusions: Visual function was decreased and the GCIPL thickness was reduced in major depressive disorder patients. The retinal neurodegenerative process in depression might be considered in patients with depression.

Local photoreceptor degeneration causes local pathophysiological remodeling of retinal neurons.

Vision loss in age-related macular degeneration (AMD) stems from disruption of photoreceptor cells in the macula, the central retinal area required for high-acuity vision. Mice and rats have no macula, but surgical insertion of a subretinal implant can induce localized photoreceptor degeneration due to chronic separation from retinal pigment epithelium, simulating a key aspect of AMD. We find that the implant-induced loss of photoreceptors in rat retina leads to local changes in the physiology of downstream retinal ganglion cells (RGCs), similar to changes in RGCs of rodent models of retinitis pigmentosa (RP), an inherited disease causing retina-wide photoreceptor degeneration. The local implant-induced changes in RGCs include enhanced intrinsic excitability leading to accelerated spontaneous firing, increased membrane permeability to fluorescent dyes, and enhanced photosensitization by azobenzene photoswitches. The local physiological changes are correlated with an increase in retinoic acid receptor-induced (RAR-induced) gene transcription, the key process underlying retinal remodeling in mouse models of RP. Hence the loss of photoreceptors, whether by local physical perturbation or by inherited mutation, leads to a stereotypical set of pathophysiological consequences in RGCs. These findings implicate RAR as a possible common therapeutic target for reversing the signal-corrupting effects of retinal remodeling in both RP and AMD.

Adaptation of retinal ganglion cell function during flickering light in the mouse

Rapid dilation of retinal vessels in response to flickering light (functional hyperemia) is a well-known autoregulatory response driven by increased neural activity in the inner retina. Little is known about flicker-induced changes of activity of retinal neurons themselves. We non-invasively investigated flicker-induced changes of retinal ganglion cell (RGC) function in common inbred mouse strains using the pattern electroretinogram (PERG), a sensitive measure of RGC function. Flicker was superimposed on the pattern stimulus at frequencies that did not generate measurable flicker-ERG and alter the PERG response. Transition from flicker at 101 Hz (control) to flicker at 11 Hz (test) at constant mean luminance induced a slow reduction of PERG amplitude to a minimum (39% loss in C57BL/6J mice and 52% loss in DBA/2J mice) 4–5 minutes after 11 Hz flicker onset, followed by a slow recovery to baseline over 20 minutes. Results demonstrate that the magnitude and temporal dynamics of RGC response induced by flicker at 11 Hz can be non-invasively assessed with PERG in the mouse. This allows investigating the functional phenotype of different mouse strains as well as pathological changes in glaucoma and optic nerve disease. The non-contact flicker-PERG method opens the possibility of combined assessment of neural and vascular response dynamics.

Elevated expression of TREK-TRAAK K2P channels in the retina of adult rd1 mice.

To examine the expression of Twik-related K+ channel 1 (TREK-1), Twik-related K+ channel 2 (TREK-2), and Twik-related arachidonic acid-stimulated K+ channel (TRAAK) in the retina of adult rd1 mice and to detect the protective roles of TREK-TRAAK two-pore-domain K+ (K2P) channels against retinal degeneration. Twenty-eight-day-old C57BL/6J mice and 28-day-old rd1 mice were used in this study. Retinal protein, retinal RNA, and embedded eyeballs were prepared from these two groups of mice. Real-time quantitative polymerase chain reaction and Western blot analyses were used to assess the gene transcription and protein levels, respectively. Retinal structures were observed using hematoxylin and eosin (H&E) staining. Immunohistochemistry was utilized to observe the retinal localization of TREK-TRAAK channels. Current changes in retinal ganglion cells (RGCs) after activation of TREK-TRAAK channels were examined using a patch-clamp technique. Compared with C57BL/6J mice, rd1 mice exhibited significantly higher retinal mRNA and protein expression levels of TREK-1, TREK-2, and TRAAK channels. In both groups, immunohistochemistry showed expression of TREK-TRAAK channels in retinal layers. After addition of the TREK-TRAAK channel agonist arachidonic acid (AA), whole-cell voltage step evoked currents were significantly higher in RGCs from rd1 mice than in RGCs from control C57BL/6J mice, suggesting that TREK-TRAAK channels were opened in RGCs from rd1 mice. TREK-TRAAK K2P channels' expression is increased in adult rd1 mice. AA induced the opening of TREK-TRAAK K2P channels in adult rd1 mice and may thus counterbalance depolarization of RGCs and protect the retina from excitotoxicity. TREK-TRAAK channels may play a protective role against retinal degeneration.

The expression and function of TRPV4 channels in primate retinal ganglion cells and bipolar cells

The transient receptor potential vanilloid 4 (TRPV4) channel may be opened by mechanical stimuli to mediate Ca2+ and Na+ influxes, and it has been suggested to mediate glaucoma retinopathy. However, it has been mostly unclear how TRPV4 activities affect the function of primate retinal ganglion cells (RGCs). We studied RGCs and bipolar cells (BCs) in the peripheral retina of the old-world primate using whole-cell current-clamp and voltage-clamp recordings, immunomarkers and confocal microscopy. RGCs were distinguished from displaced amacrine cells (ACs) by the absence of GABA and glycine immunoreactivity and possession of an axon and a large soma in the RGC layer. Strong TRPV4 signal was concentrated in medium to large somas of RGCs, and some TRPV4 signal was found in BCs (including PKCα-positive rod BCs), as well as the end feet, soma and outer processes of Mȕller cells. TRPV4 immunoreactivity quantified by the pixel intensity histogram revealed a high-intensity component for the plexiform layers, a low-intensity component for the soma layers of ACs and Mȕller cells, and both components in the soma layers of RGCs and BCs. In large RGCs, TRPV4 agonists 4α-phorbol 12,13 didecanoate (4αPDD) and GSK1016790A reversibly enhanced the spontaneous firing and shortened the delay of voltage-gated Na+ (Nav) currents under current-clamp conditions, and under voltage-clamp conditions, 4αPDD largely reversibly increased the amplitude and frequency of spontaneous excitatory postsynaptic currents. In BCs, changes in the membrane tension induced by either applying pressure or releasing the pressure both activated a transient cation current, which reversed at ~ −10 mV and was enhanced by heating from 24 °C to 30 °C. The pressure for the half-maximal effect was ~18 mmHg. These data indicate that functional TRPV4 channels are variably expressed in primate RGCs and BCs, possibly contributing to pressure-related changes in RGCs in glaucoma.

High hydrostatic pressure induces apoptosis of retinal ganglion cells via regulation of the NGF signalling pathway.

High pressure is the most important factor inducing retinal ganglion cell (RGC) apoptosis. However, the underlying mechanisms remain obscure. The present study investigated the effects of different levels of hydrostatic pressure (HP) on RGCs and the potential mechanisms involved. Primary cultured rat RGCs were exposed to five levels of HP (0, 20, 40, 60 and 80 mmHg) for 24 h. Morphological changes in RGCs were observed. The viability and apoptosis rate of RGCs were detected using a Cell Counting Kit-8 assay and Annexin V-fluorescein isothiocyanate/propidium iodide flow cytometry, respectively. Western blotting, reverse transcription-quantitative polymerase chain reaction and immunofluorescence were used to detect the expression and mRNA levels of nerve growth factor (NGF), protein kinase B (AKT), apoptosis signal-regulating kinase 1 (ASK1), forkhead box O1 (FoxO1) and cAMP response element binding protein (CREB). In the 0- and 20-mmHg groups, there were no apoptotic morphological changes. In the 40 mmHg group, parts of the cell were shrunken or disrupted. In the 60 mmHg group, neurite extension was weakened and parts of the cells were disintegrating or dying. In the 80 mmHg group, the internal structures of the cells were not visible at all. The apoptosis rates of RGCs were significantly higher and the viability rates significantly lower under 40, 60 and 80 mmHg compared with under 0 or 20 mmHg (all P<0.01). The expression and mRNA levels of NGF, AKT and CREB decreased in a dose-dependent manner in the 40-, 60- and 80-mmHg groups (all P<0.05), but those of ASK1 and FoxO1 increased in a dose-dependent manner (all P<0.05). Interestingly, the alterations to the expression and mRNA levels of CREB were significantly larger compared with the changes in ASK1 or FoxO1 in the 40-, 60- and 80-mmHg groups (all P<0.01). The results of the present study demonstrate that elevated HP of 40, 60 or 80 mmHg reduces viability and induces apoptosis in RGCs, which may occur through effects on the NGF/ASK1/FoxO1 and NGF/AKT/CREB pathways, of which the latter is more strongly affected.

Functional Changes of Retinal Ganglion Cells and Visual Pathways in Patients with Chronic Leber’s Hereditary Optic Neuropathy during One Year of Follow-up

To assess changes of retinal ganglion cells (RGCs) and visual pathways' function in patients with Leber's hereditary optic neuropathy (LHON) during 12 months of follow-up of the chronic phase. Retrospective case series. Twenty-two patients with LHON (mean age, 36.3±9.3 years) in the "chronic phase" of the disease, providing 42 eyes (LHON group) with different pathogenic mitochondrial DNA mutations (group 11778: 21 eyes; group 3460: 4 eyes; group 14484: 13 eyes; and group 14568: 4 eyes) were enrolled. Twenty-five age-similar healthy participants, providing 25 eyes, served as controls. Pattern electroretinogram (PERG) and visual evoked potentials (VEP), in response to 60' and 15' checks visual stimuli, were recorded at baseline in all subjects and after 6 and 12 months of follow-up in patients with LHON. At baseline, in all LHON eyes for each PERG and VEP parameter (amplitude and implicit time), the 95% confidence limit (CL) of test-retest variability was calculated. The PERG and VEP mean values observed in LHON eyes were compared (1-way analysis of variance [ANOVA]) with those of controls. During the follow-up, the PERG and VEP differences observed with respect to baseline were evaluated by ANOVA. Changes of individual and mean absolute values of 60' and 15' PERG amplitude and VEP amplitude and implicit time at each time point compared with baseline values in the LHON group. At baseline, mean values of PERG and VEP parameters detected in the LHON group were significantly (P < 0.01) different with respect to control values. In the LHON group, at 6 and 12 months of follow-up, the majority of eyes showed unmodified (within 95% CL) PERG and VEP values, and mean absolute values of these measures were not significantly (P > 0.01) different from baseline values. In our untreated patients with chronic LHON, with different specific pathogenic mutations, RGCs and visual pathways function were not significantly modified during 12 months of follow-up. This should be considered in the disease natural history when attempts for treatments are proposed in chronic LHON.

Downregulation of microRNA-149 in retinal ganglion cells suppresses apoptosis through activation of the PI3K/Akt signaling pathway in mice with glaucoma.

Glaucoma represents a major cause of blindness, generally associated with elevated intraocular pressure (EIOP). The aim of the present study was to investigate whether microRNA-149 (miR-149) affects retinal ganglion cells (RGCs) and the underlying mechanism based on a mouse model of chronic glaucoma with EIOP. The successfully modeled mice were administered with mimics or inhibitors of miR-149. Next, the number of RGCs, ultrastructural changes of RGCs, and purity of RGCs in the retinal tissues were detected. Moreover, the RGCs were collected and subsequently treated with 60 mmHg pressure and transfected with a series of plasmids aiding in the regulation of the expression of miR-149 and betacellulin (BTC). The levels of miR-149, BTC, phosphatidylinositol 3-kinase (PI3K), and Akt were subsequently determined. Finally, RGC viability and apoptosis were detected accordingly. Dual luciferase reporter gene assay provided validation, highlighting BTC was indeed a target gene of miR-149. The downregulation of miR-149 is accompanied by an increased number of RGCs and decreased ultrastructural RGC alterations. Additionally, downregulated miR-149 was noted to increase the levels of BTC, PI3K, and Akt in both the retinal tissues and RGCs, whereas the silencing of miR-149 was observed to promote the viability of RGC and inhibit RGC apoptosis. Taken together, the results of the current study provided validation suggesting that the downregulation of miR-149 confers protection to RGCs by means of activating the PI3K/Akt signaling pathway via upregulation of BTC in mice with glaucoma. Evidence presented indicated the promise of miR-149 inhibition as a potential therapeutic strategy for glaucoma treatment.

Neonatal inflammation induces reorganization in dendritic morphology of retinal ganglion cells but not their retinogeniculate projection in mice

Perinatal inflammatory insult in preterm babies is associated with vision impairment, but the underlying cellular mechanism is still unknown. In this study, we set out to explore whether systemic inflammatory stress affects the development of retinal ganglion cells (RGCs). Neonatal inflammation was induced by single and systemic injection of lipopolysaccharide (LPS, 1 mg/kg) at postnatal day 4 (P4). Morphological changes of RGCs were investigated by using 3D neuron reconstruction technique in Thy-1 YFPH transgenic mice at P21, of which a fraction of RGCs selectively expresses the yellow fluorescent protein (YFP). Three types (Type I, II, III) of RGCs were distinguished and classified according to the characteristic features in their dendritic field area and dendrite density. Neonatal exposure to LPS did not alter the composition of the three RGC types but induced a reorganization of dendritic architecture in the RGC Type I and II (but not Type III). The average diameter, surface area and volume of dendrites in both RGC Type I and II were increased after systemic inflammation compared with those in the control group. However, soma sizes of all three RGC types were not affected by neonatal inflammation. Meanwhile, using anterograde labeling of the retinal cells, we found that neonatal exposure to LPS also did not affect the pattern of RGC projections in the dorsal lateral geniculate nucleus of the thalamus (dLGN). These results indicate that RGC dendrite reorganization induced by neonatal inflammation may contribute to the retinal cell dysfunctions associated with systemic inflammation in premature babies.

Neuroprotective effect of Qinggan Lishui formula on retinal ganglion cell apoptosis in a microbead-induced rat chronic glaucoma model

OBJECTIVETo investigate a possible mechanism for protective effects of a decoction of the Qinggan Lishui formula (QF) on retinal ganglion cells (RGCs) in a rat model of microbead-induced chronic intraocular hypertension (COH). METHODSThe COH model was generated by injecting microbeads (superparamagnetic iron oxide) into the anterior chamber of rat eyes. QF was given by intragastric administration (gavage) once daily at a dose of 6.2 g/kg until day 28, following microbead injection. Cholera toxin B subunit (CTB) retrograde labeling and immunohistochemistry were used to evaluate changes in the number of RGCs in the retina. Terminal dUTP nick end labeling (TUNEL) staining was used to assess apoptotic changes in RGCs. RESULTSMicrobead injection induced a steady increase in intraocular pressure (IOP) of rats. Elevated IOP resulted in a progressive reduction in the number of CTB-labeled RGCs, 2-4 weeks after microbead injection. QF administration may moderately reduce IOP in the rat COH model and attenuate reduction of the number of CTB-labeled RGCs in COH rats. Furthermore, elevated IOP resulted in a progressive increase in the number of TUNEL-positive RGCs, 2–4 weeks after microbead injection, suggestive of an increase in the extent of RGC apoptosis. There was a significant reduction in the number of TUNEL-positive signals in QF-treated COH retinas, compared with untreated COH retinas. CONCLUSIONQF decoction may provide a protective effect for RGCs in COH retinas by reducing RGC loss; these effects may be mediated by inhibition of RGC apoptosis.

Early Gene Expression Profile in Retinal Ganglion Cell Layer After Optic Nerve Crush in Mice.

Optic nerve crush (ONC) induces retinal ganglion cell (RGC) death, which causes vision loss in glaucoma. To investigate early events leading to apoptosis of RGCs, we performed gene expression analysis of injured retinas in the period before RGC loss. The temporal changes of gene profiles at 0, 1, and 4 days after ONC were determined by DNA microarray. To verify the gene expression changes in RGCs, we enriched RGCs by laser-captured microdissection and performed real-time RT-PCR of 14 selected genes. In situ localization study was performed by immunohistochemistry. At 1 day and 4 days after ONC, 1423 and 2010 retinal genes were changed compared with 0 day, respectively; these genes were mainly related to apoptotic process, immune process, regulation of cell cycle, and ion transport. RT-PCR analysis revealed that expression levels of Activating transcription factor 3 (Atf3), Lipocalin 2 (Lcn2), and tumor necrosis factor receptor superfamily member 12a (Tnfrsf12a) were remarkably changed in RGC-enriched fraction within 4 days postcrush. Immunohistochemical analysis confirmed that all of these genes expressed highly in the ganglion cell layer of crushed retinas. In response to ONC, the expression of apoptotic genes was stimulated soon after crush. Atf3, Lcn2, and Tnfrsf12a might be key molecules responsible for RGC loss in glaucoma.

Curcumin Alleviates Diabetic Retinopathy in Experimental Diabetic Rats

Purpose: To investigate the potential protective effects of curcumin on the retina in diabetic rats. Methods: An experimental diabetic rat model was induced by a low dose of streptozotocin combined with a high-energy diet. Rats which had blood glucose levels ≥11.6 mmol/L were used as diabetic rats. The diabetic rats were randomly divided into 3 groups: diabetic rats with no treatment (DM), diabetic rats treated with 100 mg/kg curcumin (DM + Cur 100 mg/kg), and diabetic rats treated with 200 mg/kg curcumin (DM + Cur 200 mg/kg). Curcumin was orally administered daily for 16 weeks. After 16 weeks of administration, the rats were euthanized, and eyes were dissected. Retinal histology was examined, and the thickness of the retina was measured. Ultrastructural changes of retinal ganglion cells, inner layer cells, retinal capillary, and membranous disks were observed by electron microscopy. Malondialdehyde, superoxide dismutase, and total antioxidant capacity were measured by ELISA. Expression levels of vascular endothelial growth factor (VEGF) in retina tissues were examined by immunohistochemical staining and ELISA. Expression levels of Bax and Bcl-2 in retina tissues were determined by immunohistochemical staining and Western blotting. Results: Curcumin reduced the blood glucose levels of diabetic rats and decreased diabetes-induced body weight loss. Curcumin prevented attenuation of the retina in diabetic rats and ameliorated diabetes-induced ultrastructure changes of the retina, including thinning of the retina, apoptosis of the retinal ganglion cells and inner nuclear layer cells, thickening of retinal capillary basement membrane and disturbance of photoreceptor cell membranous disks. We also found that curcumin has a strong antioxidative ability in the retina of diabetic rats. It was observed that curcumin attenuated the expression of VEGF in the retina of diabetic rats. We also discovered that curcumin had an antiapoptotic effect by upregulating the expression of Bcl-2 and downregulating the expression of Bax in the retina of diabetic rats. Conclusions: Taken together, these results suggest that curcumin may have great therapeutic potential in the treatment of diabetic retinopathy which could be attributed to the hypoglycemic, antioxidant, VEGF-downregulating and neuroprotection properties of curcumin.

Light deprivation produces distinct morphological orchestrations on RGCs and cortical cells in a depressive-like YFP-H mouse model.

Neural physiological functions and synaptic changes underlying the pathogenesis of depression have obtained great achievements. However, neuronal morphological changes under a depressive state have not been well understood yet. Here a depressive-like YFP-H transgenic mouse model was produced by light deprivation (LD), and morphological changes of retinal ganglion cells (RGCs) and primary visual and auditory cortical layer 5 pyramidal cells (L5PCs) were investigated. Three distinct RGC subtypes were identified based on soma- and dendritic field (DF) size. RGA cells were highlighted by large soma and medium-sized to large DF. RGB cells were characterized by small- to medium-sized soma and small- to medium-sized DF. RGC cells were typical of small- to medium-sized soma and large DF. LD showed cell-type-specific morphological orchestrations on RGCs and predominantly promoted the dendritic growth of RGA cells, leaving no significant effect on RGB and RGC cells. LD produced a consistently suppressed effect on the morphology of primary visual and auditory cortical L5PCs. LD enhanced the dendritic spine density of primary visual cortical L5PCs, implying a compensation mechanism underlying morphological changes in individual cortical L5PCs. The increased morphological complexity of RGA cells and the simplified morphology of cortical L5PCs suggest a broad range of neuronal morphological "cross-modal plasticity" among different brain areas. Our observations in morphological changes of RGCs and cortical L5PCs under a depressive-like state will provide some insights into the pathogenesis of depression at a single neuronal morphological level.

Elevated IOP alters the space–time profiles in the center and surround of both ON and OFF RGCs in mouse

Glaucoma is a leading cause of blindness worldwide, and is characterized by progressive retinal ganglion cell (RGC) death. An experimental model of glaucoma has been established by elevating the intraocular pressure (IOP) via microbead occlusion of ocular fluid outflow in mice. Studies in this model have found visual dysfunction that varied with adaptational state, occurred before anatomical changes, and affected OFF RGCs more than ON RGCs. These results indicate subtle alterations in the underlying retinal circuitry that could help identify disease before irreversible RGC changes. Therefore, we looked at how RGC function was altered with elevated IOP under both photopic and scotopic conditions. We first found that responses to light offset are diminished with IOP elevation along with a concomitant decrease in receptive field center size for OFF RGCs. In addition, the antagonistic surround strength and size was reduced in ON RGCs. Furthermore, elevation of IOP significantly accelerated the photopic temporal tuning of RGC center responses in both ON and OFF RGCs. We found that some of the IOP-induced functional changes to OFF RGCs relied on ON cross-over pathways, indicating dysfunction in inner retinal circuitry. Overall, these results suggest that IOP alters multiple functions in the retina depending on the adaptational state. They provide a basis for designing multiple functional tests for early detection of glaucoma and for circuit-specific therapeutic targets in treatment of this blinding disease.

双芍护睛散抗大鼠外伤性视神经损伤的视网膜神经纤维层观察

目的：观察双芍护睛散抗大鼠外伤性视神经损伤的视网膜神经纤维层变化。方法：将实验SD大鼠60只随机抽取15只为空白对照组，其余45只造模后随机分为模型对照组、生理盐水组及中药治疗组，每组15只。空白对照组及模型对照组造模后24小时后，生理盐水组及中药治疗组(双芍护睛散)用药4周后取左眼观察视网膜神经节细胞及神经纤维层形态与神经纤维层厚度的变化。结果：模型对照组的视网膜中大量RGC肿胀，神经纤维层明显水肿增厚，其厚度与空白对照组比较有显著性差异(p < 0.05)。生理盐水组视网膜大量RGC呈空泡样变性、脱落，神经纤维层显著变薄；而中药治疗组视网膜少量RGC轻度肿胀，神经纤维层轻度变薄，其厚度与生理盐水组比较有显著性差异(p < 0.05)。结论：以养血行血法组方的双芍护睛散具有一定抗视神经的损伤，促进视网膜神经节细胞及视神经纤维修复的作用而保护视神经。 Objective: To observe the change of retinal nerve fiber layer of rats with traumatic optic nerve injury that treated by Shuang Shao Hu Jing Powder. Methods: 15 rats were randomly selected from 60 SD rats as control group, and the remaining 45 model rats were randomly divided into model control group, saline group and TCM treatment group; each group had 15 rats. Retina of the blank control group and the model control group were peeled after 24 hours, while that of the normal saline group and TCM treatment group (Shuang shao hu jing powder) were peeled after 4 weeks of treatment. Changes of retinal ganglion cells and nerve fiber layer morphology and thickness were observed. Results: In the model control group, there was a large amount of RGC in the retina, and the nerve fiber layer was significantly thickened. The thickness of the nerve fiber layer was significantly different from that of the control group (P < 0.05). A large number of RGC in the normal saline group showed vacuolar degeneration, shedding, and the nerve fiber layer became thinner; while in the treatment group, there was a slight swelling of RGC in the retina and a slight thinning of the nerve fiber layer, and there was significant difference between the treatment group and the saline group (P < 0.05). Conclusion: Shuang Shao Hu Jing Powder, with a combined function of nourishing blood to activate blood, showed certain effects of anti-optic-nerve injury, promoting retinal ganglion cells’ development and optic nerve fiber recovery, thus, protecting the optic nerve.