The retina, a tissue of the central nervous system, is vital for vision as its photoreceptors capture light and transform it into electrical signals, which are further processed before they are sent to the brain to be interpreted as images. The retina is unique in that it is continuously exposed to light and has the highest metabolic rate and demand for energy amongst all the tissues in the body. Consequently, the retina is very susceptible to oxidative stress. VDAC, a pore in the outer membrane of mitochondria, shuttles metabolites between mitochondria and the cytosol and normally protects cells from oxidative damage, but when a cell\'s integrity is greatly compromised it initiates cell death. There are three isoforms of VDAC, and existing evidence indicates that all three are expressed in the retina. However, their precise localization and function in each cell type is unknown. It appears that most retinal cells express substantial amounts of VDAC2 and VDAC3, presumably to protect them from oxidative stress. Photoreceptors express VDAC2, HK2, and PKM2-key proteins in the Warburg pathway that also protect these cells. Consistent with its role in initiating cell death, VDAC is overexpressed in the retinal degenerative diseases retinitis pigmentosa, age related macular degeneration (AMD), and glaucoma. Treatment with antioxidants or inhibiting VDAC oligomerization reduced its expression and improved cell survival. Thus, VDAC may be a promising therapeutic candidate for the treatment of these diseases.

Interaction between retinal vascular endothelial cells and neurons plays a critical role in the pathogenesis of diabetic retinopathy (DR). This study aims to compare an in vitro model over a monoculture model to simulate the neurovascular coupling under the hyperglycemic microenvironment of diabetes.
Rat retinal vascular endothelial cells (RRMECs) and ganglion cells (RGCs) were seeded mono- or co-cultured in a normal (NG, 5.5 mM) and high (HG, 75 mM) glucose concentrations culture medium. Cell viability was detected by the cell counting kit-8 (CCK-8) assay. The ability of migration and lumen formation of RRMECs were determined by scratch wound, transwell migration, and lumen formation assays. The apoptosis index of cells was calculated and detected by propidium iodide (PI)/Hoechst staining. Quantitative and morphological analysis of RGCs was performed through the labeling of RGCs by brain-specific homeobox/POU domain protein 3A (BRN3A) and anti-beta-III tubulin (TUJ1). The gene and protein expression levels of occludin (OCLN) and zonula occludens-1 (ZO-1) were evaluated by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay.
The viability, migration, and lumen formation abilities of RRMECs in the HG group significantly increased (P<0.05) in both mono- and co-culture models. Migration and lumen formation abilities of RRMECs in the co-culture with HG were lower than that in the monoculture group (P<0.05). The viability of RGCs cells with HG significantly decreased in both mono- and co-culture models (Pmono<0.001, Pco<0.001), the apoptosis index of RGCs in the co-culture with HG was higher than that in the monoculture (P=0.010). The protein and gene expression of OCLN, and ZO-1 in RRMECs significantly decreased with HG culture medium in both culture models (P<0.05). In the HG group, the protein and gene expression level of the ZO-1 and OCLN of RRMECs significantly decreased in the co-culture model than that in the monoculture model (P<0.05).
Compared with mono cell culture, the established co-culture in vitro system for diabetic neurovascular dysfunction can better stimulate the micro-environment of the retinal neurovascular unit.

Substance P (SP), a neuroprotective peptidergic neurotransmitter, is known to have immunoreactivity (IR) localized to amacrine and/or ganglion cells in a variety of species\' retinas, but it has not yet been studied in the mouse retina. Thus, we investigated the distribution and synaptic organization of SP-IR by confocal and electron microscopy immunocytochemistry in the mouse retina. SP-IR was distributed in the inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). Most of the SP-IR somas belonged to amacrine cells (2.5% of all) in the INL and their processes stratified into the S1, S3, and S5 layers of the IPL, with the most intense band in the S5 layer. Some SP-IR somas can also be observed in the GCL, which were identified as displaced amacrine cells (82%, 1269/1550) and ganglion cells (18%, 281/1550) by antibodies against AP2α and RBPMS, respectively. Such SP-IR ganglion cells (1.2% of all RGCs) can be further divided into 3 subgroups expressing SP/α-Synuclein (α-Syn), SP/GAD67, and/or SP/GAD67/α-Syn. Possible physiological and pathological roles of these ganglion cells are discussed. Further, electron microscopy evidence demonstrates that SP-IR amacrine cells receive major inputs from other SP-IR amacrine cell processes (146/242 inputs) and output mostly to SP-negative amacrine cell processes (291/673 outputs), suggesting series inhibition among amacrine cells. These results reveal for the first time an explicit distribution, novel ganglion cell features, and synaptic organization of SP-IR in the mouse retina, which is important for the future use of mouse models to study the roles of SP in healthy and diseased (including Parkinson\'s disease) retinal states.

RNAi therapy has been developed and explored for treating retinal conditions since last decades. The progression of retinal diseases including the age-related macular degeneration and glaucoma is associated with the malfunction of specific retinal cells. Therefore, to deliver therapeutic RNAi to selective retinal tissues with desired gene downregulation is crucial for the treatment of retinal diseases via RNAi therapy. Lipid-based nanoparticles are potent delivery vectors for RNAi therapeutics to achieve high gene silencing efficiency. The surface charge has been demonstrated to affect the intraocular behaviors and retinal distribution of intravitreally administered lipid nanoparticles (LNPs), which could subsequently affect the gene knockdown efficiency in specific retinal layers. Here, we evaluated three charged LNPs for their ability to deliver siRNA and facilitate gene downregulation both in vitro and in vivo. LNPs with different surface charges ranging from neutral to positive (5-34 mV) were successfully formulated. All types of charged LNPs managed gene knockdown in both mammalian cell line and primary neurons. At 48 h post intravitreal injection, neutral LNPs (6.2 mV) and mildly positive LNPs (15.9 mV) mediated limited retinal gene suppression (<10%) and the more positive LNPs (31.2 mV) led to ∼25% gene suppression in the retinal ganglion cell (RGC) layer. No gene silencing in the retinal pigmented epithelium layer was facilitated by any LNPs independent of the charges. In summary, this study has shown that positive LNPs with an optimized charge managed specific gene downregulation in the RGC layer. These RNAi carriers hold potential for the treatment of RGC-associated retinal diseases.

OBJECTIVE: To evaluate the effects of etanercept on the expression of Fas, tumor necrosis factor-alpha (TNF-α) and caspase-8 in the early stage of the apoptotic pathway in diabetic rats, and to explore the therapeutic effect of etanercept on diabetic retinopathy.
METHODS: A total of 60 Sprague-Dawley (SD) rats were randomly and evenly divided into 3 groups with 20 rats each, including control group, and diabetic groups with or without treatment. Streptozotocin (STZ)-induced diabetic rats were established for diabetic groups. Blood glucose and body weight were measured weekly. All the rats were sacrificed at the 12wk after treatment. The expressions of Fas, TNF-α and caspase-8 in rat retina were quantitatively detected by PCR and Western blot. The leakage of Evan blue was adopted to measure the retinal vascular leakage quantitatively, and to compare it among different groups. TUNEL method was used to compare the amount of apoptotic bodies quantitatively in rat retina ganglion cells under electron microscope.
RESULTS: The expressions of Fas, TNF-α and caspase-8 in each group were compared via PCR and Western blot, in which the diabetic group with treatment was lower than those without treatment (P<0.01), but all the diabetic groups were higher than the control group (P<0.01). Evans blue leakage in the diabetic treatment group was lower than those without treatment (P<0.01), but those in the control group was the lowest compared with the other two groups (P<0.01). TUNEL method showed that the apoptotic bodies of retina in the diabetic treatment group was lower than those without treatment (P<0.01), while those in the control group was the lowest compared with the other two groups (P<0.01).
CONCLUSIONS: Etanercept can effectively reduce the expression of Fas, TNF-α and caspase-8, as well as the retinal leakage and retinal cell apoptosis in diabetic rats.

OBJECTIVE: To investigate the changes in the membrane properties and synaptic stability of the rat retinal ganglion cells (RGCs) during postnatal development.
METHODS: Whole-cell patch-clamp technique was used to record the action potentials (AP) and miniature excitatory postsynaptic currents (mEPSC) of SD rat RGCs at postnatal days 7, 14 and 40. The active and passive membrane properties and the synaptic stability (measured by the amplitude, frequency, rise time and decay time of mEPSC) of the RGCs were analyzed using Patchmaster software.
RESULTS: Comparison of the RGCs in SD rats across different postnatal ages revealed significant changes in the electrophysiological characteristics of the RGCs during postnatal development. The discharge rate was significantly greater while the AP half-peak width was significantly smaller at postnatal day 15 (P15) than at P7 (P < 0.01), but were both similar between P15 and P40 (P=0.086); in terms of the passive membrane properties, the membrane time constant gradually decreased during the development. The frequency of mEPSCs increased significantly over time during postnatal development (P < 0.01), but was similar between P15 and P40 rats.
CONCLUSIONS: In SD rats, the membrane properties and synaptic stability of the RGCs undergo alterations following a specific pattern, which highlights a critical period where distinct changes occur in the electrophysiological characteristics of RGCs, followed by gradual stabilization over time. Such changes in the electrophysiological characteristics represent the basic characteristics of RGCs for visual signal processing, and understanding of this mechanism may provide insights into the exact role of the RGC in visual information processing.

Adaptive optics retinal imaging of fluorescent calcium indicators is a minimally invasive method used to study retinal physiology over extended periods of time. It has potential for discovering novel retinal circuits, tracking retinal function in animal models of retinal disease, and assessing vision restoration therapy. We previously demonstrated functional adaptive optics imaging of retinal neurons in the living eye using green fluorescent calcium indicators; however, the use of green fluorescent indicators presents challenges that stem from the fact that they are excited by short-wavelength light. Using red fluorescent calcium indicators such as jRGECO1a, which is excited with longer-wavelength light (~560 nm), makes imaging approximately five times safer than using short-wavelength light (~500 nm) used to excite green fluorescent calcium indicators such as GCaMP6s. Red fluorescent indicators also provide alternative wavelength imaging regimes to overcome cross talk with the sensitivities of intrinsic photoreceptors and blue light-activated channelrhodopsins. Here we evaluate jRGECO1a for in vivo functional adaptive optics imaging of retinal neurons using single-photon excitation in mice. We find that jRGECO1a provides similar fidelity as the established green indicator GCaMP6s.

Optic neuropathies are the leading cause of irreversible blindness and visual impairment in the developed countries, affecting more than 80 million people worldwide. While most optic neuropathies have no effective treatment, there is intensive research on retinal ganglion cell (RGC) protection and axon regeneration. We previously demonstrated potential of human periodontal ligament-derived stem cells (PDLSCs) for retinal cell replacement. Here, we report the neuroprotective effect of human PDLSCs to ameliorate RGC degeneration and promote axonal regeneration after optic nerve crush (ONC) injury. Human PDLSCs were intravitreally injected into the vitreous chamber of adult Fischer rats after ONC in vivo as well as cocultured with retinal explants in vitro. Human PDLSCs survived in the vitreous chamber and were maintained on the RGC layer even at 3 weeks after ONC. Immunofluorescence analysis of βIII-tubulin and Gap43 showed that the numbers of surviving RGCs and regenerating axons were significantly increased in the rats with human PDLSC transplantation. In vitro coculture experiments confirmed that PDLSCs enhanced RGC survival and neurite regeneration in retinal explants without inducing inflammatory responses. Direct cell-cell interaction and elevated brain-derived neurotrophic factor secretion, but not promoting endogenous progenitor cell regeneration, were the RGC protective mechanisms of human PDLSCs. In summary, our results revealed the neuroprotective role of human PDLSCs by strongly promoting RGC survival and axonal regeneration both in vivo and in vitro, indicating a therapeutic potential for RGC protection against optic neuropathies. Stem Cells 2018;36:844-855.

Recent studies revealing genetic connection of primary open angle glaucoma (POAG) and amyotrophic lateral sclerosis (ALS) have received particular attention. Exploring the evidence for common pathogenesis of these two progressive neurological disorders may assist in understanding the mechanism and searching for new treatment. Retinal nerve fiber layer (RNFL) defect and corresponding visual field (VF) impairment are well known neuropathy signs in glaucoma. In our study, thickness of certain retinal layer in ALS patients was analyzed to detect ganglion cell\'s soma and axon, and for first time visual field was examined for ALS. The correlation of retinal involvement and ALS progression were also investigated. The results were compared with those of POAG. The study may provide new knowledge for these two neurodegenerative diseases.

The present study was planned to observe the expression of heat shock protein 72 (HSP72) in retina of rats in a glaucoma model. A total of 50 Wistar rats were randomly divided into the high intraocular tension group (glaucoma model) and the sham control (sham operation) group. Glaucoma rat models were created by application of electrocoagulation on at least three groups of veins, reduction of venous return of aqueous humor and by enhancement of intraocular tension at the same time; 1, 2, 3, 4 and 8 weeks after operation, the intraocular tension of rats was observed respectively, and the expression/distribution of HSP72 in retina was assessed by immunohistochemical detection. As a result, the high intraocular tension group was found with obviously increased intraocular tension of the right eyes after operation (P<0.05), which was stable after one week. It was observed that the positive expression of HSP72 in retina gradually increased significantly with increase in intraocular tension in the rat model of glaucoma in comparison to controls. Furthermore, retinal ganglion cells (RGCs) of rats from both groups were cultured respectively, for confirmation. It was observed that the expression levels of HSP72 in the high intraocular tension group were higher in comparison to the sham control group. In conclusion, the enhanced expression of endogenous HSP72 may play an important role in glaucomatous optic neuro-protection.