Cells In Outer Nuclear Layer Research Articles

Neuronal apoptosis, axon damage and synapse loss occur synchronously in acute ocular hypertension

Retinal ganglion cells (RGCs) apoptosis and their axon degeneration are pivotal features in glaucoma. Previous studies suggest that the process of RGCs soma degeneration is distinct from axon degeneration and that both of them lead to vision loss but separately. However, since a normal visual function relies on the integrity of axon, synapse and soma in the retina, a comprehensive understanding of the changes of these neuron components in glaucoma is desired. Therefore, in an acute ocular hypertension (AOH) model in mice, we systematically evaluated retinal neuron soma, axon and synapse alteration at certain time points. We found that ocular hypertension led to a progressive apoptosis of retinal neural cells which proceeded from peripheral to central retina in the wholemount, meanwhile, started in the ganglion cell layer (GCL) and spread to the inner nuclear layer (INL) and then the outer nuclear layer (ONL) as time went on. The type of apoptotic cells was identified as RGCs in GCL, amacrine cells in INL and cone photoreceptor cells in ONL. Axon degeneration was observed at the same time as soma degenerated and also progressed from peripheral to central retina. More interestingly, accumulation of neurofilament in the soma caused by axon transport failure was detected synchronously. We also found that presynaptic and postsynaptic vesicle proteins were downregulated. Taken together, these data support a view that retinal neuronal apoptosis happens not only in RGCs, but also other neurons in laminar layers. Axon damage and synapse loss occur synchronously with soma loss in AOH. The combination of these three parameters might facilitate a systematic evaluation of the disease progression and treatment strategies in glaucoma.

Transduction Patterns of Adeno-associated Viral Vectors in a Laser-Induced Choroidal Neovascularization Mouse Model

Adeno-associated virus (AAV) vector is a promising platform technology for ocular gene therapy. Recently clinical successes to treat choroidal neovascularization (CNV) in wet type age-related macular degeneration have been reported. However, because pathologic conditions of the retina may alter the tropism of viral vectors, it is necessary to evaluate the transduction efficiency of different serotypes of AAV vectors in the retinas with CNVs. Here, we show the patterns and efficacy of transduction of AAV2, -5, and -8 vectors in a laser-induced CNV mouse model. C57BL/6J mice were subjected to unilateral laser photocoagulation on the right eye to induce CNV 5 days prior to intravitreal injection of AAV2, -5, and -8 capsids expressing EGFP. Transduction was increased around CNV lesions for all AAV capsid types, and AAV2 resulted in the highest transduction efficiency. In the absence of CNV, the AAV2 vector transduced ganglion and inner nuclear layer (INL) cells, and AAV5 and AAV8 transduced only a small proportion of cells in the retinal ganglion cell layer. CNV increased AAV2 vector expression throughout the retina and in and around CNVs; the transduced cells included retinal ganglion cells, Müller cells, cells from the INL and outer nuclear layer (ONL), photoreceptors, and retinal pigment epithelium (RPE) cells. Inflammatory cells and endothelial cells in CNVs were also transduced by AAV2. AAV5 and AAV8 were transduced in retinal ganglion, Müller, INL, ONL, and RPE cells in a localized pattern, and only endothelial cells at the surface of CNV lesions showed EGFP expression. Taken together, CNV formation resulted in enhanced transduction of AAV2, -5, and -8, and AAV2 exhibited the highest transduction efficiency in cells in CNV lesions.

Experimental oral iron administration: Histological investigations and expressions of iron handling proteins in rat retina with aging

Iron is implicated in age-related macular degeneration (AMD). The aim of this study was to see if long-term, experimental iron administration with aging modifies retinal and choroidal structures and expressions of iron handling proteins, to understand some aspects of iron homeostasis. Male Wistar rats were fed with ferrous sulphate heptahydrate (500mg/kg body weight/week, oral; elemental iron availability: 20%) from 2 months of age onward until they were 19.5 month-old. At 8, 14 and 20 months of age, they were sacrificed and serum and retinal iron levels were detected by HPLC. Oxidative stress was analyzed by TBARS method. The retinas were examined for cell death (TUNEL), histology (electron microscopy) and the expressions of transferrin, transferrin receptor-1 [TFR-1], H- and L-ferritin. In control animals, at any age, there was no difference in the serum and retinal iron levels, but the latter increased significantly in 14- and 20 month-old iron-fed rats, indicating that retinal iron accumulation proceeds with progression of aging (>14 months). The serum and retinal TBARS levels increased significantly with progression of aging in experimental but not in control rats. There was significant damage to choriocapillaris, accumulation of phagosomes in retinal pigment epithelium and increased incidence of TUNEL+ cells in outer nuclear layer and vacuolation in inner nuclear layer (INL) of 20 month-aged experimental rats, compared to those in age-matched controls. Vacuolations in INL could indicate a long-term effect of iron accumulation in the inner retina. These events paralleled the increased expression of ferritins and transferrin and a decrease in the expression of TFR-1 in iron-fed rats with aging, thereby maintaining iron homeostasis in the retina. As some of these changes mimic with those happening in eyes with AMD, this model can be utilized to understand iron-induced pathophysiological changes in AMD.

Non-Visual Photopigments Effects of Constant Light-Emitting Diode Light Exposure on the Inner Retina of Wistar Rats.

The retina is part of the central nervous system specially adapted to capture light photons and transmit this information to the brain through photosensitive retinal cells involved in visual and non-visual activities. However, excessive light exposure may accelerate genetic retinal diseases or induce photoreceptor cell (PRC) death, finally leading to retinal degeneration (RD). Light pollution (LP) caused by the characteristic use of artificial light in modern day life may accelerate degenerative diseases or promote RD and circadian desynchrony. We have developed a working model to study RD mechanisms in a low light environment using light-emitting diode (LED) sources, at constant or long exposure times under LP conditions. The mechanism of PRC death is still not fully understood. Our main goal is to study the biochemical mechanisms of RD. We have previously demonstrated that constant light (LL) exposure to white LED produces a significant reduction in the outer nuclear layer (ONL) by classical PRC death after 7 days of LL exposure. The PRCs showed TUNEL-positive labeling and a caspase-3-independent mechanism of cell death. Here, we investigate whether constant LED exposure affects the inner-retinal organization and structure, cell survival and the expression of photopigments; in particular we look into whether constant LED exposure causes the death of retinal ganglion cells (RGCs), of intrinsically photosensitive RGCs (ipRGCs), or of other inner-retinal cells. Wistar rats exposed to 200 lx of LED for 2 to 8 days (LL 2 and LL 8) were processed for histological and protein. The results show no differences in the number of nucleus or TUNEL positive RGCs nor inner structural damage in any of LL groups studied, indicating that LL exposure affects ONL but does not produce RGC death. However, the photopigments melanopsin (OPN4) and neuropsin (OPN5) expressed in the inner retina were seen to modify their localization and expression during LL exposure. Our findings suggest that constant light during several days produces retinal remodeling and ONL cell death as well as significant changes in opsin expression in the inner nuclear layer.

A Reinterpretation of Cell Transplantation: GFP Transfer From Donor to Host Photoreceptors.

The utilization of fluorescent reporter transgenes to discriminate donor versus host cells has been a mainstay of photoreceptor transplantation research, the assumption being that the presence of reporter+ cells in outer nuclear layer (ONL) of transplant recipients represents the integration of donor photoreceptors. We previously reported that GFP+ cells in the ONL of cone-GFP transplanted retinas exhibited rod-like characteristics, raising the possibility that GFP signal in recipient tissue may not be a consequence of donor cell integration. To investigate the basis for this mismatch, we performed a series of transplantations using multiple transgenic donor and recipient models, and assessed cell identity using nuclear architecture, immunocytochemistry, and DNA prelabeling. Our results indicate that GFP+ cells in the ONL fail to exhibit hallmark elements of donor cells, including nuclear hetero/euchromatin architecture. Furthermore, GFP signal does not appear to be a consequence of classic donor/host cell fusion or transfating post-transplant, but is most likely due to material exchange between donor and host photoreceptors. This transfer can be mediated by rods and cones, is bidirectional between donor and host cells, requires viable photoreceptors, occurs preferentially at sites of outer limiting membrane disruption and can be detected in second-order retinal neurons and Müller glia. Collectively, these data warrant re-evaluation of the use of lineage tracing fluorescent reporters in transplantation studies involving the retina and other CNS tissues. Furthermore, the reinterpretation of previous functional rescue data, based on material exchange, rather than cell integration, may offer a novel approach to vision rescue. Stem Cells 2017;35:932-939.

Comparative study of the effect of low-dose versus high-dose aspirin on the retina of aged male albino rats

Background Aspirin is a very popular drug worldwide. Recent studies have suggested that long-term aspirin use might be linked to wet-type acute macular degeneration. Aim This study was undertaken to investigate and compare the effects of chronic daily intake of low-dose versus high-dose aspirin on the structure of the retina in aged male albino rats for the first time. Materials and methods Twelve-month-old male albino rats were assigned to three groups: control, low-dose (2mg/kg) aspirin, and high-dose (10mg/kg) aspirin. The rats were given oral aspirin daily for 1 month. At the end of the experiment, the animals were euthanized and their eyes were enucleated and processed for paraffin blocks. Some of the paraffin sections were stained with H&E, whereas others were immunohistochemically stained with TUNEL, CD31, and glial fibrillary acidic protein markers. Histomorphometric count of ganglion cells, outer nuclear layer cells, CD31-positive cells, and TUNEL- positive cells was carried out using image analysis software. Statistical analysis of cell counts was performed. Results This study showed that chronic aspirin administration induced many degenerative changes in the aged rat retina. Stratification and vascularization of the retinal pigment epithelium was detected in addition to disruption of the photoreceptors. Apoptotic changes were detected in the inner nuclear layer and ganglion cell layer with significant decrease in cell number, which was pronounced with the high dose of aspirin. An increase in glial fibrillary acidic protein immunoreactivity of Müller cells was detected, especially in the high-dose group. Also, retinal neovascularization was clearly detected in the high-dose aspirin group. Conclusion Chronic aspirin intake is likely to produce retinal degenerative changes in aged male rat retina. High-dose aspirin was more detrimental than low dose. Results of the present experiment confirmed some clinical observations that chronic aspirin use is associated with retinal degenerative changes.

NADPH Oxidase Contributes to Photoreceptor Degeneration in Constitutively Active RAC1 Mice.

PurposeThe active form of small GTPase RAC1 is required for activation of NADPH oxidase (NOX), which in turn generates reactive oxygen species (ROS) in nonphagocytic cells. We explored whether NOX-induced oxidative stress contributes to rod degeneration in retinas expressing constitutively active (CA) RAC1.MethodsTransgenic (Tg)–CA-RAC1 mice were given apocynin (10 mg/kg, intraperitoneal), a NOX inhibitor, or vehicle daily for up to 13 weeks. Superoxide production and oxidative damage were assessed by dihydroethidium staining and by protein carbonyls and malondialdehyde levels, respectively. Outer nuclear layer (ONL) cells were counted and electroretinogram (ERG) amplitudes measured in Tg-CA-RAC1 mice. Outer nuclear layer cells were counted in wild-type (WT) mice after transfer of CA-Rac1 gene by subretinal injection of AAV8-pOpsin-CA Rac1-GFP.ResultsTransgenic-CA-RAC1 retinas had significantly fewer photoreceptor cells and more apoptotic ONL cells than WT controls from postnatal week (Pw) 3 to Pw13. Superoxide accumulation and protein and lipid oxidation were increased in Tg-CA-RAC1 retinas and were reduced in mice treated with apocynin. Apocynin reduced the loss of photoreceptors and increased the rod ERG a- and b-wave amplitudes when compared with vehicle-injected transgenic controls. Photoreceptor loss was also observed in regions of adult WT retina transduced with AAV8-pOpsin-CA Rac1-GFP but not in neighboring regions that were not transduced or in AAV8-pOpsin-GFP–transduced retinas.ConclusionsConstitutively active RAC1 promotes photoreceptor cell death by oxidative damage that occurs, at least partially, through NOX-induced ROS. Reactive oxygen species are likely involved in multiple forms of retinal degenerations, and our results support investigating RAC1 inhibition as a therapeutic approach that targets this disease pathway.

Survival and Functionality of hESC-Derived Retinal Pigment Epithelium Cells Cultured as a Monolayer on Polymer Substrates Transplanted in RCS Rats.

To determine the safety, survival, and functionality of human embryonic stem cell-derived RPE (hESC-RPE) cells seeded on a polymeric substrate (rCPCB-RPE1 implant) and implanted into the subretinal (SR) space of Royal College of Surgeons (RCS) rats. Monolayers of hESC-RPE cells cultured on parylene membrane were transplanted into the SR space of 4-week-old RCS rats. Group 1 (n = 46) received vitronectin-coated parylene membrane without cells (rMSPM+VN), group 2 (n = 59) received rCPCB-RPE1 implants, and group 3 (n = 13) served as the control group. Animals that are selected based on optical coherence tomography screening were subjected to visual function assays using optokinetic (OKN) testing and superior colliculus (SC) electrophysiology. At approximately 25 weeks of age (21 weeks after surgery), the eyes were examined histologically for cell survival, phagocytosis, and local toxicity. Eighty-seven percent of the rCPCB-RPE1-implanted animals showed hESC-RPE survivability. Significant numbers of outer nuclear layer cells were rescued in both group 1 (rMSPM+VN) and group 2 (rCPCB-RPE1) animals. A significantly higher ratio of rod photoreceptor cells to cone photoreceptor cells was found in the rCPCB-RPE1-implanted group. Animals with rCPCB-RPE1 implant showed hESC-RPE cells containing rhodopsin-positive particles in immunohistochemistry, suggesting phagocytic function. Superior colliculus mapping data demonstrated that a significantly higher number of SC sites responded to light stimulus at a lower luminance threshold level in the rCPCB-RPE1-implanted group. Optokinetic data suggested both implantation groups showed improved visual acuity. These results demonstrate the safety, survival, and functionality of the hESC-RPE monolayer transplantation in an RPE dysfunction rat model.

The Time Course of Deafness and Retinal Degeneration in a Kunming Mouse Model for Usher Syndrome.

Usher syndrome is a group of autosomal recessive diseases characterized by congenital deafness and retinitis pigmentosa. In a mouse model for Usher syndrome, KMush/ush, discovered in our laboratory, we measured the phenotypes, characterized the architecture and morphology of the retina, and quantified the level of expression of pde6b and ush2a between postnatal (P) days 7, and 56. Electroretinograms and auditory brainstem response were used to measure visual and auditory phenotypes. Fundus photography and light microscopy were used to measure the architecture and morphology of the retina. Quantitative real-time PCR was used to measure the expression levels of mRNA. KMush/ush mice had low amplitudes and no obvious waveforms of Electroretinograms after P14 compared with controls. Thresholds of auditory brainstem response in our model were higher than those of controls after P14. By P21, the retinal vessels of KMush/ush mice were attenuated and their optic discs had a waxy pallor. The retinas of KMush/ush mice atrophied and the choroidal vessels were clearly visible. Notably, the architecture of each retinal layer was not different as compared with control mice at P7, while the outer nuclear layer (ONL) and other retinal layers of KMush/ush mice were attenuated significantly between P14 and P21. ONL cells were barely seen in KMush/ush mice at P56. As compared with control mice, the expression of pde6b and ush2a in KMush/ush mice declined significantly after P7. This study is a first step toward characterizing the progression of disease in our mouse model. Future studies using this model may provide insights about the etiology of the disease and the relationships between genotypes and phenotypes providing a valuable resource that could contribute to the foundation of knowledge necessary to develop therapies to prevent the retinal degeneration in patients with Usher Syndrome.

96. An Approach to Compare Multiple Adeno-Associated Virus (AAV) Capsids Side-by-Side in a Single Eye Following Intravitreal Injection

Current gene therapy approaches for ocular diseases rely largely on the use of subretinal injection for treatment of outer nuclear layer cells of interest. While subretinal injection allows for efficient transduction of retinal cells close to the site of the injection, there is a significant risk that the injection can further damage retinas with compromised integrity. Several studies have identified new adeno-associated virus (AAV) capsid mutants that are potentially able to transduce outer nuclear layer cells following intravitreal injections. To further explore AAV vector-mediated intravitreal injection approaches for clinical translation, it is imperative to directly compare retinal transduction efficiencies between newly identified and previously published AAV mutants. Using clinically relevant large animal models, these mutant capsids, as well as other commonly used AAV serotypes, can be tested in a comprehensive manner. To this end, we propose to utilize a viral DNA/RNA barcoding approach established by our lab. In this approach, AAV capsids derived from different serotypes and mutants can be identified by DNA and RNA barcodes unique to each AAV capsid. These barcodes are contained in viral genomes and also expressed as RNA barcodes following cellular transduction. As each AAV capsid strain has a unique viral genome, a mass transduction of a number of different AAV strains can be conducted in order to directly compare transduction levels side-by-side in a single eye. To test feasibility of the barcode approach and establish proof-of-principle in the ocular gene therapy research, we produced a DNA/RNA-barcoded AAV library that contained 20 serotypes including AAV serotypes 1-11 and AAV mutants including DJ, LK03, 2i8 and 2G9. Each AAV strain expresses a pair of 12 nucleotide-long RNA barcodes unique to each strain under the control of the human U6 snRNA promoter. Seven C57BL/6J mice have been injected into vitreous humor bilaterally with the DNA/RNA-barcoded AAV library vector at doses ranging from 2 to 9 x109 vg. To date, eyes were harvested from one mouse 3 weeks post injection. Total DNA and RNA were extracted from the retina as a whole from each eye, and were subjected to DNA-PCR or reverse transcriptase (RT)-PCR to amplify viral DNA and RNA barcodes, followed by Illumina barcode sequencing. A preliminary data analysis revealed that AAV2, AAV-DJ and AAV2G9 transduced the murine retina better than other AAV strains including AAV9, the reference control AAV strain contained in the library. Although further optimization of the approach may be required to move this project forward, we plan to apply this method to assess transduction efficiencies of various AAV strains side by side in specific subsets of sorted retinal cells and in various animal species. The safer approach of intravitreal injections with the most optimal AAV vectors would significantly increase the ability to treat over 200 genetic retinal diseases and therefore the data that will be obtained from these studies will considerably benefit the field of retinal gene therapy.

The elevation of intraocular pressure is associated with apoptosis and increased immunoreactivity for nitric oxide synthase in rat retina whereas the effectiveness of retina derived relaxing factor is unaffected

Glaucoma is a progressive ocular disease that stands in the upper rank for the cause of blindness in worldwide. In the present study, we aimed to elucidate the possible disturbances occurred in the layers of retina due to an increase in intraocular pressure (IOP) and to verify the effectiveness of retina derived relaxing factor, i.e., RRF in this pathologic condition. The increase in IOP was induced by cauterization of the three of episcleral veins simultaneously in rats. After 8 weeks period, the retinas excised from the vein cauterized eyes were evaluated for the possible histopathological and ultrastructural alterations as well as for the relaxing effects on isolated bovine retinal and rat mesenteric arteries, in comparison with the retinas obtained from contralateral sham-operated eyes. In the retinas of IOP-elevated eyes, profound morphological deteriorations were determined in the ganglion and outer nuclear cell layers which were associated with an increased number of TUNEL positive cells in the ganglion and inner nuclear cell layers. Increased immunohistochemical stainings for three isoforms of nitric oxide synthase (NOS) were defined in almost all layers of the retinas of IOP-elevated eyes, in which eNOS was abundant particularly in the inner plexiform and ganglion cell layers. An irregular basal folding of retinal pigment epithelium (RPE) and an increased inter lamellar space of photoreceptor cell layer furtherly characterized the prominent degeneration of those layers in the retinas of IOP-elevated eyes. On the other hand, the relaxing effects of the retina obtained from IOP-elevated eyes were determined to be unchanged on the retinal and mesenteric arteries precontracted either with prostaglandin F2α (PGF2α, 30 μM) or potassium chloride (K+, 100 mM), when compared with the relaxations of control retina obtained from contralateral sham-operated eyes. Overall, these findings suggested that the elevation of IOP induces prominent structural changes in rat retina particularly in the ganglion and inner layers that is associated with marked apoptosis and increased immunoreactivity for NOS, while the functional effectiveness of retina derived relaxing factor, i.e., RRF is unaffected.

Sirtuin Type 1 Mediates the Retinal Protective Effect of Hydrogen-Rich Saline Against Light-Induced Damage in Rats.

Molecular hydrogen has been used as an antioxidant to treat many diseases in clinical and animal studies. However, the therapeutic mechanism of molecular hydrogen remains unclear. We previously reported mitigation of light-induced damage in the rat retina by intraperitoneal injection of hydrogen-rich saline (HRS). In the present study, we investigated whether Sirtuin Type 1 (Sirt1), a class III histone deacetylase, mediates the retinal protective effect of HRS in rats with light-induced retinal damage. Rats were treated with HRS for 5 days after intense light exposure, and then ERGs were performed and retinas were collected to evaluate the effect of HRS on Sirt1 expression. The necessity of Sirt1 for the retinal protective effect of HRS was investigated using the Sirt1 activator resveratrol, the Sirt1 inhibitor EX-527, and short interfering RNAs. In light-damaged retinas, 5 days of HRS treatment increased Sirt1 expression, mitigated a- and b-wave amplitude reduction, and decreased the reduction of outer nuclear cell layers. The Sirt1 activator resveratrol mimicked the effect of HRS in light-damaged retinas. This result supported our hypothesis that Sirt1 mediates the protective effect of HRS. Additionally, the retinal protective effect of HRS was inhibited by both the Sirt1 inhibitor EX-527 and Sirt1 targeted short interfering RNAs. Hydrogen-rich saline also increased B-cell lymphoma 2 (Bcl-2) expression and the activity of the antioxidant enzyme superoxide dismutase (SOD). Conversely, HRS decreased Bcl2-associated X protein expression, cleaved caspase-3, and oxidant-stress product malondialdehyde (MDA) in a Sirt1-dependent manner. Sirt1 mediates light-induced damage mitigation by HRS through inhibition of apoptosis and oxidant-stress.

Differential Expression of AQP1 and AQP4 in Avascular Chick Retina Exposed to Moderate Light of Variable Photoperiods.

Aquaporins (AQPs) are integral membrane proteins which maintain cellular water and ion homeostasis. Alterations in AQP expression have been reported in rod-dominated rodent retinas exposed to light. In rodents and also in birds, light of moderate intensities (700-2000lux) damages the retina, though detailed changes were not examined in birds. The aim of our study was to see if light affects cone dominated retinas, which would be reflected in expression levels of AQPs. We examined AQP1 and AQP4 expressions in chick retina exposed to 2000lux under 12h light:12h dark (12L:12D; normal photoperiod), 18L:6D (prolonged photoperiod) and 24L:0D (constant light). Additionally, morphological changes, apoptosis (by TUNEL) and levels of glutamate and GFAP (a marker of injury) in the retina were examined to correlate these with AQP expressions. Constant light caused damage in outer and inner nuclear layer (ONL, INL) and ganglion cell layer (GCL). Also, there were associated increases in GFAP and glutamate levels in retinal extracts. In normal photoperiod, AQP1 was expressed in GCL, outer part of INL and photoreceptor inner segments of. AQP4 was additionally expressed in nerve fiber layer. Immunohistochemistry and Western blotting revealed over all decreased AQP1 and AQP4 expression in constant light condition compared to those in other two groups. The elevated GFAP and glutamate levels might be involved in the reduction of AQPs in constant light group. Such decreases in AQP expressions are perhaps linked with retinal cell damage seen in constant light condition, while their relatively enhanced expression in two other conditions may help in maintaining a normal retinal architecture, indicating their neuroprotective potential.

Reduced photoreceptor death and improved retinal function during retinal degeneration in mice lacking innate immunity adaptor protein MyD88

The injury inflammatory response mediated by the innate immune system is an important contributor to neurodegeneration in the central nervous system (CNS) and retina. A major branch of the innate immune system is regulated by the Toll-like receptors (TLRs), which are receptors for endogenous damage associated molecules released from injured cells as well as pathogen-derived molecules, and interleukin-1 receptors (IL-1R), which are activated by IL-1α, IL-1β and IL-18 cytokines. TLRs and IL-1R are expressed on immune and non-immune cell types and act as first responders to cell damage, which results in tissue repair, or inflammation and apoptosis. Both TLR and IL-1R require the adaptor protein myeloid differentiation primary response gene 88 (MyD88) for signaling. Although inflammation is implicated in neuronal death in the retina, the role of MyD88-dependent TLR and IL-1R signaling in retinal degeneration is unknown. Therefore, the purpose of this study was to investigate the role of MyD88-mediated signaling in neuronal degeneration in the retinal degeneration 1 (rd1) mouse model, which exhibits a phenotype of rapid photoreceptor death and inflammation. To generate rd1 mice lacking the MyD88 gene, rd1 were bred with MyD88 knockout mice (MyD88−/−) for several generations to produce rd1/MyD88+/+ and rd1/MyD88−/− genotypes. Chemokine mRNA expression levels were analyzed by qRT-PCR, and recruitment of activated microglia was quantified by immunodetection of the IBA-1 protein. Retinal outer nuclear layer cell counts were performed to quantify photoreceptor degeneration, and retinal function was assessed using electroretinograms (ERG). Our results revealed that retinal expression of Ccl2, Ccl4, Ccl7 and Cxcl10 was reduced by 2 to 8-fold in rd1/MyD88−/− mice compared with rd1/MyD88+/+ mice (p<0.05), which coincided with attenuated microglial activation, higher numbers of photoreceptors and higher retina responses to photopic and scotopic stimuli. At later ages, rd1/MyD88−/− had reduced chemokine expression and higher photopic responses but no change in microglial recruitment compared with rd1 mice with functional MyD88. In conclusion, lack of MyD88-mediated signaling increased photoreceptor survival and retina function in rd1 mice, which implicates MyD88-mediated innate immunity pathways as an important pathogenic factor during retinal degeneration.

The neuroprotection of brain-derived neurotrophic factor in experimental retinal detachment reattached

To investigate the effect of exogenous brain-derived neurotrophic factor (BDNF) on the retinal repair after experimental retinal detachment (RD) reattached. Experimental study. Forty-eight normal rats were randomly divided into four groups:BDNF group, control group, RD reattached group and normal group. In order to detect the effects of BDNF on retinal degeneration caused by RD, the morphology of retina and the ultrastructure of retinal cells were observed by light microscopy and electron microscopy. The effect of BDNF on the apoptosis of retinal cells after RD reattached was detected by Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) technique. Data were analyzed using one-way analysis of variance (ANOVA) and secondary analysis for significance with independent-samples t-test. The average automatically reattached time was (31.3 ± 3.5) days. Compared with control group, there were less damage in out segment and inner segment, more retinal cells in outer nuclear layer (ONL) and inner nuclear layer (INL), thicker ONL of retina and better organizational structure of retina in BDNF group. The average thickness of ONL and INL of retina were (21.166 ± 3.087) µm, (23.508 ± 3.679) µm respectively in BDNF group, and (16.084 ± 2.928) µm, (12.885 ± 3.070) µm respectively in control group. The thickness of ONL and INL of retina showed a significant difference in BDNF group compared with control (P < 0.01). There were less TUNEL-positive cells in ONL of retina in BDNF group compared with control. This study indicates that the morphology of retina and the ultrastructure of retinal cells have changed, and the apoptosis of retinal cells are present after RD reattached. Exogenous BDNF can reduce retinal cells degeneration and inhibit the apoptosis of retinal cells and have a certain protection effects on the retinal damage caused by RD.

Decreased proteasomal activity causes photoreceptor degeneration in mice.

To study the retinal degeneration caused by decreased proteasomal activity in β5t transgenic (β5t-Tg) mice, an animal model of senescence acceleration. β5t-Tg mice and age-matched littermate control (WT) mice were used. Proteasomal activities and protein level of poly-ubiquitinated protein in retinal extracts were quantified. Fundus images of β5t-Tg mice were taken and their features were assessed. For histologic evaluation, the thicknesses of inner nuclear layer (INL), outer nuclear layer (ONL), and photoreceptor outer segment (OS) were measured. For functional analysis, ERG was recorded under scotopic and photopic illumination conditions. Immunofluorescence (IF) staining and TUNEL were performed to investigate the mechanism of photoreceptor degeneration. Chymotrypsin-like activity was partially suppressed in retinal tissues of β5t-Tg mice. Retinal degenerative changes with arterial attenuation were present in β5t-Tg, but not in WT mice. Inner nuclear layer thickness showed no significant change between β5t-Tg and WT mice at 1, 3, 6, and 9 months of age. By contrast, thicknesses of ONL and OS in β5t-Tg mice were significantly decreased at 3, 6, and 9 months compared with those in WT mice. Electroretinograms showed decrease of scotopic a-wave amplitude in β5t-Tg mice. The number of TUNEL-positive cells in ONL were significantly increased in β5t-Tg mice and colocalized with apoptosis-inducing factor, but not with cleaved caspase-3 and -9, indicating that the photoreceptor cell death was induced via a caspase-independent pathway. The current data showed that impaired proteasomal function causes photoreceptor degeneration.

Protective effect of light emitting diode phototherapy on fluorescent light induced retinal damage in Wistar strain albino rats

BackgroundArtificial light at night alters retinal physiology. Several studies have shown that light emitting diode phototherapy protects the retina from the damaging effects of acute light exposure. ObjectiveThe aim of this study has been to elucidate the protective effects of 670nm LED light on retinal damage induced by chronic fluorescent light in Wistar rats. MethodsMale Wistar albino rats were divided into four groups: group 1 were control (CL), group 2, 3 and 4 were exposed to fluorescent light (FL), LED preexposure+fluorescent light exposure (LL) and only LED light exposure (OL) respectively. All animals were maintained in their specific exposure regime for 30 days. Fluorescent light of 1800lx was exposed between 8 pm to 8 am. Rats were exposed to therapeutic LED light of 670nm of 9J/cm2 at 25mW/cm2 for 6min duration. Histopathological changes in the retina were studied. ResultsAnimals of the FL group showed a significant reduction in the outer nuclear layer thickness and cell count in addition to the total thickness of the retina. LL group which were exposed to 670nm LED prior to exposure to fluorescent light showed a significant decrease in the degree of damage. Conclusions670nm LED light preexposure is protective to retinal cells against fluorescent light-induced damage.

Human Retinal Progenitor Cell Transplantation Preserves Vision

Cell transplantation is a potential therapeutic strategy for retinal degenerative diseases involving the loss of photoreceptors. However, it faces challenges to clinical translation due to safety concerns and a limited supply of cells. Human retinal progenitor cells (hRPCs) from fetal neural retina are expandable in vitro and maintain an undifferentiated state. This study aimed to investigate the therapeutic potential of hRPCs transplanted into a Royal College of Surgeons (RCS) rat model of retinal degeneration. At 12 weeks, optokinetic response showed that hRPC-grafted eyes had significantly superior visual acuity compared with vehicle-treated eyes. Histological evaluation of outer nuclear layer (ONL) characteristics such as ONL thickness, spread distance, and cell count demonstrated a significantly greater preservation of the ONL in hRPC-treated eyes compared with both vehicle-treated and control eyes. The transplanted hRPCs arrested visual decline over time in the RCS rat and rescued retinal morphology, demonstrating their potential as a therapy for retinal diseases. We suggest that the preservation of visual acuity was likely achieved through host photoreceptor rescue. We found that hRPC transplantation into the subretinal space of RCS rats was well tolerated, with no adverse effects such as tumor formation noted at 12 weeks after treatment.

Protective Effect of SUN N8075, a Free Radical Scavenger, against Excessive Light-Induced Retinal Damage in Mice

Although dry age-related macular degeneration (AMD) is one of the major causes of blindness, no effective therapies are developed. In this study, we investigated the effects of SUN N8075, a radical scavenger with neuroprotective properties, against light-induced retinal damage used as the model of dry AMD in mice. After dark adaption for 24 h, we exposed the mice at 8000 lx for 3 h. We evaluated the retinal damage by recording the electroretinagram (ERG) and measuring the thickness of outer nuclear layer (ONL) at 5 d after the light exposure. Retinal apoptotic cells were also detected by terminal deoxynucleotidyl transeferase mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining, and the expression of 8-hydroxy-2-deoxyguanosine (8-OHdG) as an index for oxidative stress at 48 h after exposure to light. In ERG measurement, the intraperitoneal administration of SUN N8075 at 30 mg/kg improved the retinal dysfunction induced by the excess light exposure. In the histological evaluation, SUN N8075 inhibited the reduction of ONL thickness. In addition, SUN N8075 decreased in both numbers of TUNEL- and 8-OHdG-positive cells in ONL. These findings suggest that the systemic administration of SUN N8075 has protective effects on excess light-induced photoreceptor degeneration, via inhibition of oxidative stress.

Effect of neuroprotective statins in retina: A hypercholesterolemic rabbit model

AbstractPurpose The aim of the present study was to analyze qualitative and quantitative retinal changes in hypercholesterolemic rabbits after a low‐dosage statin treatment and to evaluate their possible neuroprotective effect on the retina.Methods New Zealand rabbits were divided into three groups: Control (G0; n=6), fed a standard diet; Hypercholesterolemic (G1, n=6), fed a 0.5% cholesterol‐enriched diet for 8 months; and Statins (G2; n=6), fed a 0.5% cholesterol‐enriched diet for 8 months plus administration of fluvastatin sodium or pravastatin sodium at a dosage of 2 mg/Kg/day each. Eyes were processed and analyzed for transmission‐electron microscopy. Semi‐thin sections were used to quantify cell number and thickness of retinal layers (excluding retinal pigment epithelium and nerve‐fiber layer) under light microscopy.Results G1 and G2, in comparison to G0, had a decreased cell number in the outer nuclear layer (ONL) (p&lt;0.05 and p&lt;0.005, respectively). No differences were detected in the inner nuclear layer or ganglion‐cell layer among the study groups. In G2, the ultrastructural cell morphology was similar to G0 and there were fewer signs of necrosis and apoptosis than in G1. Although the ONL cell number was decreased in G1, the retinal thickness in this group showed no differences in comparison to G0 due to retinal edema. Retinal thickness in G2 was reduced with respect to G1 (p&lt;0.05) because of the absence of retinal edema, and to G0 (p&lt;0.05) due to ONL cell loss.Conclusion Low‐dosage statins did not prevent ONL cell loss but preserved the normal ultrastructure of the remaining retinal neurons and avoided hypercholesterolemia‐induced retinal edema.