Subretinal Injection Of Sodium Hyaluronate Research Articles

Investigations into photoreceptor energy metabolism during experimental retinal detachment.

Retinal detachment is a sight-threatening disorder, which occurs when the photoreceptors are separated from their vascular supply. The aim of the present study was to shed light on photoreceptor energy metabolism during experimental detachment in rats. Retinal detachment was induced in the eyes of rats via subretinal injection of sodium hyaluronate. Initially, we investigated whether detachment caused hypoxia within photoreceptors, as evaluated by the exogenous and endogenous biomarkers pimonidazole and HIF-1α, as well as by qPCR analysis of HIF target genes. The results showed no unequivocal staining for pimonidazole or HIF-1α within any detached retina, nor upregulation of HIF target genes, suggesting that any reduction in pO2 is of insufficient magnitude to produce hypoxia-induced covalent protein adducts or HIF-1α stabilisation. Subsequently, we analysed expression of cellular bioenergetic enzymes in photoreceptors during detachment. We documented loss of mitochondrial, and downregulation of glycolytic enzymes during detachment, indicating that photoreceptors have reduced energetic requirements and/or capacity. Given that detachment did not cause widespread hypoxia, but did result in downregulated expression of bioenergetic enzymes, we hypothesised that substrate insufficiency may be critical in terms of pathogenesis, and that boosting metabolic inputs may preserve photoreceptor bioenergetic production and, protect against their degeneration. Thus, we tested whether supplementation with the bioavailable energy substrate pyruvate mitigated rod and cone injury and degeneration. Despite protecting photoreceptors in culture from nutrient deprivation, pyruvate failed to protect against apoptotic death of rods, loss of cone opsins, and loss of inner segment mitochondria, in situ, when evaluated at 3 days after detachment. The regimen was also ineffective against cumulative photoreceptor deconstruction and degeneration when evaluated after 4 weeks. Retinal metabolism, particularly the bioenergetic profiles and pathological responses of the various cellular subtypes still presents a considerable knowledge gap that has important clinical consequences. While our data do not support the use of pyruvate supplementation as a means of protecting detached photoreceptors, they do provide a foundation and motivation for future research in this area.

Differential Effects of Experimental Retinal Detachment on S- and M/L-Cones in Rats.

Retinal detachment is a vision-threatening condition, which occurs when the neurosensory retina is separated from its blood supply. The main purpose of this study was to examine the effect of experimental retinal detachment in rats on cone photoreceptors. Retinal detachment was induced in the eyes of rats via subretinal injection of sodium hyaluronate. Experimental detachment caused a rapid, sustained loss of short (S)- and medium/long (M/L)-wavelength cone opsins. Importantly, S-opsin+ cones were affected earlier than M/L-opsin+ cones and were affected to a greater extent than M/L-opsin+ cones throughout the duration of detachment. In comparison, to cone opsins, reductions in other cone markers-peanut agglutinin PNA and cone arrestin-were substantially less marked. These data suggest that loss of cone opsins does not reflect cone degeneration and may rather indicate prolonged downregulation of specific proteins in affected cones. This conclusion is supported by the lack of TUNEL+- cone arrestin+ double-labelled cells at the time point of greatest rod photoreceptor cell death, together with the partial recovery of cone arrestin+ cell numbers over time. Analysis of retinas that had naturally re-attached reinforced the deduction that few cones die following detachment, but also highlighted that prolonged detachment leads to deconstruction of cone segments that may not be readily reversible. Survival and functional recovery of cones following surgery for retinal detachment is vital for successful recovery of vision. The data suggest that experimental detachment in rats may offer a useful approach to model the response of S-cones to retinal detachment in humans.

Local photoreceptor cell death differences in the murine model of retinal detachment

To investigate local cell death differences in the attached and detached retina at different regions in a murine experimental retinal detachment model. Subretinal injection of sodium hyaluronate was performed in eight-week-old C57BL/6J mice. Retinal regions of interest were defined in relation to their distance from the peak of the retinal detachment, as follows: (1) attached central; (2) attached paracentral; (3) detached apex; and (4) detached base. At day 0, the outer nuclear layer cell count for the attached central, attached paracentral, detached apex, and detached base was 1247.60 ± 64.62, 1157.80 ± 163.33, 1264.00 ± 150.7, and 1013.80 ± 67.16 cells, respectively. There were significant differences between the detached base vs. attached central, and between detached base vs. detached apex at day 0. The detached apex region displayed a significant and progressive cell count reduction from day 0 to 14. In contrast, the detached base region did not show progressive retinal degeneration in this model. Moreover, only the detached apex region had a significant and progressive cell death rate compared to baseline. Immediate confounding changes with dramatic differences in cell death rates are present across regions of the detached retina. We speculate that mechanical and regional differences in the bullous detached retina can modify the rate of cell death in this model.

TRPML1 Inhibited Photoreceptor Apoptosis and Protected the Retina by Activation of Autophagy in Experimental Retinal Detachment

Objective: In this study, we used a rat model of retinal detachment (RD) to investigate the effects of transient receptor potential mucolipin 1 (TRPML1) on photoreceptor cells and the underlying mechanism. Methods: An RD model was established by subretinal injection of sodium hyaluronate, and mucolipin synthetic agonist 1 (ML-SA1) and dimethyl sulphoxide were subretinally injected after RD induction. Retinal morphology was observed using haematoxylin-eosin staining, and the apoptosis of photoreceptor cells was detected by transmission electron microscopy. Reactive oxygen species (ROS) were examined with an ROS detection kit. The retinal expression levels of TRPML1, the autophagy-related protein microtubule-associated protein 1 light chain 3 (LC3), Beclin 1, and cleaved caspase 3 were detected by Western blotting. The Morris water maze was used to test vision-dependent behaviour. Results: We found that retinal structure and the outer nuclear layer were improved and that the apoptosis of photoreceptor cells was reduced after ML-SA1 injection. The expression of ROS was reduced, and the loss of TRPML1 was inhibited after ML-SA1 treatment. The LC3-II to LC3-I ratio and Beclin 1 expression were enhanced, and cleaved caspase 3 expression was decreased after ML-SA1 treatment. Treatment with ML-SA1 also improved vision-dependent behaviour. Conclusions: Our findings suggest that ML-SA1 attenuates photoreceptor apoptosis and improves vision-dependent behaviour by activation of autophagy.

IL-33 deficiency causes persistent inflammation and severe neurodegeneration in retinal detachment

BackgroundInterleukin-33 (IL-33) belongs to the IL-1 cytokine family and resides in the nuclei of various cell types. In the neural retina, IL-33 is predominately expressed in Müller cells although its role in health and disease is ill-defined. Müller cell gliosis is a critical response during the acute phase of retinal detachment (RD), and in this study, we investigated if IL-33 was modulatory in the inflammatory and neurodegenerative pathology which is characteristic of this important clinical condition.MethodsRD was induced by subretinal injection of sodium hyaluronate into C57BL/6 J (WT) and IL-33−/− mice and confirmed by fundus imaging and optical coherence tomography (OCT). The expression of inflammatory cytokines, complement components and growth factors was examined by RT-PCR. Retinal neurodegeneration, Müller cell activation and immune cell infiltration were assessed using immunohistochemistry. The expression of inflammatory cytokines in primary Müller cells and bone marrow-derived macrophages (BM-DMs) was assessed by RT-PCR and Cytometric Bead Array.ResultsRD persisted for at least 28 days after the injection of sodium hyaluronate, accompanied by significant cone photoreceptor degeneration. The mRNA levels of CCL2, C1ra, C1s, IL-18, IL-1β, TNFα, IL-33 and glial fibrillary acidic protein (GFAP) were significantly increased at day 1 post-RD, reduced gradually and, with the exception of GFAP and C1ra, returned to the basal levels by day 28 in WT mice. In IL-33−/− mice, RD induced an exacerbated inflammatory response with significantly higher levels of CCL2, IL-1β and GFAP when compared to WT. Sustained GFAP activation and immune cell infiltration was detected at day 28 post-RD in IL-33−/− mice. Electroretinography revealed a lower A-wave amplitude at day 28 post-RD in IL-33−/− mice compared to that in WT RD mice. IL-33−/− mice subjected to RD also had significantly more severe cone photoreceptor degeneration compared to WT counterparts. Surprisingly, Müller cells from IL-33−/− mice expressed significantly lower levels of CCL2 and IL-6 compared with those from WT mice, particularly under hypoxic conditions, whereas IL-33−/− bone marrow-derived macrophages expressed higher levels of inducible nitric oxide synthase, TNFα, IL-1β and CCL2 after LPS + IFNγ stimulation compared to WT macrophages.ConclusionIL-33 deficiency enhanced retinal degeneration and gliosis following RD which was related to sustained subretinal inflammation from infiltrating macrophages. IL-33 may provide a previously unrecognised protective response by negatively regulating macrophage activation following retinal detachment.

Rapamycin Inhibited Photoreceptor Necroptosis and Protected the Retina by Activation of Autophagy in Experimental Retinal Detachment

ABSTRACTPurpose: After experimental retinal detachment (RD), the applications of caspase inhibitor z-vad-fmk (a pan-caspase inhibitor) could inhibit apoptosis, but increased receptor interacting protein (RIP)-mediated necroptosis. In this study, we investigated whether rapamycin could inhibit necroptosis and cooperate with z-vad-fmk to protect the retina after RD.Methods: RD animal models were established in Sprague–Dawley rats by subretinal injection of sodium hyaluronate and treated with subretinal injections of z-vad-fmk or z-vad-fmk combined with rapamycin. On day 3 after RD, retinas were collected and analyzed by transmission electron microscopy (TEM), ROS assay, and western blot (for beclin-1, LC-3, RIP-1, AIF). On day 7 after RD, retinas were observed by H&E staining. Vision-dependent behavior of rats was tested by the modified Morris water maze.Results: TEM and H&E staining indicated that rapamycin combined with z-vad-fmk could reduce photoreceptor necrosis and preserve the ONL thickness after RD. The modified Morris water maze test showed that vision-dependent behavior was also significantly improved in the rapamycin + z-vad-fmk group.Western Blotting results demonstrated that rapamycin promoted the activation of autophagy by promoting beclin-1 and LC-3 induction and inhibited z-vad-fmk-induced necroptosis by inhibiting RIP-1 expression. In addition, rapamycin could also inhibit ROS production and AIF release.Conclusions: These findings indicated that rapamycin is a promising therapeutic agent that inhibits z-VAD-induced necroptosis, and protects photoreceptors and improves functional outcome in combination with z-vad-fmk.

Modulation of α-adrenoceptor signalling protects photoreceptors after retinal detachment by inhibiting oxidative stress and inflammation.

Currently available treatments do not halt progression of photoreceptor death and subsequent visual impairment related to retinal detachment (RD) which is observed in various retinal disorders. This study investigated the neuroprotective effects of two adrenoceptor ligands, the α1 -adrenoceptor antagonist doxazosin and the α2 -adrenoceptor agonist guanabenz, against photoreceptor cell death in RD. We used a model of experimental RD in Brown-Norway rats induced by subretinal injection of sodium hyaluronate. Oxidative stress biomarkers and cytokine production were quantified with elisa. Protein expression levels and immunofluorescent labelling were determined in rats with RD and controls for mechanistic elucidation. The effects of systemic (i.p.) administration of doxazosin or guanabenz on photoreceptor apoptosis, retinal histology and electroretinography were evaluated in rats with RD and compared to the effects in vehicle controls. Photoreceptors were the major source of RD-induced ROS overproduction in the rat retina through the regulation of NADPH oxidase. Systemic administration of doxazosin or guanabenz decreased the RD-induced production of ROS and proinflammatory cytokines, including IL-1β and the chemokine CCL2, and suppressed retinal gliosis, resulting in attenuation of photoreceptor death and preservation of retinal structures and functions in RD. Our findings point to α-adrenoceptors as novel therapeutic targets to provide photoreceptor protection and suggest that both doxazosin and guanabenz, two FDA-approved drugs, could be further explored to treat retinal diseases.

Activation of autophagy in photoreceptor necroptosis after experimental retinal detachment.

To investigate whether photoreceptor necroptosis induced by z-VAD-FMK (pan caspase inhibitor) was involved the activation of autophagy and whether Necrostatin-1, a specific necroptosis inhibitor, could inhibit this induction of autophagy after experimental retinal detachment. Experimental retinal detachment models were created in Sprague-Dawley rats by subretinal injection of sodium hyaluronate and subretinal injections of z-VAD-FMK, vehicle or z-VAD-FMK plus Necrostatin-1. Three days after retinal detachment, morphologic changes were observed by transmission electron microscopy. In other animals, retinas were subjected to immunoprecipitation and Western Blotting, then probed with anti-RIP1, phosphoserine, LC-3II or caspase 8 antibody. It was proved by immunoprecipitation and western blotting, that photoreceptor necroptosis was mediated by caspase-8 inhibition and receptor interacting protein kinase (RIP1) phosphorylation activation. Transmission electron microscope and western blotting results indicated that photoreceptor necroptosis was involved the LC-3II and autophagosomes induction. We also discovered Necrostatin-1 could inhibit RIP1 phosphorylation and LC-3II induction. These data firstly indicate photoreceptor necroptosis is associated with the activation of autophagy. Necrostatin-1 protects photoreceptors from necroptosis and autophagy by down-regulation of RIP1 phosphorylation and LC-3II.

Strain difference in photoreceptor cell death after retinal detachment in mice.

To evaluate the potential for mouse genetic background to effect photoreceptor cell death in response to experimental retinal detachment (RD). Retinal detachment was induced in three inbred mouse strains (C57BL/6, BALB/c, and B6129SF2) by subretinal injection of sodium hyaluronate. A time course of photoreceptor cell death was assessed by TUNEL assay. Total photoreceptor cell death was analyzed through comparing the outer nuclear layer (ONL)/inner nuclear layer (INL) ratio 7 days post RD. Western blot analysis or quantitative real-time PCR (qPCR) were performed to assess cell death signaling, expression of endogenous neurotrophin, and levels of apoptosis inhibitors 24 hours after RD. Inflammatory cytokine secretion and inflammatory cell infiltration were quantified by ELISA and immunostaining, respectively. The peak of photoreceptor cell death after RD was at 24 hours in all strains. Photoreceptor cell death as well as monocyte chemoattractant protein 1 and interleukin 6 secretion at 24 hours after RD was the highest in BALB/c, followed in order of magnitude by C57BL/6 and B6129SF2. Conversely, nerve growth factor expression and ONL/INL ratio were the lowest in BALB/c. Apoptosis signaling was higher in C57BL/6, whereas necroptosis signaling was higher in C57BL/6 and BALB/c. Autophagic signaling was higher in BALB/c. X-linked inhibitor of apoptosis (XIAP) and survivin protein levels were lower in C57BL/6 and BALB/c, respectively. Macrophage/microglia infiltration was higher in C57BL/6 and BALB/c at 24 hours after RD. Photoreceptor cell death after RD was significantly different among the three strains, suggesting the presence of genetic factors that affect photoreceptor cell death after RD.

Erratum: Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Erratum: Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Protective Effects of Resveratrol in Experimental Retinal Detachment

BackgroundOxidative stress is one of the major factors that trigger photoreceptor apoptosis. To investigate whether resveratrol, a potent antioxidant and small molecule activator of the FoxO pathway, would be neuroprotective against photoreceptor cell death in a rodent model of retinal detachment.MethodsRetinal detachment was created in adult Brown Norway rats by subretinal injection of sodium hyaluronate. The animals were treated daily with vehicle or resveratrol (20mg/kg) intraperitoneal injection. Photoreceptor death was assessed by counting the number of apoptotic cells with TdT-dUTP terminal nick-end labeling (TUNEL) and measurement of the outer nuclear layer (ONL) thickness 3 days after RD. Changes in expression of FoxO1a, FoxO3a, and FoxO4 were analyzed by western blot. The activity of caspase 3, caspase 8, caspase 9, spectrin and their cleavage forms were studied.ResultsThree days after retinal detachment, caspase 3, caspase 8 and caspase 9 were significantly activated in the detached retina. Spectrin cleavage products at 120 and 145 kDa were also detected. Both caspase and calpain activation are involved in apoptotic photoreceptor cell death in detached retinas. Treatment with resveratrol increases FoxO1a, FoxO3a, and FoxO4 protein expression in detached retinas only. Resveratrol treatment decreases activation of intrinsic and extrinsic caspase apoptotic pathways triggered by RD. The number of TUNEL-positive cells decreases from 1301±51 cells/mm2 in control groups to 430±35 cells/mm2 in treatment groups (p<0.05). Resveratrol treatment also demonstrates 59% less ONL thickness loss compared to controls.ConclusionsResveratrol treatment up-regulates the FoxO family and blocks Caspase3, 8, and 9 activation. Resveratrol has the potential to be used as a novel therapeutic agent for preventing vision loss in diseases characterized by photoreceptor detachment.

Edaravone, an ROS Scavenger, Ameliorates Photoreceptor Cell Death after Experimental Retinal Detachment

To investigate whether edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a free radical scavenger, would be neuroprotective against photoreceptor cell death in a rat model of retinal detachment (RD). RD was induced in adult Brown Norway rats by subretinal injection of sodium hyaluronate. Edaravone (3, 5, or 10 mg/kg) or physiologic saline was administered intraperitoneally once a day until death on day 3 or 5. Oxidative stress in the retina was assessed by 4-hydroxynonenal staining or ELISA for protein carbonyl content. Photoreceptor death was assessed by TUNEL and measurement of the outer nuclear layer thickness. Western blot analysis and caspase activity assays were performed. Inflammatory cytokine secretion and inflammatory cell infiltration were evaluated by ELISA and immunostaining, respectively. RD resulted in increased generation of ROS. Treatment with 5 mg/kg edaravone significantly reduced the ROS level, along with a decrease in TUNEL-positive cells in the photoreceptor layer. A caspase assay also confirmed decreased activation of caspase-3, -8, and -9 in RD treated with edaravone. The level of the antiapoptotic Bcl-2 was increased in detached retinas after edaravone treatment, whereas the levels of the stress-activated p-ERK1/2 were decreased. In addition, edaravone treatment resulted in a significant decrease in the levels of TNF-α, MCP-1, and macrophage infiltration. Oxidative stress plays an important role in photoreceptor cell death after RD. Edaravone treatment may aid in preventing photoreceptor cell death after RD by suppressing ROS-induced photoreceptor damage.

Minocycline Inhibition of Photoreceptor Degeneration

To determine whether systemic minocycline can protect photoreceptors in experimental retinal detachment (RD). Retinal detachment was induced in mice by subretinal injection of sodium hyaluronate, 1.4%. In 1 experiment, mice received daily injections of minocycline (group 1) or saline (group 2). In a second experiment, mice were treated with minocycline or saline beginning 24 hours prior, immediately after, or 24 hours after experimental RD. In both experiments, photoreceptor cell survival and apoptosis were assessed by immunohistochemistry with primary antibodies against photoreceptor cell markers, rod rhodopsin, and cone opsin, and by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling. Photoreceptor cell apoptosis was detected at day 1 after experimental RD, with apoptotic cells peaking in number at day 3 and dropping by day 7. Treatment with minocycline significantly reduced the number of apoptotic photoreceptor cells associated with RD when given 24 hours before or even 24 hours after RD. Our data suggest that minocycline may be useful in the treatment of photoreceptor degeneration associated with RD, even when given up to 24 hours after RD. Use of minocycline in patients with macula-off RD may prevent photoreceptor apoptosis and glial cell proliferation, improving final visual outcomes.

Ceramide production associated with retinal apoptosis after retinal detachment

During retinal detachment, premature apoptosis of photoreceptors and a loss of optimally corrected visual acuity occur. We hypothesized that retinal cell death and generation of ceramide, a pro-apoptotic lipid, would progress as a function of time following experimental retinal detachment, and undertook to define the appropriate temporal window. Unilateral retinal detachment was induced in white New Zealand rabbits by subretinal injection of sodium hyaluronate. In experimental animals, we injected sphingosine-1-P into the vitreous 2 hours before retinal detachment. Both eyes were removed on days 1, 3 and 6 for histological and biochemical examination. The number of photoreceptors was counted in section, the level of apoptosis was assessed using the TUNEL assay, and the production of ceramide was analyzed in situ with immunohistochemistry. The concentration of ceramide was also determined on retinal homogenates using a diacylglycerol kinase assay. We confirmed that the average number of live photoreceptors decreased gradually after retinal detachment. In eyes pre-treated with sphingosine-1-P the number of apoptotic photoreceptors was significantly lower. The proportion of apoptotic photoreceptors (14%) remained constant as a function of time in the window studied. As compared to controls, the detached retina showed intense ceramide immunostaining that was prominent in the photoreceptors, but also present to a lesser extent in other retinal layers. The total concentration of intra-retinal ceramide increased by 40% on the first day and continued augmenting through the sixth day after retinal detachment. Retinal apoptosis during experimental retinal detachment is associated with in vivo production of ceramide.

Attenuated Glial Reactions and Photoreceptor Degeneration after Retinal Detachment in Mice Deficient in Glial Fibrillary Acidic Protein and Vimentin

To characterize the reactions of retinal glial cells (astrocytes and Müller cells) to retinal injury in mice that lack glial fibrillary acidic protein (GFAP) and vimentin (GFAP-/-Vim-/-) and to determine the role of glial cells in retinal detachment (RD)-induced photoreceptor degeneration. RD was induced by subretinal injection of sodium hyaluronate in adult wild-type (WT) and GFAP-/-Vim-/- mice. Astroglial reaction and subsequent monocyte recruitment were quantified by measuring extracellular signal-regulated kinase (Erk) and c-fos activation and the level of expression of chemokine monocyte chemoattractant protein (MCP)-1 and by counting monocytes/microglia in the detached retinas. Immunohistochemistry, immunoblotting, real-time quantitative polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA) were used. RD-induced photoreceptor degeneration was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and measurement of outer nuclear layer (ONL) thickness. RD-induced reactive gliosis, characterized by GFAP and vimentin upregulation, Erk and c-fos activation, MCP-1 induction, and increased monocyte recruitment in WT mice. Absence of GFAP and vimentin effectively attenuated reactive responses of retinal glial cells and monocyte infiltration. As a result, detached retinas of GFAP-/-Vim-/- mice exhibited significantly reduced numbers of TUNEL-positive photoreceptor cells and increased ONL thickness compared with those of WT mice. The absence of GFAP and vimentin attenuates RD-induced reactive gliosis and, subsequently, limits photoreceptor degeneration. Results of this study indicate that reactive retinal glial cells contribute critically to retinal damage induced by RD and provide a new avenue for limiting photoreceptor degeneration associated with RD and other retinal diseases or damage.

Effects of Nerve Growth Factor for Retinal Cell Survival in Experimental Retinal Detachment

Purpose: To investigate the neuron protective effect of recombined nerve growth factor (rNGF) on retinal cell damage induced by experimental retinal detachment. Methods: Experimental retinal detachment models were created in Sprague-Dawley rats by subretinal injection of sodium hyaluronate. Intravitreal injection of rNGF (5 μ g/eye) or phosphate-buffered saline (PBS) was separately applied every 4 days after retinal detachment. The rat eyes were then observed and sacrificed at various time points. Morphologic changes were observed by light microscopy, electron microscopy, and cell counts. Apoptosis of retinal cells was detected by TUNEL assay. Results: After retinal detachment, most eyes from NGF-treated groups showed better organized structure of retinal cells than those from the PBS-treated control groups. Cell counts indicated that the nuclei numbers in the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL) of NGF-treated groups were significantly more than those from PBS-treated control group (p < 0.05) after retinal reattachment. TUNEL-positive cells were identified in ONL, INL, and GCL. They peaked at the fourth day after retinal detachment in both the NGF-treated groups and the control groups. But the cell counts of apoptosis revealed that the NGF-treated retina had less TUNEL-positive cells than the control groups (p < 0.05). Conclusions: The results showed that intravitreal injection of exogenous NGF can protect retinal cells from degeneration and apoptosis in experimental retinal detachment. It may exert its neuroprotection effect by preventing the apoptosis of retinal cells after retinal detachment.

Triamcinolone Does Not Alter Glial Cell Activation in the Experimentally Detached Rabbit Retina

Retinal detachment induces neural and photoreceptor cell degeneration and fast activation of micro- (immune) and macroglial cells. Hypoxia caused by increased distance between the choriocapillaris and the neural retina, and retinal oedema during detachment, are factors causing gliotic responses and cell degeneration. Triamcinolone may inhibit some cellular responses that accompany hypoxia. Therefore, we investigated whether triamcinolone acetonide may be effective to reduce the gliotic alterations in the detached retina. Local retinal detachment in rabbit eyes was created by subretinal injection of sodium hyaluronate, and triamcinolone acetonide (8 mg) was applied intravitreally. Wholecell patch-clamp records from Muller cells and Ca2+ imaging from retinal wholemounts were carried out. Microglial/immune cells in the nerve-fiber layer of retinal wholemounts were labeled with Griffonia simplicifolia agglutinin (GSA) isolectin. Additionally, two morphological parameters which characterize microglial activation/immune cell infiltration were estimated: the cross-sectional area of the somata of the cells in the nerve-fiber layer and the number of cell processes which evolve from the soma. Three days after detachment, gliotic alterations were apparent in Muller cells isolated from both detached and nondetached retinal areas, as indicated by the cellular hypertrophy, by the downregulation of the plasma membrane K+ conductance, and by the upregulation of intracellular Ca2+ responsiveness to stimulation of purinergic P2Y receptors. Intravitreal triamcinolone did not alter these gliotic alterations of Muller cells. Furthermore, triamcinolone could not inhibit the immune cell activation present in detached and attached retinal areas. However, intravitreal triamcinolone led to a strong decrease in the process number of GSA lectin-positive cells from detached retinas. The results suggest that triamcinolone is ineffective to inhibit gliotic responses in the detached retina. However, the immune cell activation after detachment was partially influenced by triamcinolone.

Critical role of photoreceptor apoptosis in functional damage after retinal detachment

Purpose. Although apoptosis is assumed to play a pivotal role in retinal function loss, its mechanism and real influence on retinal function are still unclear. To investigate the relation between retinal function and apoptosis, we studied photoreceptor apoptosis in experimental retinal detachment (RD). Methods. We induced RD by subretinal injection of sodium hyaluronate in Brown Norway rats. Apoptotic photoreceptors were detected by TdT-dUTP Terminal Nick-End Labeling (TUNEL). To evaluate the function of the detached retina, electroretinograms (ERGs) were taken on day 1, 3 with corneal electrodes and full-field stimulation. Results. Apoptotic DNA fragmentation appeared 12 hours after RD, was most prominent on day 3, and decreased thereafter. The ERGs showed that the amplitudes of dark-adapted a-waves and light adapted 2Hz b-waves decreased immediately after RD and continued to decrease over time. The administration of Fas/Fc chimera recombinant protein or a caspase inhibitor, Z-VAD.fmk, failed to prevent either photoreceptor apoptosis or retinal functional damage. In contrast, brain derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) significantly impeded both apoptosis and dysfunction. The ERGs recognized the functional changes sensitively, and these ERG changes correlated well to the amount of photoreceptor apoptosis. Immunohistochemical study showed that apoptosis-inducing factor (AIF), a novel caspase-independent apoptotic factor, was relocalized from mitochondria to the nucleus in this process. Conclusions. The present results showed that apoptosis was a key phenomenon in the retinal dysfunction in RD and that this process was transmitted mainly by mitochondria-dependent pathways rather than Fas/Fas-L or downstream caspase dependent pathways.