Experimental Retinal Detachment Research Articles

Peripheral monocytes and neutrophils promote photoreceptor cell death in an experimental retinal detachment model

Photoreceptor cell death and immune cell infiltration are two major events that contribute to retinal degeneration. However, the relationship between these two events has not been well delineated, primarily because of an inadequate understanding of the immunological processes involved in photoreceptor degeneration, especially that of peripheral leukocytes that infiltrate the subretinal space and retinal tissues. In this work, we characterized the role of leukocyte infiltration within the detached retina. We observed that CD45+ CD11b+ Ly6G+ neutrophils and CD45+ CD11b+ Ly6G− Ly6C+ monocytes are the predominant peripheral immune cell populations that infiltrate the retinal and subretinal space after detachment. Selective depletion of monocytes or neutrophils using cell-specific targeting is neuroprotective for photoreceptors. These results indicate that peripheral innate immune cells contribute to photoreceptor degeneration, and targeting these immune cell populations could be therapeutic during retinal detachment.

Transcriptomic Analysis of Retinal Gene in Experimental Retinal Detachment Rats and Exploration of S100A9 and TLR4 in Human Vitreous

Purpose To screen for the differentially expressed genes in experimental retinal detachment rats, and to explore the expression of S100 calcium-binding protein A9 and Toll-like receptor 4 in the vitreous of rhegmatogenous retinal detachment patients. Methods Three rats of experimental retinal detachment and three normal rats were enrolled in the study. Transcriptomics (RNAseq) sequencing technology was used to screen differentially expressed genes in the retinas of the experimental retinal detachment group and the normal group. The selected differentially expressed genes for gene ontology and Kyoto Encyclopedia of Genes and Genomes functional enrichment analysis were performed. In addition, the vitreous of 15 patients with rhegmatogenous retinal detachment and six patients with the control group were collected. The expressions of S100 calcium-binding protein A9 and Toll-like receptor 4 were detected by Elisa, and the differences in expression levels were analyzed statistically. Results A total of 198 differentially expressed genes were screened by RNAseq sequencing, including 118 upregulated genes and 80 downregulated genes. Kyoto Encyclopedia of Genes and Genomes analysis confirmed that the most enriched pathway was the mitogen-activated protein kinase signaling pathway. Compared to the normal group, the expressions of suppressor of cytokine signaling-3, Storkhead box-2, S100 calcium-binding protein A9, Spi-1 proto-oncogene, phosphodiesterase 1B, and kinesin-light chain 1 mRNA in the retinas of the experimental retinal detachment rats were up-regulated, and the expressions of Max interacting protein 1 and the voltage-gated sodium 1 were down-regulated. Compared to the control group, the expressions of S100 calcium-binding protein A9 and Toll-like receptor 4 were upregulated by Elisa in the vitreous humor of rhegmatogenous retinal detachment patients with a statistically significant difference (p all <.05). Conclusion The differentially expressed genes of experimental retinal detachment rats were suppressor of cytokine signaling-3, Storkhead box-2, S100 calcium-binding protein A9, Spi-1 proto-oncogene, phosphodiesterase 1B, kinesin-light chain 1, Max interacting protein 1, voltage-gated sodium 1, etc. The differences of S100 calcium-binding protein A9 and Toll-like receptor 4 expressions between the rhegmatogenous retinal detachment patients and the control group were statistically significant, indicating that they may play a potential role in the inflammatory process of rhegmatogenous retinal detachment.

Metabolic Alterations Caused by Simultaneous Loss of HK2 and PKM2 Leads to Photoreceptor Dysfunction and Degeneration.

HK2 and PKM2 are two main regulators of aerobic glycolysis. Photoreceptors (PRs) use aerobic glycolysis to produce the biomass necessary for the daily renewal of their outer segments. Previous work has shown that HK2 and PKM2 are important for the normal function and long-term survival of PRs but are dispensable for PR maturation, and their individual loss has opposing effects on PR survival during acute nutrient deprivation. We generated double conditional (dcKO) mice lacking HK2 and PKM2 expression in rod PRs. Western blotting, immunofluorescence, optical coherence tomography, and electroretinography were used to characterize the phenotype of dcKO animals. Targeted and stable isotope tracing metabolomics, qRT-PCR, and retinal oxygen consumption were performed. We show that dcKO animals displayed early shortening of PR inner/outer segments, followed by loss of PRs with aging, much more rapidly than either knockout alone without functional loss as measured by ERG. Significant alterations to central glucose metabolism were observed without any apparent changes to mitochondrial function, prior to PR degeneration. Finally, PR survival following experimental retinal detachment was unchanged in dcKO animals as compared to wild-type animals. These data suggest that HK2 and PKM2 have differing roles in promoting PR neuroprotection and identifying them has important implications for developing therapeutic options for combating PR loss during retinal disease.

Attenuation of Microglial Activation and Pyroptosis by Inhibition of P2X7 Pathway Promotes Photoreceptor Survival in Experimental Retinal Detachment.

Photoreceptor (PR) death is the ultimate cause of irreversible vision loss in retinal detachment (RD). Although microglial infiltration in the subretinal space (SRS) was observed after RD, the molecular mechanism underlying microglial activation and the outcomes of infiltrating microglia remain unclear. We aimed to uncover the mechanism of initiation of microglial activation to help explore potential therapy to promote PR survival. An RD model was conducted by injecting sodium hyaluronate into SRS of C57BL/6J wild type mice. Adenosine triphosphate (ATP) was measured by a ATP Microplate Assay Kit. Bioinformatics analysis was used to evaluate the upregulated receptor relating to ATP binding in human datasets and mouse transcriptomes of RD. Expression of P2X7, its downstream signaling pathways, and microglial pyroptosis were confirmed by qPCR, WB, and immunofluorescence in vivo and in vitro. The cell viability of PR was measured by cell counting kit-8. Brilliant Blue G, a P2X7 antagonist, was subretinally or intraperitoneally injected to inhibit microglial activation in vivo and was applied for microglia cell line treatment in vitro. The decrease in microglial activation and pyroptosis was detected by immunofluorescence and WB. The protective effect on PR was measured by hematoxylin and eosin staining, TUNEL assay, and electroretinogram analysis. The results showed that extracellular ATP released in the SRS after RD triggered P2X7 activation and attracted microglia. The downstream cascade of inflammasome activation induced by P2X7 activation contributed to microglial pyroptosis and then to PR death. ATP-activated microglia led to PR death in vitro. P2X7 blockade rescued PR morphologically and functionally by inhibiting microglial activation and pyroptosis. These results elucidate that ATP-induced P2X7-mediated microglial activation leads to microglial pyroptosis, contributing to PR death. Appropriate inhibition of microglial pyroptosis might serve as a pharmacotherapeutic strategy for decreasing PR death in RD.

Microglia in Cultured Porcine Retina: Qualitative Immunohistochemical Analyses of Reactive Microglia in the Outer Retina.

A late stage of several retinal disorders is retinal detachment, a complication that results in rapid photoreceptor degeneration and synaptic damages. Experimental retinal detachment in vivo is an invasive and complicated method performed on anesthetized animals. As retinal detachment may result in visual impairment and blindness, research is of fundamental importance for understanding degenerative processes. Both morphological and ethical issues make the porcine retina a favorable organotypic model for studies of the degenerative processes that follow retinal detachment. In the cultured retina, photoreceptor degeneration and synaptic injuries develop rapidly and correlate with resident microglial cells' transition into a reactive phenotype. In this immunohistochemical study, we have begun to analyze the transition of subsets of reactive microglia which are known to localize close to the outer plexiform layer (OPL) in degenerating in vivo and in vitro retina. Biomarkers for reactive microglia included P2Ry12, CD63 and CD68 and the general microglial markers were CD11b, Iba1 and isolectin B4 (IB4). The reactive microglia markers labeled microglia subpopulations, suggesting that protective or harmful reactive microglia may be present simultaneously in the injured retina. Our findings support the usage of porcine retina cultures for studies of photoreceptor injuries related to retinal detachment.

Dynamics of components of lipid peroxidation and antioxidant defense system in rats with experimental rhematogenous retinal detachment = Динаміка компонентів перекисного окиснення ліпідів та системи антиоксидантного захисту у щурів з експериментальним регматогенним відшаруванням сітківки

Dynamics of components of lipid peroxidation and antioxidant defense system in rats with experimental rhematogenous retinal detachment = Динаміка компонентів перекисного окиснення ліпідів та системи антиоксидантного захисту у щурів з експериментальним регматогенним відшаруванням сітківки

Optimizing Retinal Thermofusion in Retinal Detachment Repair: Achieving Instant Adhesion without Air Tamponade.

Rhegmatogenous retinal detachment repair by intraoperative sealing of the tear without a tamponade agent should enable faster restoration of vision and resumption of normal activities. It avoids the need for further surgery in the case of silicone oil endotamponade. This study evaluated the retinal thermofusion (RTF) retinopexy method of subretinal space dehydration before photocoagulation to create an instantaneous intraoperative retina reattachment in a preclinical model. Preclinical study. Twenty Dutch Belt, pigmented rabbits that underwent RTF repair after experimental retinal detachment. This exvivo model quantified adhesion force between the retina and underlying retinal pigment epithelium and choroid after treatment of 1 retinal edge using postmortem porcine or human retina (6× 12 mm). We compared (1) control, (2) laser photocoagulation alone, (3) dehydration alone, and (4) dehydration followed by photocoagulation (RTF). Optimized parameters for RTF were then applied in the invivo rabbit model of retinal detachment. Animals were followed up for 14 days. For this exvivo model, we measured adhesion force and related this to tissue temperature. For the invivo study, we assessed retinal attachment using funduscopy and histologic analysis. The exvivo model showed that RTF repair produced significantly higher adhesion force than photocoagulation alone independent of dehydration method: warm (60° C) high airflow (50-70 ml/minute) or using laser wavelengths targeting water absorption peaks (1470 or 1940 nm) with coaxial low airflow (10-20 ml/minute). The latter approach produced a smaller footprint of dehydration. Application of RTF (1940-nm laser with coaxial airflow) in an invivo retinal detachment model in rabbit eyes resulted in immediate retinal adhesion, achieving forces similar to those in the exvivo experiments. Retinal thermofusion repair resulted in stable reattachment of the retina over the 2-week follow-up period. We showed that a short preliminary dehydrating laser treatment of a retinal tear margin before traditional laser photocoagulation creates an immediate intraoperative waterproof retinopexy adhesion independent of tamponade and a wound-healing response. This approach potentially will allow rapid postoperative recovery regardless of the tear location and improved vision.

Cellular remodelling of the inner retina and the retinal ganglion cells in experimental retinal detachment

AbstractPurpose: Anatomical surgical outcomes for the treatment of retinal detachment (RD) are outstanding; however, only 50% of the patients with a detached macula regain 5/10 or better vision. Thus, to preserve or restore visual function in RD, several gaps in our understanding of the pathophysiology of the disease need to be bridged. Therefore, the purpose of the current study, was to investigate the anatomical/morphological changes of retinal interneurons and retinal ganglion cells (RGCs) in an experimental model of RD.Methods: Experimental RD was induced in C57BL6/J mice by a subretinal injection of 1% sodium hyaluronate. Following RD, mice were euthanized at 1‐, 3‐, 7‐, 30‐ and 90‐days post‐surgery and eyes or retinas were isolated and proceed for sectioning or retinal whole mounts respectively. Immunofluorescence and electron microscopy studies were performed against protein kinase C alpha (PKC‐α) to target rod bipolar cells, calretinin and calbindin D to target horizontal cells and brain‐specific homeobox/POU domain protein 3A (Brn3a) and neuron‐specific class III beta‐tubulin (β‐tubulin III) to target RGCs.Results: Three days post‐RD, PKC‐α+ neurons (rod bipolar cells) began to migrate towards the outer nuclear layer (ONL), while 30 days post‐RD they had significantly migrated towards the ONL and displayed irregular distribution of their cell bodies in the inner nuclear layer. Similarly, processes of horizontal cells also migrated towards the ONL, 7 days post‐RD and became more invasive and disoriented after longer‐term detachment (30 days post‐surgery). Moreover, there was a significant increase in the intensity of calbindin D, 30‐days post‐RD. Brn3a/β‐tubulin ΙΙΙ+ RGCs were significantly fewer 30‐ and 90‐days post RD. All retinal neurons that were examined, did not show any noteworthy differences 1 day post RD.Conclusions: RD resulted in significant anatomical changes in both rod bipolar and horizontal neurons of the retina along with a notable reduction of RGCs. The cellular response of the inner retina and the RGCs of the detached retina might play an important role in the imperfect visual score of an anatomical successful surgical reattachment.

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.

Autophagy activation and photoreceptor survival in retinal detachment

We assess the effect of autophagy inhibition on photoreceptor (PR) survival during experimental retinal detachment (RD) and examine the and examine the relationship between autophagy and the expression of glycolytic enzymes HK2 and PKM2 in the retina. We find that inhibiting autophagy by genetic knock out of the autophagy activator Atg5 in rod PRs resulted in increased apoptotic and necroptotic cell death during RD, demonstrated by elevated terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells, caspase 8 activity, transcript levels of Fas receptor and RIPK3 as compared to controls. The absence of autophagy in rods resulted in downregulation of hexokinase 2 and pyruvate kinase muscle isozyme 2 levels. More than 460 proteins were identified by mass spectroscopy in autophagosomes isolated from detached retinas compared with less than 150 proteins identified in autophagosomes from attached retinas. Among various cellular compartments, proteins from cytoskeleton, cytoplasm and intracellular organelles constituted a large portion of increased autophagosome contents. These proteins represent numerous biological processes, including phototransduction, cell-cell signaling, metabolism and inflammation. Our findings suggest that competent autophagy machinery is necessary for PR homeostasis and improving PR survival during periods of nutrient deprivation.

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.

PJ34 Protects Photoreceptors from Cell Death by Inhibiting PARP-1 Induced Parthanatos after Experimental Retinal Detachment

ABSTRACT Purpose Our previous study discoveredreactive oxygen species (ROS) and apoptosis inducing factor (AIF) increased after retinal detachment. Parthanatos is a cell death form involving ROS and AIF, which is induced by poly (ADP-ribose) polymerase-1 (PARP-1). Therefore, we investigated whether PJ34 (a PARP-1 inhibitor) could inhibit parthanatos and protect the photoreceptors from cell death after retinal detachment (RD). Methods Experimental retinal detachment modelswere created in Sprague-Dawley rats by subretinal injection of sodium hyaluronate.PJ34 orDMSO were introduced into subretinal space at RD induction, respectively. The structure of retinas and the morphology of photoreceptors were observed by hematoxylin eosin (H&E) staining and transmission electron microscope (TEM). Parthanatos related proteins (PARP-1, PAR,AIF) were detected by Western blot. The vision-dependent behavior of rat was tested by Morris water maze. Results H&E staining and TEM results indicated that the structure and outer nuclear layer (ONL) thickness of retinas were preserved, and the photoreceptors death decreasedwith PJ34 treatment. Western blot showed that the expression of PARP-1, PAR and AIF were decreased withPJ34 treatment. In addition, administration of PJ34 also improved the vision-dependent behavior of rat. Conclusions These findings suggested that PJ34 is a potential therapeutic agent that attenuated photoreceptor parthanatos death in retinal detachment through inhibition of PARP-1/AIF pathway.

Hexokinase 2 is dispensable for photoreceptor development but is required for survival during aging and outer retinal stress

Photoreceptor death is the ultimate cause of vision loss in many retinal degenerative conditions. Identifying novel therapeutic avenues for prolonging photoreceptor health and function has the potential to improve vision and quality of life for patients suffering from degenerative retinal disorders. Photoreceptors are metabolically unique among other neurons in that they process the majority of their glucose via aerobic glycolysis. One of the main regulators of aerobic glycolysis is hexokinase 2 (HK2). Beyond its enzymatic function of phosphorylating glucose to glucose-6-phosphate, HK2 has additional non-enzymatic roles, including the regulation of apoptotic signaling via AKT signaling. Determining the role of HK2 in photoreceptor homeostasis may identify novel signaling pathways that can be targeted with neuroprotective agents to boost photoreceptor survival during metabolic stress. Here we show that following experimental retinal detachment, p-AKT is upregulated and HK2 translocates to mitochondria. Inhibition of AKT phosphorylation in 661W photoreceptor-like cells results in translocation of mitochondrial HK2 to the cytoplasm, increased caspase activity, and decreased cell viability. Rod-photoreceptors lacking HK2 upregulate HK1 and appear to develop normally. Interestingly, we found that HK2-deficient photoreceptors are more susceptible to acute nutrient deprivation in the experimental retinal detachment model. Additionally, HK2 appears to be important for preserving photoreceptors during aging. We show that retinal glucose metabolism is largely unchanged after HK2 deletion, suggesting that the non-enzymatic role of HK2 is important for maintaining photoreceptor health. These results suggest that HK2 expression is critical for preserving photoreceptors during acute nutrient stress and aging. More specifically, p-AKT mediated translocation of HK2 to the mitochondrial surface may be critical for protecting photoreceptors from acute and chronic stress.

Bone marrow mesenchymal stem cells enhance autophagy and help protect cells under hypoxic and retinal detachment conditions.

Our study aimed to evaluate the protective role and mechanisms of bone marrow mesenchymal stem cells (BMSCs) in hypoxic photoreceptors and experimental retinal detachment. The cellular morphology, viability, apoptosis and autophagy of hypoxic 661w cells and cells cocultured with BMSCs were analysed. In retinal detachment model, BMSCs were intraocularly transplanted, and then, the retinal morphology, outer nuclear layer (ONL) thickness and rhodopsin expression were studied as well as apoptosis and autophagy of the retinal cells. The hypoxia‐induced apoptosis of 661w cells obviously increased together with autophagy levels increasing and peaking at 8 hours after hypoxia. Upon coculturing with BMSCs, hypoxic 661w cells had a better morphology and fewer apoptosis. After autophagy was inhibited, the apoptotic 661w cells under the hypoxia increased, and the cell viability was reduced, even in the presence of transplanted BMSCs. In retina‐detached eyes transplanted with BMSCs, the retinal ONL thickness was closer to that of the normal retina. After transplantation, apoptosis decreased significantly and retinal autophagy was activated in the BMSC‐treated retinas. Increased autophagy in the early stage could facilitate the survival of 661w cells under hypoxic stress. Coculturing with BMSCs protects 661w cells from hypoxic damage, possibly due to autophagy activation. In retinal detachment models, BMSC transplantation can significantly reduce photoreceptor cell death and preserve retinal structure. The capacity of BMSCs to reduce retinal cell apoptosis and to initiate autophagy shortly after transplantation may facilitate the survival of retinal cells in the low‐oxygen and nutrition‐restricted milieu after 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.

A new multichannel method quantitating TUNEL in detached photoreceptor nuclei

Nuclear co-localization labels are critical to ocular research. Among these, the TUNEL assay has been established as a gold standard of cell death and apoptosis. While several validated computer-based methods exist to quantitate these markers, including ImageJ Retina Analysis (RA) Toolkit and ImagePro, none verify the count with the nuclear counter stain to confirm nuclear co-localization. We established a new ImageJ-based automated multichannel thresholding (MCT) method to quantitate nuclear co-localized labeling. The MCT method was validated by comparing it with the two published TUNEL analysis in TUNEL-positive photoreceptors in an experimental retinal detachment (RD) model. RDs were induced in murine eyes and cross-sectional images of TUNEL and DAPI counter stain were obtained. Images were classified as “typical” or high density ”hotspot” TUNEL regions (n = 10/group). Images were analyzed and compared between the MCT method and the published techniques including both ”standard” and ”high” settings of the RA Toolkit for detecting lower or higher TUNEL densities, respectively. Additional testing of the MCT method with built-in ImageJ thresholding algorithms was performed to produce fully automated measurements. All images were compared with Bland-Altman mean difference plots to assess the difference in counts and linear regression plots to assess correlation. Comparison between the MCT method and the ImagePro method were found to be well correlated (typical: R2 = 0.8972, hotspot: R2 = 0.9000) with minor to non-significant differences. The RA Toolkit settings were found to be mostly well correlated as well (standard/typical: R2 = 0.8036, standard/hotspot: R2 = 0.4309, high/typical: R2 = 0.7895, high/hotspot: R2 = 0.8738) but were often found to have significantly higher counts than the MCT. In conclusion, the MCT method compared favorably with validated computer-based methods of nuclear marker immunofluorescence quantitation and avoids staining artifacts through the incorporation of the nuclear counter stain to confirm positive cells.

Potential Role of Zinc Dyshomeostasis in Matrix Metalloproteinase-2 and -9 Activation and Photoreceptor Cell Death in Experimental Retinal Detachment.

We investigated whether zinc dyshomeostasis, a known mechanism of cell death in acute brain injury, contributes to the activation of matrix metalloproteinases (MMPs) and photoreceptor cell death in experimental retinal detachment (RD). RD was induced in mice by subretinal injection of 1:1 mixture of balanced salt solution and 1% sodium hyaluronate. On days 1 and 3 post RD, eyeballs were sectioned and examined for cell death (TUNEL staining), the degree of hypoxic insult (Hypoxyprobe staining), free zinc levels (TFL-Zn staining), and MMP-2 and -9 activity (gelatin zymography). In addition, we examined whether modulating extracellular zinc concentration or MMP activation in subretinal fluid affected photoreceptor cell death in RD. These changes were further examined in primary retinal cell and photoreceptor-derived cell (661W) cultures. Photoreceptor cell death peaked on day 3 post RD. Intracellular zinc markedly decreased on day 1 post RD, and subsequently accumulated on day 3. MMP-2 and -9 activity showed a concurrent increase in detached retinas. Detached retinas stained with Hypoxyprobe showed strongly positive cells, especially in the photoreceptor layer. Subretinal injection of a zinc-chelator (CaEDTA) or MMP inhibitor (GM6001, minocycline) at the time of RD significantly attenuated photoreceptor cell death in RD. Similar findings were confirmed in oxygen-glucose-deprived or zinc-exposed cell cultures. Upon RD, hypoxic retinal cells in deep layers underwent zinc dyshomeostasis, MMP activation, and ultimately death. These findings provide new insight into the possible mechanism of photoreceptor death in RD, and as such may prove useful in crafting protective measures for photoreceptor cells.

Inhibition of Mitochondrial Fission Preserves Photoreceptors after Retinal Detachment

Photoreceptor degeneration is a leading cause of visual impairment worldwide. Separation of neurosensory retina from the underlying retinal pigment epithelium is a prominent feature preceding photoreceptor degeneration in a variety of retinal diseases. Although ophthalmic surgical procedures have been well developed to restore retinal structures, postoperative patients usually experience progressive photoreceptor degeneration and irreversible vision loss that is incurable at present. Previous studies point to a critical role of mitochondria-mediated apoptotic pathway in photoreceptor degeneration, but the upstream triggers remain largely unexplored. In this study, we show that after experimental retinal detachment induction, photoreceptors activate dynamin-related protein 1 (Drp1)-dependent mitochondrial fission pathway and subsequent apoptotic cascades. Mechanistically, endogenous reactive oxygen species (ROS) are necessary for Drp1 activation invivo, and exogenous ROS insult is sufficient to activate Drp1-dependent mitochondrial fission in cultured photoreceptors. Accordingly, inhibition of Drp1 activity effectively preserves mitochondrial integrity and rescues photoreceptors. Collectively, our data delineate an ROS-Drp1-mitochondria axis that promotes photoreceptor degeneration in retinal diseased models.