Dataset Information

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

ABSTRACT:

Purpose

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.

Methods

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.

Results

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.

Conclusions

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.

SUBMITTER: Cao M

PROVIDER: S-EPMC10292671 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Publications

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

Cao Manjing M Huang Xinting X Zou Jingling J Peng Yingqian Y Wang Yanbing Y Zheng Xichen X Tang Luosheng L Zhang Lusi L

Investigative ophthalmology & visual science 20230601 7

<h4>Purpose</h4>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.<h4>Methods</h4>An RD model was conducted by inje ...[more]

PMID: 37351878

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Project description: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.

Project description:PurposePhotoreceptor (PR) death is the ultimate cause of irreversible vision loss in retinal detachment (RD). Previous studies have shown that microglia may have a dual role in RD. Nevertheless, the potential protective effects of microglia on PR are largely unknown. We aimed to uncover the phagocytic role of microglia in RD and propose a new concept to regulate PR survival.MethodsAn RD model was conducted by injecting sodium hyaluronate into the subretinal space (SRS) of C57BL/6J wild type mice. Bioinformatics analysis was used to evaluate the highly enriched pathways and terms relating to phagocytosis in human datasets and mouse transcriptomes of RD. The observation of microglial morphology was performed by immunofluorescence through cryosection and flat mount. PLX 3397 was used for microglial ablation. Phagocytosis of the outer segment (OS) by microglia was confirmed by immunofluorescence and hematoxylin and eosin staining. Expression of phagocytic markers in microglia was detected by immunofluorescence of cryosection. The PR survival was measured by TUNEL assay and hematoxylin and eosin staining. The optical coherence tomography (OCT) images through the center of the fovea in twelve patients were obtained to observe the clinic features of IS/OS dynamics after RD.ResultsThe results showed that OS went through an accumulation-clearance process after RD. Ameboid microglia accumulated in the SRS and engulfed OS. Upregulation of phagocytic markers was observed in subretinal microglia. Depletion of microglia led to failure of OS clearance and retinal ruffling, which had the same characteristics as outer retinal undulation (ORU) in some patients with RD. PR did not benefit from microglial depletion, as no morphology and thickness recovery of PR was observed in the long term.ConclusionsThese results elucidate that microglial phagocytosis of OS is a critical process after RD. Insufficient phagocytosis leads to the accumulation of OS in the SRS and PR abnormalities. Appropriate regulation of microglial phagocytosis to remove OS may be a new concept to regulate photoreceptor survival.

Dataset Information

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

Purpose

Methods

Results

Conclusions

Publications

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

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