Bloqueio do acoplamento celular após trauma mecânico na retina altera a distribuição de células em apoptose.

Abstract

Considering the recent advances in drug development and delivery, neuroprotection stands out as one of the most pursued hot topics in applied neurosciences. Accruing evidence indicates that connexin (Cx) channels in the gap junctions (GJ) are involved in neurodegeneration after injury. However, studies using KO animal models endowed apparently contradictory results in relation to the role of coupling in neuroprotection. We choose analyze the role of Cx-mediated communication in focal lesion induced by mechanical trauma in the retina, a model that allows spatial and temporal definition of the lesion with high reproducibility, permitting visualization of the focus, penumbra and adjacent areas, and generate a concentration gradient of cells in apoptosis from the focus to adjacent regions nearby the injury. Our results showed a distinct regulation of gene expression and protein levels for Cx36 and Cx43 throughout the neurodegeneration progress. The Cx36 distribution pattern did not change during the lesion progression, but the Cx43 showed disorganized pattern and upregulaton after 7 days. At the same period, glial fibrilar protein (GFAP) was upregulated in the lesion focus exposing a reactive gliosis after 7 days. Cx36 was observed close to apoptotic nuclei, revealing the presence of this protein surrounding apoptotic cells. Our results revealed that amacrine cells are coupled with health neighborhood cells by Cx36. The functional role of cell coupling was assessed employing GJ blockers and openers combined with lactate dehydrogenase (LDH) assay, a direct method for evaluating cell death/viability. Carbenoxolone (CBX), a broad-spectrum GJ blocker, reduced LDH release after 4 hours, whereas quinine, a Cx36-channel specific blocker, decreased LDH release as early as 1 hour. Furthermore, analysis of dying cell distribution confirmed that the use of GJ blockers reduced apoptosis spread. Accordingly, blockade of GJ communication during neurodegeneration with quinine, but not CBX, caused downregulation of initial and effector caspases. To summarize, we observed specific changes in Cx gene expression and protein distribution during the progress of retinal degeneration, indicating the participation of these elements in acute neurodegeneration processes. More importantly, our results revealed that direct control of GJ channels permeability may take part in reliable neuroprotection strategies aimed to rapid, fast treatment of mechanical trauma.Keywords: Retina. Connexins. Apoptosis. Caspases. GJ Blockers. Neurodegeneration. LISTA DE ABREVIATURAS E SIGLAS SN -sistema nervoso MCP -morte celular programada ROS -espécies reativas de oxigênio JC -junção comunicante Cx -conexina SNC -sistema nervoso central KO -knockout RPE -epitélio pigmentado retiniano PL -camada de fotorreceptores ONL -camada nuclear externa OPL -camada plexiforme externa INL -camada nuclear interna IPL -camada plexiforme interna GCL -camada de células ganglionares NFL -camada de axônios retinianos PFA -paraformaldeído PB -tampão fosfato BS -solução sali...