Name: Confocal Imaging of Vital and Non-Vital Pericytes in Rat Acute Brain Slices
Uploaded: 2023-08-18T15:00:00
Description: Watch this Scientific Journal Video about Confocal Imaging of Vital and Non-Vital Pericytes in Rat Acute Brain Slices at JoVE.com

confocal imaging of vital and non-vital pericytes in rat acute brain slices

Visualização da contratilidade do pericito cerebral pós-hemorragia subaracnóidea

Brain pericytes, distinguished by their slender protuberances and protruding cell bodies, encircle the microcirculation1,2. While cerebral blood flow augmentation is predominantly driven by capillary dilation, smaller arteries exhibit slower rates of dilation3. Pericyte contractility exerts influence over capillary diameter and pericyte morphology, impacting vascular dynamics4. Contraction of brain pericytes leads to capillary constriction, and in pathological scenarios, excessive contraction may impede erythrocyte flow5. Various factors, including norepinephrine released from the locus coeruleus, can induce brain pericyte contraction within capillaries6. With a regulatory role in cerebral blood flow, pericytes exhibit 20-HETE synthesis, serving as an oxygen sensor during hyperoxia7. Oxidative-nitrative stress-triggered contraction of brain pericytes detrimentally affects capillaries5. Despite both in vivo and ex vivo investigations into brain pericyte contraction8, limited knowledge persists regarding the imaging of viable and non-viable brain pericytes within brain slices.
Crucially, post-tissue fixation imaging of brain pericytes compromises their vitality and subsequent contractility assessment. Moreover, in scenarios such as neurological disorders (e.g., subarachnoid hemorrhage - SAH), transgenic labeling of brain pericytes fails to differentiate between viable and non-viable pericytes, as confirmed by our preliminary SAH-induced brain pericyte death study9. 
To surmount these challenges, we employed TO-PRO-3 to label live pericytes, while deceased ones were stained with propidium iodide (PI). We used high-resolution confocal imaging technologies to visualize viable and non-viable brain pericytes in brain slices while preserving slice activity during imaging. This article aims to present a reproducible method for imaging viable and non-viable brain pericytes in brain slices, serving as a valuable tool to probe the impact of brain pericytes on cerebral microcirculation post SAH.

Autor em destaque: Imagem de pericitos pós-subaracnóideos em roedores 

Imagem de Pericitos Cerebrais Vitais e Não Vitais em Fatias Cerebrais após Hemorragia Subaracnóidea

Our study showed a significant increase in pericytes deaths. So starting at a sick horse after SAH, it demonstrated a positive correlation between the number of low vital parasites and the time elapsed after SAH. Imaging vital and the non-vital brain parasites in brain slices following SAH and the parasite isolation techniques, have advanced the research in my field.Currently, it's quite difficult to fully distinguish between dead parasites and dead endothelial cells. Our study find contraction-dependent apoptosis in parasites after SAH. We have developed a very reliable protocol, which enables the fluorescent labeling, both functional and a non-functional brain parasite in brain sections, and the same time.

Watch this Scientific Journal Video about Confocal Imaging of Vital and Non-Vital Pericytes in Rat Acute Brain Slices at JoVE.com

Confocal Imaging of Vital and Non-Vital Pericytes in Rat Acute Brain Slices  

Imagem Confocal de Pericitos Vitais e Não Vitais em Cortes Cerebrais Agudos de Rato

Para começar, use uma pipeta plástica de Pasteur para transferir suavemente as fatias cerebrais coradas fluorescentemente para pratos confocais com fundo de vidro. Recheie estes pratos com ACSF equilibrado. Com uma malha de ferro, prenda as fatias cerebrais do rato no lugar.Em seguida, coloque as placas confocais de fundo de vidro no palco de um microscópio confocal. Posicione a fatia cerebral aguda na parte inferior do prato e perfunda com ACSF de equilíbrio durante a aquisição de imagens. Usando a objetiva 40X, visualize as células da parede da microvasculatura cortical cerebral.Em seguida, use um software compatível para capturar pilhas de imagens. Identificar microvasculatura e pericitos cerebrais com base em sua rede e morfologia, em seguida, localizar uma região específica contendo uma rede de microvasculatura cerebral em cada fatia cerebral e capturar imagens. Em condições fisiológicas normais, os pericitos cerebrais não experimentam morte celular.Pericitos viáveis que não foram corados com iodeto de propídio foram detectados. Os modelos de HAS mostraram pericitos vitais dentro da microvasculatura. Pericitos não vitais também foram detectados.Os pericitos não vitais marcados com iodeto de propídio permaneceram descolados de toda a microvasculatura.

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