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

Visualisering av hjärnans pericytkontraktilitet efter subaraknoidalblödning

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.

Författare i fokus: Avbildning av pericyter efter subaraknoidalblödning hos gnagare 

Avbildning av vitala och icke-vitala hjärnpericyter i hjärnskivor efter subaraknoidalblödning

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  

Konfokal avbildning av vitala och icke-vitala pericyter i akuta hjärnskivor hos råtta

Till att börja med, använd en Pasteur-pipett av plast för att försiktigt överföra de fluorescerande färgade hjärnskivorna till konfokala skålar med glasbotten. Fyll dessa skålar med balanserad ACSF. Fäst råtthjärnskivorna på plats med ett järnnät.Placera sedan konfokalskålarna med glasbotten på scenen i ett konfokalmikroskop. Placera den akuta hjärnskivan i botten av skålen och perfusion med jämvikts-ACSF under avbildningen. Använd 40X-objektivet för att se väggcellerna i hjärnbarkens mikrokärl.Använd sedan kompatibel programvara för att ta bildstaplar. Identifiera cerebrala mikrovaskulaturer och pericyter baserat på deras nätverk och morfologi, lokalisera sedan en specifik region som innehåller ett cerebralt mikrovaskulaturnätverk på varje hjärnskiva och ta bilder. Under normala fysiologiska förhållanden upplever hjärnpericyter inte celldöd.Livsdugliga pericyter som inte var färgade med propidiumjodid detekterades. SAH-modellerna visade vitala pericyter i mikrovaskulaturen. Icke-vitala pericyter upptäcktes också.Procidiumjodid märkt icke-vitala pericyter"förblev avskild till hela mikrokärlen.

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