Name: Labeling and Staining Rat Brain Pericytes for Fluorescent Imaging
Uploaded: 2023-08-18T15:00:00
Description: Watch this Scientific Journal Video about Labeling and Staining Rat Brain Pericytes for Fluorescent Imaging at JoVE.com

labeling and staining rat brain pericytes for fluorescent imaging

Subaraknoid Kanama Sonrası Beyin Perisit Kontraktilitesinin Görüntülenmesi

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.

Yazar Spotlight: Kemirgenlerde Subaraknoid Kanama Sonrası Perisitlerin Görüntülenmesi 

Subaraknoid Kanama Sonrası Beyin Dilimlerinde Vital ve Non-vital Beyin Perisitlerinin Görüntülenmesi

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 Labeling and Staining Rat Brain Pericytes for Fluorescent Imaging at JoVE.com

Labeling and Staining Rat Brain Pericytes for Fluorescent Imaging  

Floresan Görüntüleme için Sıçan Beyin Perisitlerinin Etiketlenmesi ve Boyanması

Perisitleri etiketlemek için, floresan boya TO-PRO-3'ü yapay beyin omurilik sıvısına veya ACSF'ye ekleyin. Şimdi taze dilimlenmiş sıçan beynini boya yüklü ACSF'de oda sıcaklığında karanlık bir ortamda 20 dakika inkübe edin. Ardından, naylon ağ süzgecini beyin dilimleriyle birlikte altı oyuklu bir plaka durulama odasına aktarın.Beyin dilimlerini haznede 10 dakika dinlendirin. Hayati olmayan perisitleri etiketlemek için, TO-PRO-3 etiketli beyin dilimlerini konjuge izolektin B4'te 37 santigrat derecede karanlıkta 30 dakika inkübe edin. İnkübasyondan sonra, beyin dilimlerini yapay beyin omurilik sıvısında 15 dakika durulayın.Ardından, beyin dilimlerini %5 karbondioksit ve %95 oksijen ile gazlanmış ACSF'ye yerleştirin. Buna, 37 santigrat derecede 37 mikromolar konsantrasyonda propidyum iyodür ekleyin. Boya alımını durdurmak ve arka plan etiketlemesini en aza indirmek için preparatları ACSF'de 15 dakika durulayın.Normal fizyolojik koşullar altında, beyin perisitleri hücre ölümü yaşamaz. Promidyum iyodür ile boyanmamış canlı perisitler tespit edildi. SAK modelleri, mikrovasküler sistem içinde hayati perisitlere sahip olmalıdır.Hayati olmayan perisitler de tespit edildi. Propidyum iyodür etiketli hayati olmayan perisitler tüm mikrovaskülatüre bağlı kaldı.

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