Name: Induction of the Murine Subarachnoid Hemorrhage Model and Brain Slice Preparation for Fluorescent Imaging
Uploaded: 2023-08-18T15:00:00
Description: Watch this Scientific Journal Video about Induction of the Murine Subarachnoid Hemorrhage Model and Brain Slice Preparation for Fluorescent Imaging at JoVE.com

induction of the murine subarachnoid hemorrhage model and brain slice preparation 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 Induction of the Murine Subarachnoid Hemorrhage Model and Brain Slice Preparation for Fluorescent Imaging at JoVE.com

Induction of the Murine Subarachnoid Hemorrhage Model and Brain Slice Preparation for Fluorescent Imaging  

Murin Subaraknoid Kanama Modelinin İndüksiyonu ve Floresan Görüntüleme için Beyin Dilimi Hazırlanması

Subaraknoid kanama veya SAK modelini oluşturmak için, anestezi uygulanmış bir sıçanın suprasellar sarnıcına bir mikroenjeksiyon iğnesi yerleştirin. Bir şırınga pompası ile, sıçanın kuyruk arterinden alınan otolog kanı suprasellar sarnıca enjekte edin. Şimdi verilen koordinatlarda sağ lateral serebral ventriküle başka bir iğne implante edin.SAH modelleri için 0.2 mililitre sıçan kuyruğu arter kanı enjekte edin. SHAM grupları için aynı hacimde izotonik salin enjekte edin. Dekapitasyondan sonra, sıçanın tüm beynini çıkarın ve buz gibi yapay beyin omurilik sıvısına veya ACSF'ye batırın.Bir vibratom ile 200 mikrometrelik akut beyin dilimleri ve buz gibi dengelenmiş ACSF hazırlayın. Beyin dilimlerini, 35 ila 37 santigrat derecede bir saklama odasında dengelenmiş ACSF'ye daldırılmış naylon bir ağ üzerine yerleştirin.

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