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

मस्तिष्क पेरिसाइट सिकुड़न के बाद Subarachnoid रक्तस्राव के दृश्य

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.

लेखक स्पॉटलाइट: इमेजिंग पेरिसाइट्स पोस्ट-सबराचोनोइड कृन्तकों में रक्तस्राव 

इमेजिंग महत्वपूर्ण और गैर महत्वपूर्ण मस्तिष्क Pericytes Subarachnoid रक्तस्राव के बाद मस्तिष्क स्लाइस में

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  

फ्लोरोसेंट इमेजिंग के लिए murine Subarachnoid रक्तस्राव मॉडल और मस्तिष्क टुकड़ा तैयारी का प्रेरण

सबराचोनोइड रक्तस्राव, या एसएएच मॉडल बनाने के लिए, एक संवेदनाहारी चूहे के सुपरसेलर सिस्टर्न में एक माइक्रोइंजेक्शन सुई डालें। एक सिरिंज पंप के साथ, suprasellar कुंढ में चूहे की पूंछ धमनी से प्राप्त ऑटोलॉगस रक्त इंजेक्षन. अब दिए गए निर्देशांक पर सही पार्श्व सेरेब्रल वेंट्रिकल में एक और सुई प्रत्यारोपित करें।एसएएच मॉडल के लिए, चूहे की पूंछ धमनी रक्त के 0.2 मिलीलीटर इंजेक्ट करें। SHAM समूहों के लिए, आइसोटोनिक खारा की समान मात्रा इंजेक्ट करें। सिर काटने के बाद, चूहे के पूरे मस्तिष्क को निकालने और उन्हें बर्फ ठंड कृत्रिम मस्तिष्क रीढ़ की हड्डी के तरल पदार्थ या एसीएसएफ में जलमग्न.एक vibratome के साथ, 200 micrometers और बर्फ ठंड संतुलित ACSF के तीव्र मस्तिष्क स्लाइस तैयार. एक नायलॉन जाल पर मस्तिष्क स्लाइस रखें, 35 से 37 डिग्री सेल्सियस पर एक भंडारण कक्ष में संतुलित एसीएसएफ में डूबे हुए।

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