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

Visualization of Brain pericyte Contractility Post-Subarachnoid Hemorrhage(뇌주막막막 출혈 후 뇌주막구 수축률의 시각화)

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.

저자 스포트라이트: 설치류의 지주막막 후 출혈 pericytes 이미징 

지주막하 출혈 후 뇌 절편에서 Vital and Non-vital Brain Pericytes 이미징

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  

Labeling and Staining Rat Brain Pericytes for Fluorescent Imaging(형광 이미징을 위한 쥐 뇌 주위 세포 라벨링 및 염색)

주위 세포를 표지하려면 인공 뇌척수액 또는 ACSF에 형광 염료 TO-PRO-3을 첨가합니다. 이제 갓 썰어낸 쥐의 뇌를 염료가 듬뿍 들어간 ACSF에 넣어 실온의 어두운 환경에서 20분 동안 배양합니다. 다음으로, 브레인 슬라이스가 있는 나일론 메쉬 스트레이너를 6웰 플레이트 헹굼 챔버로 옮깁니다.뇌 절편을 챔버에서 10분 동안 그대로 두십시오. 중요하지 않은 pericyte에 라벨을 붙이려면 TO-PRO-3 태그가 부착된 뇌 절편을 섭씨 37도에서 어두운 곳에서 30분 동안 접합 이솔렉틴 B4에 배양합니다. 배양 후 뇌 절편을 인공 뇌척수액으로 15분 동안 헹굽니다.다음으로, 5%의 이산화탄소와 95%의 산소로 가스를 주입한 ACSF에 뇌 절편을 넣습니다. 여기에 섭씨 37도에서 37마이크로몰 농도의 요오드화 프로피듐을 첨가합니다. 염료 흡수를 중단하고 배경 라벨링을 최소화하려면 ACSF에서 15분 동안 준비제를 헹굽니다.정상적인 생리적 조건에서 뇌 주위세포는 세포 사멸을 경험하지 않습니다. 프로피듐 요오드화물로 염색되지 않은 생존 가능한 주위세포가 검출되었습니다. SAH 모델은 미세혈관 내에서 중요한 주위 세포여야 합니다.비필수 주위세포(non-vital pericyte)도 검출되었습니다. 프로피듐 요오드화물(Propidium iodide)로 표지된 비생명 주위 세포(non-vital pericyte)는 전체 미세혈관에 부착된 상태로 남아 있었습니다.

Research

JoVE Journal

Neuroscience

Labeling and Staining Rat Brain Pericytes for Fluorescent Imaging

Imaging Vital and Non-vital Brain Pericytes in Brain Slices following Subarachnoid Hemorrhage

Pericytes are crucial mural cells situated within cerebral microcirculation, pivotal in actively modulating cerebral blood flow via contractility ...

연구

신경과학

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

형광 이미징을 위한 Murine Subarachnoid Hemorrhage Model 및 Brain Slice Preparation 유도

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

쥐 급성 뇌 절편에서 중요한 세포와 비생명 주위 세포의 공초점 이미징