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

Visualización de la contractilidad del pericito cerebral post-hemorragia subaracnoidea

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.

Autor destacado: Imágenes de pericitos post-hemorragia subaracnoidea en roedores 

Obtención de imágenes de pericitos cerebrales vitales y no vitales en cortes de cerebro después de una hemorragia subaracnoidea

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  

Inducción del modelo de hemorragia subaracnoidea murina y preparación de cortes de cerebro para imágenes fluorescentes

Para crear el modelo de hemorragia subaracnoidea, o HSA, se inserta una aguja de microinyección en la cisterna supraselar de una rata anestesiada. Con una bomba de jeringa, inyecte sangre autóloga adquirida de la arteria de la cola de la rata en la cisterna supraselar. Ahora implante otra aguja en el ventrículo cerebral lateral derecho en las coordenadas dadas.Para los modelos de HSA, inyecte 0,2 mililitros de sangre de la arteria de la cola de rata. Para los grupos SHAM, inyectar el mismo volumen de solución salina isotónica. Después de la decapitación, extrae todo el cerebro de la rata y sumérgela en líquido cefalorraquídeo artificial helado o ACSF.Con un vibrátomo, prepare cortes agudos de cerebro de 200 micrómetros y ACSF equilibrado helado. Coloque las rodajas de cerebro en una malla de nailon, sumergidas en ACSF equilibrado en una cámara de almacenamiento a 35 a 37 grados centígrados.

Research

JoVE Journal

Neuroscience

Induction of the Murine Subarachnoid Hemorrhage Model and Brain Slice Preparation 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 ...

Investigación

Neurociencias

Labeling and Staining Rat Brain Pericytes for Fluorescent Imaging

Marcaje y tinción de pericitos cerebrales de rata para imágenes fluorescentes

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

Imágenes confocales de pericitos vitales y no vitales en cortes cerebrales agudos de rata