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

Visualizzazione della contrattilità del pericito cerebrale dopo l'emorragia 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.

Autore in primo piano: Imaging dei periciti post-emorragia subaracnoidea nei roditori 

Imaging di periciti cerebrali vitali e non vitali in fette di cervello dopo emorragia 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  

Induzione del modello murino di emorragia subaracnoidea e preparazione della fetta di cervello per l'imaging fluorescente

Per creare l'emorragia subaracnoidea, o modello SAH, inserire un ago per microiniezione nella cisterna soprasellare di un ratto anestetizzato. Con una pompa a siringa, iniettare sangue autologo prelevato dall'arteria caudale del ratto nella cisterna soprasellare. Ora impiantare un altro ago nel ventricolo cerebrale laterale destro alle coordinate date.Per i modelli SAH, iniettare 0,2 millilitri di sangue dell'arteria caudale di ratto. Per i gruppi SHAM, iniettare lo stesso volume di soluzione fisiologica isotonica. Dopo la decapitazione, estrai l'intero cervello del ratto e immergilo in un liquido cerebrospinale artificiale ghiacciato o ACSF.Con un vibratomo, preparare fette di cervello acuto di 200 micrometri e ACSF bilanciato ghiacciato. Disporre le fette di cervello su una rete di nylon, immerse in ACSF equilibrato in una camera di conservazione a 35-37 gradi Celsius.

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 ...

Ricerca

Neuroscienze

Labeling and Staining Rat Brain Pericytes for Fluorescent Imaging

Etichettatura e colorazione dei periciti cerebrali di ratto per l'imaging fluorescente

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

Imaging confocale di periciti vitali e non vitali in fette cerebrali acute di ratto