Name: 视频: 从猪脑中纯化毛细血管 - 概念
Uploaded: 2024-08-30T13:24:07.000+00:00
Duration: 1 min 18 s
Description: 22 次观看. 来源:Nielsen， S. S. E.， et al. 建立基于猪脑内皮细胞的体外血脑屏障模型的改进方法。J. Vis. Exp. （2017 年）。 该视频演示了从新鲜猪脑中分离毛细血管的过程。该过程包括去除脑膜，从脑组织中分离灰质，均质化和过滤灰质以分离毛细血管，最后使用酶消化进一步净化毛细血管。

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Purification of Porcine Brain Capillaries
<ol>
	<li>Collect 8-10 brains from 5-6-month-old domestic pigs (e.g. from a nearby slaughterhouse) and transport them on ice to the laboratory. We recommend starting the following purification procedure within 2-3 h of termination of the animal.</li>
	<li>Place the brains in a sterile flow bench and gently wash them with 1 L PBS in a beaker placed on ice.</li>
	<li>Carefully remove meninges from one brain at a time using fine-tip forceps and transfer the meninges-free brains to another 1 L beaker with PBS placed on ice. Extending the time taken can complicate the removal. The approximate time used for each brain should be 10-15 min.</li>
	<li>Using a scalpel, scrape off grey matter from one brain at a time and transfer the isolated material to Petri dishes (8.8 cm2) containing 20 mL DMEM Nutrient Mixture F-12 (DMEM/F-12) placed on ice. Isolate as much grey matter as possible without withdrawing white matter material. For initial fragmentation, run the grey matter material through a 50-mL syringe without a needle. Continue for all the brains and pool the collected grey matter material. Extending the time taken can complicate the removal. The approximate time used for each brain should be 10-15 min.</li>
	<li>Transfer the isolated grey matter material to the grinder tube of a hand-held tissue homogenizer in a ratio of 50/50 with DMEM/F-12 media. Homogenize the material by making 8 up and down strokes with a loose pestle, followed by 8 up and down strokes with a tight pestle. Continue until all isolated material has been homogenized, then transfer the homogenate to a 500-mL bottle and dilute with DMEM/F-12 to approximately 450 mL total volume.</li>
	<li>Filter the homogenate using a 500-mL blue-cap bottle with filter holder and 140 &#181;m filter-mesh, and isolate capillaries by running the tissue through the filter. Use one filter per 50 mL of homogenate and wash each filter with DMEM/F-12 afterwards.</li>
	<li>Place the capillary-containing filters in Petri dishes with a digestion solution of trypsin/EDTA (2.5 % trypsin, 0.1 nM EDTA in PBS), collagenase CLS2 (2,000 U/mL) and DNase 1 (3,400 U/mL) in DMEM/F-12. Use 1 Petri dish (8.8 cm2, 20 mL solution) per 3 filter meshes.</li>
	<li>Place the Petri dishes at 37 &#176;C for 1 h on an orbital shaker at 180 rpm or stir them gently every 10 min. After 1 h, wash off the capillaries from the filters with suspension from the Petri dish using a 1 mL pipette.</li>
	<li>Split the suspension from the 3 dishes into 2 50-mL tubes and stop the digestion by adding 10 mL DMEM/F-12 to each 50-mL tube.</li>
	<li>Centrifuge the cell-suspensions at 250 x g, 4 &#176;C for 5 min. Aspirate the supernatants and re-suspend each pellet in 10 mL of DMEM/F-12. Add a further 20 mL of DMEM/F12 to each tube. Repeat this centrifuge step twice.</li>
	<li>Let the tubes cool down on ice for 5 min and transfer the solutions to 2 new 50 mL tubes.</li>
	<li>Centrifuge the cell-suspensions at 250 x g, 4 &#176;C for 5 minutes, and re-suspend each pellet in 8 - 10 mL (approximately 1 mL/brain used) of freezing solution consisting of 10% DMSO in FBS.</li>
	<li>Transfer the cell suspension to cryovials, using 1 mL per cryovial. On average, purification results in 1 vial per brain used, on average providing endothelial cells for 12-16 inserts. Place the vials in a freezing box at -80 &#176;C for at least 4 h up to overnight. Afterwards, store the cryovials in a cryotank with liquid nitrogen. 
	Caution: Liquid nitrogen has extremely low temperatures. Please wear appropriate protection.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM/F-12&#160;</td>
			<td>Lonza&#160;</td>
			<td>BE12-719F</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)&#160;</td>
			<td>Gibco Invitrogen&#160;</td>
			<td>10-270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Gibco Invitrogen</td>
			<td>15090-046</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>34896</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse 1</td>
			<td>Sigma-Aldrich</td>
			<td>D4513</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase CLS2</td>
			<td>Sigma-Aldrich</td>
			<td>C6885</td>
			<td></td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td></td>
			<td></td>
			<td>Made with Elga System</td>
		</tr>
		<tr>
			<td>15 ml centrifuge tubes</td>
			<td>Cellstar</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml centrifuge tubes</td>
			<td>Cellstar</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Thermo Scientific</td>
			<td>150350</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryo vials</td>
			<td>Thermo Scientific</td>
			<td>377224</td>
			<td></td>
		</tr>
		<tr>
			<td>500 ml bottle</td>
			<td>Thermo Scientific</td>
			<td>159910/159920</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpels</td>
			<td>Swann-Morten</td>
			<td>REF0211</td>
			<td>Type 24</td>
		</tr>
		<tr>
			<td>Tissue homogenizer</td>
			<td>Sigma</td>
			<td>D9188</td>
			<td></td>
		</tr>
		<tr>
			<td>140 &#956;m filters</td>
			<td>MERCK</td>
			<td>NY4H04700</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine-tip curved forceps&#160;</td>
			<td>KLS Martin&#160;</td>
			<td>12-409-12-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Broad tip forceps</td>
			<td>VWR</td>
			<td>82027-390</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter holder</td>
			<td>MERCK Milipore</td>
			<td>Swinnex-47</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml syringe</td>
			<td>Braun</td>
			<td>4617509F</td>
			<td></td>
		</tr>
	</tbody>
</table>

purifying capillaries from a porcine brain

Source: Nielsen, S. S. E., et al. <a href="https://app.jove.com/v/56277">Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells.</a> J. Vis. Exp. (2017).
This video demonstrates the isolation of capillaries from fresh porcine brains. The process involves removing the meninges, isolating the grey matter from the brain tissue, homogenizing and filtering the grey matter to isolate the capillaries, and finally using enzymatic digestion to purify the capillaries further.

Purifying Capillaries From a Porcine Brain

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Purification of Porcine Brain ...

Take a porcine brain.
Remove the meninges, the protective membrane covering the brain, to prevent meningeal cell contamination.
Scrape the grey matter, a region rich in capillaries, and collect the tissue in a culture dish containing media.
Pass the grey matter through a syringe without a needle to fragment the tissue.
Transfer the fragments to a homogenizer and homogenize to disintegrate the tissue further, releasing the brain capillaries.
Pass the homogenate through a filter with a specific pore size to retain the capillaries, allowing other brain cells to flow through.
Place the filter in a solution containing enzymes and DNase.
The enzymes detach the adherent cells, while DNase degrades contaminating DNA, facilitating the isolation of pure capillaries.
Wash the capillaries from the filter.
Add media containing serum proteins to inactivate the enzymes.&#160;
Transfer the capillaries to a tube, centrifuge, and remove the supernatant. Resuspend the pure capillaries in fresh media for further analysis.

purifying-capillaries-from-a-porcine-brain

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

22 次观看. 来源:Nielsen， S. S. E.， et al. 建立基于猪脑内皮细胞的体外血脑屏障模型的改进方法。J. Vis. Exp. （2017 年）。 该视频演示了从新鲜猪脑中分离毛细血管的过程。该过程包括去除脑膜，从脑组织中分离灰质，均质化和过滤灰质以分离毛细血管，最后使用酶消化进一步净化毛细血管。 

视频: 从猪脑中纯化毛细血管 - 概念

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves &#8212; innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

分离及脑实质小动脉插管

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

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神经科学

Protocol for Isolating the Mouse Circle of Willis

The cerebral arterial circle (circulus arteriosus cerebri) or circle of Willis (CoW) is a circulatory anastomosis surrounding the optic chiasma and hypothalamus that supplies blood to the brain and surrounding structures. It has been implicated in several cerebrovascular disorders, including cerebral amyloid angiopathy (CAA)-associated vasculopathies, intracranial atherosclerosis and intracranial aneurysms. Studies of the molecular mechanisms underlying these diseases for the identification of novel drug targets for their prevention require animal models. Some of these models may be transgenic, whereas others will involve isolation of the cerebro-vasculature, including the CoW.The method described here is suitable for CoW isolation in any mouse lineage and has considerable potential for screening (expression of genes, protein production, posttranslational protein modifications, secretome analysis, etc.) studies on the large vessels of the mouse cerebro-vasculature. It can also be used for ex vivo studies, by adapting the organ bath system developed for isolated mouse olfactory arteries.

We describe here a reproducible protocol for isolating the mouse circle of Willis.

协议隔离威利斯的鼠标圈

The cerebral arterial circle (circulus arteriosus cerebri) or circle of Willis (CoW) is a circulatory anastomosis surrounding the optic chiasma and ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

新鲜人脑组织中毛细血管的分离

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Purifying Capillaries From a Porcine Brain

成績單

Purifying Capillaries From a Porcine Brain