preparing cerebral endothelial tubes from mouse parenchymal arterioles

Preparing Cerebral Endothelial Tubes from Mouse Parenchymal Arterioles

Place the ventral brain side facing up in a chamber containing cold dissection solution to isolate parenchymal arterioles. To isolate parenchymal arterials, secure the isolated brain with steel pins and cold dissection solution in a Petri dish containing more than 50-centimeter deep charcoal-infused silicone polymer coating.
Cut a rectangle of brain tissue from both hemispheres with sharp and aligned dissection scissors around the MCA, while ensuring that the upper part of the tissue segment is past the branching point from the circle of Willis. Secure the brain tissue into the dish with the MCA facing upwards with steel pins. Carefully make a shallow incision near the pins, and remove the pia with small forceps, gently peeling from one end towards the other.
Carefully secure the isolated pia with parenchymal arterioles branched from MCA in the dish with the pins, and carefully dissect the parenchymal arterioles. Cut off any remaining distal branches and ensure the arteriole is clean with no tissue attached. Use this clean intact arterial for enzymatic digestion. Alternatively, cut each arterial into two pieces for enzymatic digestion to prepare endothelial tubes, if desired.
For partial digestion of arteriolar segments, place intact arteriolar segments into 1 milliliter of dissociation solution in a 10-milliliter glass tube containing papain, dithioerythritol, collagenase, and elastase. Incubate arteriolar segments at 34 degrees Celsius for 10 to 12 minutes.
To isolate the arteriolar endothelial tube, place the trituration pipette attached with the microsyringe injector in the dissociation solution in the chamber and position it close to one end of the digested vessel segment. Set a rate within the range of 1 to 3 nanoliters per second on the pump controller for gentle treat duration. While maintaining 100 times to 200 times magnification, withdraw the arterial segment into the pipette, and then inject to dissociate the adventitia and smooth muscle cells. Triturate the vessel segment until all smooth muscle cells are dissociated and only endothelial cells remain as an intact tube.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1.&#160;Solutions and drugs
<ol>
	<li>Physiological salt solution (PSS)
	<ol>
		<li>Prepare a minimum of 1 L of PSS using 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM N-2-hydroxyethylpiperazine-N&#39;-2-ethanesulfonic acid (HEPES), and 10 mM glucose.</li>
		<li>Prepare necessary solutions lacking CaCl2 (zero Ca2+ PSS) for dissection of cerebral arterioles and isolation of endothelial tube. 
		NOTE: Prepare all solutions in ultrapure deionized H2O, followed by filtration (0.22 &#181;m). Ensure that the final product contains a pH 7.4 with osmolality between 290 and 300 mOsm.</li>
	</ol>
	</li>
	<li>Bovine serum albumin (BSA, 10%)
	<ol>
		<li>Dissolve 1 g of the lyophilized BSA powder in a beaker of 10 mL of zero Ca2+ PSS. Cover the beaker and allow at least 2 h with slow stirring for the BSA to dissolve.</li>
		<li>Filter solution using a 10 mL syringe plus a 0.22 &#181;m filter and prepare 1 mL aliquots to be stored at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Dissection solution
	<ol>
		<li>Add 500 &#181;L of BSA (10%) to 49.5 mL of zero Ca2+ PSS for a total volume of 50 mL.</li>
		<li>Transfer the solution to a Petri dish for arteriolar dissections.</li>
	</ol>
	</li>
	<li>Dissociation solution
	<ol>
		<li>Add 5 &#181;L of CaCl2 solution (1 M) and 500 &#181;L of BSA (10%) to 49.5 mL of zero Ca2+ PSS for a total volume of 50 mL. Incubate the solution at room temperature for at least 1 h.</li>
	</ol>
	</li>
	<li>Enzymes
	<ol>
		<li>Prepare 1 mL of digestion solution with 100 &#181;g/mL papain, 170 &#181;g/mL dithioerythritol, 250 &#181;g/mL collagenase (Type H blend), and 40 &#181;g/mL elastase. 
		NOTE: Enzyme activities can vary, although successful protocols will likely require the following enzymatic activities: &#8805; 10 units/mg for papain, &#8805; 1 unit/mg for collagenase, and &#8805; 5 units/mg for elastase. 
		&#160;</li>
	</ol>
	</li>
</ol>
2.&#160;Isolation of parenchymal arterioles
NOTE: Arterioles arising from the middle cerebral arteries (MCAs) and embedded in brain parenchyma are chosen for this protocol. The arterioles are isolated as previously described11, with modification.
<ol>
	<li>Use steel pins (diameter: 0.2 mm, length: 11 to 12 mm) to secure the isolated brain in cold dissection solution in a Petri dish containing a charcoal-infused silicon polymer coating (depth &#8805; 50 cm).</li>
	<li>Using sharp and aligned dissection scissors, cut a rectangle of brain tissue (length: 5 mm, width: 3 mm) (Figure 1B) around the MCA while ensuring that the upper part of the tissue segment is past the branching point from the Circle of Willis. Cut another rectangle of brain tissue from the other hemisphere of the brain to access more arterioles if necessary.</li>
	<li>Secure the separated brain tissue segment into the dish with the MCA facing upwards (distal from the Circle of Willis) using steel pins (diameter: 0.1 mm, length: 13 to 14 mm). Carefully make a shallow incision near the pins to remove the pia with small forceps, gently peeling from one end towards the other (Figure 1C). Remove the pia from the other tissue segment to access more arterioles if needed. Carefully secure the isolated pia with parenchymal arterioles branched from MCA in the dish with the pins, and dissect the parenchymal arterioles (Figure 1D), ensuring that the arterioles are not damaged. 
	NOTE: If the arterioles are not easily pulled out with the pia from the parenchyma, use fine forceps and dig into the brain, starting at the MCA branch point from the Circle of Willis. Locate the arteriole, carefully loosen the tissue around arterioles (Figure 1E), and gently pull the arteriole out of the parenchyma, holding the upper end of the arteriole (Figure 1F). Multiple arterioles (length: ~1.5-2 mm) can be isolated from one rectangle of brain tissue.</li>
	<li>Ensure the arteriole is clean with no tissue attached to it, and cut off any remaining distal branches (Figure 2A). Use this clean, intact arteriole for enzymatic digestion. Alternatively, cut each arteriole into two pieces (length: 0.75-1 mm) for enzymatic digestion for the preparation of endothelial tubes if desired. 
	&#160;</li>
</ol>
3.&#160;Digestion of parenchymal arterioles and preparation of endothelial tubes
<ol>
	<li>Arrangement of equipment and pipettes 
	NOTE: Isolation of arterial endothelial tubes from the brain has been described previously13,18. The current protocol incorporates modifications for the isolation of endothelium from parenchymal (intracerebral) arterioles. Multiple arterioles or/and pieces of arterioles can be used together for enzymatic digestion.
	<ol>
		<li>Assemble the trituration apparatus13 for preparing endothelial tubes using an aluminum stage supporting a chamber and micromanipulators. Assemble a microscope equipped with objectives (5x-60x) and a camera connected to a computer monitor. Secure a microsyringe with a pump controller next to the stage and specimen.</li>
		<li>Secure a trituration pipette backfilled with mineral oil over the microsyringe piston. Use the microsyringe with the pump controller to withdraw the dissociation solution into the trituration pipette (~130 nL) on top of the mineral oil while taking care to avoid air bubbles in the pipette.</li>
	</ol>
	</li>
	<li>Partial digestion of arteriolar segments
	<ol>
		<li>Place intact arteriolar segments into 1 mL of dissociation solution in a 10 mL glass tube containing 100 &#181;g/mL papain, 170 &#181;g/mL dithioerythritol, 250 &#181;g/mL collagenase, and 40 &#181;g/mL elastase. Incubate arteriolar segments at 34 &#176;C for 10-12 min.</li>
		<li>After digestion, replace the enzyme solution with 3-4 mL of fresh dissociation solution at room temperature.</li>
	</ol>
	</li>
	<li>Isolation of arteriolar endothelial tube 
	&#8203;NOTE: Following digestion and replacement of the enzyme solution, transfer one or multiple partially digested segments into the dissociation solution in a superfusion chamber attached to a mobile platform. While viewing at 100x to 200x magnification, select one partially digested but unbroken vessel segment and focus on it under the microscope.
	<ol>
		<li>Place the trituration pipette attached to the microsyringe injector in the dissociation solution in the chamber and position it close to one end of the digested vessel segment. Set a rate within the range of 1-3 nL/s on the pump controller for gentle trituration.</li>
		<li>While maintaining 100x to 200x magnification, withdraw the arteriolar segment into the pipette and then eject to dissociate the adventitia and smooth muscle cells (Figure 1B). Triturate the vessel segment until all smooth muscle cells are dissociated, and only endothelial cells remain as an intact &#34;tube.&#34; 
		NOTE: If necessary during trituration, carefully use fine-tipped forceps to remove the dissociated adventitia and internal elastic lamina from the endothelial tube. Typically, only a few cycles of trituration yield an intact endothelial tube.</li>
		<li>Use micromanipulators to secure each end of the endothelial tube on the glass coverslip in the chamber with borosilicate glass pinning pipettes (Figure 2C,D).</li>
	</ol>
	</li>
	<li>Securing the arteriolar endothelial tube
	<ol>
		<li>Replace the dissociation solution with superfusion solution (PSS containing 2 mM CaCl2) while ensuring that nonendothelial material is washed out of the chamber.</li>
		<li>Transfer the secured endothelial tube in the mobile platform to the microscope rig designed for continuous superfusion and intracellular or fluorescent recordings/imaging.</li>
		<li>Apply continuous delivery of PSS during the experiment. Manually set the flow rate (5-7 mL/min) throughout the experiment using the inline flow control valve, consistent with the laminar flow, while matching the feed of solution flow to the extent of vacuum suction. Allow &#8805;5 min of PSS flow to the chamber for superfusion of the endothelial tube before acquiring background data and/or dye loading.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bath Chiller (Isotemp 500LCU)</td>
			<td>ThermoFisher Scientific</td>
			<td>13874647</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries (Pinning)</td>
			<td>Warner Instruments</td>
			<td>G150T-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries (Sharp Electrodes)</td>
			<td>Warner Instruments</td>
			<td>GC100F-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries (Trituration)</td>
			<td>World Precision Instruments (WPI), Sarasota, FL, USA</td>
			<td>1B100-4</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA: Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2: Calcium Chloride</td>
			<td>Sigma</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase (Type H Blend)</td>
			<td>Sigma</td>
			<td>C8051</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquision Digitizer</td>
			<td>Molecular Devices, Sunnyvale, CA, USA</td>
			<td>Digidata 1550A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Dish (Glass Petri with Charcoal Sylgard bottom)</td>
			<td>Living Systems Instrumentation, St. Albans City, VT, USA</td>
			<td>DD-90-S-BLK</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithioerythritol</td>
			<td>Sigma</td>
			<td>D8255</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO: Dimethyl Sulfoxide</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Elastase (porcine pancreas)</td>
			<td>Sigma</td>
			<td>E7885</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Control Valve</td>
			<td>Warner Instruments</td>
			<td>FR-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence system interface, ARC lamp &#38; power supply, hyperswitch and PMT</td>
			<td>Molecular Devices, Sunnyvale, CA, USA</td>
			<td>IonOptix Systems</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps (Fine-tipped, sharpened)</td>
			<td>FST</td>
			<td>Dumont #5 &#38; Dumont #55</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich (St. Louis, MO, USA)</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl: Potassium Chloride</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2: Magnesium Chloride</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Siskiyou</td>
			<td>MX10</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller (digital)</td>
			<td>Sutter Instruments, Novato, CA, USA</td>
			<td>P-97 or P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope (Nikon-inverted)</td>
			<td>Nikon Instruments Inc, Melville, NY, USA</td>
			<td>Ts2</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope (Nikon-inverted)</td>
			<td>Nikon Instruments Inc</td>
			<td>Eclipse TS100</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Stage (Aluminum)</td>
			<td>Siskiyou, Grants Pass, OR, USA</td>
			<td>8090P</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsyringe Pump Controller</td>
			<td>World Precision Instruments (WPI), Sarasota, FL, USA</td>
			<td>SYS-MICRO4</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl: Sodium Chloride</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Sigma</td>
			<td>P4762</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase contrast objectives</td>
			<td>Nikon Instruments Inc</td>
			<td>(Ph1 DL; 10X &#38; 20X)</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors (3 mm &#38; 7 mm blades)</td>
			<td>Fine Science Tools (or FST), Foster City, CA, USA</td>
			<td>Moria MC52 &#38; 15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors (Vannas style; 9.5 mm &#38; 3 mm blades)</td>
			<td>World Precision Instruments</td>
			<td>555640S, 14364</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature Controller (Dual Channel)</td>
			<td>Warner Instruments</td>
			<td>TC-344B or C</td>
			<td></td>
		</tr>
		<tr>
			<td>Valve Control System</td>
			<td>Warner Instruments</td>
			<td>VC-6</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Hakim, M. A., et al., <a href="https://app.jove.com/v/63463">Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma.</a> J. Vis. Exp. (2022)
This video demonstrates a method for isolating the endothelial tubes from mouse cerebral parenchyma. Through a combination of enzymatic digestion and mechanical dissociation, the process yields an intact tube with endothelial cells.

<img alt="Figure 1" src="/files/ftp_upload/22597/22597_Figure1.jpg" /> 
Figure 1: Dissection and isolation of parenchymal arterioles. (A) Isolated mouse brain with intact Circle of Willis. (B) A dissected rectangular piece of brain tissue containing the MCA (white arrow). (C) Isolation of pia from the upper part of the tissue (arteriole, blue arrow). (D) Arterioles (outlined with blue arrowheads) connected to middle cerebral arteries (yellow arrows). (E) Identified a...

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1.&#160;Solutions and drugs

	...

Secure a mouse brain, ventral side up, in a silicon polymer coated-dish containing buffer.
Dissect a tissue section from the hemisphere around the middle cerebral artery, or MCA, with the upper tissue segment extending beyond the branching point from the Circle of Willis.
Secure the tissue with the MCA facing up.
Remove the pia mater, with the arterioles, which branch from the MCA.
Secure the pia mater with the arterioles. Dissect the arteriole and remove the surrounding tissue. Discard any distal branches.
Incubate the arteriole in a proteolytic enzyme cocktail to facilitate partial digestion of the segment.&#160;
Replace the enzymes with buffer and transfer the digested segment to a chamber containing buffer on a microscope stage equipped with a trituration pipette.
Pipette the arteriole repeatedly to remove the loosened adventitia, and smooth muscle cells, resulting in an intact tube containing arteriolar endothelial cells.

10 visualizações. Preparando tubos endoteliais cerebrais a partir de arteríolas parenquimatosas de camundongos

Vídeo: Preparando tubos endoteliais cerebrais a partir de arteríolas parenquimatosas de camundongos - Experimento

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.

Isolamento e Canulação da cerebral parenquimatosa Arteríolas

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

JoVE Journal

Neurociência

Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and cardiac arrest are remarkably sexually dimorphic diseases, and both induce cerebral ischemia. However, progress in understanding the vascular cognitive impairment, and then developing sex-specific treatments, has been partly limited by challenges in investigating the brain microcirculation from mouse models in functional studies. Here, we present an approach to examine the capillary-to-arteriole signaling in an ex vivo hippocampal capillary-parenchymal arteriole (HiCaPA) preparation from mouse brain. We describe how to isolate, cannulate, and pressurize the microcirculation to measure arteriolar diameter in response to capillary stimulation. We show which appropriate functional controls can be used to validate the HiCaPA preparation integrity and display typical results, including testing potassium as a neurovascular coupling agent and the effect of the recently characterized inhibitor of the Kir2 inward rectifying potassium channel family, ML133. Further, we compare the responses in preparations obtained from male and female mice. While these data reflect functional investigations, our approach can also be used in molecular biology, immunochemistry, and electrophysiology studies.

The present manuscript details how to isolate hippocampal arterioles and capillaries from the mouse brain and how to pressurize them for pressure myography, immunofluorescence, biochemistry, and molecular studies.

Ex Vivo pressurizado Hipocampal Capilar-Parenchymal Arteriole Preparação para estudo funcional

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and ...

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies.

Isolamento de arteríolas retina para estudos de fisiologia celular Ex Vivo

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

Preparing Cerebral Endothelial Tubes from Mouse Parenchymal Arterioles

Transcrição

Preparing Cerebral Endothelial Tubes from Mouse Parenchymal Arterioles