Name: isolation and cannulation of cerebral parenchymal arterioles
Uploaded: 2016-05-23T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Cannulation of Cerebral Parenchymal Arterioles at JoVE.com

Funded by NHLBI R01HL091905 (SE), the United Leukodystrophy Foundation CADASIL research grant (FD) and AHA 15POST247200 (PWP). The authors would like to thank Samantha P. Ahchay for providing the image on Figure 1, and Dr. Gerry Herrera, Ph.D., for providing critical comments on the manuscript.

1. Cannula and Chamber Preparation
<ol>
	<li>Insert clean borosilicate glass capillaries (outer diameter: 1.2 mm; internal diameter: 0.69 mm; 10 mm in length) into the grooves of a pipette puller with a platinum filament (diameter: 100 &#181;m).</li>
	<li>Using appropriate settings, pull the capillary to generate a cannula with a long and thin tip (Figure 2) using a micropipette puller. The settings used are: Heat - 700, Pull - 100, Velocity - 50, Time - 10.</li>
	<li>Insert cannula into the holder of ...

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurovascular+signaling+in+the+brain+and+the+pathological+consequences+of+hypertension.">Neurovascular signaling in the brain and the pathological consequences of hypertension.</a> Am J Physiol Heart Circ Physiol. 306, H1-H14 (2014).</li><li>Iadecola, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurovascular+regulation+in+the+normal+brain+and+in+Alzheimer's+disease.">Neurovascular regulation in the normal brain and in Alzheimer's disease.</a> Nat Rev Neurosci. 5, 347-360 (2004).</li><li>Dabertrand, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostaglandin+E2,+a+postulated+astrocyte-derived+neurovascular+coupling+agent,+constricts+rather+than+dilates+parenchymal+arterioles.">Prostaglandin E2, a postulated astrocyte-derived neurovascular coupling agent, constricts rather than dilates parenchymal arterioles.</a> J Cereb Blood Flow Metab. 33, 479-482 (2013).</li><li>Nishimura, N., Schaffer, C. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+heterogeneity+of+endothelial+P2+purinoceptors+in+the+cerebrovascular+tree+of+the+rat.">Functional heterogeneity of endothelial P2 purinoceptors in the cerebrovascular tree of the rat.</a> Am J Physiol. 277, H893-H900 (1999).</li><li>Nagase, K., Iida, H., Dohi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+ketamine+on+isoflurane-+and+sevoflurane-induced+cerebral+vasodilation+in+rabbits.">Effects of ketamine on isoflurane- and sevoflurane-induced cerebral vasodilation in rabbits.</a> J Neurosurg Anesthesiol. 15, 98-103 (2003).</li><li>Fisher, C. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+hypertension+on+the+cerebral+circulation.">The effects of hypertension on the cerebral circulation.</a> Am J Physiol Heart Circ Physiol. 304, H1598-H1614 (2013).</li><li>Filosa, J. A., Bonev, A. D., Nelson, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+dynamics+in+cortical+astrocytes+and+arterioles+during+neurovascular+coupling.">Calcium dynamics in cortical astrocytes and arterioles during neurovascular coupling.</a> Circ Res. 95, e73-e81 (2004).</li><li>Dacey, R. G., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+rat+intracerebral+arterioles:+methods,+morphology,+and+reactivity.">A study of rat intracerebral arterioles: methods, morphology, and reactivity.</a> Am J Physiol. 243, H598-H606 (1982).</li><li>Coyne, E. F., Ngai, A. C., Meno, J. R., Winn, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+isolation+and+characterization+of+intracerebral+arterioles+in+the+C57/BL6+wild-type+mouse.">Methods for isolation and characterization of intracerebral arterioles in the C57/BL6 wild-type mouse.</a> J Neurosci Methods. 120, 145-153 (2002).</li><li>Cipolla, M. J., Smith, J., Kohlmeyer, M. M., Godfrey, J. 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Cerebral parenchymal arterioles are high resistance arterioles with few anastomoses and branches that perfuse distinct neuronal populations. These specialized blood vessels are central players in cerebrovascular autoregulation and neurovascular coupling through astrocyte-mediated vasodilation 1. The importance of these specialized blood vessels in cerebral vascular disease has been known for approximately 50 years, when the pioneering work of Dr. Miller Fisher described structural alterations in parenchymal ar...

The cerebral circulation is uniquely organized to support the metabolic demands of central neurons, cells that have limited energy stores and are consequently highly sensitive to changes in oxygen pressure and supply of necessary nutrients. As particular neuronal subpopulations becomes active when specific tasks are performed, the vasculature promotes a highly localized increase in perfusion to prevent local hypoxia and depletion of nutrients 1. This is a form of functional hyperemia known as neurovascular coupling, and is dependent on the proper operation of the neurovascular unit, composed of active neurons, astrocytes, and cerebral arteries 2. Intracerebral parenchymal arterioles, a group of blood vessels encompassing parenchymal, penetrating and pre-capillary arterioles, are centrally important for this response and it is then critical to study them individually in order to investigate neurovascular coupling 3.
Parenchymal arterioles are small (20 - 70 &#181;m internal diameter) high-resistance blood vessels that perfuse distinct neuronal populations within the brain. Branching out from pial arteries on the surface, parenchymal arterioles penetrate into the brain parenchyma at a nearly 90&#7506; angle to feed the subsurface microcirculation (Figure 1). These arterioles play a critical role in maintaining appropriate perfusion pressure as they are the most distal smooth muscle-containing vessels protecting the capillaries. In contrast to the surface pial circulation, parenchymal arterioles lack collateral branches and anastomoses, and consequently are &#34;bottlenecks&#34; of the cerebral circulation 4. As a result, dysfunction of parenchymal arterioles contributes to the development of cerebrovascular diseases such as vascular cognitive impairment and small ischemic strokes (also known as silent or lacunar strokes). Studies indicate that parenchymal arterioles dysfunction can be induced by essential hypertension 5, chronic stress 6, and is an early event in small vessel disease genetic mouse model 7. Further, experimentally-induced occlusion of single penetrating arterioles in rats is sufficient to cause small ischemic strokes that are cylindrical in shape, similar those observed in older humans 8.
In addition to these anatomical distinctions, mechanisms regulating contractile function differ between pial arteries and parenchymal arterioles. Myogenic vasoconstriction is greater in parenchymal arterioles 9, possibly because of the lack of extrinsic innervation 10, distinct modes of mechanotransduction 11, and differences in intracellular Ca2+ signaling 12,13 in vascular smooth muscle cells. Evidence suggests that endothelium-dependent vasodilator mechanisms also differ between these vascular segments, with parenchymal arteries exhibiting greater reliance on mechanisms involving Ca2+-activated K+ channels and electrotonic communication within the vascular wall compared with diffusible factors such as nitric oxide and prostacyclins 14. Therefore, data gathered in experiments using pial arteries may not necessarily apply to parenchymal arterioles, leaving a gap in our knowledge of local control of cerebral perfusion.
Despite their importance, parenchymal arterioles are vastly under-studied, primarily due to the technical challenges with isolation and mounting for ex vivo study. In this manuscript we describe a methodology to isolate and cannulate cerebral parenchymal arterioles, which can be used for pressure myography, or to isolate tissue for immunolabeling, electrophysiology, molecular biology, and biochemical analysis.

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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">artificial Cerebrospinal Fluid</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
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			<td height="20" style="height:21px;width:216px;">NaCl</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">S-640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">P217</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MgCl Anhydrous</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">M-8266</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">NaHCO3</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">S233</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">NaH2PO4</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D-(+)-Glucose</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">G2870</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">CaCl2</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">C4901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name</td>
			<td style="width:127px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isolation/ Cannulation</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereo Microscope</td>
			<td style="width:127px;">Olympus</td>
			<td style="width:119px;">SZX7</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:21px;width:216px;">Super Fine Forceps</td>
			<td style="width:127px;">Fine Science Tools</td>
			<td style="width:119px;">11252-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:216px;">Vannas Spring Scissors</td>
			<td style="width:127px;">Fine Science Tools</td>
			<td style="width:119px;">15000-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Wiretrol 50 &#956;L</td>
			<td style="width:127px;">VWR Scientific</td>
			<td style="width:119px;">5-000-1050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.2 &#956;m Sterile Syringe Filter</td>
			<td style="width:127px;">VWR Scientific</td>
			<td style="width:119px;">28145-477</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micropipette Puller</td>
			<td style="width:127px;">Sutter Instruments</td>
			<td style="width:119px;">P-97</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Borosilicate Glass O.D.: 1.2 mm, I.D.: 0.68 mm</td>
			<td style="width:127px;">Sutter Instruments</td>
			<td style="width:119px;">B120-69-10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dark Green Nylon Thread</td>
			<td style="width:127px;">Living Systems Instrumentation</td>
			<td style="width:119px;">THR-G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Linear Alignment Single Vessel Chamber</td>
			<td style="width:127px;">Living Systems Instrumentation</td>
			<td style="width:119px;">CH-1-LIN</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pressure Servo Controller with Peristaltic Pump</td>
			<td style="width:127px;">Living Systems Instrumentation</td>
			<td style="width:119px;">PS-200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Video Dimension Analyzer</td>
			<td style="width:127px;">Living Systems Instrumentation</td>
			<td style="width:119px;">VDA-10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Four Channel Recorder with LabScribe 3 Recording and Analysis Software</td>
			<td style="width:127px;">Living Systems Instrumentation</td>
			<td style="width:119px;">DAQ-IWORX-404</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Heating Unit</td>
			<td style="width:127px;">Warner Instruments</td>
			<td style="width:119px;">64-0102</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Automatic Temperature Controller</td>
			<td style="width:127px;">Warner Instruments</td>
			<td style="width:119px;">TC-324B</td>
			<td></td>
		</tr>
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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.

Figure 5A shows a representative constriction of mouse PAs to 60 mM KCl aCSF to evaluate the integrity of the preparation. PAs should constrict between 15 - 30% in the presence of 60 mM KCl. If the constriction is below 15%, discard the PA and cannulate another one, as it suggests that the arteriole was damaged during the isolation and cannulation process.
Figure 5B illustrates PA constriction t...

Watch this Scientific Journal Video about Isolation and Cannulation of Cerebral Parenchymal Arterioles at JoVE.com

Isolation and Cannulation of Cerebral Parenchymal Arterioles

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.

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

The overall goal of this procedure is to isolate and mount cerebral arterioles for studies of vascular function and structure using pressure myography. This method can be used to answer key questions in the cerebrovascular field, such as, how did cerebral microvasculature quickly adapts to changes in intraluminal pressure or nutrient demand or during pathological conditions. The main advantage of this technique is that it allows for pressurization of the arteriole in addition of intraluminal pressure, which are important physiological determinants of vascular function.To begin, using a micropipette puller with the appropriate settings, such as those listed here, pull capillaries to generate cannulas. After inserting a cannula into the holder of a myograph chamber and aligning it, under a dissecting microscope, use forceps to carefully break the tip to the desired diameter, such as 10 micrometers as shown here. Fill both cannulas with cerebrospinal fluid or aCSF containing 1.8 millimolar calcium, and use five to 20 milliliters of calcium free aCSF supplemented with 1%BSA and 10 micromolar diltiazem to fill the chamber.Store the chamber at four degrees Celsius until just before the cannulation. After isolating a mouse brain according to the text protocol, under the dissecting scope, locate the circle of Willis and the middle cerebral artery, or MCA, branching from it. Using sharp and aligned Vannas spring scissors, cut a small rectangle around the MCA.The top part of the rectangle should be past a branching point from the circle of Willis. Then, with the Vannas spring scissors, perform an undercut into the depth of the tissue for approximately two to three millimeters. Using insect pins, pin down the most distal end of the brain slice into the dissecting dish, with the MCA facing upwards.Make a shallow incision to cut the pia near the pin, being careful not to cut too deep into the underlying cerebral cortex. Next, with small forceps, carefully grab each side of the pia and start peeling the pia from the cortex. If necessary, hold onto the MCA ensuring that the surrounding pial membrane is not damaged.Close to the circle of Willis, resistance against peeling will increase. Take extra care at this point, because the longer parenchymal arterioles will be in this region. Continue to peel until the pia containing the MCA is free from the cortex.Care and patience should be taken during this step, as the arterioles are very fragile and sudden movements or pulling with too much force will damage them. Once the pia is free, place it carefully on top of the pad on the dissecting dish, with the surface opposite the parenchymal arterioles facing down. With Vannas spring scissors, free the dissected arterioles from the MCA by bluntly cutting the ends of the vessels.After preparing sutures and securing two onto each cannula, use 10%BSA to coat a glass micropipette, and with calcium free aCSF supplemented with 1%BSA, rinse it off. Move 50 microliters of calcium free aCSF supplemented with 1%BSA into the glass micropipette. Then, pull up a parenchymal arteriole, or PA, and transfer to the pressure myography chamber.Push the plunger in one fluid motion to dispense the parenchymal arteriole into the chamber to prevent it from sticking to the internal chamber of the glass micropipette. With two super fine forceps, open the lumen of the PA, taking care to only touch the very end of the vessel. Then, using the forceps to hold the edges of the PA, carefully pull the vessel onto the cannula.Next, loosen the two sutures on the cannula, and after spacing them slightly apart on the vessel, pull them toward the operator while tightening them on the vessel. Make sure that any branches are tied off before closing the opposite end of the PA.These are the most difficult steps. Opening up and sliding the arteriole onto the cannula requires practice.When tying up the knot, hold both sides evenly as to not break the tip of the cannula. Then, turn the chamber around and close the opposite end of the PA as a blind-sac by opening the ties on the opposite cannula and passing it onto the arteriole. Slowly close the knot in such a way that the PA will be tied to the side of the cannula.Avoid a longitudinal stretch of the arteriole. Carefully transfer the chamber to the stage of the microscope to be used for recording vessel diameter. Attach the chamber onto the microscope's stage, connecting a temperature probe and inlet and outlets for perfusion to the correct tubes.Pressurize the arteriole to 40 millimeters mercury intraluminal pressure for mouse parenchymal arterioles, or 50 millimeters mercury for rat arterioles. To carry out an agonist induced constriction experiment, after preparing the agonist solution and adding it to the PA chamber according to the text protocol, incubate the vessel in the solution for 10 minutes to allow the luminal diameter to reach a steady state. Then, record the diameter of the arteriole.After washing out the agonist and adding a drug to induce maximum dilation, record the passive diameter of the arteriole. This figure shows a representative constriction of mouse PAs to 60 millimolar potassium chloride in aCSF to evaluate the integrity of the preparation. PA should constrict between 15 to 30 percent in the presence of 60 millimolar potassium chloride.Illustrated here is the PA constriction in response to incubation with increasing concentrations of the Thromboxane A2 analog, U-46619 into the superfusing bath. The data can be presented as a change in diameter or as a percent vasoconstriction to potassium chloride, which normalizes the change in diameter by the maximum constriction to 60 millimolar potassium chloride. In this figure, a representative tracing of the lumen diameter of a PA in a myogenic reactivity experiment is shown.Stepwise increases in intraluminal pressure cause a graded constriction of PAs in the presence of 1.8 millimolar extracellular calcium. Incubation of the same PA with aCSF without calcium and supplemented with 10 micromolar diltiazem plus two millimolar EGTA abolishes myogenic tone generation, and the lumen diameter of the PA increases according to intraluminal pressure, which is the same as the passive lumen diameter of the arteriole. While attempting this technique, it's important to remember to keep your shoulders relaxed as to prevent pain.And once this technique is mastered, it should take no longer than 30 minutes from the isolation up to the cannulation of the arteriole. Following this method, other techniques like imaging of intracellular calcium can be used to answer additional questions, such as the role of intracellular calcium in control of microvascular tone. After its development, this technique has paved the way for researchers in the field of vascular biology to explore the physiological control and pathological changes of the intercerebral vasculature in many organ systems, including humans and rodents.After watching this video, you should have a good understanding of how to isolate and cannulate a cerebral parenchymal arteriole.

Research

JoVE Journal

Neuroscience

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

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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 ...