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>
	<li>Dunn, K. M., 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=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. B., Friedman, B., Lyden, P. D., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetrating+arterioles+are+a+bottleneck+in+the+perfusion+of+neocortex.">Penetrating arterioles are a bottleneck in the perfusion of neocortex.</a> Proc Natl Acad Sci U S A. 104, 365-370 (2007).</li><li>Pires, P. W., Jackson, W. F., Dorrance, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+myogenic+tone+and+structure+of+parenchymal+arterioles+by+hypertension+and+the+mineralocorticoid+receptor.">Regulation of myogenic tone and structure of parenchymal arterioles by hypertension and the mineralocorticoid receptor.</a> Am J Physiol Heart Circ Physiol. 309, H127-H136 (2015).</li><li>Longden, T. A., Dabertrand, F., Hill-Eubanks, D. C., Hammack, S. E., Nelson, M. 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Y., 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=The+smallest+stroke:+occlusion+of+one+penetrating+vessel+leads+to+infarction+and+a+cognitive+deficit.">The smallest stroke: occlusion of one penetrating vessel leads to infarction and a cognitive deficit.</a> Nature neuroscience. 16, 55-63 (2013).</li><li>Cipolla, M. J., 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=Increased+pressure-induced+tone+in+rat+parenchymal+arterioles+vs.+middle+cerebral+arteries:+role+of+ion+channels+and+calcium+sensitivity.">Increased pressure-induced tone in rat parenchymal arterioles vs. middle cerebral arteries: role of ion channels and calcium sensitivity.</a> Journal of applied physiology. 117, 53-59 (2014).</li><li>Hamel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+nerves+and+the+regulation+of+cerebrovascular+tone.">Perivascular nerves and the regulation of cerebrovascular tone.</a> Journal of applied physiology. 100, 1059-1064 (2006).</li><li>Brayden, J. E., Li, Y., Tavares, M. 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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. 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+arterial+lesions+underlying+lacunes.">The arterial lesions underlying lacunes.</a> Acta Neuropathol. 12, 1-15 (1968).</li><li>Brown, W. R., Moody, D. M., Thore, C. R., Anstrom, J. A., Challa, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+changes+in+the+white+mater+in+dementia.">Microvascular changes in the white mater in dementia.</a> J Neurol Sci. 283, 28-31 (2009).</li><li>Pires, P. W., Dams Ramos, C. M., Matin, N., Dorrance, A. 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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			<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>
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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>
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			<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>
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			<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>
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			<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>
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			<td height="21" style="height:21px;width:216px;">Isolation/ Cannulation</td>
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			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;width:216px;">Stereo Microscope</td>
			<td style="width:127px;">Olympus</td>
			<td style="width:119px;">SZX7</td>
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			<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>
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		<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>
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		<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>
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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 ...

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