Name: the ex vivo isolated skeletal microvessel preparation for investigation of vascular reactivity
Uploaded: 2012-04-28T14:00:00
Description: Watch this Scientific Journal Video about The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity at JoVE.com

This work was supported by the American Heart Association (EIA 0740129N) and NIH T32 HL90610.

1. Prior to the Experiment
<ol>
 <li>Prior to the experiment day, glass capillary tubes of the appropriate dimensions for the station are pulled into micropipettes (either a horizontal or vertical puller can be used). The tip diameter can be readily adjusted depending on the vessel being isolated, although we generally use a diameter range between 50-150 &mu;m. These are then bent to the appropriate configuration for the microvessel station following heating over a butane flame. Micropipette tips are physically broken ...

<ol>
	<li>Goodwill, A. G., Frisbee, S. J., Stapleton, P. A., James, M. E., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Chronic+Anticholesterol+Therapy+on+Development+of+Microvascular+Rarefaction+in+the+Metabolic+Syndrome.">Impact of Chronic Anticholesterol Therapy on Development of Microvascular Rarefaction in the Metabolic Syndrome.</a> Microcirculation. , 1-18 (2009).</li><li>Goodwill, A. G., James, M. E., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+vascular+thromboxane+generation+impairs+dilation+of+skeletal+muscle+arterioles+of+obese+Zucker+rats+with+reduced+oxygen+tension.">Increased vascular thromboxane generation impairs dilation of skeletal muscle arterioles of obese Zucker rats with reduced oxygen tension.</a> Am. J. Physiol. Heart Circ. Physiol. 295, H1522-H1528 (2008).</li><li>Samora, J. B., Frisbee, J. C., Boegehold, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth-dependent+changes+in+endothelial+factors+regulating+arteriolar+tone.">Growth-dependent changes in endothelial factors regulating arteriolar tone.</a> Am. J. Physiol. Heart Circ. Physiol. 292, H207-H214 (2007).</li><li>Samora, J. B., Frisbee, J. C., Boegehold, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+emerges+as+a+regulator+of+tone+in+skeletal+muscle+arterioles+during+juvenile+growth.">Hydrogen peroxide emerges as a regulator of tone in skeletal muscle arterioles during juvenile growth.</a> Microcirculation. 15, 151-161 (2008).</li><li>Samora, J. B., Frisbee, J. C., Boegehold, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+myogenic+responsiveness+of+skeletal+muscle+arterioles+with+juvenile+growth.">Increased myogenic responsiveness of skeletal muscle arterioles with juvenile growth.</a> Am. J. Physiol. Heart Circ. Physiol. 294, 2344-2351 (2008).</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. Heart Circ. Physiol. 243, H598-H606 (1982).</li><li>Fredricks, K. T., Liu, Y., Lombard, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+extraparenchymal+resistance+arteries+of+rat+skeletal+muscle+to+reduce+PO2.">Response of extraparenchymal resistance arteries of rat skeletal muscle to reduce PO2.</a> Am. J. Physiol. 267, H706-H715 (1994).</li><li>Durand, M. J., Raffai, G., Weinberg, B. D., Lombard, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin-(1-7)+and+low-dose+angiotensin+II+infusion+reverse+salt-induced+endothelial+dysfunction+via+different+mechanisms+in+rat+middle+cerebral+arteries.">Angiotensin-(1-7) and low-dose angiotensin II infusion reverse salt-induced endothelial dysfunction via different mechanisms in rat middle cerebral arteries.</a> Am. J. Physiol. Heart Circ. Physiol. 299, H1024-H1033 (2010).</li><li>LeBlanc, A. J., Cumpston, J. L., Chen, B. T., Frazer, D., Castranova, V., Nurkiewicz, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+inhalation+impairs+endothelium-dependent+vasodilation+in+subepicardial+arterioles.">Nanoparticle inhalation impairs endothelium-dependent vasodilation in subepicardial arterioles.</a> J. Toxicol. Environ. Health A. 72, 1576-1584 (2009).</li><li>Jernigan, N. L., LaMarca, B., Speed, J., Galmiche, L., Granger, J. P., Drummond, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+salt+enhances+benzamil-sensitive+component+of+myogenic+constriction+in+mesenteric+arteries.">Dietary salt enhances benzamil-sensitive component of myogenic constriction in mesenteric arteries.</a> Am. J. Physiol. Heart Circ. Physiol. 294, H409-H420 (2008).</li><li>Stapleton, P. A., Goodwill, A. G., James, M. E., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+mechanisms+of+endothelium-dependent+dilation+in+skeletal+muscle+arterioles+with+genetic+hypercholesterolemia.">Altered mechanisms of endothelium-dependent dilation in skeletal muscle arterioles with genetic hypercholesterolemia.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 293, R1110-R1119 (2007).</li><li>Goodwill, A. G., Stapleton, P. A., James, M. E., d'Audiffret, A. C., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+arachidonic+acid-induced+thromboxane+generation+impairs+skeletal+muscle+arteriolar+dilation+with+genetic+dyslipidemia.">Increased arachidonic acid-induced thromboxane generation impairs skeletal muscle arteriolar dilation with genetic dyslipidemia.</a> Microcirculation. 15, 621-631 (2008).</li><li>Baumbach, G. L., Hadju, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+and+composition+of+cerebral+arterioles+in+renal+and+spontaneously+hypertensive+rats.">Mechanics and composition of cerebral arterioles in renal and spontaneously hypertensive rats.</a> Hypertension. 21, 816-826 (1993).</li><li>Uchida, E., Bohr, D. F., Hoobler, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+studying+isolated+resistance+vessel+from+rabbit+mesentery+and+brain+and+their+responses+to+drugs.">A method for studying isolated resistance vessel from rabbit mesentery and brain and their responses to drugs.</a> Circ. Res. 4, 525-536 (1967).</li><li>Davis, M. J., Kuo, L., Chilian, W. M., Muller, J. M. I. s. o. l. a. t. e. d., Barker, J. H., Anderson, G. L., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+23.">Chapter 23.</a> Isolated, perfused microvessels. In: Clinically Applied Microcirculation Research. 32, 435-456 (1995).</li><li>Lombard, J. H., Liu, Y., Fredricks, K. T., Bizub, D. M., Roman, R. J., Rusch, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+and+mechanical+responses+of+rat+middle+cerebral+arterieal+to+reduced+PO2+and+prostacyclin.">Electrical and mechanical responses of rat middle cerebral arterieal to reduced PO2 and prostacyclin.</a> Am. J. Physiol. 276, H509-H516 (1994).</li></ol>

The protocol presented describes the isolation, removal and double cannulation of a skeletal muscle microvessel, although this general technique can be readily applied to most tissues. For the current manuscript, the term "arteriole" has been used by the authors to describe a resistance vessel ranging between 70-120 &mu;m in diameter under resting active tone, which is also a major contributor to the regulation of perfusion resistance to an organ or tissue.
With some modifications, this syste...

<table border="1">
	<tbody>
		<tr>
			<td>Reagents and Equipment</td>
			<td>Company</td>
			<td>Comments/Catalogue #</td>
		</tr>
		<tr>
			<td>Vessel Chamber</td>
			<td>Custom</td>
			<td>Dave Eick (MCW)</td>
		</tr>
		<tr>
			<td>Heated Circulating Water Bath</td>
			<td>PolyScience and Haake</td>
			<td>Haake DC 10</td>
		</tr>
		<tr>
			<td>Pipets</td>
			<td>Frederick Haer &amp; Co.</td>
			<td>Capillary Tubing 2.0 mm OD x 1.0 mm ID (27-33-1)</td>
		</tr>
		<tr>
			<td>Pressure Monitor</td>
			<td>World Precision Instruments</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Water Jacketed Reservoir</td>
			<td>Custom</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>External Light Source</td>
			<td>World Precision Instruments</td>
			<td>Novaflex</td>
		</tr>
		<tr>
			<td>Pipet Puller</td>
			<td>MicroData Instruments</td>
			<td>PMP102 Micropipet Puller</td>
		</tr>
		<tr>
			<td>Full complement of surgical tools</td>
			<td>Fine Science Tools</td>
			<td>Dumont</td>
		</tr>
		<tr>
			<td>Ultra Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>Inox #5</td>
		</tr>
		<tr>
			<td>Silk Suture Thread</td>
			<td>Ethilon</td>
			<td>#10-0 or 9-0</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>Olympus SZ-11</td>
		</tr>
		<tr>
			<td>Analog Video Calipers</td>
			<td>Boeckeler</td>
			<td>Via Controller (Via-100)</td>
		</tr>
		<tr>
			<td>High Resolution Analog Camera</td>
			<td>Panasonic</td>
			<td>GP-MF 602</td>
		</tr>
		<tr>
			<td>Oxygen Tank</td>
			<td>Regional</td>
			<td>21% balance nitrogen and 5% CO2 balance nitrogen</td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td>Tygon</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Drain Pump</td>
			<td>Cole Parmer Instrument Co.</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Modified Rat PSS</td>
			<td>See recipe below</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Van Breemen's Relaxant PSS</td>
			<td>See recipe below</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table 1. A list of the major components of isolated microvessel station setup presented in the Figures.
<table border="1">
	<tbody>
		<tr>
			<td>Modified Rat PSS Recipe </td>
			<td>To make two liters of PSS </td>
			<td>20X Salt Stock (2L)</td>
			<td>20X Buffer Stock (2L) </td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>&nbsp;</td>
			<td>278.0 g</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>&nbsp;</td>
			<td>14.0 g</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgSO4-7H2O</td>
			<td>&nbsp;</td>
			<td>11.5 g</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2-H2O</td>
			<td>&nbsp;</td>
			<td>9.4 g</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>80.8 g</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>0.4 g</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>0.28 g</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>1.98 g</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>20x Salt Stock</td>
			<td>100 mL</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>20x Buffer Stock</td>
			<td>100 mL</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>1800 mL</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table 2. Recipe for standard physiological salt solution (PSS) used in the isolated microvessel protocols.
Comments on Recipe: Make 2 L of Salt Stock and 2 L of Buffer Stock. These can be refrigerated when not being used, but shake them well and often before preparing PSS. The additional ingredients are added at the time of preparation of final PSS.
<table border="1">
	<tbody>
		<tr>
			<td>Van Breemen's Relaxant PSS </td>
			<td>To make 2 liters of PSS </td>
			<td>20X Salt Stock (1L)</td>
			<td>20X Buffer Stock (1L) </td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>&nbsp;</td>
			<td>107.4 g</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>&nbsp;</td>
			<td>7.0 g</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgSO4-7H2O</td>
			<td>&nbsp;</td>
			<td>5.76 g</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgCl2-6H2O</td>
			<td>&nbsp;</td>
			<td>81.32 g</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>40.4 g</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>0.2 g</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>15.22</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>0.28 g</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>1.98 g</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>20x Salt Stock</td>
			<td>100 mL</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>20x Buffer Stock</td>
			<td>100 mL</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>1800 mL</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table 3. Recipe for Van Breemen's relaxant physiological salt solution (PSS) used in the isolated microvessel protocols under conditions of zero active tone.
Comments on Recipe: Make 1 L of Salt Stock and 1 L of Buffer Stock. These can be refrigerated when not being used, but shake them well and often before preparing PSS. The additional ingredients are added at the time of preparation of final relaxant PSS.

the ex vivo isolated skeletal microvessel preparation for investigation of vascular reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

Watch this Scientific Journal Video about The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity at JoVE.com

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

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