The project described was supported by Award Numbers R01 HL064761, R25 HL059868, 1SC1 HL099139 and P20 MD000175 form National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

1. Isolation of rat mesenteric small artery
<ol>
 <li>Anesthetize animal with isoflurane in a closed chamber and wipe the abdomen with alcohol.</li>
 <li>Perform a mid-line laparotomy to expose mesenteric bed.</li>
 <li>Using scissors, remove about 85 cm of intestine with feeding vasculature with the superior mesenteric artery. Cut the proximal end of intestinal section close to the pylorus and the distal end near the ileo-coecal junction. Segments of the intestine are isolated from rats that are deeply anesthetized with isoflurane and euthanized by open-chest cardiac puncture.</li>
 <li>Placed excised section in a coated petri dish containing PSS and perform the dissection of the mesenteric artery at room temperature.</li>
 <li>Pin down the proximal end of the intestine on the right hand side and pin out the remainder of the intestineina counter-clockwise direction (i.e.proximal end on the left feeding vasculature on the far side of the intestine).</li>
 <li>Dissect out the branch II and III segments together with a piece of the proximal segment.</li>
 <li>Dissect out the vein and isolate the artery (with V-shaped branch point) and clean it by removing the adipose and connective tissue. Avoid direct contact with the artery by pulling gently with forceps and cutting through the membrane of the connective tissue.</li>
</ol>
2. Mounting of vessel
<ol>
 <li>Cut two short segments (&asymp; 4 cm) of a 40 &mu;m tungsten-free stainless steel wire and, with a fine forceps,insert one into the lumen of each artery taking care not to damage the endothelium. Use the wire tip to open the lumen if necessary. Blood streaming out of the vessel is a good sign the lumen is opened.</li>
 <li>Fill the myograph chamber with physiological salt solution (PSS; mM: 115 NaCl, 4.7 KCl, 1.4 MgSO4.7H2O, 5 NaHCO3, 1.2 K2HPO4, 1.1 Na2HPO4, 1.0 CaCl2, 20 HEPES and 5 glucose, pH 7.4) with ascorbic acid (100 &mu;M) at 37 &deg;C and using forceps carefully transfer the threaded vessel segment from the petri dish into the chamber at room temperature and transfer the excised proximal vessel segment to the myograph chamber and pull the end along the wire to feed it into the vessel. Avoid stretching the vessel.</li>
 <li>Secure the near end of the wire counter-clockwise under the near fixing screw on the right-hand jaw connected to the micrometer. Catch the free end of the wire with forceps and secure it clockwise under the far fixing screw on the right-hand jaw. Ensure that the vessel segment along the wire is situated in the gap between the jaws without making any contact with the jaw itself.</li>
 <li>Screw the jaws apart, and align the second wire parallel with the vessel and insert it into the far end of the lumen. Gently feed the wire through the lumen of the vessel segment in one motion using the already mounted wire as a guide. Hold the wire about 1 cm from the vessel to avoid stretching it during the maneuver, and avoid touching the endothelium.</li>
 <li>Screw the jaws together and ensure that the second mounting wire moves underneath the first one secured on the right-hand jaw.</li>
 <li>Secure the near end of the second wire in a clockwise direction under the near fixing screw of the left-hand jaw connected to the transducer.</li>
 <li>Secure the far end of the wire under the fixing screw on the left-hand jaw and tighten to stretch the wire.</li>
 <li>Once mounting is complete, reset the motor in the "Mounting Menu" and start normalization of the vessel.</li>
</ol>
3. Normalization
The Auto Dual Wire Myograph System- 510A has an automated normalization function, which is assessed from the "Normalization" menu and allows the vessel to be stretched to a normalized internal circumference by a standardized procedure according to the manufacturer's protocol after equilibration for 30 min at 37 &deg;C. An exponential curve is then fitted to the internal circumference pressure data. The procedure defines the lumen diameter (d100) that the artery would have had in vivo when relaxed and under a transmural pressure of 100 mmHg1.The normalization parameters for rat mesenteric artery are as follows:
<ol>
 <li>Target transmural pressure = 13.3 kPa (100 mm Hg).</li>
 <li>Time = 60 sec; Duration of each of the normalization steps.</li>
 <li>IC1/IC100 = 0.9 (IC1 = normalized internal circumference, IC100 = internal circumference corresponding to target pressure.</li>
 <li>Eyepiece calibration 2*&Delta; (mm/ocular division); 2 delta is for programming reasons.</li>
</ol>
Measure the length of the mounted mesenteric artery using the microscope eye-piece readings when the hairlines are over the far and near ends of the mounted vessel segment. Briefly, the length of the mounted vessel segment is measured at maximum magnification with a calibrated ocular eye-piece in the dissection microscope. The eye-piece reading with hair-line over the far end of the segment (a1) and the near end of the segment (a2) in ocular divisions are measured and recorded. These values, with the micrometer readings, before and after stretching the vessel are recorded and entered into the menus for the program to calculate the fitted curve and the internal diameter corresponding to the a target transmural pressure of 100 mm Hg
In our studies, the arteries were set to the lumen diameter of d1 = 0.9 x d100, where active force development is maximal. Active force development of &ge; 10 mN in rat mesenteric arteries is considered optimum for experiments to proceed. Tissues with lower active force were discarded.
4. Measurement of responses
After normalization, the mechanical and functional properties of vessels are re-activated by performing a "standard start", which involves challenging vessels with repeated applications (normally 2 or 3) of 5 &mu;M phenylephrine (PE) to obtain reproducible contractions. A typical "standard start" in our laboratory consists of a series of stimulations and washout periods as follows:
<ol>
 <li>Replace myograph chamber cover and start aeration with 95% air and 5% CO2.</li>
 <li>Fill chamber with fresh PSS (containing 100 &mu;M ascorbic acid)</li>
 <li>Contract vessel with 5 &mu;M PE for 5 min and wash 4 times with PSS buffer.</li>
 <li>After the last wash, refill chamber with PSS and wait 3 min before repeating steps (ii) and (iii).</li>
</ol>
After the standard start, the artery is ready for the experiment. Relaxations of contracted vessels are assessed by cumulative addition of increasing concentrations of CaCl2 (0.5 - 4 mM). When inhibitors are used vessels are pre-incubated with the compounds in the myograph chamber for 20 min and present during the assays.
5. Data analysis
The "Normalization" data for vessels and force tracings, directly converted to text data,were analyzed with the SigmaPlot 11.0 graphing program (Systat Software, Point Richmond, CA) and plotted as shown in Figure 1. Concentration-response data, calculated from the tracings, were analyzed by determining EC50 values from experimental data fitted to a four-parameter logistic function in the Pharmacology menu of the program. Comparisons between groups and within groups were carried out by One Way Analysis of Variance (ANOVA) and differences with p &lt; 0.05 are considered significant.
6. Representative results:
<table border="1">
 <tbody>
 <tr>
 <th>
 Parameters</th>
 <th>
 Wistar</th>
 <th>
 Dahl Salt-Sensitive</th>
 <th>
 Dahl Salt-Resistant</th>
 </tr>
 <tr>
 <td>L100 (&mu;m)</td>
 <td>83.26 &plusmn; 2.33</td>
 <td>91.52 &plusmn; 4.67</td>
 <td>117.45 &plusmn; 8.43*</td>
 </tr>
 <tr>
 <td>X1</td>
 <td>604.21 &plusmn; 41.97</td>
 <td>752.24 &plusmn; 85.25</td>
 <td>745.84 &plusmn; 110.09</td>
 </tr>
 <tr>
 <td>r2 </td>
 <td>0.99 &plusmn; 0.003</td>
 <td>0.97 &plusmn; 0.008</td>
 <td>0.97 &plusmn; 0.007</td>
 </tr>
 </tbody>
</table>
*The difference between L100 values for Wistar and DR are statistically significant. Shapiro-Wilk Normality Test passed (p = 0.588); Equal Variance Test passed (p = 0.237).
L100 = Internal diameter of the mounted segment corresponding to the target transmural pressure.
X1 = The micro positioner setting required to stretch the mounted vessel to its normalized internal circumference (i.e. the value of the micrometer reading at which experiments were performed).
r2 = Regression coefficient for fit of (Xi, Yi) to an exponential curve.
NB: The detailed procedure is described in the manual for the 510A Myograph System6.
Table 1. Sample Read-Out of normalization parameters for mesenteric artery segments from Wistar, DS and DR from Basic program shown in the "Normalization Menu". Values are means (&plusmn; SEM) of 5 animals. *Differences between L100 values for Wistar and DS are statistically significant (p &lt; 0.05).
<table border="1">
 <tbody>
 <tr>
 <th>
 Wistar</th>
 <th>
 DS</th>
 <th>
 DR</th>
 </tr>
 <tr>
 <td>14.2 &plusmn; 0.6</td>
 <td>25.8 &plusmn; 2.7*</td>
 <td>20.9 &plusmn; 1.3*</td>
 </tr>
 </tbody>
</table>
Table 2. Developed tensions(mN) in mesenteric arteries isolated from Wistar, DS and DR rats,following applications of 5 &mu;M PE to each vessel. Values reported are means (&plusmn; SEM) of 6-8 animals. *Significantly different from Wistar controls (p &lt; 0.05).
<img alt="Figure 1" src="/files/ftp_upload/3119/3119fig1.jpg" /> 
Figure 1. Ca2+e-induced relaxation of a PE-contracted mesenteric artery from a Wistar rat mounted in a wire myograph in physiological salt solution containing 1 mM Ca2+. The artery was equilibrated for 30 min at 37 &deg;C with constant aeration and responses to Ca2+e determined. A representative force tracing on addition of 5 &mu;M PE followed by cumulative additions of Ca2+ is shown.
<img alt="Figure 2" src="/files/ftp_upload/3119/3119fig2.jpg" /> 
Figure 2. Ca2+e-induced relaxation of a PE-contracted mesenteric artery from a DS rat mounted in a wire myograph in physiological salt solution containing 1 mM Ca2+. The artery was equilibrated for 30 min at 37 &deg;C with constant aeration and responses to Ca2+e determined. A representative force tracing on addition of 5 &mu;M PE followed by cumulative additions of Ca2+ is shown. Ca2+ relaxation was severely compromised in this tissue compared to those from Wistar and DR rats.
<img alt="Figure 3" src="/files/ftp_upload/3119/3119fig3.jpg" /> 
Figure 3. Ca2+e-induced relaxation of a PE-contracted mesenteric artery from a DR rat mounted in a wire myograph in physiological salt solution containing 1 mM Ca2+. The artery was equilibrated for 30 min at 37 &deg;C with constant aeration and responses to Ca2+e determined. A representative force tracing on addition of 5 &mu;M PE followed by cumulative additions of Ca2+ is shown.
<img alt="Figure 4" src="/files/ftp_upload/3119/3119fig4.jpg" /> 
Figure 4. A. [Ca2+]e-response curves. B. Bar chart showing EC50 values for relaxation determined by fitting the data to a four parameter logistic curve. C. CaR expression in mesenteric arcades isolated from DR and DS rats.

<ol>
	<li>Mulvany, M. J., Halpern, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+vascular+smooth+muscle+cells+in+situ.">Mechanical properties of vascular smooth muscle cells in situ.</a> Nature. 260, 617-619 (1976).</li><li>Bevan, J. A., Osher, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+direct+method+for+recording+tension+changes+in+the+wall+of+small+blood+vessels+in+vitro.">A direct method for recording tension changes in the wall of small blood vessels in vitro.</a> Agents Actions. 2, 257-260 (1972).</li><li>Mulvany, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedures+for+investigating+of+small+vessels+using+small+vessel+myograph.">Procedures for investigating of small vessels using small vessel myograph.</a> DMT Danish Myo Technology. , (2004).</li><li>Angus, J. A., Wright, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+to+study+the+pharmacodynamics+of+isolated+large+and+small+blood+vessels.">Techniques to study the pharmacodynamics of isolated large and small blood vessels.</a> J. Pharmacol. Toxicol. Methods. 44, 395-407 (2000).</li><li>Halpern, W., Mulvany, M. J., Warshaw, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+smooth+muscle+cells+in+the+walls+of+arterial+resistance+vessels.">Mechanical properties of smooth muscle cells in the walls of arterial resistance vessels.</a> J. Physiol. 275, 85-101 (1978).</li><li>Lindhorst, J., Alexander, N., Blignaut, J., Rayner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+hypertension+between+blacks+and+whites:+an+overview.">Differences in hypertension between blacks and whites: an overview.</a> Cardiovasc. J. Afr. 18, 241-247 (2007).</li><li>Eley, S. L., Allen, C. M., Williams, C. L., Bukosi, R. D., Pointer, 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=Action+of+thiazide+on+renal+interstitial+calcium.">Action of thiazide on renal interstitial calcium.</a> Am. J. Hypertens. 21, 814-819 (2008).</li><li>Palmer, C. E., Rudd, M. A., Bujoski, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+interstitial+Ca2++during+sodium+loading+of+normotensive+and+Dal-salt+hypertensive+rats.">Renal interstitial Ca2+ during sodium loading of normotensive and Dal-salt hypertensive rats.</a> Am. J. Hypertens. 16, 771-776 (2003).</li><li>Hurwitz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeostatic+control+of+plasma+calcium+concentration.">Homeostatic control of plasma calcium concentration.</a> Crit. Rev. Biochem. Mol. Biol. 31, 41-100 (1996).</li><li>Brown, E. M., MacLeod, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+sensing+and+extracellular+calcium+signaling.">Extracellular sensing and extracellular calcium signaling.</a> Physiol. Rev. 81, 239-297 (2001).</li><li>Breitwieser, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+calcium+as+an+integrator+of+tissue+function.">Extracellular calcium as an integrator of tissue function.</a> Int. J. Biochem. Cell Biol. 40, 1467-1480 (2008).</li><li>Mupanomunda, M. M., Wang, Y., Bukoski, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+chronic+sensory+denervation+on+Ca2+-induced+relaxation+of+isolated+mesenteric+resistance+arteries.">Effect of chronic sensory denervation on Ca2+-induced relaxation of isolated mesenteric resistance arteries.</a> Am. J. Physiol. 274, 1655-1661 (1998).</li><li>Mupanomunda, M. M., Ishioka, N., Bukoski, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstitial+Ca2++undergoes+dynamic+changes+sufficient+to+stimulate+nerve-dependent+Ca2+-induced+relaxation.">Interstitial Ca2+ undergoes dynamic changes sufficient to stimulate nerve-dependent Ca2+-induced relaxation.</a> Am. J. Physiol. 276, 1035-1042 (1999).</li><li>Mupanomunda, M. M., Tian, B., Ishioka, N., Bukoski, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+interstitial+Ca2+.">Renal interstitial Ca2+.</a> Am. J. Physiol. Renal Physiol. 278, F644-F649 (2000).</li><li>Awumey, E. M., Hill, S. K., Diz, D. I., Bukoski, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochrome+P-450+metabolites+of+2-arachidonoylglycerol+play+a+role+in+Ca2+-induced+relaxation+of+rat+mesenteric+arteries.">Cytochrome P-450 metabolites of 2-arachidonoylglycerol play a role in Ca2+-induced relaxation of rat mesenteric arteries.</a> Am. J. Physiol. Heart Circ. Physiol. 294, 2363-2370 (2008).</li><li>Chen, W., Bergsman, J. B., Wang, X., Gilkey, G., Pierpoint, C. R., Daniel, E. A., Awumey, E. M., Dauban, P., Dodd, R. H., Ruat, M., Smith, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+external+calcium+signaling+involves+the+calcium-sensing+receptor+in+neocortical+nerve+terminals.">Presynaptic external calcium signaling involves the calcium-sensing receptor in neocortical nerve terminals.</a> PloS One. 5, e8563-e8563 (2010).</li><li>Bukoski, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+perivascular+sensory+nerve+Ca2++receptor+and+blood+pressure+regulation:+a+hypothesis.">The perivascular sensory nerve Ca2+ receptor and blood pressure regulation: a hypothesis.</a> Am. J. Hypertens. 11, 1117-1123 (1998).</li><li>Bukoski, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+Ca2++and+blood+pressure:+evidence+that+Ca2+-sensing+receptor+activated+sensory+nerve+dilator+activity+couples+changes+in+interstitial+Ca2++with+vascular.">Dietary Ca2+ and blood pressure: evidence that Ca2+-sensing receptor activated sensory nerve dilator activity couples changes in interstitial Ca2+ with vascular.</a> 16, 218-221 (2001).</li></ol>

No conflicts of interest declared.

Hypertension is a leading cause of cardiovascular, cerebral and renal morbidity/mortality. The occurrence of hypertension is high in the population and salt-sensitive hypertension is particularly high in the aging population, and more prevalent in blacks than whites. This is believed to be due to the tendency of blacks to retain sodium in their kidneys9. Salt-sensitivity is a major contributing factor to kidney disease and is associated with endothelial dysfunction, but the mechanism is not fully understood. R...

<table border="1">
	<tbody>
		<tr>
			<th>Name of equipment/ reagent	</th>
			<th>Company	</th>
			<th>Catalogue number	</th>
			<th>Comments(optional)</th>
		</tr>
		<tr>
			<td>Auto Dual Wire Myograph System-510A	DMT-USA, Inc.</td>
			<td>Atlanta, GA.</td>
			<td>100151</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML866/" target="_blank">PowerLab/4SP Data Acquisition System</a></td>
			<td><a href="http://www.adinstruments.com/" target="_blank">ADInstruments</a>, Colorado Springs, CO.</td>
			<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML866/" target="_blank">ML750</a></td>
			<td>New models with 4-16 input channels are available.</td>
		</tr>
		<tr>
			<td>Dell Dimension XPS Gen 4 Computer	</td>
			<td>Dell</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stemi SV II (Apo) Dissection Microscope with Ocular	</td>
			<td>Carl Zeiss International.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Wistar, Dahl salt-sensitive, Dahl salt-resistant rats	</td>
			<td>Harlan Sprague Dawley, Indianapolis, IN.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rodent chow	</td>
			<td>Harland Tekland, Madison, WI.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phenylephrine</td>
			<td>Sigma-Aldrich, St. Louis, MO.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Other chemicals</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>All chemicals used were of the purest grades available commercially.</td>
		</tr>
	</tbody>
</table>

mesenteric artery contraction and relaxation studies using automated wire myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 &mu;m in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 &mu;m. The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2. The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3.The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors.
We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca2+e). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca2+-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca2+e. Tissues from Wistar rats showed higher Ca2+-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca2+e-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS.
The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side6,7,8.

Watch this Scientific Journal Video about Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography at JoVE.com

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 &mu;m, independently.

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between ...

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28.4K Views.  North Carolina Central University, Durham. An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 μm, independently.

Video: Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic response is a key regulator of local blood flow. In isobaric conditions small resistance arteries develop sustained constriction known as myogenic tone (MT), which is a major determinant of systemic vascular resistance (SVR). Hence, ex vivo&#160;pressurized preparations of small resistance arteries are major tools to study microvascular function in near-physiological states. To achieve this, a freshly isolated intact segment of a small resistance artery (diameter ~260 &#956;m) is mounted onto two small glass cannulas and pressurized. These arterial preparations retain most in vivo characteristics and permit assessment of vascular tone in real-time. Here we provide a detailed protocol for assessing vasoactivity in pressurized small resistance mesenteric arteries from rats; these arteries develop sustained vasoconstriction - approximately 25% of maximal diameter - when pressurized at 70 mmHg. These arterial preparations may be used to study the effect of investigational compounds on relationship between intra-arterial pressure and vasoactivity and determine changes in microvascular function in animal models of various diseases.

Pressure myography is used to assess vasoactivity of small arteries that develop sustained constriction when pressurized. This manuscript provides a detailed protocol to assess in isolated segments of small mesenteric arteries from rats, vasoactivity and the effect of intraluminal pressure on vascular diameter.

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic ...

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent&#160;neointima for studies of restenosis.

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

Mechanical Control of Relaxation Using Intact Cardiac Trabeculae

Diastolic dysfunction is a common phenotype across cardiovascular disease presentations. In addition to elevated cardiac stiffness (elevated left ventricular end-diastolic pressure), impaired cardiac relaxation is a key diagnostic indicator of diastolic dysfunction. While relaxation requires the removal of cytosolic calcium and deactivation of sarcomeric thin filaments, targeting such mechanisms has yet to provide effective treatments. Mechanical mechanisms, such as blood pressure (i.e., afterload), have been theorized to modify relaxation. Recently, we showed that modifying the strain rate of a stretch, not afterload, was both necessary and sufficient to modify the subsequent relaxation rate of myocardial tissue. The strain rate dependence of relaxation, called the mechanical control of relaxation (MCR), can be assessed using intact cardiac trabeculae. This protocol describes the preparation of a small animal model, experimental system and chamber, isolation of the heart and subsequent isolation of a trabecula, preparation of the experimental chamber, and experimental and analysis protocols. Evidence for lengthening strains in the intact heart suggests that MCR might provide new arenas for better characterization of pharmacological treatments, along with a method to assess myofilament kinetics in intact muscles. Therefore, studying the MCR may elucidate a path to novel approaches and new frontiers in the treatment of heart failure.

Rapid myocardial and cardiac relaxation is essential for normal physiology. Mechanical relaxation mechanisms are now known to be dependent on strain rate. This protocol provides an overview of the acquisition and analysis of experiments to further study the mechanical control of relaxation.

Diastolic dysfunction is a common phenotype across cardiovascular disease presentations. In addition to elevated cardiac stiffness (elevated left ...

Multimodality Diagnosis of Mesenteric Ischemia

Early diagnosis of mesenteric ischemia remains challenging because mesenteric ischemia presents with no key symptoms or physical findings, and no laboratory data specifically indicates intestinal tissue ischemic status before necrosis develops. While computed tomography is the standard for diagnostic imaging, there are several limitations: (1) repeated assessments are associated with increased radiation exposure and risk of renal damage; (2) the computed tomography findings can be misleading because necrosis occasionally occurs despite opacified mesenteric arteries; and (3) computed tomography is not necessarily available within the golden time of salvaging the intestines for those patients in the operating room or at a place far from the hospital. This article describes a challenge to overcome such limitations using ultrasonography and near-infrared light, including clinical studies. The former is capable of providing not only morphologic and kinetic information of the intestines but also perfusion of the mesenteric vessels in real-time without transferring the patient or exposing them to radiation. Transesophageal echocardiography enables precise assessment of mesenteric perfusion in the OR, ER, or ICU. Representative findings of mesenteric ischemia in seven aortic dissection cases are presented. Near-infrared imaging with indocyanine green helps visualize the perfusion of vessels and intestinal tissues although this application requires laparotomy. Findings in two cases (aortic aneurysm) are shown. Near-infrared spectroscopy demonstrates oxygen debt in the intestinal tissue as digital data and can be a candidate for early detection of mesenteric ischemia without laparotomy. The accuracy of these assessments has been confirmed by intraoperative inspections and postoperative course (prognosis).

This article presents a multimodal approach that aims to overcome the limitations of traditional methods in detecting mesenteric ischemia and preventing bowel necrosis. The presented technique offers a promising solution by combining state-of-the-art ultrasonography with cutting-edge near-infrared light technologies.

Early diagnosis of mesenteric ischemia remains challenging because mesenteric ischemia presents with no key symptoms or physical findings, and no ...

Rodent Model of Intestinal Ischemia-Reperfusion Injury via Occlusion of the Superior Mesenteric Artery

Intestinal ischemia-reperfusion injury (IRI) is associated with a myriad of conditions in both veterinary and human medicine. Intestinal IRI conditions, such as gastric dilatation volvulus (GDV), mesenteric torsion, and colic, are observed in animals such as dogs and horses. An initial interruption of blood flow causes tissues to become ischemic. Although necessary to salvage viable tissue, subsequent reperfusion can induce further injury. The main mechanism responsible for IRI is free radical formation upon reperfusion and reintroduction of oxygen into damaged tissue, but there are many other components involved. The resulting local and systemic effects often impart a poor prognosis. 
Intestinal IRI has been the subject of extensive research over the past 50 years. An in vivo rodent model in which the base of the superior mesenteric artery (SMA) is temporarily ligated is currently the most common method used to study intestinal IRI. Here, we describe a model of intestinal IRI utilizing isoflurane anesthesia in 21% O2 medical air that yields reproducible injury, as demonstrated by consistent histopathology of the small intestines. Tissue injury was also assessed in the colon, liver, and kidneys.

We describe how to generate a widely used surgical model of intestinal ischemia-reperfusion injury (IRI) in rodents. The procedure involves occlusion of the superior mesenteric artery followed by the restoration of blood flow. This model is useful for studies investigating occlusive causes of intestinal IRI in both veterinary and human medicine.

Intestinal ischemia-reperfusion injury (IRI) is associated with a myriad of conditions in both veterinary and human medicine. Intestinal IRI conditions, ...