Authors would like to acknowledge Drs. Paul Huang and Dmitriy Atochin for use of DMT pressure myograph and Drs. Binglan Yu and Chong Lei for providing mice fed with high fat diet or standard diet.
Sources of Funding
This work was supported by Scientist Development Grant 10SDG2610313 from the American Heart Association (to E. S. Buys), and an Eleanor and Miles Shore 50th Anniversary Fellowship program for Scholars in Medicine from Harvard Medical School (to E. S. Buys).

1. Preparation of Solution
<ol>
	<li>500X EDTA stock: weigh 500 mg EDTA-Na2&bull;2H2O and dissolve in 50 ml deionized water. Store at room temperature.</li>
	<li>KCl depolarizing solution.
	<ol>
		<li>Prepare K10X stock solution: 3.69 g NaCl, 18.64 g KCl, 0.36 g MgSO4 anhydrous, 0.41 g KH2PO4, 0.46 g CaCl2&bull;2H2O. Dissolve in 250 ml deionized water. Store at room temperature.</li>
		<li>For 100 ml final KCl depolarizing solution 1X: Dissolve 0.21 NaHCO3, 0.18 g glucose in 90 ml deionized water. Add 10 ml of K10X stock solution. Add 95 &mu;l of 500X EDTA solution. Mix well. Store at 4 &deg;C. Use within one week.</li>
	</ol>
	</li>
	<li>Prepare fresh HEPES-PSS solution on the day of experiment (For 1 liter).
	<ol>
		<li>6.96 g NaCl, 0.35 g KCl, 0.288 g MgSO4&bull;7H2O, 0.1605 g KH2PO4, 2.38 g HEPES. Dissolve in 100 ml deionized water. Mark as flask A.</li>
		<li>0.312 g CaCl2&bull;2H2O. Dissolve in 100 ml deionized water. Mark as flask B.</li>
		<li>1.25 g NaHCO3, 0.991 g glucose. Dissolve in 98 ml deionized water. Mark as flask C.</li>
	</ol>
	</li>
	<li>Add 2 ml of 500X EDTA in flask C. Take 700 ml deionized water in a big flask. Pour contents of flask A, B and C in big flask with continuous vortexing. Adjust the pH to 7.4 with NaOH.</li>
</ol>
2. Drugs Used
<ol>
	<li>Phenylephrine (10-2 M): Dissolve 20.37 mg in 10 ml deionized water. Aliquot 1 ml and store at -80 &deg;C. Serially dilute to obtain 10-3, 10-4, 10-5, and 10-6 mol/L on the day of experiment.</li>
	<li>Acetylcholine (10-2 M): Dissolve 18.17 mg in 10 ml deionized water. Aliquot 1 ml and store at -80 &deg;C. Serially dilute to obtain 10-3, 10-4, 10-5, and 10-6 mol/L on the day of experiment.</li>
</ol>
3. Mice
Male WT and sGC&alpha;1-/- mice on the S6 and B6 background were studied throughout this study. The sGC&alpha;1-/- were generated as described previously 4,5. Housing and procedures involving experimental animals (mice) were approved by the Subcommittee on Research Animal Care of Massachusetts General Hospital, Boston, MA.
4. Measurement of Vascular Reactivity in Mesenteric Artery
<ol>
	<li>Euthanize the mice with pentobarbital (200 mg/kg, i.p.). Open the abdominal cavity and dissect out the mesenteric tissue. Isolate and dissect a mesenteric artery of 3rd order free of connective and adipose tissue, and place the artery in ice-cold HEPES-PSS solution pre-equilibrated with 95% O2 / 5% CO2 for 15 min. Cut rings of 2-3 mm length with no branching from the mesenteric artery. Differentiating between arteries and veins at micro vessels level is problematic when there is no blood in the vessels. Therefore, we recommend identifying the vessel while it is still intact in the animal before dissecting it out.</li>
	<li>Fill in the glass cannulas in the pressure myograph chamber (DMT Model 110P&amp;11P version 1.31, Figure 2) with HEPES-PSS solution with the help of a 10 ml syringe through inlet and outlet valves. Filling of cannula should be done gently and carefully as excessive pressure can damage the fragile transducer connected to cannulas. After filling cannulas close both inlet and outlet valves tightly.</li>
	<li>Mount one end of the vessel onto right cannula and carefully tie it with one fine strand of nylon suture. With the help of a syringe, flush and fill in the vessel with HEPES solution via inlet valve. Bring in the left cannula closer to the vessel and mount the other end of vessel onto it. Tie it with nylon suture. Fill the chamber with up to 10 ml with HEPES solution. Check for the leak by gently pushing HEPES solution via inlet valve with the help of a syringe.</li>
	<li>Install the chamber on the myograph and under the video camera. Start the oxygen and heat (37 &deg;C) in the chamber. Connect the inlet valve with tube P1 from the first HEPES solution reservoir, while the valve is closed. Let any bubble and solution pass through the tube until there are no bubbles in the tube. Open the valve.</li>
	<li>Connect the left valve of the chamber with tube P2 coming from pressure regulator. Again check for any bubbles. There should not be any bubbles or leakage in the entire system.</li>
	<li>Go to the pressure menu on the myo-interface panel or in the software and turn ON the pump while turning OFF the flow.</li>
	<li>Open the program and click COLLECT. Vessel should be seen on the screen now (Figure 3). The vessel is visualized with a camera fitted on a microscope, allowing monitoring and analysis of the vessel lumen, vessel diameter, and wall thickness.</li>
	<li>Gradually raise the interluminal pressure via the P1 valve by selecting P1 pressure in the myo-interface panel or in the program and enter the pressure value as follows; 5 mmHg &rarr; 10 &rarr; 20 &rarr; 40 &rarr; 60 mmHg. Vessel sometime tends to twist or convolute while pressure is increasing; adjust the tension or strain accordingly with the help of vertical or longitudinal micropositioner (Figure 2). Equilibrate the vessel at 60 mmHg and 37 &deg;C at least for 45 min. Change the bath solution once with pre-warmed HEPES during equilibration.</li>
	<li>Apply 10 ml of KCl depolarizing solution, prewarmed at 37 &deg;C, to the bath to fully depolarize smooth muscle cells and achieve maximum constriction. After stable constriction, rinse the bath with HEPES solution 3 times every 10 min.</li>
	<li>Check the viability of the vessel and integrity of the endothelium by stable and reproducible responses to the addition of phenylephrine (10-5 M) and acetylcholine (10-5 M). Rinse 3 times with HEPES solution every 10 min.</li>
	<li>Add phenylephrine (10-5 M) to preconstrict the vessel, and when a stable constriction has been obtained, perform a cumulative concentration- vasodilation response curve (CRC) by sequential addition of increasing doses of acetylcholine (10-9, 3 x 10-9, 10-8, 3 x 10-8, 10-7, 3 x 10-7, 10-6, 3 x 10-6, and 10-5 M). After last dose, rinse 3 times with HEPES solution every 10 min before starting new CRC.</li>
	<li>Determine the passive lumen diameter by applying Ca2+-free PSS containing 2 mM EGTA. From the recorded tracings measure lumen diameter for each dose response (Figure 4), which will be used to calculate all parameters.</li>
	<li>Express all acetylcholine relaxation responses as percentage change in lumen diameter after phenylephrine preconstriction compared with the difference between calcium-free diameter and diameter after phenylephrine constriction, using the following equation:</li>
	<li>Percentage dilation = 100% x [(Dx - Di)/(DCa-free - Di)], where D is the measured lumen diameter and x, i, and Ca-free denote arterial diameters at each dose of agonist (x), initial diameter following phenylephrine constriction (i), and in Ca2+-free buffer (Ca-free).</li>
</ol>
5. Statistical Analysis
All continuous measurements are expressed as mean &plusmn;SEM. The vascular reactivity in mesenteric arteries is analyzed by repeated-measures two-way ANOVA. In all cases, P &lt; 0.05 is considered statistically significant.

<ol>
	<li>Bridges, L. E., Williams, C. L., Pointer, M. A., Awumey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenteric+artery+contraction+and+relaxation+studies+using+automated+wire+myography.">Mesenteric artery contraction and relaxation studies using automated wire myography.</a> J. Vis .Exp. (55), e3119 (2011).</li><li>Arribas, S. M., Daly, C. J., McGrath, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+vascular+remodeling+by+confocal+microscopy.">Measurements of vascular remodeling by confocal microscopy.</a> Methods Enzymol. 307, 246-273 (1999).</li><li>Lei, C., Yu, B., 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=Inhaled+Nitric+Oxide+Attenuates+the+Adverse+Effects+of+Transfusing+Stored+Syngeneic+Erythrocytes+in+Mice+with+Endothelial+Dysfunction+after+Hemorrhagic+Shock.">Inhaled Nitric Oxide Attenuates the Adverse Effects of Transfusing Stored Syngeneic Erythrocytes in Mice with Endothelial Dysfunction after Hemorrhagic Shock.</a> Anesthesiology. , (2012).</li><li>Buys, E. S., Cauwels, A., 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=sGC&#945;1&#946;1+attenuates+cardiac+dysfunction+and+mortality+in+murine+inflammatory+shock+models.">sGC&#945;1&#946;1 attenuates cardiac dysfunction and mortality in murine inflammatory shock models.</a> Am. J. Physiol. Heart Circ. Physiol. 297 (2), H654-H663 (2009).</li><li>Buys, E. S., Sips, P., 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=Gender-specific+hypertension+and+responsiveness+to+nitric+oxide+in+sGC&#945;1+knockout+mice.">Gender-specific hypertension and responsiveness to nitric oxide in sGC&#945;1 knockout mice.</a> Cardiovasc. Res. 79 (1), 179-186 (2008).</li><li>Panza, J. A., Quyyumi, A. A., Brush, J. E., Epstein, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+endothelium-dependent+vascular+relaxation+in+patients+with+essential+hypertension.">Abnormal endothelium-dependent vascular relaxation in patients with essential hypertension.</a> N. Engl. J. Med. 323 (1), 22-27 (1990).</li><li>Huang, P. L., Huang, Z., 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=Hypertension+in+mice+lacking+the+gene+for+endothelial+nitric+oxide+synthase.">Hypertension in mice lacking the gene for endothelial nitric oxide synthase.</a> Nature. 377 (6546), 239-242 (1995).</li><li>Friebe, A., Koesling, 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+function+of+NO-sensitive+guanylyl+cyclase:+what+we+can+learn+from+genetic+mouse+models.">The function of NO-sensitive guanylyl cyclase: what we can learn from genetic mouse models.</a> Nitric Oxide. 21 (3-4), 149-156 (2009).</li><li>Ehret, G. B., Munroe, P. B., 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=Genetic+variants+in+novel+pathways+influence+blood+pressure+and+cardiovascular+disease+risk.">Genetic variants in novel pathways influence blood pressure and cardiovascular disease risk.</a> Nature. 478 (7367), 103-109 (2011).</li><li>Buys, E. S., Raher, 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=Genetic+modifiers+of+hypertension+in+soluble+guanylate+cyclase+alpha1-deficient+mice.">Genetic modifiers of hypertension in soluble guanylate cyclase alpha1-deficient mice.</a> J. Clin. Invest. 122 (6), 2316-2325 (2012).</li><li>Kauffenstein, G., Laher, I., Matrougui, K., Guerineau, N. C., Henrion, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+role+of+G+protein-coupled+receptors+in+microvascular+myogenic+tone.">Emerging role of G protein-coupled receptors in microvascular myogenic tone.</a> Cardiovascular Research. 95 (2), 223-232 (2012).</li><li>Ruilope, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertension+in+2010:+Blood+pressure+and+the+kidney.">Hypertension in 2010: Blood pressure and the kidney.</a> Nat. Rev. Nephrol. 7 (2), 73-74 (2011).</li><li>Michael, S. K., Surks, H. K., 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=High+blood+pressure+arising+from+a+defect+in+vascular+function.">High blood pressure arising from a defect in vascular function.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (18), 6702-6707 (2008).</li><li>Mendelsohn, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+hypertension,+the+kidney+is+not+always+the+heart+of+the+matter.">In hypertension, the kidney is not always the heart of the matter.</a> J. Clin. Invest. 115 (4), 840-844 (2005).</li></ol>

The authors have nothing to disclose.

Mice are the experimental model of choice for many investigators, in part because of the possibility to introduce genetic modifications, thereby generating mouse models for human pathophysiology. The vasoactive status of small resistance but not of larger conduit vessels largely defines the regulation of blood flow throughout the vascular system 11. The size of resistance arteries in small animals, such as mice, prevents the use of a wire myograph to study microvascular function. Pressure myographs not only overcome this limitation but also allow for the measurement of vascular function under near physiological conditions.
We previously reported that male mice deficient in the sGC&alpha;1 are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) 5 but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6) 4. We hypothesized that this strain-related disparity in blood pressure is due to the differences in vascular reactivity between sGC&alpha;1-/- mice on the B6 and S6 backgrounds. Using a pressure myograph, we studied endothelium-dependent vasorelaxation responses in 3rd order mesenteric arteries. Acetylcholine is a muscarinic receptor agonist that binds to its receptor in the endothelium to activate endothelial nitric oxide synthase (eNOS) via increasing intracellular Ca2+ concentration. NO can subsequently activate sGC in the underlying smooth muscle cells layer to induce relaxation.
Using pressure myography to compare the ability of endothelial derived NO to vasorelax mesenteric arteries isolated from WT and sGC&alpha;1-/- mice on the S6 and B6 background, we identified differences in vascular reactivity between sGC&alpha;1-/- mice on the B6 and S6 backgrounds 10. sGC&alpha;1-deficiency was associated with impaired endothelium-dependent vasorelaxation in both S6 and B6 mice. However, this impairment was greater in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. The decreased ability of acetylcholine to induce vasorelaxation in S6 mice likely contributes to the increase in blood pressure observed in sGC&alpha;1-/-S6 mice but not in sGC&alpha;1-/-B6 mice. Although elevated blood pressure is often attributed to renal abnormalities 12, these results strengthen the emerging hypothesis that primary abnormalities of vascular smooth muscle tone, for example as associated with impaired NO-cGMP signaling 10,13, can directly contribute to the development of systemic hypertension 14.
In conclusion, the pressure myograph technique is a very powerful tool in the hands of the vascular physiologist or pharmacologist and can be used to analyze micro vessel function. Our study demonstrates the applicability of pressure myography in detecting underlying vascular abnormalities in a model of systemic hypertension.

Pressure myograph systems are used to measure the physiological function and properties of small arteries, veins and other vessels. An intact small segment of an artery or vein is mounted onto two small glass cannulas and pressurized to a suitable luminal pressure, allowing the vessel to maintain most of its in vivo characteristics (Figures 1 and 2). The near physiological condition in a pressure myograph reflects the in vivo behavior of the vascular bed, allowing the investigation of intrinsic properties (e.g. myogenic tone) of isolated vessels. Some of the advantages of pressure myography over wire myography, where muscle contraction is assessed by direct mechanical coupling to a force transducer 1, include (i) that micro resistance arteries, which define the overall resistance developed in vascular system, can be studied, whereas wire myograph is limited to larger conduit arteries, (ii) that the risk to damage the endothelium is reduced since no wires need to be passed through the vessel lumen, (iii) that the natural shape of the vessel is better maintained, and (iv) that vessel dimension can be studied at a wide range of pressures or shear stress 2.
Studying micro vessels can be more informative than studying larger conduit arteries to help understand the pathophysiology and molecular mechanisms that contribute to altered vascular tone in cardiovascular diseases such as hypertension. For example, impairment of endothelial function associated with feeding mice a high fat diet for 8 weeks could be demonstrated in 2nd-order mesenteric arteries 3 but not in aortic rings (Figure 3). Another advantage of pressure myography is that intrinsic myogenic constriction of the pressurized vessel is present, and that the role and function of the endothelium in this phenomenon can be studied. Here, we describe the use of a pressure myograph to study vascular reactivity of mouse mesenteric 3rd order resistance arteries in a setting of impaired nitric oxide (NO)-cGMP signaling.

<table border="1">
		<tbody>
		 <tr>
			<td>Name</td>
			<td>Company</td>
			<td>Cataloge No.</td>
		 </tr>
		 <tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>BP358</td>
		 </tr>
		 <tr>
			<td>CaCl2 (2H2O)</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
		 </tr>
		 <tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
		 </tr>
		 <tr>
			<td>MgSO4 </td>
			<td>Fisher Scientific</td>
			<td>M65-500</td>
		 </tr>
		 <tr>
			<td>KH2PO4 </td>
			<td>Sigma</td>
			<td>P3786</td>
		 </tr>
		 <tr>
			<td>NaHCO3 </td>
			<td>Fisher Scientific</td>
			<td>BP328</td>
		 </tr>
		 <tr>
			<td>NaOH</td>
			<td>Fisher Scientific</td>
			<td>S318</td>
		 </tr>
		 <tr>
			<td>D-Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
		 </tr>
		 <tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>BP121</td>
		 </tr>
		 <tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
		 </tr>
		 <tr>
			<td>Phenylephrine</td>
			<td>Acros Organics</td>
			<td>AC20724</td>
		 </tr>
		 <tr>
			<td>Acetylcholine</td>
			<td>Sigma</td>
			<td>A6625</td>
		 </tr>
		 <tr>
			<td>Pressure Myograph System</td>
			<td>DMT</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

assessing murine resistance artery function using pressure myography

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
	To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the &alpha;1 subunit of soluble guanylate cyclase (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6). Using pressure myography, we demonstrate that sGC&alpha;1-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice, likely contributing to the higher blood pressure in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. 
	Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

NO is centrally involved in maintenance of blood pressure homeostasis both in humans 6 and in animal models 7. The ability of NO to control vascular smooth muscle relaxation is mediated by soluble guanylate cyclase (sGC), a heme-containing heterodimeric enzyme that generates cGMP 8. Recently, a blood pressure-modifying genetic variant was identified in a locus that contains the sGC&alpha;1 and sGC&beta;1 genes, illustrating the relevance of sGC in regulating blood pressure in humans 9. Interestingly, male mice deficient in the &alpha;1 subunit of sGC (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) 5 but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6) 4. We used pressure myography to study vascular relaxation in response to acetylcholine in mesenteric resistance arteries isolated from WT and sGC&alpha;1-/- mice on the S6 and B6 backgrounds and preconstricted with phenylephrine (Figure 4). We studied resistance arteries because they define ultimate resistance and compliance of the vascular system and thus directly regulate blood pressure. sGC&alpha;1-deficiency was associated with a decreased ability of a cetylcholine to induce vascular relaxation, regardless of the genetic background of the mice studied (Figure 4 and 5). However, endothelium-dependent relaxation was more markedly impaired in sGC&alpha;1-/- mice on the S6 background than in sGC&alpha;1-/- mice on the B6 background (Figure 5) 10. Together, these findings suggest that the decreased sensitivity of the vasculature to endothelial-dependent relaxation may contribute to the strain-specific hypertension in sGC&alpha;1-/- mice.
<img alt="Figure 1" src="/files/ftp_upload/50328/50328fig1.jpg" /> 
Figure 1. Schematic depicting the various parts of the pressure myograph chamber (DMT). Inset shows a mounted vessels on cannulas in the chamber. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50328/50328fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/50328/50328fig2.jpg" /> 
Figure 2. Representative images, captured during video recording, of a 2nd order mouse mesenteric artery under (A) basal and (B) phenylephrine-induced constriction. The outer diameter and vessel lumen are delineated by green and red lines, respectively. The traces in the right panels represent the signal intensity, measured in the area defined by the blue box in the images on the left, for outer diameter (green arrows) and lumen diameter (red arrows).
<img alt="Figure 3" src="/files/ftp_upload/50328/50328fig3.jpg" /> 
Figure 3. Endothelial dysfunction in mice fed with high-fat diet for 8 weeks was not apparent in aortic rings. Endothelial function was measured by the vasorelaxation response induced by acetylcholine ( 10-9 to 10-5 M) in phenylephrine ( 10-5 M) pre-contracted aortic rings. Standard diet, wild-type mice fed a standard diet (n=8); High-fat diet, wild-type mice fed a high-fat diet for 4-6 weeks (n=13).
<img alt="Figure 4" src="/files/ftp_upload/50328/50328fig4.jpg" /> 
Figure 4. Representative original tracings of a dose response curve of acetylcholine on phenylephrine-proconstricted 2nd order mesenteric arteries isolated from a WTB6 (A) and a sGC&alpha;1-/-S6 mouse (B). Arrows represent cumulative addition of increasing doses of acetycholine ( 10-9 to 10-5 M). The X axis represents time (in seconds), the Y axis represents lumen diameter (in &mu;M). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50328/50328fig4large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 5" src="/files/ftp_upload/50328/50328fig5.jpg" /> 
Figure 5. The strain-specific hypertension in sGC&alpha;1-/- mice is associated with greater impairment of vascular reactivity in sGC&alpha;1-/-S6 mice than in sGC&alpha;1-/-B6 mice. The ability of acetylcholine ( 10-9 to 10-5) to induce relaxation is quantitated in phenylephrine-precontracted ( 10-5 M) mesenteric arteries from male wild-type (WT, closed symbols, full line) and sGC&alpha;1-/- (open symbnols, dashed line) mice on the B6 (circles) or S6 (squares) genetic background. Acetycholine-induced vascular relaxation is impaired to a greater extent in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. N=3,5,3 and 6 for sGC&alpha;1-/-S6, sGC&alpha;1-/-B6, WTS6, respectively. &dagger;*P &lt; 0.05 vs sGC&alpha;1-/-B6, P &lt; 0.05 vs. sGC&alpha;1-/- of the same genetic background. Adapted form Buys, et al. (2012).

Watch this Scientific Journal Video about Assessing Murine Resistance Artery Function Using Pressure Myography at JoVE.com

Assessing Murine Resistance Artery Function Using Pressure Myography

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure myography.

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

assessing-murine-resistance-artery-function-using-pressure-myography

Nanyang Technological University

Research

JoVE Journal

Biology

21.9K Views.  Massachusetts General Hospital, Harvard Medical School. In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGCα1-/- mice using pressure myography.

Video: Assessing Murine Resistance Artery Function Using Pressure Myography

Measuring Blood Pressure in Mice using Volume Pressure Recording, a Tail-cuff Method

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume. A specially designed differential pressure transducer and an occlusion tail-cuff measure the total blood volume in the tail without the need to obtain the individual pulse signal. Special attention is afforded to the length of the occlusion cuff in order to derive the most accurate blood pressure readings. VPR can easily obtain readings on dark-skinned rodents, such as C57BL6 mice and is MRI compatible. The CODA system provides you with measurements of six (6) different blood pressure parameters; systolic and diastolic blood pressure, heart rate, mean blood pressure, tail blood flow, and tail blood volume. Measurements can be made on either awake or anesthetized mice or rats. The CODA system includes a controller, laptop computer, software, cuffs, animal holders, infrared warming pads, and an infrared thermometer. There are seven different holder sizes for mice as small as 8 grams to rats as large as 900 grams.

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. This tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume.

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50 can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50 using the same methodology.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

Assessing Cerebral Autoregulation via Oscillatory Lower Body Negative Pressure and Projection Pursuit Regression

The process by which cerebral perfusion is maintained constant over a wide range of systemic pressures is known as &#8220;cerebral autoregulation.&#8221; Effective dampening of flow against pressure changes occurs over periods as short as ~15 sec and becomes progressively greater over longer time periods. Thus, slower changes in blood pressure are effectively blunted and faster changes or fluctuations pass through to cerebral blood flow relatively unaffected. The primary difficulty in characterizing the frequency dependence of cerebral autoregulation is the lack of prominent spontaneous fluctuations in arterial pressure around the frequencies of interest (less than ~0.07 Hz or ~15 sec). Oscillatory lower body negative pressure (OLBNP) can be employed to generate oscillations in central venous return that result in arterial pressure fluctuations at the frequency of OLBNP. Moreover, Projection Pursuit Regression (PPR) provides a nonparametric method to characterize nonlinear relations inherent in the system without a priori assumptions and reveals the characteristic non-linearity of cerebral autoregulation. OLBNP generates larger fluctuations in arterial pressure as the frequency of negative pressure oscillations become slower; however, fluctuations in cerebral blood flow become progressively lesser. Hence, the PPR shows an increasingly more prominent autoregulatory region at OLBNP frequencies of 0.05 Hz and below (20 sec cycles). The goal of this approach it to allow laboratory-based determination of the characteristic nonlinear relationship between pressure and cerebral flow and could provide unique insight to integrated cerebrovascular control as well as to physiological alterations underlying impaired cerebral autoregulation (e.g., after traumatic brain injury, stroke, etc.).

Cerebral perfusion is maintained across a range of pressures via cerebral autoregulation. However, characterizing autoregulation requires prominent pressure fluctuations at regulated frequencies. The described protocol will show how oscillatory lower body negative pressure can generate pressure fluctuations to provide data for projection pursuit regression for quantification of the autoregulatory curve.

The process by which cerebral perfusion is maintained constant over a wide range of systemic pressures is known as &#8220;cerebral autoregulation.&#8221; ...

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative ...

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular systolic and diastolic function. Measures of ventricular contractility and compliance are obtained through examination of cardiac response to changes in afterload and preload. These techniques were originally developed nearly three decades ago to measure cardiac function in large mammals and humans. The application of these analyses to small mammals, such as mice, has been accomplished through the optimization of microsurgical techniques and creation of conductance catheters. Conductance catheters allow for estimation of the blood pool by exploiting the relationship between electrical conductance and volume. When properly performed, these techniques allow for testing of cardiac function in genetic mutant mouse models or in drug treatment studies. The accuracy and precision of these studies are dependent on careful attention to the calibration of instruments, systematic conduct of hemodynamic measurements and data analyses. We will review the methods of conducting pressure-volume loop experiments using a conductance catheter in mice.

Cardiac pressure-volume loop analysis is the most comprehensive way to measure cardiac function in the intact heart. We describe a technique to perform and analyze cardiac pressure volume loops, using conductance catheters.

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular ...

Intracavernosal Pressure Recording to Evaluate Erectile Function in Rodents

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. Rodents are employed by many studies to research the physiology/pathology of erectile function. Erectile function in rodents can be evaluated by measuring the intracavernosal pressure (ICP). In practice, ICP can be monitored following electrical stimulation of the cavernous nerves (CNs). The arterial pressure of the carotid artery (the mean arterial pressure) is used as the reference for ICP. Using ICP recording protocols, many key parameters of erectile function can be measured from the ICP response curve. The ICP measurement provides more information than the apomorphine-induced penile erection test, and is cheaper than telemetric monitoring of the corpus spongiosum penis, making this method the most popular one to evaluate erectile function. However, compared to the easily-performed APO-induced erectile function test, successful ICP recordings require attention to detail, practice, and adherence to the operation method. In this work, an introduction to ICP recording in rats is provided to complement the procedure efficiently.

Intracavernosal pressure recording (ICP) is an important method to evaluate the erectile function of experimental animals. Here, a detailed protocol is demonstrated for the recording procedure of ICP by catheterizing the crura penis and then electrically stimulating the cavernous nerves in rats.

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. ...