Sandeep Khurana is supported by NIH (K08DKO81479). Vikrant Rachakonda is supported by (T32DK067872).

The examples shown here are from experiments approved by IACUC at Georgia Regents University - Protocol No: # 2011-0408
1. Preparation of Reagents
<ol>
	<li>Prepare dissection solution stock: For 500 ml of stock dissection solution (5x), dissolve 21.18 g NaCl, 0.875 g KCl, 0.739 g MgSO4, 1.049 g MOPS and 0.019 g EDTA in 450 ml of Milli-Q water. Adjust pH to 7.3-7.4 using 1 N NaOH. Make up the volume to 500 ml with Milli-Q water. Stock solution can be stored up to 7-10 days. See Table 1 for a list of chemicals and their vendors. See Table 2 for concentration in mM.</li>
	<li>Prepare working dissection solution: Prepare fresh working dissection solution every day. For 100 ml working solution, dissolve 0.091 g glucose, 0.016 g NaH2PO4 and 0.022 g sodium pyruvate in 79.8 ml of Milli-Q water. Add 0.2 ml 1M CaCl2 and 20 ml dissection solution stock to bring volume to 100 ml.</li>
	<li>Prepare physiological salt solution (PSS): To prepare 1,000 ml PSS, dissolve 0.365 g KCl, 6.545 g NaCl, 0.296 g MgSO4, 0.163 g KH2PO4, 2.072 g glucose, 2.184 g NaHCO3 and 2.383 g HEPES in 950 ml of Milli-Q water. Adjust pH to 7.3-7.4 using 1 N NaOH. Make up the volume to 1,000 ml with Milli-Q water. Remove 2 ml of solution and replace it with 2 ml of 1 M CaCl2. (Fresh PSS needs to be prepared daily)</li>
	<li>Prepare calcium (Ca2+) free PSS: For 100 ml PSS without Ca2+, dissolve 0.036 g KCl, 0.654 g NaCl, 0.029 g MgSO4, 0.016 g KH2PO4, 0.207 g glucose, 0.218 g NaHCO3, 0.238 g HEPES, 0.015 g EGTA and 0.0026 g Sodium nitroprusside (SNP) in 95 ml of Milli-Q water. Adjust pH to 7.3-7.4 using 1 N NaOH. Make up the volume to 100 ml with Milli-Q water.</li>
</ol>
2. Preparation of Glass Cannulas
<ol>
	<li>Pull glass pipettes to generate the 100-150 &#956;m tipped cannulas using a pipette puller as per manufacturer&#39;s guidelines.</li>
	<li>Bevel the glass cannula tips using a microelectrode beveller, fire polish them and bend the glass cannula tips by ~45&#176; using a heater probe.</li>
	<li>Load the cannulas into micropipette holder and attach the micropipette holder on to the perfusion chamber.</li>
</ol>
3. Preparation of Perfusion Chamber
<ol>
	<li>Rinse perfusion chamber with Milli-Q water followed by dissection solution for 5 min each. Load the chamber with 2 ml of dissection solution.</li>
	<li>Suction dissection solution through cannula using 10 ml syringe and fill carefully the entire cannula and the attached tubing without any bubbles. Apply suction gently to prevent generation of bubbles.</li>
	<li>Prepare two sutures with a half-knot each using blunt forceps. Since ophthalmic monofilament nylon sutures (10-0, 0.2 metric) are used to prepare the knots that are only 1-2 mm in diameter, dissection microscope may be needed.</li>
	<li>Visualizing under dissection microscope, use dissection forceps to load both cannulas with partially closed suture knots slightly away from the tip. Later these knots will be slid carefully onto the cannulated arterial ends and closed completely.</li>
</ol>
4. Collection of Mesenteric Artery Arcade from Sprague-Dawley Rats
<ol>
	<li>Seek approval of the local Institutional Animal Care and Use Committee (IACUC) before conducting these experiments. House animals in the animal facility with controlled temperature and lighting and allow free access to water and a commercial rodent chow.</li>
	<li>Anesthetize rats by intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). Confirm deep anesthesia&#160;by toe-pinch and if needed administer additional anesthetics.</li>
	<li>After confirming the surgical anesthesia, euthanize the animal by decapitation. Follow AAALAC guidelines for utilizing appropriate methods for animal euthanasia.</li>
	<li>Use a dissection scissor and a forceps to perform a mid-line laparotomy from pelvis&#160;to sternum. This is done in two steps: first, incise the skin and second, incise the underlying muscle layer. Care must be taken not to injure the intra-abdominal organs.</li>
	<li>Cut the proximal end of intestine close to the pylorus and the distal end close to the ileo-cecal junction. Tie both ends separately to prevent leak of chyme and feces thus avoiding contamination of extracellular bathing solution. Incise the mesentery at its base near the feeding vasculature i.e., superior mesenteric artery and transfer the entire small intestinal mesenteric bed to a 50 ml beaker containing ice-cold dissection solution.</li>
	<li>Allow harvested tissue to stay in ice cold dissection solution for 5 min and rinse with fresh dissection solution to get rid of blood.</li>
</ol>
5. Isolation and Cannulation of 4th Order Mesenteric Artery
<ol>
	<li>Pin down the proximal end of the intestine on the right hand side in a sylgard-coated dish. Extend the remaining intestine in a counter clockwise path, pinning the segment down to spread the mesentery and exposing the blood vessels (Figure 1). Note: We isolate arterial segments at room temperature. Otherwise we place the mesenteric arcade containing dish on ice. Some labs, including those at our institution, use chiller units to dissect arteries at 4 &#176;C.</li>
	<li>Under a stereo zoom microscope dissect out 3rd and 4th order small mesenteric arteries (~260 &#956;m) parallel to the small intestine using small scissors.&#160;First dissect away all the covering fat. Then dissect out the vein and isolate the artery with V-shaped branch point. Be careful not to puncture the selected segment. Start dissecting the fat near a 2nd order branch and find the way to 3rd or 4th order vessels.
	<ol>
		<li>Note: Arteries and veins can be distinguished based on their wall thickness - arterial wall is thicker than vein&#39;s. Moreover, when adjoining connective tissue is pulled gently perpendicular to the vessels, veins collapse readily while arteries don&#39;t. Since arteries with lumen diameter &#60;400 &#956;m are major sites of systemic vascular resistance, for this protocol we used 4th order rat mesenteric arteries (lumen diameter &#60;300 &#956;m).</li>
	</ol>
	</li>
	<li>Isolate a 4-5 mm section of artery parallel to the small intestine. Visualize all the 5th order branches embedding into small intestine and cut them slightly away from the origin of branches and preserve a portion. These preserved portions of branches serve as holding sites (with dissection forceps) for transferring arterial segments to a perfusion chamber and, subsequently guide their cannulation.</li>
	<li>Then cut the arterial segments by making 2 incisions distal to the 5th order branches on each side of the artery and transfer it to the perfusion chamber (see Figure 1C and legend).</li>
	<li>Cannulate one end of the vessels on one of glass micropipette (diameter: 100-150 &#956;m) using dissection forceps by holding the tips of the arterial segment with dissection forceps. Slide the previously loaded partially closed suture onto the cannulated end and secure it. Note: Proximal end of the artery may be cannulated onto the glass cannula that is connected to servo-controlled pressure-regulating device to mimic in situ environment.</li>
	<li>Attach a dissection solution loaded 10 ml syringe to the stopcock connected to this cannula such that dissection solution in the tubing connecting the cannula and stopcock merges with that in the syringe. Gently raise the syringe. The gravitational force on the solution will remove the intra vascular blood from the open end of the vessel. After removing the intra-arterial blood, close the stopcock. 
	Note: Alternatively, attach the stopcock to the pressure controller, turn it on and gently increase the pressure to 5-10 mm Hg to achieve the same result.</li>
	<li>Tie the distal end of the vessels onto a second glass cannula by carefully bringing the other cannula as close as possible to the untied end of the arterial segment. Slide the previously loaded partially closed suture onto the cannulated end and secure it. Care must be taken not tug or pull on the arterial segments. Make sure that stopcocks attached to both cannulas are closed.</li>
	<li>Transfer perfusion chamber on to the stage of inverted microscope equipped with live video recording.</li>
	<li>Connect the stopcock of cannula tied to the proximal end of arterial segment to a servo-controlled pressure-regulating device and make sure that stopcock attached to the other cannula remains closed to maintain stable intraluminal pressure.</li>
	<li>Next, attach the vacuum tubing to the suction port and the perfusion tubing to the perfusion port of the chamber. 
	Note: Beveled needle port is used for suction and blunt needle port for perfusion.</li>
	<li>Start perfusion of vessel with warm PSS through single inline solution heater (37 &#176;C, equilibrated with gas mixture: 5% CO2, 5% O2&#160;and 90% N to maintain neutral pH and adequate oxygenation17) at 2 ml/min using a peristaltic pump. Turn the vacuum on as well. Place a thermistor in the chamber to monitor temperature continuously.</li>
	<li>As the temperature of PSS in the chamber nears ~37 &#176;C (usually within 5 min), slowly increase intraluminal pressure from 20 to 100 mmHg and check vessels for leaks. This is done using the automatic pressure setting of pressure regulator. Discard vessels with leak and replace with another segment. The vessels with leaks will not be able to hold the pressure.</li>
	<li>Assess the arterial segment for bends while maintaining the pressure at 100 mmHg. Using the screw-lever, move the cannula to straighten the arterial segment. Do not over stretch the arterial segments; the goal is to mimic in vivo arterial segment length.</li>
	<li>Reduce the pressure to 70 mmHg (to mimic in vivo pressure in the mesenteric arcade18) and allow the arterial segment to stabilize and develop myogenic tone. Arteries may be pressurized variably (40-70 mmHg) according to experimental strategy and vascular bed. A previously published review provides an excellent review of variability in MT in arterial segments from different vascular beds8.</li>
</ol>
6. Measurement of Arterial Diameter
<ol>
	<li>View arteries at 10X objective on a microscope equipped with a monochrome video charge-coupled device camera. Measure luminal diameter using video frame grabber and real-time edge-detection system. A list of equipment used is provided in Table 3.</li>
	<li>Monitor and record vessel diameter continuously.</li>
	<li>Observe for development of MT. Note: We observed that in rat mesenteric resistance arteries, at 70 mmHg, development of MT is characterized by ~20% decrease in diameter. MT varies according to vascular bed and animal species.</li>
	<li>Confirm vascular viability by assessing vasoconstrictor and vasodilator responses to 1 &#956;M phenylephrine (Phe) and 1 &#956;M acetylcholine (ACh).</li>
	<li>At the end of each experiment, determine passive diameter (PD) by incubating arteries in Ca2+-free PSS for 20 min.</li>
</ol>
7. Myogenic Response
<ol>
	<li>Reduce the pressure to 20 mm Hg and allow the diameter to stabilize. Increase the intraluminal pressure in incremental steps (20, 40, 60, 80 and 100) and at each pressure step allow arteries to achieve a stable diameter (usually within 5 min).</li>
	<li>Reduce the intraluminal pressure to 20 mmHg and incubate the arterial segment in Ca2+-free PSS containing 0.39 mM EGTA and 0.1 mM SNP. Allow the arterial diameter to stabilize (usually 15 min).</li>
	<li>Repeat the pressure-step response in Ca2+-free PSS containing 0.39 mM EGTA and 0.1 mM SNP.</li>
</ol>
8. Interpretation of Results and Calculation Of Data
<ol>
	<li>Calculate the MT as the percent difference in diameter observed for Ca2+-containing versus Ca2+-free PSS at each pressure according to following calculation: 
	<img alt="Equation 1" fo:src="/files/ftp_upload/50997/50997eq1highres.jpg" src="/files/ftp_upload/50997/50997eq1.jpg" /></li>
	<li>For arteries undergoing vasomotion the diameter may be calculated by averaging the plateau phase for 1 min. Express the collected data as percent of maximal relaxation (%PD) according to the relationship: %PD = 100 x [&#916;D/PD]; &#916;D is the difference between the diameter before and after addition of any investigational compound (e.g. Phe); PD is passive diameter (also the maximal diameter).</li>
</ol>

<ol>
	<li>Christensen, K. L., 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=Location+of+resistance+arteries.">Location of resistance arteries.</a> J Vasc Res. 38, 1-12 (2001).</li><li>Rietzschel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidized+low-density+lipoprotein+cholesterol+is+associated+with+decreases+in+cardiac+function+independent+of+vascular+alterations.">Oxidized low-density lipoprotein cholesterol is associated with decreases in cardiac function independent of vascular alterations.</a> Hypertension. 52, 535-541 (2008).</li><li>Hill, M. A., Ege, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+and+passive+mechanical+properties+of+isolated+arterioles+from+STZ-induced+diabetic+rats.+Effect+of+aminoguanidine+treatment.">Active and passive mechanical properties of isolated arterioles from STZ-induced diabetic rats. Effect of aminoguanidine treatment.</a> Diabetes. 43, 1450-1456 (1994).</li><li>Osol, G., Cipolla, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+myogenic+and+adrenergic+mechanisms+in+isolated,+pressurized+uterine+radial+arteries+from+late-pregnant+and+nonpregnant+rats.">Interaction of myogenic and adrenergic mechanisms in isolated, pressurized uterine radial arteries from late-pregnant and nonpregnant rats.</a> Am J Obstet Gynecol. 168, 697-705 (1993).</li><li>Cipolla, M. J., McCall, A. L., Lessov, N., Porter, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reperfusion+decreases+myogenic+reactivity+and+alters+middle+cerebral+artery+function+after+focal+cerebral+ischemia+in+rats.">Reperfusion decreases myogenic reactivity and alters middle cerebral artery function after focal cerebral ischemia in rats.</a> Stroke. 28, 176-180 (1997).</li><li>Ren, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+myogenic+response+in+the+afferent+arteriole+of+spontaneously+hypertensive+rats.">Enhanced myogenic response in the afferent arteriole of spontaneously hypertensive rats.</a> Am J Physiol Heart Circ Physiol. 298, H1769-H1775 (2010).</li><li>Hayashi, K., Epstein, M., Saruta, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+myogenic+responsiveness+of+the+renal+microvasculature+in+experimental+hypertension.">Altered myogenic responsiveness of the renal microvasculature in experimental hypertension.</a> J Hypertens. 14, 1387-1401 (1996).</li><li>Schubert, R., 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=The+myogenic+response:+established+facts+and+attractive+hypotheses.">The myogenic response: established facts and attractive hypotheses.</a> Clin Sci (Lond). 96, 313-326 (1999).</li><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. , (2011).</li><li>Buus, N. H., Vanbavel, E., 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=Differences+in+Sensitivity+of+Rat+Mesenteric+Small+Arteries+to+Agonists+When+Studied+as+Ring+Preparations+or+as+Cannulated+Preparations.">Differences in Sensitivity of Rat Mesenteric Small Arteries to Agonists When Studied as Ring Preparations or as Cannulated Preparations.</a> British Journal of Pharmacology. 112, 579-587 (1994).</li><li>Falloon, B. J., Stephens, N., Tulip, J. R., Heagerty, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+small+artery+sensitivity+and+morphology+in+pressurized+and+wire-mounted+preparations.">Comparison of small artery sensitivity and morphology in pressurized and wire-mounted preparations.</a> Am J Physiol. 268, H670-H678 (1995).</li><li>Schubert, R., Wesselman, J. P. M., Nilsson, H., 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=Noradrenaline-induced+depolarization+is+smaller+in+isobaric+compared+to+isometric+preparations+of+rat+mesenteric+small+arteries.">Noradrenaline-induced depolarization is smaller in isobaric compared to isometric preparations of rat mesenteric small arteries.</a> Pflugers Archiv-European Journal of Physiology. 431, 794-796 (1996).</li><li>Khurana, S., Raina, H., Pappas, V., Raufman, J. P., Pallone, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+deoxycholylglycine,+a+conjugated+secondary+bile+acid,+on+myogenic+tone+and+agonist-induced+contraction+in+rat+resistance+arteries.">Effects of deoxycholylglycine, a conjugated secondary bile acid, on myogenic tone and agonist-induced contraction in rat resistance arteries.</a> PLoS One. 7, e32006 (2012).</li><li>Bayliss, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+local+reactions+of+the+arterial+wall+to+changes+of+internal+pressure.">On the local reactions of the arterial wall to changes of internal pressure.</a> J Physiol. 28, 220-231 (1902).</li><li>Hughes, J. M., Bund, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+myogenic+properties+of+the+spontaneously+hypertensive+rat.">Arterial myogenic properties of the spontaneously hypertensive rat.</a> Exp Physiol. 87, 527-534 (2002).</li><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>Raina, H., Zacharia, J., Li, M., Wier, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+by+Ca2+/calmodulin+of+an+exogenous+myosin+light+chain+kinase+in+mouse+arteries.">Activation by Ca2+/calmodulin of an exogenous myosin light chain kinase in mouse arteries.</a> J Physiol. 587, 2599-2612 (2009).</li><li>Fenger-Gron, J., Mulvany, M. J., Christensen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenteric+blood+pressure+profile+of+conscious,+freely+moving+rats.">Mesenteric blood pressure profile of conscious, freely moving rats.</a> J Physiol. 488 (Pt 3), 753-760 (1995).</li><li>Davis, M. J., Hill, 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=Signaling+mechanisms+underlying+the+vascular+myogenic+response).">Signaling mechanisms underlying the vascular myogenic response).</a> Physiol Rev. 79, 387-423 (1999).</li><li>Osmond, J. M., Mintz, J. D., Dalton, B., Stepp, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity+increases+blood+pressure,+cerebral+vascular+remodeling,+and+severity+of+stroke+in+the+Zucker+rat.">Obesity increases blood pressure, cerebral vascular remodeling, and severity of stroke in the Zucker rat.</a> Hypertension. 53, 381-386 (2009).</li><li>Ge, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenously+produced+20-HETE+modulates+myogenic+and+TGF+response+in+microperfused+afferent+arterioles.">Endogenously produced 20-HETE modulates myogenic and TGF response in microperfused afferent arterioles.</a> Prostaglandins Other Lipid Mediat. 102-103, 42-48 (2013).</li><li>Ryan, 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=Placental+ischemia+impairs+middle+cerebral+artery+myogenic+responses+in+the+pregnant+rat.">Placental ischemia impairs middle cerebral artery myogenic responses in the pregnant rat.</a> Hypertension. 58, 1126-1131 (2011).</li><li>Bagi, 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=Type+2+diabetic+mice+have+increased+arteriolar+tone+and+blood+pressure:+enhanced+release+of+COX-2-derived+constrictor+prostaglandins.">Type 2 diabetic mice have increased arteriolar tone and blood pressure: enhanced release of COX-2-derived constrictor prostaglandins.</a> Arterioscler Thromb Vasc Biol. 25, 1610-1616 (2005).</li></ol>

Authors have no financial conflicts.

Critical steps, troubleshooting and modifications
In a typical isobaric vessel preparation, the artery is pressurized at 70 mmHg between two glass cannulas perfused with warm (37 &#176;C) PSS. After 30-45 min, arteries develop MT, characterized by spontaneous decrease in diameter that stabilizes in 20-30 min. The resistance arteries from various vascular beds develop variable MT. For example rat resistance mesenteric arteries develop MT ~25% of PD, while cremastric arteries may achieve MT ~40% of...

Small resistance arteries are major determinants of SVR and play an important role in pathophysiology of many diseases1,2. Conditions such as diabetes3, pregnancy4, ischemia-reperfusion5,obesity and hypertension6,7 are frequently associated with altered microvascular function. Vascular myography can not only provide important insights into changes in microvascular function in various diseases but also help identify therapeutic targets and evaluate the efficacy of vasoactive compounds. Vascular function has been studied using isolated small arteries under isometric or isobaric vessel conditions8. Detailed description of isometric myography is provided elsewhere9. However there are differences in data obtained from isometric versus isobaric preparations10-12. Since pressurized arterial preparations allow the study of microvascular function in near-physiological conditions, the obtained findings may correlate better with in vivo behavior of the vascular bed8,13.
In 1902 Bayliss first described the effect of transmural pressure on vascular diameter14. He observed in small resistance arteries from various vascular beds of rabbits, cats and dogs that a decrease in pressure was followed by vasodilation, and an increase in pressure was followed by vasoconstriction. This phenomenon is known as myogenic response. Bayliss and subsequent investigators observed that in isobaric conditions small resistance arteries develop sustained constriction known as MT15,16. Both myogenic response and MT can be assessed by using pressure myography (PM) technique. PM is used primarily to determine vasoactivity of small arteries, veins and other vessels. In addition to assessing the effect of vasoactive compounds on vascular diameter, PM - as the name indicates - is used to assess intravascular pressure-mediated changes on vascular diameter. Over the last few decades advances in computer software, which enhanced video microscopy and glass pipette pulling, have made PM easier to perform. However, dissection of viable intact segments of small blood vessels remains tedious and sometimes challenging. Here we outline a detailed protocol to study myogenic response in small mesenteric resistance arteries isolated from rats.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Chemical</td>
		 </tr>
		 <tr>
			<td>Acetylcholine</td>
			<td>Sigma Aldrich</td>
			<td>A6625</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Calcium chloride (CaCl2)</td>
			<td>Sigma Aldrich</td>
			<td>223506</td>
			<td></td>
		 </tr>
		 <tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G5767</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetra acetic acid (EGTA)</td>
			<td>Sigma Aldrich</td>
			<td>E3889</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ethylene diamine tetra acetic acid (EDTA)</td>
			<td>Sigma Aldrich</td>
			<td>E9884</td>
			<td></td>
		 </tr>
		 <tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3784</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Magnesium sulfate (MgSO4)</td>
			<td>Sigma Aldrich</td>
			<td>M7506</td>
			<td></td>
		 </tr>
		 <tr>
			<td>MOPS</td>
			<td>Sigma Aldrich</td>
			<td>M5162</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Phenylephrine</td>
			<td>Sigma Aldrich</td>
			<td>P6126</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma Aldrich</td>
			<td>P3911</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Potassium phosphate (KH2PO4)</td>
			<td>Sigma Aldrich</td>
			<td>P5655</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium bicarbonate (NaHCO3 )</td>
			<td>Sigma Aldrich</td>
			<td>S6014</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma Aldrich</td>
			<td>S7653</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma Aldrich</td>
			<td>S5881</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium nitroprusside</td>
			<td>Sigma Aldrich</td>
			<td>13451</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium phosphate monobasic monohydrate (NaH2PO4)</td>
			<td>Sigma Aldrich</td>
			<td>S9638</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium pyruvate</td>
			<td>Sigma Aldrich</td>
			<td>P8574</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1.</td>
		 </tr>
		 <tr>
		 <td></td>
		 <td></td>
		 <td></td>
		 <td>
 <table border="1">
		<tbody>
		 <tr>
			<td>Physiological salt solution (1000 ml)</td>
			<td> mM</td>
			<td></td>
		 </tr>
		 <tr>
			<td>KCl</td>
			<td>4.9</td>
			<td>0.365 g</td>
		 </tr>
		 <tr>
			<td>NaCl</td>
			<td>112</td>
			<td>6.545 g</td>
		 </tr>
		 <tr>
			<td>MgSO4.7H2O</td>
			<td>1.2</td>
			<td>0.296 g</td>
		 </tr>
		 <tr>
			<td>KH2PO4</td>
			<td>1.2</td>
			<td>0.163 g</td>
		 </tr>
		 <tr>
			<td>Glucose</td>
			<td>11.5</td>
			<td>2.072 g</td>
		 </tr>
		 <tr>
			<td>NaHCO3</td>
			<td>26</td>
			<td>2.184 g</td>
		 </tr>
		 <tr>
			<td>HEPES</td>
			<td>10</td>
			<td>2.383 g</td>
		 </tr>
		 <tr>
			<td>CaCl2</td>
			<td>2</td>
			<td>2 ml (1M stock)</td>
		 </tr>
		 <tr>
			<td>De-ionized water</td>
			<td></td>
			<td>998 ml</td>
		 </tr>
		 <tr>
			<td>Ca2+ free physiological salt solution (100 ml)</td>
			<td> mM</td>
			<td></td>
		 </tr>
		 <tr>
			<td>KCl</td>
			<td>4.9</td>
			<td>0.036 g</td>
		 </tr>
		 <tr>
			<td>NaCl</td>
			<td>112</td>
			<td>0.645 g</td>
		 </tr>
		 <tr>
			<td>MgSO4.7H2O</td>
			<td>1.2</td>
			<td>0.029 g</td>
		 </tr>
		 <tr>
			<td>KH2PO4</td>
			<td>1.2</td>
			<td>0.016 g</td>
		 </tr>
		 <tr>
			<td>Glucose</td>
			<td>11.5</td>
			<td>0.207 g</td>
		 </tr>
		 <tr>
			<td>NaHCO3</td>
			<td>26</td>
			<td>0.218 g</td>
		 </tr>
		 <tr>
			<td>HEPES</td>
			<td>10</td>
			<td>0.238 g</td>
		 </tr>
		 <tr>
			<td>EGTA </td>
			<td>0.39</td>
			<td>0.015 g</td>
		 </tr>
		 <tr>
			<td>Sodium nitroprusside </td>
			<td>0.1</td>
			<td>0.0026 g</td>
		 </tr>
		 <tr>
			<td>De-ionized water</td>
			<td></td>
			<td>100 ml</td>
		 </tr>
		 <tr>
			<td>Dissection solution, stock (500 ml)</td>
			<td> mM</td>
			<td></td>
		 </tr>
		 <tr>
			<td>NaCl</td>
			<td>145</td>
			<td>21.18 g</td>
		 </tr>
		 <tr>
			<td>KCl</td>
			<td>4.7</td>
			<td>0.875 g</td>
		 </tr>
		 <tr>
			<td>MgSO4</td>
			<td>1.2</td>
			<td>0.739 g</td>
		 </tr>
		 <tr>
			<td>MOPS</td>
			<td>2</td>
			<td>1.049 g</td>
		 </tr>
		 <tr>
			<td>EDTA</td>
			<td>0.02</td>
			<td>0.019 g</td>
		 </tr>
		 <tr>
			<td>De-ionized water</td>
			<td></td>
			<td>500 ml</td>
		 </tr>
		 <tr>
			<td>Working dissection solution (100 ml)</td>
			<td> mM</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dissection solution stock</td>
			<td>20 ml</td>
		 </tr>
		 <tr>
			<td>Glucose</td>
			<td>1.2</td>
			<td>0.091 g</td>
		 </tr>
		 <tr>
			<td>NaH2PO4</td>
			<td>5</td>
			<td>0.016 g</td>
		 </tr>
		 <tr>
			<td>Sodium pyruvate</td>
			<td>2</td>
			<td>0.022 g</td>
		 </tr>
		 <tr>
			<td>CaCl2</td>
			<td>2</td>
			<td>0.2 ml (1M stock)</td>
		 </tr>
		 <tr>
			<td>De-ionized water</td>
			<td></td>
			<td>79.8 ml</td>
		 </tr>
	 </tbody>
</table>
 </td>
	 </tr>
		 <tr>
		 <td></td>
		 <td></td>
		 <td></td>
		 <td>Table 2. Composition of Experimetnal solutions</td>
	 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>CCD Monochrome Camera</td>
			<td>The imaging Source</td>
			<td>DMK 21AU04</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Single inline solution heater</td>
			<td>Warner Instruments</td>
			<td>64-0102</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Thermistor</td>
			<td>Warner Instruments</td>
			<td>64-0108</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dual automatic temperature controller</td>
			<td>Warner Instruments</td>
			<td>TC-344B</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Flaming/Brown micropipette puller</td>
			<td>Sutter Instruments </td>
			<td>P-97</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fluorescence System Interface</td>
			<td>IonOptix</td>
			<td>model FSI-700</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Forceps and scissors </td>
			<td>World Precision Instruments</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ion Wizard-Core and Analysis</td>
			<td>IonOptix</td>
			<td>Ion Wizard 6.0</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Laboratory tubing</td>
			<td>Silastic </td>
			<td>508-005</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Male Sprague Dawley rat</td>
			<td>Harlan Laboratories</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Master flex console drive</td>
			<td>Cole-parmer</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Milli-Q Plus Ultrapure Water System</td>
			<td>Millipore</td>
			<td>ZD5211584</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ophthalmic monofilament nylon suture </td>
			<td>Ethicon </td>
			<td>9007G</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Photometry and Dimensioning Microscope</td>
			<td>Motic</td>
			<td>AE31</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pressure Servo Controller with peristaltic pump and pressure transducer</td>
			<td>Living Systems Instrumentation</td>
			<td>PS-200</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Stereomicroscope</td>
			<td>Nikon Instruments Inc</td>
			<td>SMZ660</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Vessel Chamber</td>
			<td>Living Systems Instrumentation</td>
			<td>CH-1</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dissection dish</td>
			<td>Living Systems Instrumentation</td>
			<td>DD-90-S</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Thin Wall Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>TW120-6</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Microforge</td>
			<td>Stoelting</td>
			<td>51550</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 3.</td>
		 </tr>
	 </tbody>
</table>

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.

Schematic representation of a typical pressure myograph set-up is shown in Figure 1. The two ends of the vessel are cannulated with a glass micropipette and secured with sutures on both sides. Via tubing and an open stopcock, one cannula is connected to a servo-controlled pressure-regulator; the other cannula is connected to a closed stopcock. The chamber is perfused with PSS and vascular diameter changes are observed by an inverted microscope connected to a CCD camera.
The ar...

Watch this Scientific Journal Video about Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography at JoVE.com

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

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 ...

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16.7K Views.  Georgia Regents University. 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.

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

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.

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 ...

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

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 ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale behind this technique is the direct observation of the mechanical response of the TM to acute IOP elevation. Prior to scanning, IOP is measured at baseline and during IOP elevation. The limbus is scanned by spectral-domain optical coherence tomography at baseline and during IOP elevation (ophthalmodynamometer (ODM) applied at 30 g force). Scans are processed to enhance visualization of the aqueous humor outflow pathway using ImageJ. Vascular landmarks are used to identify corresponding locations in baseline and IOP elevation scan volumes. Schlemm canal (SC) cross-sectional area (SC-CSA) and SC length from anterior to posterior along its long axis are measured manually at 10 locations within a 1 mm segment of SC. Mean inner to outer wall distance (short axis length) is calculated as the area of SC divided by its long axis length. To examine the contribution of adjacent tissues to the effect IOP elevations, measurements are repeated without and with smooth muscle relaxation with instillation of tropicamide. TM migration into SC is resisted by TM stiffness, but is enhanced by the support of its attachment to adjacent smooth muscle within the ciliary body. This technique is the first to measure the living human TM response to pressure elevation in situ under physiological conditions within the human eye.

Trabecular meshwork (TM) migration into Schlemm&#8217;s canal space can be induced by acute pressure elevation by ophthalmodynamometer, and observed by spectral domain optical coherence tomography. The goal of this method is to quantify the morphometric response of the living outflow tract to acute pressure elevation in living tissues in situ.

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Laparoscopic Technique for Serial Collection of Liver and Mesenteric Lymph Nodes in Macaques

The mesenteric lymph nodes (MLN) and the liver are exposed to microbes and microbial products from the gastrointestinal (GI) tract, making them immunologically unique. The GI tract and associated MLN are sites of early viral replication in human immunodeficiency virus (HIV) infection and the MLN are likely important reservoir sites that harbor latently-infected cells even after prolonged antiretroviral therapy (ART). The liver has been shown to play a significant role in immune responses to lentiviruses and appears to play a significant role in clearance of virus from circulation. Nonhuman primate (NHP) models for HIV and Acquired Immunodeficiency Syndrome (AIDS) closely mimic these aspects of HIV infection and serial longitudinal sampling of primary sites of viral replication and the associated immune responses in this model will help to elucidate critical events in infection, pathogenesis, and the impact of various intervention strategies on these events. Current published techniques to sample liver and MLN together involve major surgery and/or necropsy, which limits the ability to investigate these important sites in a serial fashion in the same animal. We have previously described a laparoscopic technique for collection of MLN. Here, we describe a minimally invasive laparoscopic technique for serial longitudinal sampling of liver and MLN through the same two port locations required for the collection of MLN. The use of the same two ports minimizes the impact to the animals as no additional incisions are required. This technique can be used with increased sampling frequency compared to major abdominal surgery and reduces the potential for surgical complications and associated local and systemic inflammatory responses that could complicate interpretation of results. This procedure has potential to facilitate studies involving NHP models while improving animal welfare.

Here, we describe a minimally invasive laparoscopic technique for serial sampling of liver and mesenteric lymph nodes (MLN) in macaques that allows for increased sampling frequency, and reduces the potential for surgical complications when compared to performing a laparotomy.

The mesenteric lymph nodes (MLN) and the liver are exposed to microbes and microbial products from the gastrointestinal (GI) tract, making them ...

A Cell Culture Model of Resistance Arteries

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and signaling processes that can control vascular tone and blood pressure. As it is a projection from either the endothelial or smooth muscle cell, and due to its small size (on average, an area of ~1 &#181;m2), the MEJ is difficult to study in isolation. However, we have developed a cell culture model called the vascular cell co-culture (VCCC) that allows for in vitro MEJ formation, endothelial cell polarization, and dissection of signaling proteins and processes in the vascular wall of resistance arteries. The VCCC has a multitude of applications and can be adapted to suit different cell types. The model consists of two cell types grown on opposite sides of a filter with 0.4 &#181;m pores in which the in vitro MEJs can form. Here we describe how to create the VCCC via plating of cells and isolation of endothelial, MEJ, and smooth muscle fractions, which can then be used for protein isolation or activity assays. The filter with intact cell layers can be fixed, embedded, and sectioned for immunofluorescent analysis. Importantly, many of the discoveries from this model have been confirmed using intact resistance arteries, underscoring its physiological relevance.

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...