We thank Dr. Wei Qi He (Soochow University, Suzhou, China) and Dr. Yan Ning Qiao (Shaanxi Normal University, Xi&#39;an, China) for the technical assistance. This work was supported by the National Natural Science Foundation of China (Grant 31272311, 81373295 and 81473420) and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (Grant No. ysxk-2016).

Animal manipulations were approved by the Institutional Animal Care and Use Committee (IACUC) of the Model Animal Research Center of Nanjing University.
1. Solution Preparation
<ol>
	<li>Prepare HEPES-Tyrode solution (H-T) using 137.0 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2&#8729;6H2O, 5.6 mM D-glucose, and 10 mM HEPES, pH 7.3-7.4.</li>
	<li>Prepare HEPES-Tyrode solution without calcium (Ca2+-free H-T) using 140.6 mM NaCl, 2.7 mM KCl, 1 mM MgCl2&#8729;6H2O, 5.6 mM D-glucose, and 10 mM HEPES, pH 7.3-7.4.</li>
	<li>Prepare HEPES-Tyrode solution using 124 mM KCl (High K+) using 15.7 mM NaCl, 124.0 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2&#8729;6H2O, 5.6 mM D-glucose, and 10 mM HEPES, pH 7.3-7.4.</li>
</ol>
2. Experiment Preparation
<ol>
	<li>Preheat H-T and High-K+ solutions using a 37 &#176;C water bath.</li>
	<li>Turn on the myograph system, data acquisition hardware and computer.</li>
	<li>Carefully fill all myograph chambers with 5 mL of H-T solution each.</li>
	<li>Fill two Petri dishes with 20 mL of 4 &#176;C H-T and Ca2+-free H-T solutions, respectively, and store on ice.</li>
	<li>Fill a 10-cm coated Petri dish with 20 mL of H-T solution, and maintain it at room temperature.</li>
</ol>
3. Mouse Mesenteric Artery Dissection
<ol>
	<li>Euthanize an 8-12-week-old C57BL/6J female or male mouse by cervical dislocation. Pin the mouse down with its abdomen facing up.</li>
	<li>Moisten the abdomen with 70% ethanol. Then, cut the skin with scissors along the ventral midline from the groin, and make incisions from the start of the first incision downwards to the legs on both sides. Pull the skin back on both sides; make similar incisions to open the peritoneum.</li>
	<li>Using scissors, cut the oesophagus, the colon and other connective tissues to completely isolate the gastrointestinal tract with feeding vasculature from the body.</li>
	<li>With forceps, move the isolated segment into the dish containing the cold H-T prepared in step 2.4, and gently rinse the tissue in H-T solution several times to wash off the blood.</li>
	<li>Transfer the isolated segment into the coated Petri dish prepared in step 2.5, and perform mesenteric artery dissection at room temperature.</li>
	<li>Smooth out the stomach, jejunum, ileum and caecum in a clockwise direction, and pin the stomach and caecum on the left and right-hand side, respectively.</li>
	<li>Stretch the mesenteric vasculature bed and fix the intestine with pins to expose the dissected mesenteric arteries. 
	Note: Under these conditions, the arteries are on top of the veins.</li>
	<li>Turn on the transmission light source of a stereoscopic microscope, and dissect arteries under the microscope. Make sure that the entire tissue is immersed in the solution.</li>
	<li>Clamp the adipose tissues around the arteries with forceps, and isolate the arteries by cutting off all the connective tissues with dissection scissors. Avoid injuring the arteries.</li>
</ol>
4. Arterial Mounting
<ol>
	<li>Transfer and immerse the mesenteric artery tree into the cold Ca2+-free H-T solution (prepared in step 2.4) by clamping the excess arteries with forceps.</li>
	<li>Cut off a 1.4-mm portion of the artery proximal to intestinal wall of a mesenteric arcade, and use two forceps to open both sides of this artery segment carefully.</li>
	<li>Prepare two segments of stainless steel wire 2.5 cm in length, and place them into the same dish.</li>
	<li>Gently clamp one end of the artery using forceps, and carefully insert two wires into the lumen of the artery one by one with help of another forceps. Ensure that the wires are kept straight and do not touch the endothelium.</li>
	<li>Using two forceps, clamp the two steel wires outside of the threaded vessel simultaneously, and carefully transfer the vessel from the Petri dish to a myograph chamber previously filled with H-T solution (step 2.3).</li>
	<li>Screw the jaws apart to make space for mounting. Clamp both sides of one of the two inserted wires using two forceps, and place the vessel in the jaw gap (Figure 1A).</li>
	<li>Wrap both sides of the clamped wire around screws of the jaw connected to the micrometer (Figure 1B).</li>
	<li>Fix the left-hand screw by twisting clockwise. Straighten the wire using right-hand forceps, then fix the right-hand screw by twisting clockwise (Figure 1C). Make sure that the vessel is always inside the jaw gap, but do not touch the jaw to avoid damage.</li>
	<li>Close the two jaws using the micrometer (Figure 1D). Make sure the two jaws are close enough but that they do not touch each other and that the unfixed wire is on the top of the fixed wire.</li>
	<li>Using the right-hand forceps, carefully fold the unscrewed wire at the corner of the jaw connected to force transducer, and wrap it clockwise around the right-side screw (Figure 1E). Then, fix the screw. Repeat this step on the left side of the wire and fix the left-side screw (Figure 1F).</li>
	<li>Move the jaws slightly apart by carefully rotating the micrometer (Figure 1G). Avoid stretching the vessel. Use forceps to move the wire at the micrometer side to the horizontal plane of the wire at the transducer side. Carefully rotate the micrometer so that the gap between the two jaws can just accommodate the two wires.</li>
	<li>Repeat Steps 4.2 &#8211; 4.11 to mount arteries onto the other chambers. Connect all the chambers to the equipment, cover the chambers, attach the 100% oxygen supply and a temperature probe, and start heating to 37 &#176;C. Open the charting software and press the Start button on the Chart View window to start recording.</li>
	<li>Equilibrate for about 20 min.</li>
</ol>
5. Normalization
Note: In order to standardize the experimental conditions and to obtain reliable physiological responsiveness of vessels, a normalization procedure is necessary15. According to the relationship between the active force and internal circumference of the vessel, the wire myograph system has a standard normalization program to assess the internal circumference (IC) of the mounted vessel5,8,9. Briefly, to calculate IC (&#181;m), read the micrometer and input the value as the X value and the transducer output force, i.e., resting wall tension (mN/mm), as the Y value. The program will return a fitted curve of (X, Y) and calculate the IC corresponding to a transmural pressure of 100 mmHg (IC100). The vessel is set to the normalized internal circumference (IC1) when the active responsiveness is maximal.
<ol>
	<li>Set forces to zero for all channels on the device, and equilibrate for another 1-2 min.</li>
	<li>Select Normalization settings from the &#8220;DMT menu, and set up the parameters as follows: 
	Eyepiece calibration (mm/div): 0.36; Target pressure (kPa): 13.3; IC1/IC100: 0.9; Online averaging time (seconds): 2; Delay time (seconds): 60. Click the OK button to close the DMT Normalization Settings window.</li>
	<li>Select the channel of interest from the DMT menu to open a DMT normalization window for the corresponding channel. Enter the constant values into the window as follows: Tissue end-points a1: 0.1; Tissue end-points a2: 4; Wire diameter (&#181;m): 40. The window displays the calculated vessel length as 1.40 mm.</li>
	<li>Read the micrometer of the appropriate tissue chamber. Enter the value into the Micrometer reading box, and click Add Point button. This value is the initial value of X (X0). After a 60 s delay time, the window displays the force and the effective pressure (ERTP) corresponding to this micrometer value. Simultaneously, the Micrometer reading box becomes active.</li>
	<li>Stretch the vessel being normalized by turning the micrometer in a counter-clockwise direction. Enter the micrometer value into the Micrometer reading box, and click Add Point button. Wait for a delay time of 60 s again.</li>
	<li>Repeat step 5.5, continue to stretch the vessel, and add micrometer values until the window displays the value of &#8220;Micrometer X1&#8221;, which is the calculated micrometer setting required to stretch the vessel to its IC1.</li>
	<li>Set the micrometer to X1 value. 
	Note: The normalized tension is usually 1-2 mN.</li>
</ol>
6. Artery Contraction Recording
Note: All the solutions, including H-T and High-K+ solution used in this section, were prepared in step 2.1.
<ol>
	<li>After normalization, equilibrate the vessel in the chamber for 15-20 min. 
	Note: These is no need to change the solution in this step.</li>
	<li>Challenge the vessel with High-K+ solution twice.
	<ol>
		<li>To challenge the vessel, replace H-T solution with 5 mL of High-K+ solution to induce contraction for 10 min, followed by washing with 5 mL of H-T solution 3-4 times. 
		Note: Typical contraction has a maximal force over 3 mN and a constant sustained force around 2.5 mN12. If the first challenge generates a maximal force below 2.5 mN or the sustained force decreases with time or the second challenge generates a much lower force than the first-time dose, the vessel is discarded and will not be used for further investigation.</li>
	</ol>
	</li>
	<li>Challenge the vessel with 5 mL of High-K+ solution to induce contraction. After 5 min, add 0.5 &#181;L of the neoliensinine stock solution (10 mM in DMSO)14 into the chamber to relax the vessel at a final concentration of 1 &#181;M neoliensinine.</li>
	<li>When the force is stable (this usually takes several minutes), add another 0.5 &#181;L of neoliensinine stock solution into the chamber to increase the concentration to 2 &#181;M. Add 1 &#181;L of the stock solution each time to increase the concentration to 4, 6, 8 and 10 &#181;M to generate the dose-response curve. 
	Note: The stock and working concentrations vary among drugs.</li>
</ol>

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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=Contractile+Properties+of+Small+Arterial+Resistance+Vessels+in+Spontaneously+Hypertensive+and+Normotensive+Rats.">Contractile Properties of Small Arterial Resistance Vessels in Spontaneously Hypertensive and Normotensive Rats.</a> Circulation Research. 41 (1), 19-26 (1977).</li><li>Mulvany, M. J., Nyborg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+increased+calcium+sensitivity+of+mesenteric+resistance+vessels+in+young+and+adult+spontaneously+hypertensive+rats.">An increased calcium sensitivity of mesenteric resistance vessels in young and adult spontaneously hypertensive rats.</a> British Journal of Pharmacology. 71 (2), 585-596 (1980).</li><li>Mulvany, M. 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The authors have nothing to disclose.

Hypertension is a widespread public health challenge due to its severe complications, including cardiovascular and kidney diseases16. Understanding the pathogenesis of hypertension and exploring more anti-hypertensive drugs has become an urgent task in this field. Blood pressure is generated and maintained by peripheral resistance of the circulation. According to Poiseuille&#39;s Law, the relatively small arteries generate a large proportion of circulatory resistance and serve as the dominant prod...

The small vessel myograph system used here was for measuring the isometric contraction of small resistance vessels with internal diameters ranging from 100 to 400 &#181;m. Isolated small vessels (about 2 mm long) were inserted by two 40-&#181;m diameter wires and were then mounted on the micrometer-side and transducer-side jaws sequentially. This myograph technique was first suggested in 19721 and then developed primarily by Mulvany and his colleagues2,3,4,5,6. It is now a mature technique with stable equipment, easy performance and a standard normalization procedure7,8,9. We utilized this method with some modifications for measurements in the mouse mesenteric artery.
Vascular smooth muscle lines the walls of almost all blood vessels. Their fundamental function is to generate forces through contraction in response to various stimuli. The normal contractility of vascular smooth muscle is essential for blood pressure regulation and nutrition supplement10. Abnormal regulation of blood pressure results in a variety of diseases, including hypertension, heart failure and ischaemia. Several studies have suggested that abnormal blood pressure is always associated with dysfunctional vascular smooth muscle contractility7,11,12,13. The myograph method allows investigation of isometric contractility of mouse vessels induced by various stimuli including vasoconstrictors, inhibitors and drugs. Successful measurements of contraction will help us understand the mechanisms of blood pressure maintenance and the pathogenesis of vascular smooth muscle-associated diseases and to explore novel therapeutic approaches.
Many Chinese herbs have been widely used for clinical treatment of vascular diseases; however, their effective ingredients usually remain unknown. Thus, isolation and identification of the effective components is very important for the development of novel drugs. Multi-wire myograph technology offers a simple approach for screening active components in herbs. We have reported several studies using the small vessel myograph system to investigate mouse mesenteric artery contraction and identified natural compounds with anti-hypertension activity12,13,14. Here, we describe the detailed protocol for the myograph method and assess the relaxant effect of neoliensinine isolated from embryos of lotus seed (Nelumbo nucifera Gaertn.)14.

<table><tbody><tr><td>Multi wire myograph system</td><td>DMT</td><td>610-M</td><td></td></tr><tr><td>Stainless steel wire</td><td>DMT</td><td>400447</td><td></td></tr><tr><td>Geuder dissection scissor</td><td>DMT</td><td>400431</td><td></td></tr><tr><td>Dumont forceps</td><td>DMT</td><td>300413</td><td></td></tr><tr><td>PowerLab/8SP</td><td>ADInstruments</td><td>ML785</td><td></td></tr><tr><td>Software</td><td>ADInstruments</td><td>LabChart 5</td><td></td></tr><tr><td>NaCl</td><td>SigmaAldrich</td><td>S5886</td><td></td></tr><tr><td>KCl</td><td>SigmaAldrich</td><td>P5405</td><td></td></tr><tr><td>CaCl2</td><td>SigmaAldrich</td><td>C4901</td><td></td></tr><tr><td>MgCl2&amp;middot;6H2O</td><td>SigmaAldrich</td><td>M2393</td><td></td></tr><tr><td>D-Glucose</td><td>SigmaAldrich</td><td>G6152</td><td></td></tr><tr><td>HEPES</td><td>Sangon Biotech</td><td>A100511-0250</td><td></td></tr><tr><td>NaOH</td><td>SigmaAldrich</td><td>S8045</td><td></td></tr><tr><td>DMSO</td><td>SigmaAldrich</td><td>D2650</td><td></td></tr></tbody></table>

isometric contractility measurement of the mouse mesenteric artery using wire myography

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

We measured the isometric contractility of mouse mesenteric artery using a multi-wire myograph system and assessed the relaxant effect of neoliensinine purified from embryos of lotus seed (Nelumbo nucifera Gaertn.)14. The mouse mesenteric resistance artery was isolated, cleaned of connective tissues and cut into 1.4-mm segments. The artery segment was inserted by two steel wires in Ca2+-free H-T solution in a Petri dish, and then the segment was...

Watch this Scientific Journal Video about Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography at JoVE.com

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

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

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

isometric-contractility-measurement-mouse-mesenteric-artery-using

Research

JoVE Journal

Medicine

14.7K Views.  Nanjing University. De draad myograph techniek wordt gebruikt om vasculaire vlotte spier functies en de scherm nieuwe drugs te onderzoeken. Wij rapporteren een gedetailleerd protocol voor het meten van de isometrische contractility van de muis mesenterische slagader en voor het screenen van de nieuwe skeletspierrelaxantia van vasculaire glad spierweefsel.

Video: Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

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 Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

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

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

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

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.

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  ...

Biology

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic ...

Measurement of Endothelium-Dependent Vasorelaxation in the Mouse Thoracic Aorta Using Tensometric Small Volume Chamber Myography

Small volume chamber tensometric myography is a commonly used technique to evaluate the vascular contractility of small and large blood vessels in laboratory animals and small arteries isolated from human tissue. The technique allows researchers to maintain isolated blood vessels in a tightly controlled and standardized (near-physiological) setting, with the option of adjusting to various environmental factors, while challenging the isolated vessels with different pharmacological agents that can induce vasoconstriction or vasodilation. The myograph chamber also provides a platform to measure vascular reactivity in response to various hormones, inhibitors, and agonists that may impact the function of smooth muscle and endothelial layers separately or simultaneously. The blood vessel wall is a complex structure consisting of three different layers: the intima (endothelial layer), media (smooth muscle and elastin fibers), and adventitia (collagen and other connective tissue). To gain a clear understanding of the functional properties of each layer, it is critical to have access to an experimental platform and system that would allow for a combinational approach to study all three layers simultaneously. Such an approach demands access to a semi-physiological condition that would mimic the in vivo environment in an ex vivo setting. Small volume chamber tensometric myography has provided an ideal environment to evaluate the impact of environmental cues, experimental variables, or pharmacological agonists and antagonists on vascular properties. For many years, scientists have used the tensometric myograph technique to measure endothelial function and smooth muscle contractility in response to different agents. In this report, a small volume chamber tensometric myograph system is used to measure endothelial function in the isolated mouse aorta. This report focuses on how small volume chamber tensometric myography can be used to evaluate the functional integrity of the endothelium in small segments of a large artery such as the thoracic aorta.

The present protocol describes the concepts and technical application of the tensometric myograph technique using a multi-chamber myograph system in the experimental ex vivo assessment of mouse aortic endothelial function.

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

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