Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography
September 22nd, 2011
An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 μm, independently.
This procedure uses doll salt sensitive and salt resistant rats to compare the reactivity of mesenteric resistance arteries under phenylephrine tension to extracellular calcium. This is accomplished by first isolating the mesenteric arcade from animals and dissecting out the resistance artery branches.Next. The first of two stainless steel wires is threaded through a short segment of the dissected artery and the vessel is mounted into the graph chamber.
Then the second stainless steel wire is threaded through the vessel lumen parallel to the first wire. Next, the mounted vessel is stretched to a normalized internal circumference. The final step of the procedure is to reactivate the vessel by challenging it with repeated applications of phenylephrine and relaxing it with cumulative applications of calcium.
Ultimately, the results show changes in tension in the vessel, which can be converted into a calcium response curve and used to determine EC 50 values. For comparison. We first had the idea for using this method when we discovered that the nev network of rat Masonary batteries expressed a gpro copper calcium sens receptor.
That can be studied in the same way as bitter AIC receptors, which are activated by agonist. Generally, individuals new to this method may struggle because the endothelium and smooth muscle layers is small. Vessels are easily damaged.
If this occurs, the mechanical properties of these vessels may be compromised. To begin, prepare the work area and surgical instruments. Place an anesthetized animal on its back and wipe the abdomen with alcohol.
After performing a midline laparotomy, use scissors to remove about 85 centimeters of intestine, including the superior mesenteric artery. Cut the proximal end close to the pylori and the distal end near the ileocecal junction. Place the excised section into a coated Petri dish containing physiological salt solution or PSS at room temperature.
Pin down the proximal end of the intestine to the right and then pin the remainder of the intestine in a counterclockwise direction. Next, dissect away a segment of the mesenteric artery containing branch two and three and a piece of the intestine from the proximal end. Identify the V-shaped branch point of the mesenteric artery and dissect it away from the vein.
Use forceps to gently pull away adipose and connective tissue to avoid direct contact with the artery. To begin mounting the vessel cut two artery segments about two millimeters long. The tip of a four centimeter 40 micrometer tungsten free stainless steel wire may be used to open the lumen of the artery.
Then use fine forceps to insert the wire into the lumen of each artery. Taking care not to damage the endothelium, fill the myo graft chamber with PSS at 37 degrees Celsius. Use forceps to transfer the threaded vessel segment into the chamber.
Next, transfer the excised proximal vessel segment to the myo graph chamber and pull the end along the wire to feed it into the vessel. Avoid stretching the vessel. Secure the near end of the wire counterclockwise under the near fixing screw on the right hand jaw connected to the micrometer.
Catch the free end of the wire with forceps and secure it clockwise under the far fixing screw on the right hand jaw, ensure that the vessel segment along the wire is situated in the gap between the jaws without making any contact with the jaw itself. Screw the jaws apart. Align the second wire parallel with the vessel and insert it into the far end of the lumen.
Gently feed the wire through the lumen of the vessel segment in one motion using the already mounted wire as a guide. Hold the wire about one centimeter from the vessel to avoid stretching it during the maneuver and avoid touching the endothelium. Screw the jaws together and ensure that the second mounting wire moves underneath the first one secured on the right hand.
Jaw secure the near end of the second wire in a clockwise direction under the near fixing screw of the left hand jaw connected to the transducer. Secure the far end of the wire under the fixing screw on the left hand jaw and tighten to stretch the wire. Once mounting is complete, allow 30 minutes for equilibration at 37 degrees Celsius.
After equilibration, the motor is reset from the mounting menu and the normalization procedure, which stretches the vessel to a normalized internal circumference is initiated. To begin open the normalization menu and set the target transmural pressure to 13.3 kilopascals. Next, set the duration of each of the normalization steps to 60 seconds.
The IC one to IC 100 ratio should equal 0.9 and the IP calibration should be set to two star delta. Measure the length of the mounted mesenteric artery using the microscope eyepiece readings when the hair lines are over the far and neurons of the vessel segment. After normalization, the vessels are reactivated by performing a standard start consisting of a series of stimulations and washout periods.
First, replace the myo chamber cover and start aeration with 95%air and 5%carbon dioxide. Fill the chamber with fresh PSS containing 100 micromolar ascorbic acid. Contract the vessel with five micromolar fennel, rine, or PE for five minutes and wash four times with PSS buffer After the last wash, refill the chamber with PSS and wait three minutes before refilling the chamber with PSS and contracting with PE.As shown previously, the artery is now ready for the experiment.
Measure the relaxation of contracted vessels by cumulatively adding increasing concentrations of calcium chloride. This table shows that the basal tensions following phenylephrine application varied between wistar DS or doll salt sensitive and DR or doll salt resistant rats. This trace from a wistar rat shows relaxation of PE contracted arteries by cumulative additions of calcium relaxation with severely compromised in tissues from DS rats compared to Wistar rats and compared to Dr.Rats as seen here, the differences in calcium sensitivity can be seen in the calcium response curves and again when calculating the EC 50 values.
Finally, DS rats show a reduction in the expression of the calcium sensing receptor in arteries compared to Dr.Rats Once mastered. This technique can be done in two hours if performed properly. After watching this video, you should have a good understanding of how to compare vascular reactivity of small me enteric attri to exogenous compound under isometric conditions.
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Overview
This article describes an automated myography method for measuring force in isolated mesenteric arteries. The technique utilizes a Mulvany-Halpern Auto Dual Wire Myograph 510A to assess responses to phenylephrine and extracellular calcium, enabling consistent isometric response measurements in small vessels.
Key Study Components
Area of Science
- Neuroscience
- Vascular Biology
- Myography Techniques
Background
- Mesenteric arteries play a crucial role in regulating blood flow.
- Understanding their reactivity is important for cardiovascular research.
- Phenylephrine is a common agonist used to study vascular responses.
- Calcium levels are critical for vascular smooth muscle contraction.
Purpose of Study
- To develop a reliable method for measuring force in small mesenteric arteries.
- To compare the reactivity of salt-sensitive and salt-resistant rats.
- To evaluate the effects of phenylephrine and calcium on vascular responses.
Methods Used
- Isolation of mesenteric arcade from rats.
- Dissection of resistance artery branches.
- Mounting arteries in a myograph chamber using stainless steel wires.
- Application of phenylephrine and calcium to assess vascular reactivity.
Main Results
- Consistent force measurements were achieved in isolated arteries.
- Differences in reactivity were observed between salt-sensitive and salt-resistant rats.
- Phenylephrine effectively induced tension in the arteries.
- Calcium application resulted in relaxation of the vessels.
Conclusions
- The automated myography method provides a reliable approach for studying vascular responses.
- This technique can enhance understanding of mesenteric artery function.
- Findings may have implications for cardiovascular disease research.
Frequently Asked Questions
Executive Industry Relevance
Automated wire myography of mesenteric arteries enables precise, quantitative assessment of vascular reactivity under isometric conditions, supporting early-stage target validation in cardiovascular research. This approach provides predictive confidence for mechanistic de-risking of vascular targets and informs portfolio decisions in hypertension and vascular dysfunction programs. The method's reproducibility and quantitative outputs facilitate robust cross-comparison of compound effects and genetic models.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of vascular smooth muscle receptor function and signaling pathways.
- Supports biological de-risking by quantifying contractile and relaxation responses to agonists and antagonists.
- Facilitates predictive confidence in target selection for vascular and cardiovascular indications.
Screening & Assay Development
- Prepares validated ex vivo vessel systems for compound screening and mechanistic studies.
- Delivers standardized, reproducible, and quantitative tension measurements for assay development.
- Enables side-by-side comparison of pharmacological agents and genetic models in a scalable format.
Translational & Preclinical Research
- Aligns ex vivo vascular reactivity data with disease-relevant models of hypertension and vascular dysfunction.
- Supports translational biomarker identification by linking receptor expression to functional outcomes.
- Provides continuity from early discovery through preclinical validation of vascular targets.
Pipeline & Workflow Integration
This automated myography method integrates into the discovery-to-preclinical continuum, bridging early mechanistic studies with translational research in vascular biology.
- Discovery Biology: Quantifies vessel contractility and relaxation to test hypotheses about receptor function and signaling.
- Screening: Provides assay-ready, reproducible vessel preparations for compound evaluation and potency ranking.
- Analytics: Generates EC50 values and response curves for robust statistical comparison across conditions and models.
- Translational Research: Links ex vivo functional data to in vivo disease models and biomarker strategies.
- Enterprise Reuse: Establishes a standardized platform for ongoing vascular target and pathway interrogation.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in vascular target validation.
- Operational Value: Delivers standardized, reproducible, and scalable vessel reactivity assays.
- Strategic Value: Informs go/no-go decisions and reduces late-stage biological risk in cardiovascular portfolios.
- Portfolio Impact: Enables risk-adjusted prioritization of vascular targets and compounds.
Implementation Considerations
- Requires expertise in vascular dissection and ex vivo tissue handling.
- Needs access to automated wire myography instrumentation and analytical software.
- Demands rigorous cross-team standardization of vessel mounting and normalization procedures.
- Adaptation may be needed for different vessel types or disease models.
- Small vessel size increases risk of mechanical damage, impacting assay reliability.
