This study describes isolating rat intrapulmonary artery and vascular smooth muscle cells. Several experimental protocols were employed, such as using the organ bath technique to investigate vascular response of the intrapulmonary artery. Intrapulmonary arteries and vascular smooth muscle cells are excellent models to study vascular physiology and pathophysiology.
Adaptations of this technique could evaluate vascular responses of any drugs, extracts or phytochemicals. Mounting the isolated intrapulmonary arterial ring in an organ bath enables the use of isolated intrapulmonary arteries to evaluate a drug's role in modulating various diseases, including pulmonary hypertension. The experimental protocols are technically feasible, but the crucial steps are complicated and essential for success, so having a visual demonstration makes it easier to understand and follow.
Demonstrating the procedure will be Kittiwoot To-on, a master's degree student in physiology from the Cardiovascular Research Unit Laboratory, Department of Physiology, Faculty of Medical Science, Naresuan University. Begin by slicing a single lobe of the lung with 11-centimeter scissors and placing it on a nine-centimeter Petri dish with the medial side, or root of the lung, facing upward. Observe and identify the alignment of the vein, bronchia, and artery from top to bottom.
Cut open the bronchus longitudinally with scissors. Then use the 11-centimeter forceps to grab the tip of the bronchus. Gently dissect and remove the bronchus and veins out of the lung.
Use the forceps to grab the tip of the intrapulmonary artery and carefully dissect it out of the lung tissue with scissors. Keep the isolated intrapulmonary artery in cold Krebs solution until the organ bath assembly is set up. After isolating the intrapulmonary artery, cut open the main branch of the intrapulmonary artery longitudinally with the 11-centimeter scissors and cut into two-millimeter strips.
Immerse the intrapulmonary artery strips in DM, then incubate the strips for one hour at four degrees Celsius in DM containing one milligram per milliliter papain, 0.04%BSA and 0.4 millimolar DTT. Then incubate at 37 degrees Celsius for 15 minutes. Add one milligram per milliliter of collagenase Type IA in DM, and again incubate at 37 degrees Celsius for five minutes.
Transfer the tissues into fresh DM and disperse by gentle trituration using a glass Pasteur pipette. Keep triturating until isolated vascular smooth muscle cells become visible in the bathing solution under the microscope. Isolate intrapulmonary artery as demonstrated earlier and cut the main branch of the intrapulmonary artery into rings of approximately two millimeters in length.
Affix the intrapulmonary artery rings in organ bath chambers by threading them onto two 40-micrometer-diameter stainless steel wires. Attach stainless steel wires mounted with intrapulmonary artery rings to the isometric force transducers connected to the data acquisition device and the computer system installed with the suitable physiological software for data recording and analysis. Then gently raise the tension of the intrapulmonary artery ring to one gram.
Allow the vessel segments to equilibrate for about 45 minutes at a resting tension of one gram. During the equilibration period, ensure that the Krebs solution is regularly changed every 15 minutes. After equilibration, test the viability of the vessels by measuring their vasoconstriction to high extracellular potassium solution.
Assess the presence or absence of endothelium by computing the relaxation response to acetylcholine in rings precontracted with PE.Mechanically remove the endothelium by gently rubbing the inside of the vessel with a small wire to induce denudation. Then equilibrate the arterial rings for 30 minutes before the start of the test experiments. Investigate the relaxant effect of the plant extract by precontracting intrapulmonary artery rings with PE.Then carefully add 0.1 to 1000 micrograms per milliliters of the plant extract cumulatively to endothelium intact rings and endothelium-denuded rings to induce vasorelaxation and acquire a concentration-dependent response curve.
Ensure that the effective DMSO used as a solvent is also evaluated similarly to serve as a negative control. Evaluate the vasorelaxant mechanism of action of the plant extract as mentioned in the text manuscript. Then after the contractions to PE stabilize, add cumulative concentrations of the plant extract.
Present the effects of plant extract as percentage relaxation of the intrapulmonary artery rings in the presence of inhibitors compared to the response of the intrapulmonary artery rings without inhibitors and construct the concentration response curve. In endothelium intact intrapulmonary artery, the plant extract elicited a concentration-dependent response of vasorelaxation. Eradication of endothelium profoundly reduced the vasorelaxation induced by the plant extract as reflected by the increase in the EC50 by 2.2 fold, inhibition of eNOS and EDHF evidently decreased the vasorelaxant response to the plant extract.
This shifted the concentration response curve to the right and increased the EC50 without altering the E max values. On the contrary, Indomethacin showed no effect on the vasorelaxant response to the plant extract. In the endothelium-denuded intrapulmonary artery rings, calcium-activated potassium channel blockers decreased the vasorelaxant response to the plant extract.
While blockers of voltage-gated or ATP-sensitive potassium channels showed no deviation. Preincubation with the plant extract inhibited the calcium-chloride-induced contraction. The endothelium-denuded intrapulmonary artery rings preincubated with calcium-free Krebs solution and PE rendered a transient contraction.
In comparison to the vehicle, the plant extracts significantly reduced the contraction induced by PE.During the procedure, one must clearly identify vein, bronchia, and artery early on. Cutting or damaging the artery will reduce its length and affect the vascular responses. This technique can be adapted to investigate compounds that might serve as prototypes for treatments of pulmonary vascular diseases such as pulmonary arterial hypertension.
