Experimental Procedure
Measurements of the contraction of aortic smooth muscle are performed according to accepted protocols. Young adult (>10 weeks) male Sprague Dawley rats, 175g to 250g, are euthanized and the aortas are isolated. Euthanasia is performed according to an IACUC approved protocol. The thoracic aorta is cleaned of fatty deposits and cut into four rings, each 5 mm in length. These aortic rings are attached to an isometric force transducer, placed in a temperature controlled tissue bath, and bathed in oxygenated Krebs-Ringer bicarbonate solution (KRB) at 37oC (see figure above). The KRB is continually gassed with 95% O2-5% CO2 to maintain pH and oxygenation. Tissues are mounted with 3.0 g resting tension (optimum) and allowed to equilibrate until the tension is maintained. Tissues are tested by contraction with 3 x 10-7 M norepinephrine (NE) and relaxation with 10-5 M carbachol (muscarinic acetylcholine agonist). This procedure is repeated once to insure the stability of the smooth muscle and intactness of the endothelium. Rings that perform poorly in test contractions (> 1.0 g active tension [contraction] or < 50% relaxation) are discarded.
In the initial screening, the herbal medicines were tested for their effect on these NE-induced contraction. All four tissues were contracted with 3 x 10-7 M NE. Sufficient time was allowed for the tonic phase of the contraction to achieve equilibrium. 0.300 ml of the HM infusion was added to two of the four baths and a blank solution added to the two control baths. Again, sufficient time is allowed for equilibration of the tissue. The bath was then washed out and the tissues are allowed to recover. When the tissues returned to the original resting tension, they were contracted with 3 x 10-7 M NE, to determine if they reach a level of tension comparable to before exposure to the HM (adequate washout of its effect). If the contraction was normal, the tissues were exposed to 10-5 M carbachol to insure that the endothelium was still functioning normally. If the tissues had recovered properly (i.e., responding similar to before exposure to the HM), the baths were washed out and then the next HM tested. Each HM was tested in at least two additional aortas, resulting in the HM being tested in at least six rings from three different animals.
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