Male Sprague Dawley rats (320-350 g body weight), kept in university experimental animal facilities under standard animal housing conditions were anesthetized by ether and killed instantly by cervical dislocation under the university regulation for the use of experimental animals. The thoracic aorta from 1-2 rats in each experiment were quickly removed and rinsed with Ringer’s solution to remove blood and blood clots.
The entire thoracic aorta with intact PVFT and partially fat-denuded aorta are shown in figure 13
Fat intact (F +) and fat denuded (F -) aortic rings were obtained from the same rat and studied in parallel in each experiment. The loosely attached connective tissues were gently removed with tweezers and scissors. Then the vessel was cut into ring segments of 3 - 4 mm in width. The fatty tissue strips remained either intact or denuded. The composition of the Ringer’s solution was (mM): NaCl, 119; NaHCO3, 25; KCl, 4.7; KHCO3, 1.2; MgSO4, 1.2; CaCl2, 1.6; and Glucose, 1.1. Ringer’s solution was kept at 36.5 C and pH 7.3. For prolonged experiments and during equilibration, the Ringer’s solutions were replaced every 20-30 minutes. At the end of the experiment, the wet weights of aortic rings before and after removal of PVFT were measured in order to estimate the amount of fat tissues expressed as tissue wet weight..
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Figure 13 :
The rat thoracic aorta with fat tissue intact ( F +) and fat tissue denuded (F -)2. Methods
Contractility measurements
General conditions: After tissue dissection, all the aortic rings were equilibrated in a 5 ml organ bath solution for an hour with a pre-determined stable 2 g (usually 1.5-2.5g) optimal resting tension. After equilibration, the rat aortic rings were then stimulated with 80 mM KCl 2-3 times until they reached a stable plateau contraction. The tension is expressed as a percentage of the steady-state tension (100%) (77). At the end of the experiment (usually after 5-6 hours), the contractile responses to 80 mM KCl remained about 80-90% of the original KCl contraction. Some aortic rings were denuded of fat tissues which were then transferred to and incubated in aerated Ringer’s solution for approximately 2 hours before use (to release the adipocyte – derived factors). The clear solution after removal of the fat tissue just before use is operationally termed “ F solution” (Fat solution). These F solutions were either keep at room temperature or heated to boiling (to deactivated or destroy any heat sensitive factors) and subsequently cooled down to room temperature before adding to fat-denuded aortic rings. (Figure 14)
Unless otherwise indicated, most experiments were performed in endothelium-denuded ring preparations. The functional intactness of endothelium was checked against the degree of endothelium-dependent relaxation to 3 uM CCh after reaching plateau contraction in response to 1 uM PE In most cases, the endothelium – dependent relaxation reached a magnitude of > 60% of the PE contractile response.
Relaxation to CCh of < 20% of the PE contractile response was considered to be endothelium denuded.
Preparation of F solution: Rat’s thoracic aortic rings, divided into 6 strips, usually weighing from 9.0 mg to 12 mg in each strip. Removed of fat tissue from those aortic rings and the fat tissue were transferred to a Petric dish containing 20 ml of Ringer’s solution with continuous aeration. The incubation time of at least 2 hrs was allowed to ensure a high release of adipocyte-derived factors (ADF), For control groups, fat denuded aortic rings were stimulated with PE or KCl to construct the concentration-contraction curves. For the test groups, 2 different ways were used to apply the fat solutions.
were stimulated with PE or KCl to construct the dose response curves. Figure 15, tracing 1
(2) Fat-denuded aortic rings were stored with 5 ml of Ringer’s solution, then increase the concentration of PE or KCl and added to it under the dose response curves.
When a suboptimal contractile level were obtained, the Ringer’s solution were replaced by the same volume of F-solution which contained the same concentration of PE or KCl. Figure 15, tracing 2
Figure 14 : The preparation and transfer of the solution pre-incubated with PVAT to
the organ baths. First, fat tissues were removed from the thoracic aorta of the rat, and then transferred to a Patrick dish containing 20 ml of Ringer’s solution and let stand with aeration for at least 2 hrs. The solution following the removal of fat tissue byFigure 15 :
Two methods in transfer fat solution to the F- aortic rings. One is to transfer fat solution into the organ bath, after 30 minutes incubation, the rings were stimulated with PE or KCl.(tracing 1) Another way is to stimulate with PE or KCl . When the contractile reaches the plateau, the fat solution containing the same concentration of PE or KCl was introduced (tracing 2)In some study groups, the fat tissues were placed in 2.5 ml (half volume of organ bath) of distilled water. After 2-hr incubation, the F-solution was heated till boiling. When the solution was cooled to room temperature, 2.5 ml of doubly concentrated Ringer’s solution was added and stored at a temperature of 36.5 C with continuous aeration shortly before use. This protocol helped minimize the change of the pH of the solution following heating, which tends to reduce the HCO3¯ in the solution causing altalination.
3. Chemicals
The phenylephrine (PE), potassium chloride (KCl), carbachol (CCh) and L-N-nitro-L-arginine methyl ester (L-NAME) were all purchased from Sigma chemical Co. (St.
Louis, Mo USA). They were all dissolved in distilled water. PE was a 10 mM stock solution. KCl stock solution of 3 M. CCh stock solution of 330 mM. L-NAME stock solution of 30 mM
4. Statistical analysis
The results were expressed as the mean ± standard error of the mean (SEM). The Student’s t test or ANOVA test was used when comparing two or multiple values as appropriate: n represents the number of rats. A value of P<0.05 is considered statistically significance.