Sprague Dawley male rats weighing between 350 to 500 g were killed with cervical dislocation method. All the procedures were carried out in conformity with the animal handling guidance by Home Office Schedule 1.
The thorax was opened and the thoracic aorta was isolated from the dissected rat as quickly as possible. Other adherent tissues (connective tissues) were removed from the aorta and then, the aorta was sectioned into 6 - 8 mm long fragments which are put into Krebs solution immediately.
Since the aim of the experiment is to determine the mechanism of action of a nitric oxide donor, SNP, interference from other endogenous endothelium substances should be avoided to prevent false results. The endothelium was physically removed by lightly rubbing the aorta with a thin cord put through the lumen of the aorta. After the endothelium of the aorta is denuded, the aortic ring was positioned in the organ bath.
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The aortic ring was held up by two parallel L-shaped wires, one in which was connected to an isometric force transducer (UF1 dynamometer ranges from 0-55 g, LCM Systems Ltd, England, UK) and another to a movable rack for basal tension adjustment. Tension when the smooth muscle of the aorta ring contracts or relax will be detected by the transducer which is connected to the amplifier 1000 x (CED 1902, Cambridge Electro- nic Design, Cambridge, UK). PCI cards in the computer then translates analogue input into digital signals. This is done by an analogue converter (NI - DAQmx PCI - 6221, National Instruments, UK) and data is then recorded in data acquisition software, Chart (v4.9.0 University of Strathclyde, Glasgow, UK). The held up aortic ring was immersed in Krebs solution contained in a 20 ml organ chamber. Krebs solution was maintained at pH 7.4 and was constantly aerated with carbogen (95% oxygen, 5% carbon dioxide) (supplier) at 37 °C.
The organ bath was gassed with air as well. Initially, the aortic rings were tensed to a stable basal strain of 1.0 g before left to equilibrate for 20-30 minutes. Krebs solution in the bath chamber was replaced every 15-20 minutes intervals.
To qualitatively assess whether the endothelium is entirely removed, acetylcholine (1 x 10-6 M) were introduced into the bath chamber after the aortic ring was pre-contracted with phenylephrine at concentration 1 x 10-6 M. If there was any measurable relaxation detected, the aortic rings were discarded. This is to eliminate the effect of endogenous substances produced by the endothelium during the experiment which may influence the outcome of the experiment.
Determination of EC50 for contraction with phenylephrine
When the tension of the aortic ring is stable at the baseline, phenylephrine was administered into the bath chamber in graded concentration starting with the lowest concentration first (10-9 to 10-5). The subsequent concentration is loaded when tension measured seems to stabilise. Dilutions were done with two different stock solutions. Dilutions done were summarized in the following table.
Stock solution (M)
Final concentration in 20 ml bath chamber (M)
Volume of stock solution to be added into 20 ml bath chamber (µl)
1 x 10-9
3 x 10-9
1 x 10-8
3 x 10-8
1 x 10-7
3 x 10-7
1 x 10-6
3 x 10-6
1 x 10-5
3 x 10-5
1 x 10-4
3 x 10-4
After repeating this process on six independent tissues, a cumulative concentration-response (% contraction) curve was plotted. The concentration of agent required to generate half of the maximum response (EC50) was established following log transformation of the normalized concentration vs response curves and is interpreted as negative logarithms (pEC50 = - log EC50) of the average values for individual tissue. Results were expressed as mean ± standard error of the mean.
Determination of time-dependent differences in the response of aortic rings
This step is vital to make sure that there are no time-dependent changes in the response generated by the tissue so that it will affect the reliability of the data obtained. After the aortic ring has been pre-contracted with phenylephrine at EC50, different concentration of SNP (10-9 to 10-4) were introduced into the bath chamber in increasing concentrations. The relaxation caused by SNP was correlated with the amount of contraction caused by phenylephrine. Percentage reversal of depolarization (contraction caused by phenylephrine) when SNP is administered into the bath chamber will be calculated. A cumulative concentration-response (% relaxation) curve was then plotted. The tissue is then washed with Krebs solution 3 times before the process is repeated again on the same tissue. Differences in the ec50 for both set of processes on the same aortic ring were evaluated by paired t-test. This process is repeated on 3 different tissues for consistency. Statistical significance is fixed at p < 0.05.
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Determination of mechanism of action of SNP
To investigate the potential mechanism of action of SNP which is suggested to be related to SERCA, a selective inhibitor of SERCA is introduced into the organ bath. The aortic ring was pre-contracted with phenylephrine and was introduced to graded concentration of SNP as done before. The aortic ring is then washed several times before CPA (10 microM) was introduced and left for 30 minutes before a second introduction of phenylephrine with a concentration that will produce sufficient degree of contraction (3.0 x 10-7 M). Subsequently, relaxation was induced by SNP by increasing the dose gradually as done before. Concentration-response curves were plotted showing the differences in the relaxation when CPA is absent or present. Results were presented numerically and graphically. The magnitude of any detectable parallel shift in the concentration-response (% relaxation) curve by CPA was expressed as 'log unit shift'.
Log unit shift = negative log IC50 (control) - negative log IC50 (inhibitor present)
Differences in the readings for both set of processes on the same aortic ring were evaluated by paired t-test before normalization of data and Mann Whitney Test after normalization. Statistical significance between mean values was evaluated using the one-way variance analysis (ANOVA). Statistical significance is fixed at p < 0.05.