We present a method for isolating intestinal tubes and assessing the impact of drug on their tension, frequency, and amplitude in which this method offers a valuable approach for researchers in investigating intestinal tubes. The current experimental challenge is maintaining the consistency and the stability of technical methods to ensure reliable and reproducible scientific research outcomes, particularly considering the sensitivity and reliability inherited in biological systems. This protocol offers advantages such as simplicity, economical feasibility, easy control of experimental conditions, minimal influencing factors, high reproducibility and accurate results.
To begin, prepare a physiological salt solution or PSS. Saturate the solution by bubbling with a mixed gas of 95%oxygen and 5%carbon dioxide. Maintain the pH values of the solution between 7.38 and 7.42 with two millimolar sodium hydroxide.
Pre-cool, one third of the PSS to four degrees Celsius. Then prewarm the remaining solution to 37 degrees Celsius for subsequent experiments. Now gather Petri dishes filled with four degrees Celsius, PSS, surgical tweezers, and scissors.
Quickly place the stomach and intestine tubes in a Petri dish filled with four degrees Celsius PSS saturated with 95%oxygen and 5%carbon dioxide. Locate the duodenum in the Polaris of the stomach. Using tweezers, delicately lift the adjacent tissue and carefully trim it away from the intestine's edge with scissors.
Divide the intestine into one to two centimeter segments. To begin, turn on the in vitro tissue perfusion system. Adjust the instrument's bath temperature to 37 degrees Celsius.
Then place the 37 degrees Celsius physiological salt solution or PSS into the bath. Prepare a 15 centimeter surgical suture and soak it in four degrees Celsius PSS saturated with 95%oxygen and 5%carbon dioxide. Secure one end of the intestinal canal with the suture and use a steel needle hook to secure the other end.
Next, install the intestinal tube. Mount the segment with the steel needle hook at the bottom of the bath, and attach the other end of the surgical line to the transducer. Turn on the gas switch to allow bubbles to emerge in the bath.
Now open the data acquisition software and click on Start to ensure the corresponding path signal is being recorded. Rotate the spiral axis of the bath counterclockwise to relax the intestinal tubes to their natural state. Click on setup zero, all inputs to ensure that the software sets the intestinal tube's initial tension to zero grams.
Then rotate the spiral axis of the bath clockwise to pull the tension value to one gram and stabilize it in the 37 degrees Celsius PSS for 30 minutes. Observe the rhythmic spontaneous contraction waves in the software as this indicates a sufficient response. Next, add the test drug to the bath to study its effect on intestinal tube function.
Afterward, stop the data acquisition software and perform data analysis using the software. To edit the data board and select the analysis parameters, click on Window data pad and choose the average tension for the channel. Select the contraction curve before administration and click Add to data pad.
Then select the contraction curve after administration and click Add to data pad. The average tension value before and after administration will appear on the data pad in turn. Then click on Window data pad to copy data to other statistical analysis software for further analysis.
Analyze other parameters such as average amplitude, average frequency, and integral area under the curve. Replace the average tension with the respective parameter. Afterward, select the curve, click on Edit copy lab chart data to copy the data to drawing software for visualization.
The study examined the effects of various agents on the tension of isolated intestinal tub rings. Acetylcholine significantly enhanced tension in these tubes. Conversely, atropine inhibited the tension caused by acetylcholine.
Similarly, barium chloride also enhanced tension. On the other hand, both epinephrine and nifedipine inhibited tension in the isolated intestinal tube rings.
