ISOLATED RABBIT JEJUNUM PREPARATION
Introduction
The jejunum of the rabbit may be kept alive for many hours after removal from the animal if it is placed in a suitable saline medium and supplied with oxygen and glucose. It shows rhythmical contractions superimposed on a background of tonic (steady) contraction. The preparation consists not only of the circular and longitudinal smooth muscle layers, but also a network of nerve cells and fibres (intrinsic nerve supply) organised into submucousal and myenteric plexus, the latter being situated between the two muscle coats. In situ the gut also receives an innervation from the parasympathetic and sympathetic nervous systems (extrinsic nerves). These make synapses with the intrinsic plexus and may also supply the smooth muscle directly the exact organisation being uncertain but probably varying in different parts of the gut. The tonic and rhythmic contraction seen in the isolated rabbit jejunum preparation are clearly independent of the extrinsic nerves. Peristalsis is dependent on the integrity of the intrinsic contractions of the gut. The action of the extrinsic nerves can also be mimicked by the chemical transmitter substance known to be released from the postganglionic sympathetic and parasympathetic nerve terminals. These transmitter substances are thought to produce their effects by combining with receptor sites on the effector organs which, in the gut, may be either on smooth muscle cells or on nerve elements of the intrinsic plexus.
This experiment illustrates that the actions of the known transmitter substances (or similar substances) released by the extrinsic postganglionic autonomic nerves on the non-sphincteric part of the transmitters can also be demonstrated. The effects of drugs which block gut receptor sites for the chemical transmitters can also be demonstrated.
Method
The apparatus consists of an electrically heated tank which is maintained at 37oC by moving the control knob and by switching it on or off. This needs constant attention for consistent experimental result. In the tank is the bath which can be filled with saline solution by releasing the spring clip on the aspirator. The bath may be drained by releasing the clip on the drainage tube beneath the tank. Dipping into the bath is a glass tube with its lower end hook-shaped; this serves both for passing a slow steam of 02 and C02 (95%:5%) through the saline and for the attachment of the lower end of the segment of gut. Above the bath is a side writing lever.
The saline solution used is a modification by Tyrode of that originally devised by Ringer for mammalian tissues.
You will use Tyrode's solution for this experiment, as it is more suitable for mammalian tissues. It differs from the Ringer's solution (used for the frog experiments) in several respects. The composition of three physiological solutions which you use in this course is given in the table:-
SALTSRINGERTYRODEKREBS
%w/v%w/v% w/v
NaCl0.60.80.7
CaCl20.010.020.028
KCl0.00750.020.035
MgCl2-0.010.03
NaHCO30.010.10.21
NaH2PO4-0.0050.016
KH2P04--0.0162
MgSO4--0.0294
Glucose-0.1(0.2)
Procedure
Fill the gut bath with Tyrode's solution. Turn on the oxygen supply and adjust the flow so that a gentle stream of bubbles passes through the solution. Have ready your instruments, needle and thread, and a bowl of cold tyrode's solution. A piece of rabbit's jejunum will be given to you. Drop it into the cold Tyrode's solution in the bowl. If the solution becomes dirty with the contents of the gut, change it, repeating the procedure if necessary. Cut off about an inch of jejunum and with the aid of a needle, pass ligatures through the wall about 3/8 inch from each end. Tie each one in a loose loop so as not to crush the gut or occlude its lumen. Leave a long thread at one end and tie the other end close to the hook on the end of the oxygen tube. These procedures should be done quickly to avoid drying out of the tissue. Lower the tube with the gut attached into the bath and fasten the long ligature to the side writing lever so that the lever is horizontal. The lever is then correctly adjusted. Check that the gut is vertical and not rubbing on the side of the bath. Move the whole bath apparatus so that the side writing lever makes contact with the drum on the kymograph.
Methods of adding chemical substances ('drugs')
Caution
Be careful when using drugs not to get them mixed with each other and always wash out one drug from the gut bath before adding another unless you have a particular reason for wanting more than one drug in the bath at once. Similarly always use a clean pipette. It will be impossible to draw any conclusions from your experiments if you do not observe these precautions.
1.Take a short record of normal activity.
2.Without stopping the drum add the stated quantity of drug to the bath.
3.Mark on the drum the point at which the drug was added.
4.When the DRUG has produced its maximum effect, stop the drum, drain the bath, and refill with fresh Tyrode's solution.
5.After a short pause empty the bath and refill again. If necessary repeat after 3 minutes until the preparation has recovered its normal activity.
Order of adding the drugs.
This sequence must be followed. Read through each section before proceeding, and make a separate list of the drugs to be added so that you are familiar with the procedure to be followed.
1.Action of the agonist drugs alone. The aim of this section is to determine the normal effect of the gut on these drugs.
(a)Adrenaline hydrochloride 1 cc. of a 10-5g/cc solution.
N.B. If the adrenaline solution is pink or brown, reject it and get a fresh supply.
(b)Acetylcholine hydrochloride 1 cc. of a 10-5g/cc solution.
N.B. This solution rapidly looses its activity. If you suspect that this has happened, get some fresh solution.
(c)Barium chloride 1 cc of a 10-3g/cc solution.
2.The antagonist action of atropine sulphate
(a)Add 1 cc of 10-5 g/cc solution of atropine sulphate.
i.Is there any effect? Does the gut return to normal?
(b)Without washing out, add 1 cc of 10-5 g/cc acetylcholine hydrochloride solution. When the effect has occurred wash out.
i.Does acetylcholine now have any effect?
ii.Atropine blocks the action of acetylcholine directly on the muscle.
(c)Add 1 cc of 10-5 g/cc solution of atropine sulphate without washing out, add 1 cc 10-3 g/cc barium chloride. Wash out when the maximum effect has occurred.
i.Is there any change in the action of barium chloride?
ii.In view of this result, is atropine a specific blocking drug for acetylcholine at the nerve smooth muscle junction or does it seem to block other drugs acting on the muscle?
(d)Add 1 cc of 10-5 g/cc atropine sulphate then add 1 cc of 10-5 g/cc adrenaline hydrochloride. When the maximum effect has occurred, wash out.
i.Is there any change in the action of adrenaline?
ii.From 2 (c) ii would you expect atropine to have any effect on the action of adrenaline?
3.The action of adrenergic blocking agents
(a)Add/cc of a 10-4 g/cc solution of Dibenyline (pheno benzamine).
i. With reference to your previous tracing, compare the effect of adrenaline alone and in the presence of Dibenyline.
ii.Debenyline is an alpha adrenoreceptor blocker. What is the effect brought about by adrenaline through the alpha adrenoreceptor on the gut? How do you account for the remaining activity?
(b)Add/cc of 10-4 g/cc solution of Inderal (propranolol). 15 seconds later add 1 cc of 10-5 g/cc adrenaline hydrochloride.
i.Compare the effects of adrenaline alone and in the presence of Inderal.
ii.Inderal is a beta adrenoreceptor blocker. What is the effect brought about by adrenaline through the beta adrenoreceptors on gut. How do you account for the remaining activity.
(c)From your experiment make an assessment of which type of adrenoreceptor is predominant in this tissue assuming that adrenaline acts equally on each type of receptor.
YOUR REPORT
METHOD. As described in the schedule.
RESULTS
Describe exactly the results of your experiment under the appropriate tracing, which should be stuck into your practical work.
CONCLUSION
Give a detailed account of the action (and site of action) of each agonist and antagonist drug you have investigated, using the questions in the schedule to help you. Explain all possible sources of error.
ANSWER THE FOLLOWING QUESTIONS:-
(a)Explain what is meant by the nocotinic and muscarinic actions of acetylcholine.
(b)List the actions known to the mediated via alpha and beta adrenoreceptors and other smooth muscles (outside the gut) and cardiac muscle.