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:SALTS
NaCl
CaCl2
KCl
MgCl2
RINGER
%w/v
0.6
0.01
0.0075
-
TYRODE
%w/v
0.8
0.02
0.02
0.01
KREBS
% w/v
0.7
0.028
0.035
0.03
NaHCO3
NaH2PO4
KH2P04
MgSO4
Glucose
0.01
-
0.1
0.005
0.1
0.21
0.016
0.0162
0.0294
(0.2)
Diagram of the apparatus
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.
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.
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?
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.
(b)
2.
3.
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.