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Perfusion of the central canal and subarachnoid space of the cat and rabbit spinal cord in vivo
PERFUSION OF THE CENTRAL CANAL AND SUBARACHNOID SPACE OF THE CAT AND RABBIT SPINAL CORD IN I/IT/O I. K. M. MORTON,* C. J. STAG@ and R. A. WEBSTER Department
of Pharmacology,
University
College
London,
(Accepted 24 March
Gower
Street, London
WClE
6BT
1976)
Summary~Methods have been developed which allow perfusion of the central canal (or subarachnoid space) of the cat and rabbit spinal cord in uiuo for several hours without any significant loss of cord function. Changes in neuronal activity, as evidenced by ventral root recording and electromyography, could be induced by the addition of drugs (strychnine, mephenesin) and neurotransmitters (acetylcholine) to the perfusate. The detection of acetylcholine in the perfusate in amounts that were increased by stimulation of neural inputs to the cord suggests that the method is suitable for the collection of transmitters released in the spinal cord. Central canal perfusion has the advantage of allowing tracts. independent stimulation of ventral and dorsal roots and descending
of substances to neurones in the central nervous system and the collection of transmitters released by neural stimulation necessitates circumvention of the blooddbrain barrier. The development of iontophoretic techniques has overcome this problem with respect to the application of transmitters but their collection still presents many problems. Small glass or Perspex cylinders or ‘cups’ (McIntosh and Oborin, 1953) applied to the surface of the brain have enabled a number of workers to study the release of acetylcholine (ACh) from the cerebra1 cortex and other parts of the brain, and Feldberg and Sherwood (1954) have described methods for perfusing the ventricular spaces of the brain. There are no comparable methods for studying release in the spinal cord although the accessibility of peripheral nerves, dorsal roots and descending tracts for stimulation makes activation of certain pathways relatively easy. Perfusion of the cord vasculature rapidly leads to loss of neuronal activity (Kuno and Rudomin, 1966) and there still remains a diffusional barrier. Edery and LevingeY (1971) perfused the subarachnoid space of the cat by simply piercing the dura with fine needles at two levels (e.g. thoracic and sacral) but they were unable to detect a reproducible stimulated release of transmitters The superfused hemisected frog or toad cord has been used to show the ,release of acetylcholine (Mitchell and Phillis, 1962). but there is probably reduced synaptic transmission in this preparation especially in polysynaptic pathways. This paper describes in some detail the development of a method for perfusing the central canal or subarachnoid space of the cat and rabbit cord in viuo (see also Morton. Stagg and Webster, 1969). We have The application
* Present address: t Present address: St. Albans, England.
King’s College, London. Union International Research
Centre,
shown that the cord retains a high level of tivity, that responds to drugs added to the fluid, for several hours, and that stimulation inputs leads to the appearance of putative ters in the perfusate.
reflex acperfusion of neural transmit-
METHODS
General Experiments were performed on rabbits and cats of either sex and mainly in the weight range 2-3 kg. Animals were anaesthetised with halothane, after induction, in the case of rabbits, with intravenous thiopentone sodium (25 mg/‘kg). The trachea, left common carotid artery and right jugular vein were then cannulated and the right common carotid artery was ligated. Immobilisation of the animal was achieved by attaching clamps to each femur, the lateral processes of the third lumbar vertebra and across the ischial tuberosities of the pelvic girdle. The clamps were secured to pillars on a heavy T-slotted myograph table. Laminectomy was carried out on the dorsal aspect from L4-S2 and the sciatic nerves exposed. Decerebration at the mid-collicular level (rabbit) or spinalisation at Cl (cats) was then performed. Unless muscle activity was to be recorded the animals were injected intravenously with either tubocurarine chloride (0.5-l mg every 30 min) or gallamine triethiodide (4-8 mg every 30 min) and maintained on artificial respiration. Perfusion of the central canal Perfusion of the central canal with artificial cerebrospinal fluid (CSF), as used by Feldberg and Fleischhauer (1960), was achieved by inserting a 25.S.W.G. (0.51 mm outside diameter) stainless steel needle into the canal at L4/5 and cannulating the
I. K. M. MORTOKC. J.
2
canal at the sacral end. The needle formed the descending arm of a perspex T-piece connector (Fig. I) which was mounted in a micromanipulator (Narashige). Artificial CSF was supplied at a rate of 0.06 ml/min by a slow infusion apparatus (C. F. Palmer) through a second arm of the connector after being warmed to 37’C. The pressure in the system was recorded through the third arm with a transducer on a polygraph (Bell & Howell) and monitored (Grass). After cutting the dura mater the needle was positioned on the surface of the cord along the midline at L4. but clear of the central dorsal vein, The dorsal columns of a small length (5 mm) of cord were then retracted at Sl to expose the central canal and a short length of fine thread passed around the cord just above (cephalic to) the opening. The needle was next lowered into the cord to a tip depth of approx. 3.5 mm at an angle of 60 --80 to the horizontal. The depth of penetration was varied until flow was obtained at the sacral end with minimal pressure (less than 10mm Hg) in the perfusion system. When flow had been established a length of polyethylene tubing (Portex PP50), drawn to a tip diameter of approximately I mm, was inserted into the canal. In some cases this was secured by the thread previously placed around the cord. The tubing was held in the correct plane by means of cotton wool which was secured under 2 or 3 stainless steel needles pushed through the muscle on either side of the cord. When flow was established at a satisfactory pressure and recovery rate, the cord was covered with a thin layer of cotton wool moistened in artificial CSF. If ventral root recording was to be attempted. a layer of liquid paraffin was used. Perfusion of the suharuchnoid .spc~e Only cats were used in these experiments because of their greater ability to withstand the surgery and
and R. A.
STAW
WEBSTER
the tougher nature of the dura mater. After dorsal laminectomy (L4- S3) two cuts were made in the dura at L4 up to 8 mm apart (at right angles to the cord) so as to leave the ventral half of the dura intact. The portion of cord beneath the opening in the dura was then removed. The ends of a length of fine stainless steel wire were passed through a piece of 2 mm inside diameter stainless steel tube bent to a right angle and the loop of wire protruding formed into a circle of the same diameter as the inside of the dura mater. This loop was then positioned completely around the outside of the cord caudal to the section but inside the dura mater. The loop was passed 4&6.Omm down the subarachnoid space. and when traction was applied to the free ends of the wire. the loop closed to cut cleanly through the cord. This allowed a small section to be withdrawn from within the intact dura mater. A Perspex cannula of similar diameter to the cord (4.&6.0mm) was tied into the dura with a silk ligature. Cannulation of the subarachnoid space at its sacral end was carried out in much the same fashion except that cannulae as small as 2.0 mm diam were required. The perfusion system was connected to the thoracic cannula to give a flow of 0.12 ml/min at a pressure below 10mm Hg. The area of cord perfused in both cases contained the motoneurones and afferent inputs for both sciatic and femoral nerves.
Assays of ACh-like activity were carried out using either the clam (Mrrcemria mrcermriu) heart preparation as described by Florey (1967) or the leech (Hirudo medicinah) dorsal muscle. according to the method of Macintosh and Perry (1950). In both cases contractions were recorded auxotonically by interposing a light spiral spring between the preparation and an isometric force transducer (Grass FT03). cinder
I
I
PERFUSATE COLLECTED FOR ANALYSIS
L4
L5
L6
L7
SI
52
I
I ORIGINS OF FEMORAL AN0 SCIATIC NERVES
Fig. 1. Diagrammatic
presentation of system for perfusion of the subarachnoid space (A) and central canal (B) of the cat spinal cord in tko. Artificial cerebrospinal fluid (stippled area) was passed from an infusion pump through a heating coil to the needle (canal perfusion) or cannula (subarachnoid). The pressure in the system was recorded with a pressure transducer and monitored with a pen recorder.
Spinal cord perfusion favourable conditions the clam assay was able to detect ACh at bath concentrations in the range lo- “-lO- lo M but gave a very steep doseeresponse relationship. The leech preparation rarely detected ACh at concentrations less than lo-* M but was more easily manageable. Since the composition of the artificial CSF differed from that of the media used to bathe these preparations, it was particularly necessary to provide adequate controls when a significant amount of the former was added to the organ bath. Stimulating and recording procedures Contractions of the left gastrocnemius muscle were recorded by connecting the tendon to an isometric force transducer (Grass, FTlO). The electromyogram (EMG) was obtained with two small (20 SWG) stainless steel needles inserted 10mm apart beneath the skin above the gastrocnemius muscle, and amplified for display on a Grass polygraph. Discharges in filaments dissected from ventral roots L7 or Sl were recorded from one side of bipolar platinum electrodes, the other being either earthed or balanced with a crushed root filament. The electrodes were connected to paired cathode followers and the output displayed on an oscilloscope. Compound potentials were recorded from a whole or a substantial part of a ventral root suspended across both electrodes, generally with the part over the distal electrode crushed. RESULTS Demonstration qf the functional integrity of‘ the spinal cord during pelCfilsion experiments R#e.x acticity. Experiments were performed to assess the functional state of the spinal cord during prolonged perfusion by both the central canal and the subarachnoid routes. The physiological state of the cord was monitored by comparing the size of directly- and reflexly-induced muscle twitches. The isometric tension developed in the gastrocnemius group of muscles on stimulation of the medial gastrocne- _ mius nerve (direct) and central end of the cut sural nerve (reflex) was displayed on a polygraph. Figure 2 shows the results of two such comparisons where muscle activity was evoked at regular intervals over the course of 9@150 min. It can be seen that the threshold voltage for stimulation and tension responses were not constant for reflex stimulation. However, these fluctuations were reflected by similar variations in the directly evoked responses and so they would not seem to be due to changes within the spinal cord but rather to some peripheral alteration common to both pathways. Thus, there was no evidence of reduced function in the cord. Ventral root recording. Compound potentials reflect activity in a large number of neurones but- they are not reliably quantitated over prolonged periods of time as the amplitude may change spontaneously through alterations in electrode conductivity caused
3
by blood or fluid. Discharges in ventral root filaments reflect activity in a smaller number of neurones but there was ample evidence from such experiments that could be spontaneous and evoked discharges recorded over 34 hr of central canal perfusion. Although no systematic analysis of electrolyte balance between the perfused artificial CSF and nervous tissue was made, analysis of the fluid by flame photometry for potassium before and after perfusion through the central canal (both in the resting state and on electrical stimulation of the sciatic nerve), showed no marked change in the level of this ion. This point is of importance in order to eliminate the possibility that changes in the activity of perfusates on bioassay were not due to alterations in potassium content of the assay fluid. It was also found from an analysis of perfusates on a Technicon autoanalyser that they contained most of the amino acids found in natural CSF taken from the animals. but often only in trace amounts. There was no salt loss, or gain of fluid during perfusion, and no oedema. The histological picture of the cord was normal apart from some damage immediately around the input needle and sacral cannula. Blood-free perfusates were always obtained with canal perfusions and generally with the subarachnoid route. Changes in neuronal activity induced by substances added to the perfusion medium Figure 3 shows the effect on compound potentials of perfusion of 12pg/min through the central canal of the rabbit.
0
30
60
90
Time
(min)
120
150 0
ventral root strychnine Stimulation
x,
60
Time
(min)
90
Fig. 2. Tests of functional integrity of the cat spinal cord during central canal (A) and subarachnoid (B) perfusion. Voltage threshold of stimulated nerve (upper graph) and twitch strength of gastrocnemious muscle (lower graph) are shown for both direct stimulation of the motor nerve (M) and reflex activation through sural nerve stimulation (W----a). Although the response to reflex stimulation (2.5 g/mm trace) was always less than that for direct stimulation (12.5 g/mm) there was no change in response over the period of perfusion.
I. K. M. MORTON. C. J. STACG and R. A. WEBSTER
CONTROL
-14\J
14v
12v
IOV
3
IOmV
STRYCHNINE
. 12v
IOV Medial
.
14v
14v
Sural stimulation
lOmsec
gastrocnemius stimulation
Fig. 3. Effect of strychnine on a compound ventral root potentials recorded during perfusion of the central canal of the spinal cord of a decerebrate rabbit. Stimulation of the central end of the nerve to medial gastrocnemious produced a stimulus related monosynaptic component. After strychnine (12 pg/min) the same stimuli also produced polysynaptic activity and there was an increased polysynaptic response to sural nerve stimulation.
A
I
2
; .
i
\
1 1 .
1 .
1
I
.
.
B &
2-
--c.
.
1
.
,
:
C
.
.
.
.
012
set
Fig. 4. Electromyographic response to perfusion of strychnine through the central canal of the spinal cord of a decerebrate rabbit. In control studies (A), stimulation of one sural nerve produced ipsilateral activity (1) but no contralateral effect (2). Strychnine perfusion (12 pg min) increased ipsilateral activity and initiated activity in the contralateral muscle. (B, C, D at 2, 5 and 10min respectively after start of strychnine perfusion).
Spinal cord of the ipsilateral medial gastrocnemius nerve produced a monosynaptic spike, whereas sural nerve stimulation gave polysynaptic discharges. It can be seen that strychnine increased the amplitude of both monosynaptic and polysynaptic potentials, and furthermore introduced a polysynaptic component to the response seen on medial gastrocnemius stimulation. The marked change in polysynaptic activity indicates that the inte~eL]rones, which are most susceptible to asphyxia, remained functiona during cord perfusion. Evidence of exaggerated neuronal activity was also seen in electromyogram (EMG) recordings during perfusion of the central canal with strychnine. Figure 4 is representative of experiments where perfusion of 12pg/min strychnine in every case resulted in increased EMG activity within 1 min of it entering the cord. The gradual onset of increased muscle activity can be seen in the figure; s~ntaneous and evoked activity both increased markedly and activity appeared in the contralateral muscle. This increased activity reached a peak in 5-10min and died away slowly when strychnine perfusion was discontinued unless terminated by mephenesin given either intravenously (20mg/kg) or into the perfusion medium (1 m&ml). These results show that strychnine can penetrate to spinal neurones from the canal. Similar results were obtained when strychnine was added to subarachnoid perfusates although the response was not as great. Acetylcholine (lo-” g/ml) in the presence of eserine (0.2 mg/kg, i.v.) also modified compound ventral root potentials, when perfused through the central canal, causing a general increase in polysynaptic activity (Fig. 5) and variable effects on the monosynaptic component. Release of acetylcholine into perfusates. Some evidence was obtained for the presence of ACh in perfu-
CONTROL
ESERINE (0.2mg/kqi.v.)
5
perfusion
ACh (KPg/ml) 2Omin
sates after passage through the central canal. In these experiments eserine sulphate was added to the perfusion fluid in a concentration of lo-” g/ml and also injected intravenously (0.1 mg/kg). Five experiments, in which the central canal was perfused, provided samples of perfusate which possessed negative inotropic activity on the clam heart and contracted the leech muscle. The contractile activity on the leech muscle was abolished by the addition to the bath of tubocurarine chloride (lo-’ g/ml). In one instance a simple matching assay was carried out with acetylcholine, indicating activity equivalent to approx. 90 ng ACh in 1.6 ml control perfusate collected over a 30 min period and 120 ng ACh in the perfusate collected during a similar period of continuous stimulation of the sciatic nerve (5 V, 0.4 msec, 10 Hz).
DISCUSSION
These experiments show that the central canal (and subarachnoid space) of the cat and rabbit spinal cord can be perfused for a number of hours without any significant loss of neuronal activity. There is a diffusion of substances from canal to neurones and from neurones to canal. Thus, addition of strychnine and mephenesin to the perfusates increased and decreased cord activity respectively. as evidenced by ventral root recording and electromyography, and changes were also obtained in compound ventral root potentials after perfusion of acetylcholine. The appearance of acetylcholine in the perfusates is evidence for the movement of transmitters through the cord to the canal, and autoradiography has also shown that C3H]glycine and [‘Hlnoradrenaline distribute within the cord after perfusion through the central canal (Dennison. this laboratory unpublished observations).
ACh Wq/ml) 30min
NORMAL CSF IOmin
Fig. 5. Effect of acetylcholine on compound ventral root potentials recorded in the spinalised cat during perfusion of the central canal of the spinal cord. Responses are shown to stimulation of both sural and gastrocnemious nerves at 8, 10 and 12 V. After eserine (0.2 mg kg. i.v.) the addition of acetylcholine to the perfusion fluid (10-6gml) caused increased polysynaptic activity after 20 or 30min of perfusion but had variable effects on the monosynaptic spike. After returning to normal arti~ciai CSF there was some reduction in the polysynaptic spike within 10 min.
6
1. K. M. MORTON,C. J. STAGG and R. A. WEBSTER
Perfusion of the central canal has many advantages over subarachnoid perfusion. The latter is more complicated surgically; it prevents stimulation of the dorsal roots (since the dura cannot be cut) and there is less chance of stimulating descending tracts. In addition, dye studies have shown that the flow pattern is very uneven and that large tracts on the surface of the cord may not come into direct contact with the fluid because of the barrier presented by the roots around the lower part of the cord or the tightness of the dura. The response to strychnine shows that the method can be used to apply drugs directly to the spinal cord although it lacks the precision of iontophoresis. Also. clear results may not be obtained with many compounds, particularly transmitters, unless degradation can be stopped. Thus, we found that acetylcholine could increase polysynaptic activity but only in the presence of eserine. Perfusion of the central canal is more likely to find use in studying the release of neurotransmitters than is subarachnoid perfusion, since the length of cord perfused contains a large number of identical neurones, synapses and synaptic arrangements that may be more or less specifically activated by various types of stimulation, e.g. dorsal roots, ventral roots or destending tracts. There are obviously diffusional and metabolic barriers for released substances to cross before they reach the canal but the detection of acetylcholine in amounts that can be increased by stimulation of sciatic nerves is encouraging and further studies on this and the amino acids are in progress.
Acknowledyemrnts-The authors are greatly indebted to Mr. F. Ballhatchet for the construction of the myograph table and other apparatus. The hard work and technical assistance of Mr. I. M. Jones is much appreciated. C.J.S. was an MRC scholar.
REFERENCES Edery, H. and Levinger, I. M. (1971). Acetylcholine release into the perfused intermeningeal spaces of the cat spinal cord. Neuropharmacoloyy 10: 239-246. Feldberg. W. and Fleischhauer. K. (1960). Penetration of bromophenol blue from the perfused cerebral ventricles into the brain tissue. J. Phvsiol.. Land. 150: 452462. Feldberg, W. and Sherwood, S: (1954). Injections of drugs into the lateral ventricle of the cat. J. Phq‘siol., Lend. 123: 148-167. Florey, E. (1967). The clam heart bioassay for acetylcholine. Camp. Eiochem. Physiol. 20: 365. K uno, M. and Rudomin. P. (1966). The release of acetylcholine
from the spinal
cord
of the cat by antidromic
stimulation of motor nerves. J. Physiol., Land. 187: 177_~193. McIntosh, F. C. and Oborin, P. E. (1953). Release of acetylcholine from intact cerebral cortex. XIXth International Physiological Congress. pp. 58(t581. Abstract. McIntosh, F. C. and Perry, W. (1951). Biological estimation of acetylcholine. In: Methods in Medical Research. Vol. 3 (Gerrard, R. W., Ed.) pp. 78-92. The Year Book Publisher. Chicago. Mitchell, J. F. and Phi]lis, J, W. (1962). Cholinergic transmission in the frog spinal cord. Br. J. Pharmac. 19: 534-543. Morton, I. K. M. M., Stagg, C. J. and Webster, R. A. (1969). Perfusion of the sub-arachnoid space and central canal of the mammalian spinal cord. J. Physiol. 202: 72P.
of Pharmacology,
University
College
London,
(Accepted 24 March
Gower
Street, London
WClE
6BT
1976)
Summary~Methods have been developed which allow perfusion of the central canal (or subarachnoid space) of the cat and rabbit spinal cord in uiuo for several hours without any significant loss of cord function. Changes in neuronal activity, as evidenced by ventral root recording and electromyography, could be induced by the addition of drugs (strychnine, mephenesin) and neurotransmitters (acetylcholine) to the perfusate. The detection of acetylcholine in the perfusate in amounts that were increased by stimulation of neural inputs to the cord suggests that the method is suitable for the collection of transmitters released in the spinal cord. Central canal perfusion has the advantage of allowing tracts. independent stimulation of ventral and dorsal roots and descending
of substances to neurones in the central nervous system and the collection of transmitters released by neural stimulation necessitates circumvention of the blooddbrain barrier. The development of iontophoretic techniques has overcome this problem with respect to the application of transmitters but their collection still presents many problems. Small glass or Perspex cylinders or ‘cups’ (McIntosh and Oborin, 1953) applied to the surface of the brain have enabled a number of workers to study the release of acetylcholine (ACh) from the cerebra1 cortex and other parts of the brain, and Feldberg and Sherwood (1954) have described methods for perfusing the ventricular spaces of the brain. There are no comparable methods for studying release in the spinal cord although the accessibility of peripheral nerves, dorsal roots and descending tracts for stimulation makes activation of certain pathways relatively easy. Perfusion of the cord vasculature rapidly leads to loss of neuronal activity (Kuno and Rudomin, 1966) and there still remains a diffusional barrier. Edery and LevingeY (1971) perfused the subarachnoid space of the cat by simply piercing the dura with fine needles at two levels (e.g. thoracic and sacral) but they were unable to detect a reproducible stimulated release of transmitters The superfused hemisected frog or toad cord has been used to show the ,release of acetylcholine (Mitchell and Phillis, 1962). but there is probably reduced synaptic transmission in this preparation especially in polysynaptic pathways. This paper describes in some detail the development of a method for perfusing the central canal or subarachnoid space of the cat and rabbit cord in viuo (see also Morton. Stagg and Webster, 1969). We have The application
* Present address: t Present address: St. Albans, England.
King’s College, London. Union International Research
Centre,
shown that the cord retains a high level of tivity, that responds to drugs added to the fluid, for several hours, and that stimulation inputs leads to the appearance of putative ters in the perfusate.
reflex acperfusion of neural transmit-
METHODS
General Experiments were performed on rabbits and cats of either sex and mainly in the weight range 2-3 kg. Animals were anaesthetised with halothane, after induction, in the case of rabbits, with intravenous thiopentone sodium (25 mg/‘kg). The trachea, left common carotid artery and right jugular vein were then cannulated and the right common carotid artery was ligated. Immobilisation of the animal was achieved by attaching clamps to each femur, the lateral processes of the third lumbar vertebra and across the ischial tuberosities of the pelvic girdle. The clamps were secured to pillars on a heavy T-slotted myograph table. Laminectomy was carried out on the dorsal aspect from L4-S2 and the sciatic nerves exposed. Decerebration at the mid-collicular level (rabbit) or spinalisation at Cl (cats) was then performed. Unless muscle activity was to be recorded the animals were injected intravenously with either tubocurarine chloride (0.5-l mg every 30 min) or gallamine triethiodide (4-8 mg every 30 min) and maintained on artificial respiration. Perfusion of the central canal Perfusion of the central canal with artificial cerebrospinal fluid (CSF), as used by Feldberg and Fleischhauer (1960), was achieved by inserting a 25.S.W.G. (0.51 mm outside diameter) stainless steel needle into the canal at L4/5 and cannulating the
I. K. M. MORTOKC. J.
2
canal at the sacral end. The needle formed the descending arm of a perspex T-piece connector (Fig. I) which was mounted in a micromanipulator (Narashige). Artificial CSF was supplied at a rate of 0.06 ml/min by a slow infusion apparatus (C. F. Palmer) through a second arm of the connector after being warmed to 37’C. The pressure in the system was recorded through the third arm with a transducer on a polygraph (Bell & Howell) and monitored (Grass). After cutting the dura mater the needle was positioned on the surface of the cord along the midline at L4. but clear of the central dorsal vein, The dorsal columns of a small length (5 mm) of cord were then retracted at Sl to expose the central canal and a short length of fine thread passed around the cord just above (cephalic to) the opening. The needle was next lowered into the cord to a tip depth of approx. 3.5 mm at an angle of 60 --80 to the horizontal. The depth of penetration was varied until flow was obtained at the sacral end with minimal pressure (less than 10mm Hg) in the perfusion system. When flow had been established a length of polyethylene tubing (Portex PP50), drawn to a tip diameter of approximately I mm, was inserted into the canal. In some cases this was secured by the thread previously placed around the cord. The tubing was held in the correct plane by means of cotton wool which was secured under 2 or 3 stainless steel needles pushed through the muscle on either side of the cord. When flow was established at a satisfactory pressure and recovery rate, the cord was covered with a thin layer of cotton wool moistened in artificial CSF. If ventral root recording was to be attempted. a layer of liquid paraffin was used. Perfusion of the suharuchnoid .spc~e Only cats were used in these experiments because of their greater ability to withstand the surgery and
and R. A.
STAW
WEBSTER
the tougher nature of the dura mater. After dorsal laminectomy (L4- S3) two cuts were made in the dura at L4 up to 8 mm apart (at right angles to the cord) so as to leave the ventral half of the dura intact. The portion of cord beneath the opening in the dura was then removed. The ends of a length of fine stainless steel wire were passed through a piece of 2 mm inside diameter stainless steel tube bent to a right angle and the loop of wire protruding formed into a circle of the same diameter as the inside of the dura mater. This loop was then positioned completely around the outside of the cord caudal to the section but inside the dura mater. The loop was passed 4&6.Omm down the subarachnoid space. and when traction was applied to the free ends of the wire. the loop closed to cut cleanly through the cord. This allowed a small section to be withdrawn from within the intact dura mater. A Perspex cannula of similar diameter to the cord (4.&6.0mm) was tied into the dura with a silk ligature. Cannulation of the subarachnoid space at its sacral end was carried out in much the same fashion except that cannulae as small as 2.0 mm diam were required. The perfusion system was connected to the thoracic cannula to give a flow of 0.12 ml/min at a pressure below 10mm Hg. The area of cord perfused in both cases contained the motoneurones and afferent inputs for both sciatic and femoral nerves.
Assays of ACh-like activity were carried out using either the clam (Mrrcemria mrcermriu) heart preparation as described by Florey (1967) or the leech (Hirudo medicinah) dorsal muscle. according to the method of Macintosh and Perry (1950). In both cases contractions were recorded auxotonically by interposing a light spiral spring between the preparation and an isometric force transducer (Grass FT03). cinder
I
I
PERFUSATE COLLECTED FOR ANALYSIS
L4
L5
L6
L7
SI
52
I
I ORIGINS OF FEMORAL AN0 SCIATIC NERVES
Fig. 1. Diagrammatic
presentation of system for perfusion of the subarachnoid space (A) and central canal (B) of the cat spinal cord in tko. Artificial cerebrospinal fluid (stippled area) was passed from an infusion pump through a heating coil to the needle (canal perfusion) or cannula (subarachnoid). The pressure in the system was recorded with a pressure transducer and monitored with a pen recorder.
Spinal cord perfusion favourable conditions the clam assay was able to detect ACh at bath concentrations in the range lo- “-lO- lo M but gave a very steep doseeresponse relationship. The leech preparation rarely detected ACh at concentrations less than lo-* M but was more easily manageable. Since the composition of the artificial CSF differed from that of the media used to bathe these preparations, it was particularly necessary to provide adequate controls when a significant amount of the former was added to the organ bath. Stimulating and recording procedures Contractions of the left gastrocnemius muscle were recorded by connecting the tendon to an isometric force transducer (Grass, FTlO). The electromyogram (EMG) was obtained with two small (20 SWG) stainless steel needles inserted 10mm apart beneath the skin above the gastrocnemius muscle, and amplified for display on a Grass polygraph. Discharges in filaments dissected from ventral roots L7 or Sl were recorded from one side of bipolar platinum electrodes, the other being either earthed or balanced with a crushed root filament. The electrodes were connected to paired cathode followers and the output displayed on an oscilloscope. Compound potentials were recorded from a whole or a substantial part of a ventral root suspended across both electrodes, generally with the part over the distal electrode crushed. RESULTS Demonstration qf the functional integrity of‘ the spinal cord during pelCfilsion experiments R#e.x acticity. Experiments were performed to assess the functional state of the spinal cord during prolonged perfusion by both the central canal and the subarachnoid routes. The physiological state of the cord was monitored by comparing the size of directly- and reflexly-induced muscle twitches. The isometric tension developed in the gastrocnemius group of muscles on stimulation of the medial gastrocne- _ mius nerve (direct) and central end of the cut sural nerve (reflex) was displayed on a polygraph. Figure 2 shows the results of two such comparisons where muscle activity was evoked at regular intervals over the course of 9@150 min. It can be seen that the threshold voltage for stimulation and tension responses were not constant for reflex stimulation. However, these fluctuations were reflected by similar variations in the directly evoked responses and so they would not seem to be due to changes within the spinal cord but rather to some peripheral alteration common to both pathways. Thus, there was no evidence of reduced function in the cord. Ventral root recording. Compound potentials reflect activity in a large number of neurones but- they are not reliably quantitated over prolonged periods of time as the amplitude may change spontaneously through alterations in electrode conductivity caused
3
by blood or fluid. Discharges in ventral root filaments reflect activity in a smaller number of neurones but there was ample evidence from such experiments that could be spontaneous and evoked discharges recorded over 34 hr of central canal perfusion. Although no systematic analysis of electrolyte balance between the perfused artificial CSF and nervous tissue was made, analysis of the fluid by flame photometry for potassium before and after perfusion through the central canal (both in the resting state and on electrical stimulation of the sciatic nerve), showed no marked change in the level of this ion. This point is of importance in order to eliminate the possibility that changes in the activity of perfusates on bioassay were not due to alterations in potassium content of the assay fluid. It was also found from an analysis of perfusates on a Technicon autoanalyser that they contained most of the amino acids found in natural CSF taken from the animals. but often only in trace amounts. There was no salt loss, or gain of fluid during perfusion, and no oedema. The histological picture of the cord was normal apart from some damage immediately around the input needle and sacral cannula. Blood-free perfusates were always obtained with canal perfusions and generally with the subarachnoid route. Changes in neuronal activity induced by substances added to the perfusion medium Figure 3 shows the effect on compound potentials of perfusion of 12pg/min through the central canal of the rabbit.
0
30
60
90
Time
(min)
120
150 0
ventral root strychnine Stimulation
x,
60
Time
(min)
90
Fig. 2. Tests of functional integrity of the cat spinal cord during central canal (A) and subarachnoid (B) perfusion. Voltage threshold of stimulated nerve (upper graph) and twitch strength of gastrocnemious muscle (lower graph) are shown for both direct stimulation of the motor nerve (M) and reflex activation through sural nerve stimulation (W----a). Although the response to reflex stimulation (2.5 g/mm trace) was always less than that for direct stimulation (12.5 g/mm) there was no change in response over the period of perfusion.
I. K. M. MORTON. C. J. STACG and R. A. WEBSTER
CONTROL
-14\J
14v
12v
IOV
3
IOmV
STRYCHNINE
. 12v
IOV Medial
.
14v
14v
Sural stimulation
lOmsec
gastrocnemius stimulation
Fig. 3. Effect of strychnine on a compound ventral root potentials recorded during perfusion of the central canal of the spinal cord of a decerebrate rabbit. Stimulation of the central end of the nerve to medial gastrocnemious produced a stimulus related monosynaptic component. After strychnine (12 pg/min) the same stimuli also produced polysynaptic activity and there was an increased polysynaptic response to sural nerve stimulation.
A
I
2
; .
i
\
1 1 .
1 .
1
I
.
.
B &
2-
--c.
.
1
.
,
:
C
.
.
.
.
012
set
Fig. 4. Electromyographic response to perfusion of strychnine through the central canal of the spinal cord of a decerebrate rabbit. In control studies (A), stimulation of one sural nerve produced ipsilateral activity (1) but no contralateral effect (2). Strychnine perfusion (12 pg min) increased ipsilateral activity and initiated activity in the contralateral muscle. (B, C, D at 2, 5 and 10min respectively after start of strychnine perfusion).
Spinal cord of the ipsilateral medial gastrocnemius nerve produced a monosynaptic spike, whereas sural nerve stimulation gave polysynaptic discharges. It can be seen that strychnine increased the amplitude of both monosynaptic and polysynaptic potentials, and furthermore introduced a polysynaptic component to the response seen on medial gastrocnemius stimulation. The marked change in polysynaptic activity indicates that the inte~eL]rones, which are most susceptible to asphyxia, remained functiona during cord perfusion. Evidence of exaggerated neuronal activity was also seen in electromyogram (EMG) recordings during perfusion of the central canal with strychnine. Figure 4 is representative of experiments where perfusion of 12pg/min strychnine in every case resulted in increased EMG activity within 1 min of it entering the cord. The gradual onset of increased muscle activity can be seen in the figure; s~ntaneous and evoked activity both increased markedly and activity appeared in the contralateral muscle. This increased activity reached a peak in 5-10min and died away slowly when strychnine perfusion was discontinued unless terminated by mephenesin given either intravenously (20mg/kg) or into the perfusion medium (1 m&ml). These results show that strychnine can penetrate to spinal neurones from the canal. Similar results were obtained when strychnine was added to subarachnoid perfusates although the response was not as great. Acetylcholine (lo-” g/ml) in the presence of eserine (0.2 mg/kg, i.v.) also modified compound ventral root potentials, when perfused through the central canal, causing a general increase in polysynaptic activity (Fig. 5) and variable effects on the monosynaptic component. Release of acetylcholine into perfusates. Some evidence was obtained for the presence of ACh in perfu-
CONTROL
ESERINE (0.2mg/kqi.v.)
5
perfusion
ACh (KPg/ml) 2Omin
sates after passage through the central canal. In these experiments eserine sulphate was added to the perfusion fluid in a concentration of lo-” g/ml and also injected intravenously (0.1 mg/kg). Five experiments, in which the central canal was perfused, provided samples of perfusate which possessed negative inotropic activity on the clam heart and contracted the leech muscle. The contractile activity on the leech muscle was abolished by the addition to the bath of tubocurarine chloride (lo-’ g/ml). In one instance a simple matching assay was carried out with acetylcholine, indicating activity equivalent to approx. 90 ng ACh in 1.6 ml control perfusate collected over a 30 min period and 120 ng ACh in the perfusate collected during a similar period of continuous stimulation of the sciatic nerve (5 V, 0.4 msec, 10 Hz).
DISCUSSION
These experiments show that the central canal (and subarachnoid space) of the cat and rabbit spinal cord can be perfused for a number of hours without any significant loss of neuronal activity. There is a diffusion of substances from canal to neurones and from neurones to canal. Thus, addition of strychnine and mephenesin to the perfusates increased and decreased cord activity respectively. as evidenced by ventral root recording and electromyography, and changes were also obtained in compound ventral root potentials after perfusion of acetylcholine. The appearance of acetylcholine in the perfusates is evidence for the movement of transmitters through the cord to the canal, and autoradiography has also shown that C3H]glycine and [‘Hlnoradrenaline distribute within the cord after perfusion through the central canal (Dennison. this laboratory unpublished observations).
ACh Wq/ml) 30min
NORMAL CSF IOmin
Fig. 5. Effect of acetylcholine on compound ventral root potentials recorded in the spinalised cat during perfusion of the central canal of the spinal cord. Responses are shown to stimulation of both sural and gastrocnemious nerves at 8, 10 and 12 V. After eserine (0.2 mg kg. i.v.) the addition of acetylcholine to the perfusion fluid (10-6gml) caused increased polysynaptic activity after 20 or 30min of perfusion but had variable effects on the monosynaptic spike. After returning to normal arti~ciai CSF there was some reduction in the polysynaptic spike within 10 min.
6
1. K. M. MORTON,C. J. STAGG and R. A. WEBSTER
Perfusion of the central canal has many advantages over subarachnoid perfusion. The latter is more complicated surgically; it prevents stimulation of the dorsal roots (since the dura cannot be cut) and there is less chance of stimulating descending tracts. In addition, dye studies have shown that the flow pattern is very uneven and that large tracts on the surface of the cord may not come into direct contact with the fluid because of the barrier presented by the roots around the lower part of the cord or the tightness of the dura. The response to strychnine shows that the method can be used to apply drugs directly to the spinal cord although it lacks the precision of iontophoresis. Also. clear results may not be obtained with many compounds, particularly transmitters, unless degradation can be stopped. Thus, we found that acetylcholine could increase polysynaptic activity but only in the presence of eserine. Perfusion of the central canal is more likely to find use in studying the release of neurotransmitters than is subarachnoid perfusion, since the length of cord perfused contains a large number of identical neurones, synapses and synaptic arrangements that may be more or less specifically activated by various types of stimulation, e.g. dorsal roots, ventral roots or destending tracts. There are obviously diffusional and metabolic barriers for released substances to cross before they reach the canal but the detection of acetylcholine in amounts that can be increased by stimulation of sciatic nerves is encouraging and further studies on this and the amino acids are in progress.
Acknowledyemrnts-The authors are greatly indebted to Mr. F. Ballhatchet for the construction of the myograph table and other apparatus. The hard work and technical assistance of Mr. I. M. Jones is much appreciated. C.J.S. was an MRC scholar.
REFERENCES Edery, H. and Levinger, I. M. (1971). Acetylcholine release into the perfused intermeningeal spaces of the cat spinal cord. Neuropharmacoloyy 10: 239-246. Feldberg. W. and Fleischhauer. K. (1960). Penetration of bromophenol blue from the perfused cerebral ventricles into the brain tissue. J. Phvsiol.. Land. 150: 452462. Feldberg, W. and Sherwood, S: (1954). Injections of drugs into the lateral ventricle of the cat. J. Phq‘siol., Lend. 123: 148-167. Florey, E. (1967). The clam heart bioassay for acetylcholine. Camp. Eiochem. Physiol. 20: 365. K uno, M. and Rudomin. P. (1966). The release of acetylcholine
from the spinal
cord
of the cat by antidromic
stimulation of motor nerves. J. Physiol., Land. 187: 177_~193. McIntosh, F. C. and Oborin, P. E. (1953). Release of acetylcholine from intact cerebral cortex. XIXth International Physiological Congress. pp. 58(t581. Abstract. McIntosh, F. C. and Perry, W. (1951). Biological estimation of acetylcholine. In: Methods in Medical Research. Vol. 3 (Gerrard, R. W., Ed.) pp. 78-92. The Year Book Publisher. Chicago. Mitchell, J. F. and Phi]lis, J, W. (1962). Cholinergic transmission in the frog spinal cord. Br. J. Pharmac. 19: 534-543. Morton, I. K. M. M., Stagg, C. J. and Webster, R. A. (1969). Perfusion of the sub-arachnoid space and central canal of the mammalian spinal cord. J. Physiol. 202: 72P.