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Functional changes in undamaged sciatic nerves and spinal cord of mice following nerve damage
EXPERIMENTAL
NEUROLOGY
65, %7-607 (1979)
Functional Changes in Undamaged Spinal Cord of Mice following RICHARD Department
A. GERREN
of Aerospace
AND MARVIN
Engineering Boulder,
Received
September
21.
Sciatic Nerves Nerve Damage
Sciences, Colorado
1978;
revision
and
W. LUTTGES’ University
of Colorado,
80309
receir,ed
April
25,
1979
The effects of sciatic nerve crush on electrophysiologic characteristics of undamaged contralateral sciatic nerve and spinal cord were studied in mice. Composite responses were elicited from both sciatic nerve and spinal cord by single- or paired-pulse stimulation. Conduction velocities of the initial components were not different in undamaged contralateral and control nerve. Nerve recovery, characterized by amplitude ratios of paired-pulse responses, yielded a depressionfacilitation-depression pattern in control sciatic nerves with increasing intervals between pulses. Recovery in undamaged contralateral nerves did not show this pattern. Compared to controls, response amplitude ratios from undamaged contralateral nerves were either depressed or facilitated depending on the time elapsing between ipsilateral nerve crush and subsequent testing. Upon removal of spinal influences by nerve transection, the pattern of contralateral nerve recovery more closely followed the recovery pattern of control nerve. Composite cross-cord responses (CCRsj were obtained from one sciatic nerve after stimulation of the opposite sciatic nerve. The area under both CCRs elicited by paired-pulse stimulation was compared to the area under a CCR elicited by single-pulse stimulation. For control cord, CCRs were facilitated with short paired-pulse separation intervals. With longer interpulse intervals, CCRs were depressed. CCRs from animals receiving nerve crush were either depressed (6 days postcrushj or facilitated (10 days postcrushj across all pulse separation intervals compared to control. The most significant treatment effects were found in tests of pathways from damaged nerve to undamaged contralateral nerve in 6-day postcrush Abbreviations: CONTRA-contralateral, RAR-relative amplitude ratio, CCR-crosscord response. ’ We wish to thank J. Button for assistance in preparing the manuscript and W. Bank for technical assistance. The work was supported, in part, by the International Chiropractors Association and the National Institutes of Health grant NINCDS 1 POINS-12226. R. A. Gerren is a Stanley Aviation Fellow. 587 0014-4886/79/090587-21$02.00/0 Copyright All rights
0 1979 by Academic Press. Inc. of reproduction in any form reserved.
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preparations. The results suggest that a spinal mechanism may be responsible for the phasic alterations in electrophysiologic characteristics of both undamaged contralateral nerve and spinal cord. The data have implications for the possibility of functionally altered contralateral motoneurons being part of this spinal mechanism.
INTRODUCTION Many investigators have studied the functional (12,14,22,28,29,33) and structural (21, 22, 36) alterations which occur in peripheral nerves after various types of experimental nerve damage. Other investigators studied the effects of these damage-induced changes on the ipsilateral spinal pathways and motoneuron cell bodies (7, 9, 20, 34). Several of these investigators (9,34) compared the spinal pathway responses obtained from the side with the damaged nerve to those obtained from the undamaged contralateral side. The underlying assumption of such experiments was that contralateral nerve remained unchanged. However, detailed investigations were not carried out comparing nerves contralateral to damaged nerves with nerves contralateral to undamaged nerves. Detailed investigations of cross spinal pathways were initially undertaken by Sherrington (32). Since then, many investigators have observed that ipsilateral nerve stimulation alters the contralateral monosynaptic response (19, 25, 26). The alterations of the contralateral monosynaptic response may be due to either ipsilateral input coming directly to contralateral motoneurons (l-3, 27), to contralateral interneurons (13), or from presynaptic inhibition of contralateral afferent fibers (6, 10, 11). Because the contralateral spinal pathways normally seem to be influenced by input from the ipsilateral side, it seems reasonable that disruption of that input, through nerve damage, may alter the functional and structural properties of the contralateral side. Recently, Luttges et al. (22) compared the electrophoretic protein patterns obtained from contralateral undamaged mouse sciatic nerves and sciatic nerves from mice receiving no nerve damage. They found differences in the protein patterns obtained from these two different types of controls. These changes were attributed to possible compensatory effects initiated as a consequence of the dependence on the single undamaged leg for hind limb locomotion. These observations suggest that structural alterations arise in contralateral nerves as a consequence of increased nerve use, decreased damaged nerve influences, or altered metabolic activity. The present study focused on changes in neurophysiologic indexes for mouse contralateral sciatic nerves and spinal cord after ipsilateral nerve crush. Altered neurophysiologic indexes in contralateral nerve occurred
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FIG. 1. Schematic representation of electrode configurations used for various tests listed in Table 1. Also shown are sites of nerve crush and nerve cut (in appropriate experiments).
only with paired-pulse stimulation and seemed to result from phasic biasing of contralateral nerve by a spinal mechanism. The phasic effects in nerves appeared closely related to a spinal mechanism which produced demonstrable phasic changes in crossed spinal pathways. Even in the absence of spinal influences, subtle neurophysiologic changes were resolved in contralateral compared to normal nerves. The changes in the nerve, itself, remain to be investigated. METHODS Animals. Both male and female HS mice (23) were randomly selected from group (eight mice) cages. Except during surgical preparation or neurophysiologic testing, mice were providedad libitum access to food and water. Nerve Damage. Two groups of mice, experimental and control, were used. Mice in the experimental group were prepared by systematically crushing either left or right sciatic nerve. For surgery, all mice were anesthetized with injections of pentobarbital sodium (90 mg/kg body weight, i.p.). The skin over one thigh was carefully slit and the underlying musculature exposed. The muscles immediately surrounding the sciatic nerve were carefully retracted, leaving the nerve exposed. In experimental mice the nerve was squeezed 20 s under considerable pressure using fine mosquito forceps. The crushed portion (1
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to 2 mm) was inspected to determine whether or not perineurial integrity remained. If perineurial integrity was disrupted, the animal was not used. The wound was cleaned with 0.15 N saline and was sutured closed with stainless-steel (9 mm) wound clips. After the operative procedure, the experimental mice were placed in group (three mice) cages. At least 2 days elapsed before neurophysiological testing to minimize any lingering anesthetic or operative effects. Experimental Procedures. All mice were prepared for neurophysiological testing by anesthetization with chloral hydrate (700 mg/kg body weight, i.p). The anesthetized mice were placed on a water-heated aluminum plate and a rectal thermister was used to maintain the body temperature at 38.5”C. The skin overlying both sciatic nerves was cut away carefully. The muscles surrounding the sciatic nerves were tied back with silk sutures, leaving the nerves fully exposed. Both nerves were covered with pools of warm mineral oil to prevent dessication. Bipolar stimulating electrodes (300-pm platinum wire) and bipolar recording electrodes (250~pm tungsten wire with tips etched to approximately 150 pm) were placed on the sciatic nerves in one of several configurations (Fig. 1, Table 1). Stimuli (0.25-ms duration, single or paired pulses) were produced by a square wave generator and delivered to the test preparation via an isolation transformer with diode suppression. Evoked responses were obtained by differential AC preamplification (Argonaut Model L042). Evoked responses were either averaged (Princeton Applied Research, Waveform Eductor) and recorded (Sanborn Model 150) graphically or were recorded directly on film (DuMont Kymographic Camera). Sciatic Nerves. Two neurophysiological tests were carried out on sciatic nerve. Test I (Table 1, Fig. 1) was used to determine the conduction velocity of the initial component of responses evoked in control, damaged, and undamaged contralateral (CONTRA) sciatic nerve. Both damaged and CONTRA nerves were investigated on days 3 through 10 and 30 days after nerve crush. Single-pulse stimulation (0.5 Hz) was of sufficient voltage (3 to 6 V) to elicit suprathreshold responses. For each sciatic nerve, 10 suprathreshold responses were averaged. The latency for each averaged response was measured between the onset of the stimulus artifact and the initial response deflection from baseline. The conduction velocity of the initial component in any one preparation was obtained by dividing the latency into the measured distance between the stimulating and recording electrodes (7 to 9 mm). Conduction velocities were obtained from three control nerves, 27 CONTRA nerves (three at each postcrush test day), and several damaged nerves at various intervals after nerve crush. To determine whether control and CONTRA conduction velocities were significantly different, data from these preparations were subjected to a
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TABLE Electrical Characterizations
DAMAGE
591
1
of Sciatic Nerve and Spinal Cord
Electrode configuration Stimulating
Recording
Stimulus type
Structure
Condition”
3
4
Single pulse
Sciatic nerve
Conduction velocity
2
1
CON and CONTRA Damaged
II
3
4
Paired pulse
Sciatic nerve
CON and CONTRA; intact and severed
RAR’
III
2
3
Single pulse Paired pulse
Spinal cord Ib
CON and EXP
Single pulse Paired pulse
Spinal cord IS’
EXP
Test I
IV
3
2
Measure
Reference CCRd Test CCR” Reference CCRd Test CCR”
n CON-sciatic nerves or spinal cord in mice receiving no nerve crush, CONTRAsciatic nerves contralateral to damaged sciatic nerves, EXP-spinal cord in mice receiving nerve damage, intact and severed-electrical characterizations were obtained from CON and CONTRA nerves before and after nerve transection from cord. * Spinal cord I-nerve crush-induced changes in spinal pathways from damaged nerve atferents to CONTRA nerve, spinal cord II-nerve crush-induced changes in spinal pathways from CONTRA nerve atferents to damaged nerve. c RAR-sciatic nerve relative response ratio. Ten responses to paired stimuli were averaged for each nerve. The average amplitude of the second response was divided into the average amplitude of the first response to obtain the RAR. An RAR was obtained at each pulse separation interval (3, 10, 25, 50, 100, and 250 ms). d Reference CCR (cross-cord response)-the average area of the first component of 10 cross-cord responses elicited by single-pulse stimulation, test CCR-the combined first component areas from 10 consecutive cross-cord responses, elicited by paired-pulse stimulation, were averaged. The test CCR at each pulse separation interval (5, 10, 25, 50, 100, and 250 ms) was divided by the reference CCR from the same preparation, which gave a CCR area ratio.
one-way analysis of variance. A one-tailed test was used to determine significance based on expectations from previous work (16). Test II (Table 1, Fig. 1) was used to investigate response recovery in control and CONTRA nerve. This was accomplished by using paired-
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pulses of equal duration (0.25 ms) and intensity (suprathreshold). Suprathreshold voltage (3 to 6 V) was determined by single-pulse stimulation. Paired-pulse separation intervals of 3, 10,25,50, 100, and 250 ms were tested. For each nerve, 10 paired-pulse stimuli (one pair every 2 s) at each separation interval were delivered. The 10 elicited responses were averaged and the amplitude of the initial component of these averaged responses measured. The initial component amplitude of the averaged first response was divided into that of the second, for each pulse separation interval. This resulted in a relative amplitude ratio (RAR) used to characterize nerve recovery. RARs across all pulse separation intervals were obtained from six control nerves and 27 CONTRA nerves (three at each postcrush test day). After completion of the above tests for intact nerve preparations, each nerve was severed with fine iris scissors within 10 mm of the spinal cord (Fig. 1). Care was taken to prevent severance of any major blood vessel or displacement of the stimulating electrodes. Nerve severance from the spinal cord was done to remove spinal influences over nerve responsiveness. The severed nerves were tested in the same manner as intact nerve. Ten paired-pulses were given at each separation interval and the averaged first component amplitudes of first and second responses used to calculate the RARs. The RARs from each nerve type (CONTRA and control), condition (before and after severance), and pulse separation interval were subjected to a three-way analysis of variance. Additionally, CONTRA nerve data at each postcrush test day were compared directly to control nerve data using a two-way analysis of variance. These simple comparisons were made separately for data obtained before and after nerve transection. Spinal Cord. Two neurophysiological tests were used to determine if sciatic nerve crush affected transmission in cross-spinal pathways. Test III (Table 1, Fig. 1) was focused on studies of the pathways from damaged nerve afferents to the CONTRA nerve. This electrode arrangement also was used for control cord. In test IV (Table 1, Fig. l), the pathways from CONTRA nerve afferents to damaged nerve were studied. Single-pulse stimuli (0.25 ms duration; 0.5 Hz) of increasing intensity were delivered until a stable cross-cord response (CCR) was obtained. CCRs elicited from 10 consecutive stimuli were averaged. For each preparation, a single-pulse, average CCR was used as the reference response. Paired-pulse stimuli of the reference intensity were delivered at various pulse separation intervals (5, 10,25, 50, 100, and 250 ms). CCRs elicited from 10 paired-pulse stimuli (test CCRs) at each pulse separation interval were averaged. The area under both test CCRs, whether superimposed (short separation intervals) or separate, was measured and divided by the area under the single-pulse,
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reference CCR. This gave a CCR area ratio at each pulse separation interval. A CCR area ratio equal to 2 indicates that no interaction between paired CCRs occurred. CCR area ratios were obtained from three control, three 6-day postcrush and three IO-day postcrush nerve preparations. Datafrom both tests III and IV were obtained for 6- and lo-day preparations. CCR area ratios from all preparations were compared using a two-way analysis of variance. Also, CCR area ratios from each postcrush test day similarly were compared to control CCR area ratios. RESULTS After recovery from anesthesia, all experimental mice demonstrated severely impaired motor function in the leg with the crushed nerve. Although the mice soon appeared to compensate during locomotion with the undamaged leg, debilitation remained noticeable for as long as 30 days. Inspection of the crushed nerves revealed damage identical to that described previously by Luttges et al. (22). The damage consisted of swelling and discoloration distal to the site of the crush. The proximal portion of the nerve was slightly swollen, but discoloration was not as obvious. The undamaged CONTRA nerves, in contrast, appeared quite normal. Sciatic Nerve. The suprathreshold responses elicited by single-pulse stimulation in test I (Table 1) were similar in waveform appearance for all CONTRA and control nerves. All responses had at least a triphasic waveform, characteristic of bipolar recordings of responses propagated for short distances in the low-threshold, large myelinated fiber population. Occasionally, populations of smaller, slower conducting fibers responded to the suprathreshold stimulation, as indicated by the appearance of more and later response components. However, only the first, triphasic waveform was evaluated in the present study. For all nerve preparations, the latency between onset of stimulus artifact and the initial response deflection from baseline was used, together with measured distances along the nerves between stimulating and recording electrodes, to determine largest fiber conduction velocities. Under our test conditions, the conduction velocities for different preparations was 15 to 25 m/s, and were similar to those observed previously (16, 22). Conduction velocities observed in damaged nerves (6 to 10 m/s) were substantially slower than those in control nerves at all postcrush days tested and were similar to those reported earlier (22). No significant differences (Z’ > 0.25) were observed between the conduction velocities of control and CONTRA nerves, regardless of the number of days elapsing between crush and testing.
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1 I 1 I IO 25 51 100 PULSESEPARATION INTERVAL (mstc)
BEFORE AFTER I 250
B
’
’
20Omsec
I
’
200 msec
500 msec
’
’
FIG. 2. Time course of response recovery for mouse sciatic nerve. A-average response amplitude ratios from six control nerves. Data obtained before and after nerve transection from the spinal cord are plotted separately. B-representative sciatic nerve responses elicited by paired-pulse stimulation for the various test intervals.
Suprathreshold responses elicited by the first of the paired stimuli in test II, were similar in amplitude and latency for all CONTRA and control nerves. However, suprathreshold responses elicited by the second of the paired stimuli were noticeably different across various interpulse intervals. Variance in the amplitude of the second response compared to the first was
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seen in both CONTRA and control nerves. Complete quantification was undertaken to examine these amplitude changes in more detail. For each control nerve, the amplitude of the response elicited by the first of the paired stimuli was divided into the amplitude of the response elicited by the second, at each interpulse interval. This resulted in a RAR across all separation intervals for each preparation. The average RARs across separation interval for control sciatic nerve are plotted in Fig. 2A. Representative responses from which the average RARs were obtained are shown in Fig. 2B. At the shortest separation interval (3 m/s), average second response amplitudes were depressed relative to the first. In contrast, second response amplitudes were facilitated compared to the first across longer separation intervals (10 and 25 ms). At the longest separation intervals (50, 100, and 250 ms), second response amplitudes were either equal to or slightly lower than first response amplitudes. These variations in RAR for control nerve were highly significant (P < 0.005) for the entire range of separation intervals tested. After severing each control nerve close to the spinal cord (-10 mm), another series of paired-pulse responses was elicited from the nerve distal to the site of transection. Responses were analyzed by the same methods. The average RARs for these data also are shown in Fig. 2A. Again, the variations in the RAR for the entire range of separation intervals are highly significant (P < 0.005). There is no significant difference (P > 0.20), however, between RARs for control nerves before and after the removal of spinal influences. Paired-pulse data were obtained before and after nerve transection, from CONTRA nerve in animals subjected to nerve crush 3 through 10 or 30 days previously. The average RAR at each separation interval for each day postcrush was subtracted from the average control RAR. This gave the relative deviation of CONTRA RARs from the control values reported above. Calculations were done separately for both before and after transection and are summarized in the three-dimensional plots of Figs. 3A and B, respectively. Unshaded portions of the plotted averages represent those CONTRA RARs larger than the control RARs (most notably at 6 and 10 days postcrush), and shaded portions represent those with smaller RARs. Deviations from control, indicated by more exaggerated convolutions, are quite pronounced if CONTRA nerve-spinal cord integrity is preserved. Pooling the data for each day postcrush and averaging all separation intervals results in Fig. 4A, which depicts the results of tests both before and after nerve transection. The most pronounced deviations from control occurred at 4,6,7, 8,9, and 30 days postcrush for RARs obtained before nerve transection. Statistical analysis reveals that 4, 6, 7, 9, and 30 day
FIG. 3. Effects of nerve crush on undamaged contralateral response amplitude ratios (RARs). Average RARs from three nerves at each postcrush test day were subtracted from average control RARs to obtain the relative deviation from control. Calculations were done separately for before (A) and after (B) nerve transection RARs. Unshaded portions of the plots represent contralateral RARs larger than control and shaded portions represent those with lower values.
A
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AFTER
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NOllvIA3a
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DAMAGE
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postcrush RARs were significantly to highly significantly different (P < 0.005, 0.025, 0.005, 0.025, and 0.05, respectively). After nerve transection, only 3-, lo-, and 30-day postcrush RARs appeared to differ from control values, but statistical analysis did not corroborate such differences (P > 0.05, 0.05, and 0.05, respectively). Before the nerve was severed from the spinal cord, the amplitudes of the first and second responses, potentially, were determined by both the electrical characteristics of the nerve bundle and by influences originating in the spinal cord. Transection of the nerve from the cord removed the potential spinal influences. Therefore, subtraction of the data obtained from control and CONTRA nerves after nerve transection from those obtained before nerve transection should give an indication of the effect nerve crush has on spinal influences on the CONTRA nerve. Those calculations are shown in Fig. 4B. No significant difference (P > 0.20) was observed between control RARs measured before and after nerve transection. In contrast, 3-, 6-, and 7-day postcrush CONTRA RARs appear to have been significantly affected by spinal influences (P = 0.05, 0.05, and 0.05, respectively). Thus, it appears that nerve crush induced the following hierarchy of neural alterations: Striking changes occurred in the damaged nerve. In intact preparations, significant changes also occurred in undamaged CONTRA nerve. Removal of spinal influences by nerve transection eliminated most of the significant postcrush changes in the CONTRA nerve. Modest postcrush changes may have persisted in CONTRA nerve even in the absence of spinal influences. Spinal Cord. Based on the demonstrable nerve changes which appeared to originate in the spinal cord after nerve crush, the spinal cord-sciatic nerve interactions were subjected to further experimental characterization. Paired-pulse stimuli having interpulse intervals of 5, 10, 25, 50, 100 and 250 ms were delivered (0.5 Hz) to one sciatic nerve and responses were recorded from the opposite sciatic nerve (test III, Table 1 and Fig. 1). Several investigators (1, 2, 19) showed that ipsilateral ventral root stimulation has no effect on the contralateral monosynaptic response. Therefore, no attempt was made to preclude antidromic ventral root volleys in cross-cord investigations. Representative control CCRs from mice with no experimental sciatic nerve damage are shown in Fig. 5A. The average inflection latency for control CCRs elicited by single suprathreshold stimuli was 3.94 t 0.36 ms. The area under the CCR obtained by single-pulse stimulation was used as the reference for comparisons of all subsequent paired-pulse evoked CCRs (test CCRs). Figure 5B shows the CCR area ratios of control spinal cord preparations obtained by dividing the reference CCR area into the area of the combined test CCRs, at each separation interval. Control CCRs were
NERVE AND CORD FUNCTION
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U
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---
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BEFORE AFTER
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411 -
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DAYS POST CRUSH 4. Effects of nerve crush on undamaged contralateral response amplitude ratios. A-experimental values were subtracted from control values and averaged for each pulse separation interval. B-data obtained after have been subtracted from data obtained before nerve transection. FIG.
facilitated (area ratio >2.0) with short (5 and 10 ms) interpulse intervals. Depressed CCRs (ratio <2.0) were characteristic of longer (25,50, 100,250 ms) interpulse intervals. These variations in the control CCR were highly significant (P < 0.005) for the range of interpulse intervals tested. Two experimental groups of animals receiving unilateral nerve crush 6 or
GERREN AND LUTTGES
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’
ZDB msec
’
’
zoo msec
i
FIG. 5. Effects of nerve crush on composite cross-cord responses (CCRs). A-representative responses elicited for typical test intervals. B-first component area ratios of CCRs recorded from contralateral nerve after stimulation of damaged nerve. C-area ratios of CCRs recorded from damaged nerve after contralateral nerve stimulation.
10 days previously were subjected to similar tests. The 6-day postcrush group was used because previous nerve testing suggested that at this time spinal influences dominated the changes occurring in undamaged CONTRA compared to control nerve. Conversely, the IO-day postcrush group was chosen because the changes observed in undamaged CONTRA compared to control nerve appeared unaffected by spinal influences at this postcrush time (Figs. 4A and B). In the first set of experiments (test III), paired-pulse stimuli were delivered to the damaged nerve and CCRs recorded from the CONTRA nerve. The average CCR inflection latencies for 6- and lo-day postcrush preparations were found to be 6.63 + 1.09 and 5.02 + 0.72 ms, respectively. These values represent increases over the average control CCR inflection latency (3.94 k 0.36 ms) of 68.3 and 27.4%, respectively. These average inflection latencies were not significantly different (P > 0.05) from control values with the small number of observations used. The CCR area ratios obtained from test III are shown, for 6- and IO-day postcrush groups, in Fig. 5B. CCR area ratios for the experimental groups are seen to vary across pulse separation intervals in a pattern similar to that of the control group. However, the CCR area ratios from the 6-day experimental group, overall, were significantly decreased (P < 0.025) from control values. CCR area ratios from the lo-day postcrush experimental group are not significantly different (P > 0.25) from control values.
NERVE
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SIiRC
1.0
2.0
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P---G
CONTROL 6 DAY
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10 DAY
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so
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1.0 t
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1
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PULSE SEPARATIONINTERVAL(msec) FIG.
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In the second set of experiments (test IV), paired-pulse stimuli were delivered to the CONTRA nerve and CCRs recorded from the damaged nerve. The CCR area ratios for 6- and IO-day postcrush groups, together with control values, are shown in Fig. 5C. Although differences in the CCR area ratios between these experimental groups and the control group seemed to exist in this test paradigm, neither 6- nor lo-day postcrush CCR area ratios varied significantly (P > 0.05 and P = 0.1, respectively) from control values. Thus, nerve crush induced changes in CCRs under the present test conditions. Modest changes occurred in the electrical characterizations of spinal cord-damaged nerve interactions for both experimental groups
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tested. Significant changes occurred in spinal cord-CONTRA nerve interactions for the 6-day postcrush experimental group. At 10 days postcrush, only modest changes occurred in spinal cord-CONTRA nerve interactions. DISCUSSION Mouse sciatic nerves contralateral to crushed sciatic nerves were normal in appearance upon gross inspection. Both their size and color was apparently within the limits of control nerves from mice with no nerve damage. Neurophysiologic responses elicited by single electrical pulses at suprathreshold levels were similar whether recorded from CONTRA or control nerves. No significant differences were observed in either the threshold or conduction velocity of the largest fibers. Significant differences were detected, however, when CONTRA nerves were subjected to paired-pulse stimulation of suprathreshold intensity. In control nerve the response elicited by the second stimulus varied significantly in amplitude compared to the first, for all pulse separation intervals tested. With short interpulse intervals, the relative size of the second response was decreased. With longer interpulse intervals, the relative size of the second response was larger than the first. At the longest interpulse intervals, the relative size of the second response was again decreased, although not to the extent observed with short interpulse intervals. The time course of these amplitude changes (depression-facilitation-depression) followed the combined time courses of the relative refractory period, and the supernormal and subnormal periods, associated with the negative and positive afterpotentials as described by Gasser and Grundfest (15). The ratios of the first and second response amplitudes from CONTRA nerve were quite different from the control ratios for all postcrush test days. For pulse separation intervals, the relative amplitudes of all second responses generally were decreased compared to control values, except at 6 and 10 days postcrush when a relative enhancement of the second response was observed (cf. Fig. 3A). In addition, the pulse separation time course found for control nerve response ratios (depression-facilitationdepression) was not characteristic of CONTRA nerve. On the basis of these and previous observations (22), it is tempting to suggest that CONTRA nerves undergo structural alterations which affect functional properties, such as the relative recovery, supernormal, and subnormal periods. The nerve transection results cast doubt upon such facile interpretations and suggest that the spinal cord plays a major, though not exclusive, role in the observed nerve changes.
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When CONTRA nerves were severed within 10 mm of the spinal cord, it was assumed that participatory spinal influences on the nerve also were removed. Tests done immediately after nerve transection revealed response ratios resembling those obtained from control nerves. Accordingly, the ratios were quite different from those obtained just prior to nerve transection. It is unlikely that such differences in response ratios before and after nerve transection were caused by transection artifact rather than the removal of spinal influences. Control experiments showed that control nerve response ratios did not change after nerve transection whether proximal or distal to the stimulation and recording sites. The observed changes of CONTRA nerve characteristics after the removal of spinal influences suggests that CONTRA nerves may be biased by a spinal mechanism, and that such a bias is responsible for the significant changes in the electrical characteristics of CONTRA nerve. This bias of intact CONTR nerve seems to be phasic in nature, in that it appears only after a conditioning stimulus and does not for the 2-s period between sets of pulses. Such a postulated phasic bias does not appear to be shared by control nerve. Thus, some spinal mechanism appears to be altered, either directly or indirectly, by the experimental crush procedures. It should be noted that minor functional alterations may be associated with the CONTRA nerve itself. The data from several postcrush groups, obtained after CONTRA nerve transection, approached statistical significance (P < 0.1). Only the large variance between animals in each treatment group prevented the demonstration of significance. This variance might be reduced in future studies by severing the nerve instead of subjecting it to acute crush. However, the variance may be related simply to differences in the responses of each experimental animal to physiologic trauma or insult. These possibilities remain to be studied. Changes in the electrical characterization of crossed spinal pathways for animals subjected to unilateral nerve crush were detected by using paired-pulse stimulation of suprathreshold intensity. For control spinal cord, the combined area under the test CCRs elicited by paired-pulse stimulation varied significantly for the interpulse intervals tested compared with the area under the reference CCR elicited by a single pulse. With short inter-pulse intervals, a significant facilitation of the test CCR area was observed. Significant depression occurred with longer interpulse intervals. A similar time course (facilitation-depression) was observed previously for variations in the amplitude of monosynaptic test reflexes, as a consequence of applying conditioning volleys of CONTRA nerves at various intervals preceding these test reflexes (3). The facilitation is probably due to a combination of increased transmitter release (35), residual transmitter in the synaptic clefts (8), and interconnections between
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motoneuron synergists (4, 5, 17). The depression observed at longer separation intervals is probably related to after-hyperpolarization of motoneurons (8, 20) or to a long-lasting late hyperpolarization associated with multineuronal pathways (30). Such a scheme has been demonstrated in other systems (18). Of the two experimental groups subjected to CCR investigation of crossed spinal pathways from crushed nerve to CONTRA nerve, only the 6-day postcrush group differed significantly from the control group. Stimulation of the CONTRA nerve with recording from the crushed nerve gave no significant difference from control values. Although the 6-day postcrush CCR ratios followed a trend for the interpulse intervals similar to the control, the ratios were generally smaller. In addition, the average latency of the CCR was longer for 6-day postcrush animals. Several investigators (7, 9, 34) observed that peripheral nerve or nerve root damage led to increased latencies of ipsilateral monosynaptic responses. Those authors hypothesized that afferent signals were rerouted through pathways containing one or more interneurons as a consequence of nerve damage. A similar rerouting of input after nerve crush could explain the observed latency increase in experimental CCRs in this study. Huizar er af. (20) observed that after-hyperpolarization in spinal motoneurons increased in amplitude and duration after nerve damage. A similar alteration of after-hyperpolarization in CONTRA motoneurons may be responsible for both the phasic changes in CONTRA nerve and experimental cord from 6-day postcrush animals. Such a simple explanation may not be reliable based on the data obtained from CONTRA nerves. The enhancement seen in the RARs from 6-day postcrush animals is atypical; the more general effect of nerve crush is a depression of the RARs compared to the control values. This, however, does not rule out the possibility that polarization changes in CONTRA motoneurons may underlie the changes in the electrical characteristics of undamaged CONTRA nerve and crossed spinal pathways. Recent evidence (Gerren and Luttges, in preparation) suggests that CONTRA motoneurons are structurally altered by nerve crush. It is unlikely that these changes observed in CONTRA nerves and spinal cord are due to the operative procedure without nerve crush. This laboratory has routinely compared normal and sham-operated control animals and found no difference between these two different control preparations. Thus, it appears that the damage-induced changes in electrical characteristics of CONTRA nerve as well as crossed spinal pathways to CONTRA nerve may be explained by the same spinal mechanism. All evidence from the present study suggests that the CONTRA motoneurons are the most likely candidates for alteration.
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DAMAGE
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Alteration of CONTRA motoneurons may arise as a consequence of either spontaneous or elicited input from the damaged limb being rerouted through different crossed spinal pathways. Selective inactivation of largest afferent pathways may occur, because altered input from group I afferents has been shown to decrease the efficiency of transmission across synapses in monosynaptic pathways ipsilateral to the damaged side (7,9, 34). Other pathways associated with smaller afferents may be spared if synapses in these pathways exist in triad arrangements (24, 31), which maintain afferent synaptic efficiency. Histological, pharmacological, and biochemical characterizations have recently been utilized (Gerren and Luttges, in preparation) to yield direct evidence of motoneuron alterations. Extracellular recordings from CONTRA motoneurons also may provide corroboration of the postulated changes. These additional data can be instrumental in establishing the observations of the present study as a model for the induction by nerve damage of functional and, perhaps, structural alterations in the spinal cord. REFERENCES 1. BAXENDALE, R. H., AND J. R. ROSENBERG. 1976. Crossed reflexes evoked by selective activation of muscle spindle primary endings in the decerebrate cat. Brain Res. 115: 324-347. 2.
BAXENDALE, R. H., AND J. R. ROSENBERG. 1977. Crossed reflexes evoked by selective activation of tendon organ afferent axons in the decerebrate cat. Brain Res. 127: 323-326.
BOSEMARK, B. 1966. Some aspects on the crossed extensor reflex in relation to motoneurons supplying fast and slow contracting muscles. Pages 261-268 in R. GRANIT, Ed., Muscular Aflerents and Mofor Control. Almqvist Wiksell, Stockholm. 4. CULLHEIM, S., AND J. 0. KELLERTH. 1976. Combined light and electron microscopical tracing of lumbar motor axon collaterals and their synaptic terminals in the cat, after intracellular injection of horseradish peroxidase. Acra. Physiol. &and. 98 (SuppI.): 3.
440.
5. CULLHEIM, S., AND J. 0. KELLERTH. 1976. Combined light and electron microscopic tracing of neurons, including axons and synaptic terminals, after intracellular injection of horseradish. Neurosci. Lett. 2: 307-313. 6. DEVANANDAN, M. S., B. HOLMQVIST, ANDT. YOKOTA. 1965. Presynapticdepolarization of group I muscle afferents by contralateral afferent volleys. Acta. Physiol. &and. 63: 46-54.
DOWNMAN, C. B. B., J. C. ECCLES, AND A. K. MCINTYRE. 1953. Functional changes in chromatolysed motoneurons. J. Camp. Neurol. 98: 9-36. 8. ECCLES, J. C. 1965. The excitatory responses of spinal neurones. Pages 1-34 in J. C. ECCLES AND J. P. SCHADE, Eds., Progress in Brain Research Vol. XII: Physiology of Spinal Neurons. Elsevier, Amsterdam/London/New York. 9. ECCLES, J. C., AND A. K. MCINTYRE. 1953. The effects of disuse and of activity on mammalian spinal reflexes. J. Physiol. (London) 121: 492-516. 10. ECCLES, R. M.. B. HOLMQVIST, AND P. E. VOORHOEVE. 1964. Presynaptic inhibition from contralateral cutaneous afferent fibres. Acta. Physiol. S-and. 62: 464-473. 7.
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ECCLES,
R. M., B. HOLMQVIST, AND P. E. VOORHOEVE. of cutaneous afferents by volleys in contralateral
12.
1946. A study of nerve degeneration and 147: 550-581. FEDINA, L., H. HULTBORN, AND M. ILLERT. 1975. Facilitation from contralateral primary afferents of interneuronal transmission of the Ia inhibitory pathway to motoneurons. Acta. Physiol. Stand. 94: 198-221. GASSER, H. S., AND J. ERLANGER. 1929. The role of fibre size in the establishment of a nerve block by pressure or cocaine. Am. J. Physiol. 88: 581-591. GASSER, H. S., AND H. GRUNDFEST. 1936. Action and excitability in mammalian A fibers. Am. J. Physiol. 117: 113-133. GERREN, R. A., D. E. GROSWALD, AND M. W. LUTTGES. 1976. Triethyltin toxicity as a model for degenerative disorders. Pharmacol. Biochem. Behav. 5: 299-307. GOGAN, P., J. P. GUERITAUD, G. HORCHOLLE-BOSSAVIT, AND S. TYC-DUMONT. 1977. Direct excitatory interactions between spinal motoneurones of the cat. J. Physiol.
Stand.
1964. Presynaptic depolarization muscle afferents. Acta. Physiol.
62: 474-484.
ERLANGER,
J.,
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M.
SCHOEPFLE.
regeneration. Am. J. Physiol.
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18. GUTNICK, M. J., AND D. A. PRINCE. 1972. Thalamocortical relay neurons: Antidromic invasion of spikes from a cortical epileptogenic focus. Science 176: 424-426. 19. HOLMQVIST, B. 1961. Crossed spinal reflex actions evoked by volleys in somatic afferents. Acra. Physiol. Stand. 52 (Suppl.): 181. 20. HUIZAR, P., M. KUNO, AND Y. MIYATA. 1975. Electrophysiological properties of spinal motoneurones of normal and dystrophic mice. J. Physio/. (London) 248: 23 l-246. 21. JOSEPH, B. S. 1973. Somatofugal events in Wallerian degeneration: A conceptual overview. Brain Res. 59: 1-18. 22. LUTTGES, M. W., P. T. KELLY, AND R. A. GERREN. 1976. Degenerative changes in mouse sciatic nerves: Electrophoretic and electrophysiologic characterizations. Exp. Neural. 50: 706-733. 23. MCCLEARN, G. E., J. WILSON, AND W. MEREDITH. 1970. The use of isogenic and heterogenic mouse stocks in behavioral research. Pages 3-22 in G. LINDZEY AND D. THIESSEN, Eds., Contributions to Behnrior-Genetic Analysis: The Mouse as a Prototype. Appleton-Century-Crofts, New York. 24. PAPPAS, G. D. 1975. Ultrastructural basis of synaptic transmission. Pages 19-30 in D. B. TOWER AND R. 0. BRADY, Eds., The Nervous System: The Basic Neurosciences. Raven Press, New York. 25. PERL, E. R. 1958. Crossed reflex effects evoked by activity in myelinated afferent fibers of muscle. J. Neurophysiol. 21: 101- 112. 26. PERL, E. R. 1959. Effects of muscle stretch on excitability of contralateral motoneurones. J. Physiol.
(London)
145: 193-203.
27. ROSENBERG, M. E. 1970. Synaptic connexions of alpha extensor motoneurones with ipsilateral and contralateral cutaneous nerve. J. Physiol. (London) 207: 231-255. 28. ROSENBLUETH, A., AND E. W. DEMPSEY. 1939/40. A study of Wallerian degeneration. Am.
J. Ph.vsio/.
128: 19-30.
29. ROSENBLUETH, A., AND E. C. DELPOZO. 1943. The centrifugal course of Wallerian degeneration. Am. .I. Physiol. 139: 247-254. 30. SCHOMBURG. E. D., H. M. MEINCK, J. HAUSTEIN, AND J. ROESLER. 1978. Functional organization of the spinal reflex pathways from forelimb afferents to hindlimb motoneurons in the cat. Brain Res. 139: 21-33. 3 1. SHEPHERD, G. M. 1974. The Synaptic Organization oj’the Brain:An Introduction. Oxford Univ. Press, London/Toronto.
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C. S. 1970. Flexion-reflex ofthe limb, crossed extension-reflex, and reflex stepping and standing. J. Physiol. (London) 40: 28- 121. 33. STRAIN, R. E., JR., AND W. H. OLSON., 1975. Selective damage of large diameter peripheral nerve fibers by compression: an application of Laplace’s law. Exp. Neural. 32. SHERRINGTON,
47: 68-80. 34. VERA, C. L.,
AND J. V. Luco. 1958. Synaptic transmission in spinal cord during Walletian degeneration of dorsal root fibers. J. Neurophysiol. 21: 334-344. 35. WEIGHT, F. F. 1974. Physiological mechanisms of synaptic modulation. Pages 929-941 in F. 0. SCHMITT AND F. G. WORDEN, Eds.. The Neurosciences: ThirdStudy Program. The MIT Press, Cambridge/London. 36. WEISS, P., AND H. B. HISCOE. 1948. Experiments on the mechanism of nerve growth. J.
Exp. Zool.
197: 315-395.
NEUROLOGY
65, %7-607 (1979)
Functional Changes in Undamaged Spinal Cord of Mice following RICHARD Department
A. GERREN
of Aerospace
AND MARVIN
Engineering Boulder,
Received
September
21.
Sciatic Nerves Nerve Damage
Sciences, Colorado
1978;
revision
and
W. LUTTGES’ University
of Colorado,
80309
receir,ed
April
25,
1979
The effects of sciatic nerve crush on electrophysiologic characteristics of undamaged contralateral sciatic nerve and spinal cord were studied in mice. Composite responses were elicited from both sciatic nerve and spinal cord by single- or paired-pulse stimulation. Conduction velocities of the initial components were not different in undamaged contralateral and control nerve. Nerve recovery, characterized by amplitude ratios of paired-pulse responses, yielded a depressionfacilitation-depression pattern in control sciatic nerves with increasing intervals between pulses. Recovery in undamaged contralateral nerves did not show this pattern. Compared to controls, response amplitude ratios from undamaged contralateral nerves were either depressed or facilitated depending on the time elapsing between ipsilateral nerve crush and subsequent testing. Upon removal of spinal influences by nerve transection, the pattern of contralateral nerve recovery more closely followed the recovery pattern of control nerve. Composite cross-cord responses (CCRsj were obtained from one sciatic nerve after stimulation of the opposite sciatic nerve. The area under both CCRs elicited by paired-pulse stimulation was compared to the area under a CCR elicited by single-pulse stimulation. For control cord, CCRs were facilitated with short paired-pulse separation intervals. With longer interpulse intervals, CCRs were depressed. CCRs from animals receiving nerve crush were either depressed (6 days postcrushj or facilitated (10 days postcrushj across all pulse separation intervals compared to control. The most significant treatment effects were found in tests of pathways from damaged nerve to undamaged contralateral nerve in 6-day postcrush Abbreviations: CONTRA-contralateral, RAR-relative amplitude ratio, CCR-crosscord response. ’ We wish to thank J. Button for assistance in preparing the manuscript and W. Bank for technical assistance. The work was supported, in part, by the International Chiropractors Association and the National Institutes of Health grant NINCDS 1 POINS-12226. R. A. Gerren is a Stanley Aviation Fellow. 587 0014-4886/79/090587-21$02.00/0 Copyright All rights
0 1979 by Academic Press. Inc. of reproduction in any form reserved.
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preparations. The results suggest that a spinal mechanism may be responsible for the phasic alterations in electrophysiologic characteristics of both undamaged contralateral nerve and spinal cord. The data have implications for the possibility of functionally altered contralateral motoneurons being part of this spinal mechanism.
INTRODUCTION Many investigators have studied the functional (12,14,22,28,29,33) and structural (21, 22, 36) alterations which occur in peripheral nerves after various types of experimental nerve damage. Other investigators studied the effects of these damage-induced changes on the ipsilateral spinal pathways and motoneuron cell bodies (7, 9, 20, 34). Several of these investigators (9,34) compared the spinal pathway responses obtained from the side with the damaged nerve to those obtained from the undamaged contralateral side. The underlying assumption of such experiments was that contralateral nerve remained unchanged. However, detailed investigations were not carried out comparing nerves contralateral to damaged nerves with nerves contralateral to undamaged nerves. Detailed investigations of cross spinal pathways were initially undertaken by Sherrington (32). Since then, many investigators have observed that ipsilateral nerve stimulation alters the contralateral monosynaptic response (19, 25, 26). The alterations of the contralateral monosynaptic response may be due to either ipsilateral input coming directly to contralateral motoneurons (l-3, 27), to contralateral interneurons (13), or from presynaptic inhibition of contralateral afferent fibers (6, 10, 11). Because the contralateral spinal pathways normally seem to be influenced by input from the ipsilateral side, it seems reasonable that disruption of that input, through nerve damage, may alter the functional and structural properties of the contralateral side. Recently, Luttges et al. (22) compared the electrophoretic protein patterns obtained from contralateral undamaged mouse sciatic nerves and sciatic nerves from mice receiving no nerve damage. They found differences in the protein patterns obtained from these two different types of controls. These changes were attributed to possible compensatory effects initiated as a consequence of the dependence on the single undamaged leg for hind limb locomotion. These observations suggest that structural alterations arise in contralateral nerves as a consequence of increased nerve use, decreased damaged nerve influences, or altered metabolic activity. The present study focused on changes in neurophysiologic indexes for mouse contralateral sciatic nerves and spinal cord after ipsilateral nerve crush. Altered neurophysiologic indexes in contralateral nerve occurred
NERVE AND CORD FUNCTION
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DAMAGED SIDE
FIG. 1. Schematic representation of electrode configurations used for various tests listed in Table 1. Also shown are sites of nerve crush and nerve cut (in appropriate experiments).
only with paired-pulse stimulation and seemed to result from phasic biasing of contralateral nerve by a spinal mechanism. The phasic effects in nerves appeared closely related to a spinal mechanism which produced demonstrable phasic changes in crossed spinal pathways. Even in the absence of spinal influences, subtle neurophysiologic changes were resolved in contralateral compared to normal nerves. The changes in the nerve, itself, remain to be investigated. METHODS Animals. Both male and female HS mice (23) were randomly selected from group (eight mice) cages. Except during surgical preparation or neurophysiologic testing, mice were providedad libitum access to food and water. Nerve Damage. Two groups of mice, experimental and control, were used. Mice in the experimental group were prepared by systematically crushing either left or right sciatic nerve. For surgery, all mice were anesthetized with injections of pentobarbital sodium (90 mg/kg body weight, i.p.). The skin over one thigh was carefully slit and the underlying musculature exposed. The muscles immediately surrounding the sciatic nerve were carefully retracted, leaving the nerve exposed. In experimental mice the nerve was squeezed 20 s under considerable pressure using fine mosquito forceps. The crushed portion (1
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to 2 mm) was inspected to determine whether or not perineurial integrity remained. If perineurial integrity was disrupted, the animal was not used. The wound was cleaned with 0.15 N saline and was sutured closed with stainless-steel (9 mm) wound clips. After the operative procedure, the experimental mice were placed in group (three mice) cages. At least 2 days elapsed before neurophysiological testing to minimize any lingering anesthetic or operative effects. Experimental Procedures. All mice were prepared for neurophysiological testing by anesthetization with chloral hydrate (700 mg/kg body weight, i.p). The anesthetized mice were placed on a water-heated aluminum plate and a rectal thermister was used to maintain the body temperature at 38.5”C. The skin overlying both sciatic nerves was cut away carefully. The muscles surrounding the sciatic nerves were tied back with silk sutures, leaving the nerves fully exposed. Both nerves were covered with pools of warm mineral oil to prevent dessication. Bipolar stimulating electrodes (300-pm platinum wire) and bipolar recording electrodes (250~pm tungsten wire with tips etched to approximately 150 pm) were placed on the sciatic nerves in one of several configurations (Fig. 1, Table 1). Stimuli (0.25-ms duration, single or paired pulses) were produced by a square wave generator and delivered to the test preparation via an isolation transformer with diode suppression. Evoked responses were obtained by differential AC preamplification (Argonaut Model L042). Evoked responses were either averaged (Princeton Applied Research, Waveform Eductor) and recorded (Sanborn Model 150) graphically or were recorded directly on film (DuMont Kymographic Camera). Sciatic Nerves. Two neurophysiological tests were carried out on sciatic nerve. Test I (Table 1, Fig. 1) was used to determine the conduction velocity of the initial component of responses evoked in control, damaged, and undamaged contralateral (CONTRA) sciatic nerve. Both damaged and CONTRA nerves were investigated on days 3 through 10 and 30 days after nerve crush. Single-pulse stimulation (0.5 Hz) was of sufficient voltage (3 to 6 V) to elicit suprathreshold responses. For each sciatic nerve, 10 suprathreshold responses were averaged. The latency for each averaged response was measured between the onset of the stimulus artifact and the initial response deflection from baseline. The conduction velocity of the initial component in any one preparation was obtained by dividing the latency into the measured distance between the stimulating and recording electrodes (7 to 9 mm). Conduction velocities were obtained from three control nerves, 27 CONTRA nerves (three at each postcrush test day), and several damaged nerves at various intervals after nerve crush. To determine whether control and CONTRA conduction velocities were significantly different, data from these preparations were subjected to a
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TABLE Electrical Characterizations
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1
of Sciatic Nerve and Spinal Cord
Electrode configuration Stimulating
Recording
Stimulus type
Structure
Condition”
3
4
Single pulse
Sciatic nerve
Conduction velocity
2
1
CON and CONTRA Damaged
II
3
4
Paired pulse
Sciatic nerve
CON and CONTRA; intact and severed
RAR’
III
2
3
Single pulse Paired pulse
Spinal cord Ib
CON and EXP
Single pulse Paired pulse
Spinal cord IS’
EXP
Test I
IV
3
2
Measure
Reference CCRd Test CCR” Reference CCRd Test CCR”
n CON-sciatic nerves or spinal cord in mice receiving no nerve crush, CONTRAsciatic nerves contralateral to damaged sciatic nerves, EXP-spinal cord in mice receiving nerve damage, intact and severed-electrical characterizations were obtained from CON and CONTRA nerves before and after nerve transection from cord. * Spinal cord I-nerve crush-induced changes in spinal pathways from damaged nerve atferents to CONTRA nerve, spinal cord II-nerve crush-induced changes in spinal pathways from CONTRA nerve atferents to damaged nerve. c RAR-sciatic nerve relative response ratio. Ten responses to paired stimuli were averaged for each nerve. The average amplitude of the second response was divided into the average amplitude of the first response to obtain the RAR. An RAR was obtained at each pulse separation interval (3, 10, 25, 50, 100, and 250 ms). d Reference CCR (cross-cord response)-the average area of the first component of 10 cross-cord responses elicited by single-pulse stimulation, test CCR-the combined first component areas from 10 consecutive cross-cord responses, elicited by paired-pulse stimulation, were averaged. The test CCR at each pulse separation interval (5, 10, 25, 50, 100, and 250 ms) was divided by the reference CCR from the same preparation, which gave a CCR area ratio.
one-way analysis of variance. A one-tailed test was used to determine significance based on expectations from previous work (16). Test II (Table 1, Fig. 1) was used to investigate response recovery in control and CONTRA nerve. This was accomplished by using paired-
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pulses of equal duration (0.25 ms) and intensity (suprathreshold). Suprathreshold voltage (3 to 6 V) was determined by single-pulse stimulation. Paired-pulse separation intervals of 3, 10,25,50, 100, and 250 ms were tested. For each nerve, 10 paired-pulse stimuli (one pair every 2 s) at each separation interval were delivered. The 10 elicited responses were averaged and the amplitude of the initial component of these averaged responses measured. The initial component amplitude of the averaged first response was divided into that of the second, for each pulse separation interval. This resulted in a relative amplitude ratio (RAR) used to characterize nerve recovery. RARs across all pulse separation intervals were obtained from six control nerves and 27 CONTRA nerves (three at each postcrush test day). After completion of the above tests for intact nerve preparations, each nerve was severed with fine iris scissors within 10 mm of the spinal cord (Fig. 1). Care was taken to prevent severance of any major blood vessel or displacement of the stimulating electrodes. Nerve severance from the spinal cord was done to remove spinal influences over nerve responsiveness. The severed nerves were tested in the same manner as intact nerve. Ten paired-pulses were given at each separation interval and the averaged first component amplitudes of first and second responses used to calculate the RARs. The RARs from each nerve type (CONTRA and control), condition (before and after severance), and pulse separation interval were subjected to a three-way analysis of variance. Additionally, CONTRA nerve data at each postcrush test day were compared directly to control nerve data using a two-way analysis of variance. These simple comparisons were made separately for data obtained before and after nerve transection. Spinal Cord. Two neurophysiological tests were used to determine if sciatic nerve crush affected transmission in cross-spinal pathways. Test III (Table 1, Fig. 1) was focused on studies of the pathways from damaged nerve afferents to the CONTRA nerve. This electrode arrangement also was used for control cord. In test IV (Table 1, Fig. l), the pathways from CONTRA nerve afferents to damaged nerve were studied. Single-pulse stimuli (0.25 ms duration; 0.5 Hz) of increasing intensity were delivered until a stable cross-cord response (CCR) was obtained. CCRs elicited from 10 consecutive stimuli were averaged. For each preparation, a single-pulse, average CCR was used as the reference response. Paired-pulse stimuli of the reference intensity were delivered at various pulse separation intervals (5, 10,25, 50, 100, and 250 ms). CCRs elicited from 10 paired-pulse stimuli (test CCRs) at each pulse separation interval were averaged. The area under both test CCRs, whether superimposed (short separation intervals) or separate, was measured and divided by the area under the single-pulse,
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DAMAGE
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reference CCR. This gave a CCR area ratio at each pulse separation interval. A CCR area ratio equal to 2 indicates that no interaction between paired CCRs occurred. CCR area ratios were obtained from three control, three 6-day postcrush and three IO-day postcrush nerve preparations. Datafrom both tests III and IV were obtained for 6- and lo-day preparations. CCR area ratios from all preparations were compared using a two-way analysis of variance. Also, CCR area ratios from each postcrush test day similarly were compared to control CCR area ratios. RESULTS After recovery from anesthesia, all experimental mice demonstrated severely impaired motor function in the leg with the crushed nerve. Although the mice soon appeared to compensate during locomotion with the undamaged leg, debilitation remained noticeable for as long as 30 days. Inspection of the crushed nerves revealed damage identical to that described previously by Luttges et al. (22). The damage consisted of swelling and discoloration distal to the site of the crush. The proximal portion of the nerve was slightly swollen, but discoloration was not as obvious. The undamaged CONTRA nerves, in contrast, appeared quite normal. Sciatic Nerve. The suprathreshold responses elicited by single-pulse stimulation in test I (Table 1) were similar in waveform appearance for all CONTRA and control nerves. All responses had at least a triphasic waveform, characteristic of bipolar recordings of responses propagated for short distances in the low-threshold, large myelinated fiber population. Occasionally, populations of smaller, slower conducting fibers responded to the suprathreshold stimulation, as indicated by the appearance of more and later response components. However, only the first, triphasic waveform was evaluated in the present study. For all nerve preparations, the latency between onset of stimulus artifact and the initial response deflection from baseline was used, together with measured distances along the nerves between stimulating and recording electrodes, to determine largest fiber conduction velocities. Under our test conditions, the conduction velocities for different preparations was 15 to 25 m/s, and were similar to those observed previously (16, 22). Conduction velocities observed in damaged nerves (6 to 10 m/s) were substantially slower than those in control nerves at all postcrush days tested and were similar to those reported earlier (22). No significant differences (Z’ > 0.25) were observed between the conduction velocities of control and CONTRA nerves, regardless of the number of days elapsing between crush and testing.
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0
s
1.5 A
X a t es
1.0 -
z E
*---a
E .s & P z
1 3
1 I 1 I IO 25 51 100 PULSESEPARATION INTERVAL (mstc)
BEFORE AFTER I 250
B
’
’
20Omsec
I
’
200 msec
500 msec
’
’
FIG. 2. Time course of response recovery for mouse sciatic nerve. A-average response amplitude ratios from six control nerves. Data obtained before and after nerve transection from the spinal cord are plotted separately. B-representative sciatic nerve responses elicited by paired-pulse stimulation for the various test intervals.
Suprathreshold responses elicited by the first of the paired stimuli in test II, were similar in amplitude and latency for all CONTRA and control nerves. However, suprathreshold responses elicited by the second of the paired stimuli were noticeably different across various interpulse intervals. Variance in the amplitude of the second response compared to the first was
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seen in both CONTRA and control nerves. Complete quantification was undertaken to examine these amplitude changes in more detail. For each control nerve, the amplitude of the response elicited by the first of the paired stimuli was divided into the amplitude of the response elicited by the second, at each interpulse interval. This resulted in a RAR across all separation intervals for each preparation. The average RARs across separation interval for control sciatic nerve are plotted in Fig. 2A. Representative responses from which the average RARs were obtained are shown in Fig. 2B. At the shortest separation interval (3 m/s), average second response amplitudes were depressed relative to the first. In contrast, second response amplitudes were facilitated compared to the first across longer separation intervals (10 and 25 ms). At the longest separation intervals (50, 100, and 250 ms), second response amplitudes were either equal to or slightly lower than first response amplitudes. These variations in RAR for control nerve were highly significant (P < 0.005) for the entire range of separation intervals tested. After severing each control nerve close to the spinal cord (-10 mm), another series of paired-pulse responses was elicited from the nerve distal to the site of transection. Responses were analyzed by the same methods. The average RARs for these data also are shown in Fig. 2A. Again, the variations in the RAR for the entire range of separation intervals are highly significant (P < 0.005). There is no significant difference (P > 0.20), however, between RARs for control nerves before and after the removal of spinal influences. Paired-pulse data were obtained before and after nerve transection, from CONTRA nerve in animals subjected to nerve crush 3 through 10 or 30 days previously. The average RAR at each separation interval for each day postcrush was subtracted from the average control RAR. This gave the relative deviation of CONTRA RARs from the control values reported above. Calculations were done separately for both before and after transection and are summarized in the three-dimensional plots of Figs. 3A and B, respectively. Unshaded portions of the plotted averages represent those CONTRA RARs larger than the control RARs (most notably at 6 and 10 days postcrush), and shaded portions represent those with smaller RARs. Deviations from control, indicated by more exaggerated convolutions, are quite pronounced if CONTRA nerve-spinal cord integrity is preserved. Pooling the data for each day postcrush and averaging all separation intervals results in Fig. 4A, which depicts the results of tests both before and after nerve transection. The most pronounced deviations from control occurred at 4,6,7, 8,9, and 30 days postcrush for RARs obtained before nerve transection. Statistical analysis reveals that 4, 6, 7, 9, and 30 day
FIG. 3. Effects of nerve crush on undamaged contralateral response amplitude ratios (RARs). Average RARs from three nerves at each postcrush test day were subtracted from average control RARs to obtain the relative deviation from control. Calculations were done separately for before (A) and after (B) nerve transection RARs. Unshaded portions of the plots represent contralateral RARs larger than control and shaded portions represent those with lower values.
A
NERVE
AND
CORD
FUNCTION
lOUIN
AFTER
WOW
UNILATERAL
NOllvIA3a
3~11~13~
DAMAGE
597
598
GERREN
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postcrush RARs were significantly to highly significantly different (P < 0.005, 0.025, 0.005, 0.025, and 0.05, respectively). After nerve transection, only 3-, lo-, and 30-day postcrush RARs appeared to differ from control values, but statistical analysis did not corroborate such differences (P > 0.05, 0.05, and 0.05, respectively). Before the nerve was severed from the spinal cord, the amplitudes of the first and second responses, potentially, were determined by both the electrical characteristics of the nerve bundle and by influences originating in the spinal cord. Transection of the nerve from the cord removed the potential spinal influences. Therefore, subtraction of the data obtained from control and CONTRA nerves after nerve transection from those obtained before nerve transection should give an indication of the effect nerve crush has on spinal influences on the CONTRA nerve. Those calculations are shown in Fig. 4B. No significant difference (P > 0.20) was observed between control RARs measured before and after nerve transection. In contrast, 3-, 6-, and 7-day postcrush CONTRA RARs appear to have been significantly affected by spinal influences (P = 0.05, 0.05, and 0.05, respectively). Thus, it appears that nerve crush induced the following hierarchy of neural alterations: Striking changes occurred in the damaged nerve. In intact preparations, significant changes also occurred in undamaged CONTRA nerve. Removal of spinal influences by nerve transection eliminated most of the significant postcrush changes in the CONTRA nerve. Modest postcrush changes may have persisted in CONTRA nerve even in the absence of spinal influences. Spinal Cord. Based on the demonstrable nerve changes which appeared to originate in the spinal cord after nerve crush, the spinal cord-sciatic nerve interactions were subjected to further experimental characterization. Paired-pulse stimuli having interpulse intervals of 5, 10, 25, 50, 100 and 250 ms were delivered (0.5 Hz) to one sciatic nerve and responses were recorded from the opposite sciatic nerve (test III, Table 1 and Fig. 1). Several investigators (1, 2, 19) showed that ipsilateral ventral root stimulation has no effect on the contralateral monosynaptic response. Therefore, no attempt was made to preclude antidromic ventral root volleys in cross-cord investigations. Representative control CCRs from mice with no experimental sciatic nerve damage are shown in Fig. 5A. The average inflection latency for control CCRs elicited by single suprathreshold stimuli was 3.94 t 0.36 ms. The area under the CCR obtained by single-pulse stimulation was used as the reference for comparisons of all subsequent paired-pulse evoked CCRs (test CCRs). Figure 5B shows the CCR area ratios of control spinal cord preparations obtained by dividing the reference CCR area into the area of the combined test CCRs, at each separation interval. Control CCRs were
NERVE AND CORD FUNCTION
AFTER UNILATERAL
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facilitated (area ratio >2.0) with short (5 and 10 ms) interpulse intervals. Depressed CCRs (ratio <2.0) were characteristic of longer (25,50, 100,250 ms) interpulse intervals. These variations in the control CCR were highly significant (P < 0.005) for the range of interpulse intervals tested. Two experimental groups of animals receiving unilateral nerve crush 6 or
GERREN AND LUTTGES
600
’
ZDB msec
’
’
zoo msec
i
FIG. 5. Effects of nerve crush on composite cross-cord responses (CCRs). A-representative responses elicited for typical test intervals. B-first component area ratios of CCRs recorded from contralateral nerve after stimulation of damaged nerve. C-area ratios of CCRs recorded from damaged nerve after contralateral nerve stimulation.
10 days previously were subjected to similar tests. The 6-day postcrush group was used because previous nerve testing suggested that at this time spinal influences dominated the changes occurring in undamaged CONTRA compared to control nerve. Conversely, the IO-day postcrush group was chosen because the changes observed in undamaged CONTRA compared to control nerve appeared unaffected by spinal influences at this postcrush time (Figs. 4A and B). In the first set of experiments (test III), paired-pulse stimuli were delivered to the damaged nerve and CCRs recorded from the CONTRA nerve. The average CCR inflection latencies for 6- and lo-day postcrush preparations were found to be 6.63 + 1.09 and 5.02 + 0.72 ms, respectively. These values represent increases over the average control CCR inflection latency (3.94 k 0.36 ms) of 68.3 and 27.4%, respectively. These average inflection latencies were not significantly different (P > 0.05) from control values with the small number of observations used. The CCR area ratios obtained from test III are shown, for 6- and IO-day postcrush groups, in Fig. 5B. CCR area ratios for the experimental groups are seen to vary across pulse separation intervals in a pattern similar to that of the control group. However, the CCR area ratios from the 6-day experimental group, overall, were significantly decreased (P < 0.025) from control values. CCR area ratios from the lo-day postcrush experimental group are not significantly different (P > 0.25) from control values.
NERVE
AND
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FUNCTION
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S-Continued.
In the second set of experiments (test IV), paired-pulse stimuli were delivered to the CONTRA nerve and CCRs recorded from the damaged nerve. The CCR area ratios for 6- and IO-day postcrush groups, together with control values, are shown in Fig. 5C. Although differences in the CCR area ratios between these experimental groups and the control group seemed to exist in this test paradigm, neither 6- nor lo-day postcrush CCR area ratios varied significantly (P > 0.05 and P = 0.1, respectively) from control values. Thus, nerve crush induced changes in CCRs under the present test conditions. Modest changes occurred in the electrical characterizations of spinal cord-damaged nerve interactions for both experimental groups
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tested. Significant changes occurred in spinal cord-CONTRA nerve interactions for the 6-day postcrush experimental group. At 10 days postcrush, only modest changes occurred in spinal cord-CONTRA nerve interactions. DISCUSSION Mouse sciatic nerves contralateral to crushed sciatic nerves were normal in appearance upon gross inspection. Both their size and color was apparently within the limits of control nerves from mice with no nerve damage. Neurophysiologic responses elicited by single electrical pulses at suprathreshold levels were similar whether recorded from CONTRA or control nerves. No significant differences were observed in either the threshold or conduction velocity of the largest fibers. Significant differences were detected, however, when CONTRA nerves were subjected to paired-pulse stimulation of suprathreshold intensity. In control nerve the response elicited by the second stimulus varied significantly in amplitude compared to the first, for all pulse separation intervals tested. With short interpulse intervals, the relative size of the second response was decreased. With longer interpulse intervals, the relative size of the second response was larger than the first. At the longest interpulse intervals, the relative size of the second response was again decreased, although not to the extent observed with short interpulse intervals. The time course of these amplitude changes (depression-facilitation-depression) followed the combined time courses of the relative refractory period, and the supernormal and subnormal periods, associated with the negative and positive afterpotentials as described by Gasser and Grundfest (15). The ratios of the first and second response amplitudes from CONTRA nerve were quite different from the control ratios for all postcrush test days. For pulse separation intervals, the relative amplitudes of all second responses generally were decreased compared to control values, except at 6 and 10 days postcrush when a relative enhancement of the second response was observed (cf. Fig. 3A). In addition, the pulse separation time course found for control nerve response ratios (depression-facilitationdepression) was not characteristic of CONTRA nerve. On the basis of these and previous observations (22), it is tempting to suggest that CONTRA nerves undergo structural alterations which affect functional properties, such as the relative recovery, supernormal, and subnormal periods. The nerve transection results cast doubt upon such facile interpretations and suggest that the spinal cord plays a major, though not exclusive, role in the observed nerve changes.
NERVE
AND
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FUNCTION
AFTER
UNILATERAL
DAMAGE
603
When CONTRA nerves were severed within 10 mm of the spinal cord, it was assumed that participatory spinal influences on the nerve also were removed. Tests done immediately after nerve transection revealed response ratios resembling those obtained from control nerves. Accordingly, the ratios were quite different from those obtained just prior to nerve transection. It is unlikely that such differences in response ratios before and after nerve transection were caused by transection artifact rather than the removal of spinal influences. Control experiments showed that control nerve response ratios did not change after nerve transection whether proximal or distal to the stimulation and recording sites. The observed changes of CONTRA nerve characteristics after the removal of spinal influences suggests that CONTRA nerves may be biased by a spinal mechanism, and that such a bias is responsible for the significant changes in the electrical characteristics of CONTRA nerve. This bias of intact CONTR nerve seems to be phasic in nature, in that it appears only after a conditioning stimulus and does not for the 2-s period between sets of pulses. Such a postulated phasic bias does not appear to be shared by control nerve. Thus, some spinal mechanism appears to be altered, either directly or indirectly, by the experimental crush procedures. It should be noted that minor functional alterations may be associated with the CONTRA nerve itself. The data from several postcrush groups, obtained after CONTRA nerve transection, approached statistical significance (P < 0.1). Only the large variance between animals in each treatment group prevented the demonstration of significance. This variance might be reduced in future studies by severing the nerve instead of subjecting it to acute crush. However, the variance may be related simply to differences in the responses of each experimental animal to physiologic trauma or insult. These possibilities remain to be studied. Changes in the electrical characterization of crossed spinal pathways for animals subjected to unilateral nerve crush were detected by using paired-pulse stimulation of suprathreshold intensity. For control spinal cord, the combined area under the test CCRs elicited by paired-pulse stimulation varied significantly for the interpulse intervals tested compared with the area under the reference CCR elicited by a single pulse. With short inter-pulse intervals, a significant facilitation of the test CCR area was observed. Significant depression occurred with longer interpulse intervals. A similar time course (facilitation-depression) was observed previously for variations in the amplitude of monosynaptic test reflexes, as a consequence of applying conditioning volleys of CONTRA nerves at various intervals preceding these test reflexes (3). The facilitation is probably due to a combination of increased transmitter release (35), residual transmitter in the synaptic clefts (8), and interconnections between
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motoneuron synergists (4, 5, 17). The depression observed at longer separation intervals is probably related to after-hyperpolarization of motoneurons (8, 20) or to a long-lasting late hyperpolarization associated with multineuronal pathways (30). Such a scheme has been demonstrated in other systems (18). Of the two experimental groups subjected to CCR investigation of crossed spinal pathways from crushed nerve to CONTRA nerve, only the 6-day postcrush group differed significantly from the control group. Stimulation of the CONTRA nerve with recording from the crushed nerve gave no significant difference from control values. Although the 6-day postcrush CCR ratios followed a trend for the interpulse intervals similar to the control, the ratios were generally smaller. In addition, the average latency of the CCR was longer for 6-day postcrush animals. Several investigators (7, 9, 34) observed that peripheral nerve or nerve root damage led to increased latencies of ipsilateral monosynaptic responses. Those authors hypothesized that afferent signals were rerouted through pathways containing one or more interneurons as a consequence of nerve damage. A similar rerouting of input after nerve crush could explain the observed latency increase in experimental CCRs in this study. Huizar er af. (20) observed that after-hyperpolarization in spinal motoneurons increased in amplitude and duration after nerve damage. A similar alteration of after-hyperpolarization in CONTRA motoneurons may be responsible for both the phasic changes in CONTRA nerve and experimental cord from 6-day postcrush animals. Such a simple explanation may not be reliable based on the data obtained from CONTRA nerves. The enhancement seen in the RARs from 6-day postcrush animals is atypical; the more general effect of nerve crush is a depression of the RARs compared to the control values. This, however, does not rule out the possibility that polarization changes in CONTRA motoneurons may underlie the changes in the electrical characteristics of undamaged CONTRA nerve and crossed spinal pathways. Recent evidence (Gerren and Luttges, in preparation) suggests that CONTRA motoneurons are structurally altered by nerve crush. It is unlikely that these changes observed in CONTRA nerves and spinal cord are due to the operative procedure without nerve crush. This laboratory has routinely compared normal and sham-operated control animals and found no difference between these two different control preparations. Thus, it appears that the damage-induced changes in electrical characteristics of CONTRA nerve as well as crossed spinal pathways to CONTRA nerve may be explained by the same spinal mechanism. All evidence from the present study suggests that the CONTRA motoneurons are the most likely candidates for alteration.
NERVE AND CORD FUNCTION
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DAMAGE
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Alteration of CONTRA motoneurons may arise as a consequence of either spontaneous or elicited input from the damaged limb being rerouted through different crossed spinal pathways. Selective inactivation of largest afferent pathways may occur, because altered input from group I afferents has been shown to decrease the efficiency of transmission across synapses in monosynaptic pathways ipsilateral to the damaged side (7,9, 34). Other pathways associated with smaller afferents may be spared if synapses in these pathways exist in triad arrangements (24, 31), which maintain afferent synaptic efficiency. Histological, pharmacological, and biochemical characterizations have recently been utilized (Gerren and Luttges, in preparation) to yield direct evidence of motoneuron alterations. Extracellular recordings from CONTRA motoneurons also may provide corroboration of the postulated changes. These additional data can be instrumental in establishing the observations of the present study as a model for the induction by nerve damage of functional and, perhaps, structural alterations in the spinal cord. REFERENCES 1. BAXENDALE, R. H., AND J. R. ROSENBERG. 1976. Crossed reflexes evoked by selective activation of muscle spindle primary endings in the decerebrate cat. Brain Res. 115: 324-347. 2.
BAXENDALE, R. H., AND J. R. ROSENBERG. 1977. Crossed reflexes evoked by selective activation of tendon organ afferent axons in the decerebrate cat. Brain Res. 127: 323-326.
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C. S. 1970. Flexion-reflex ofthe limb, crossed extension-reflex, and reflex stepping and standing. J. Physiol. (London) 40: 28- 121. 33. STRAIN, R. E., JR., AND W. H. OLSON., 1975. Selective damage of large diameter peripheral nerve fibers by compression: an application of Laplace’s law. Exp. Neural. 32. SHERRINGTON,
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