J Neurophysiol
87: 1763–1771, 2002; 10.1152/jn.00305.2001.
Changes in Crossed Spinal Reflexes After Peripheral Nerve
Injury and Repair
ANTONI VALERO-CABRÉ AND XAVIER NAVARRO
Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain
Received 16 April 2001; accepted in final form 13 December 2001
Axotomy of peripheral nerves severs sense organs and muscles from their innervating sensory and motor nerve fibers,
respectively. Similarly, the spinal cord reflexes evoked from
intact sensory afferents may be abolished. Spinal cord synapses
are detached (Brännström et al. 1992a) by the action of glial
cells reacting to the injury (Aldskogius and Kozlova 1998) so
that spinal cord reflexes are completely abolished thereafter
(Mendell 1984). When adequate repair procedures are applied,
axons regrow distally reestablishing functional connections
with peripheral organs. In the spinal cord, motoneuron dendritic arborization is reextended, synaptic button stripping is
reverted and sensory-motor synapses become functional again
(Brännström et al. 1992b; Mendell 1984; Valero and Navarro
2001). However, distal organ reinnervation by regenerating
axons does not guarantee the restitution of normal spinal cord
reflex function. Motor and sensory neurons show lower depolarization thresholds after injury and regeneration evoking excitatory postsynaptic potentials of higher amplitude than controls after afferent stimulation (Eccles et al. 1958; Gustafsson
1979; Horch and Lisney 1981). Moreover, central reorganization induced by nerve lesion changes the laminar projections of
afferent fibers (Shortland and Fitzgerald 1994; Woolf et al.
1992) and alters the somatotopy of the body representation at
the spinal cord level (Fitzgerald 1985; Koerber et al. 1994). In
adult animals, all these changes persist for a long time after
reinnervation and may impair motor unit recruitment and control of movement (Cope and Clark 1993; Milner-Brown et al.
1974) and account in part for the poor clinical outcome
achieved after severe nerve injuries (Cope and Clark 1993; De
Medinaceli 1988; Gramsbergen et al. 2000; Wasserschaff
1990).
Previous studies have investigated spinal cord reflexes after
peripheral nerve injuries in newborn and postnatal animals.
Despite the substantial rearrangement of innervation after neonatal injuries, withdrawal reflexes easily recovered and attained a near to normal spatial organization (Holmberg and
Schouenborg 1996a). Spinal reflexes appeared to be facilitated,
yielding higher amplitude and longer lasting bursts than controls after neonatal nerve crush (Navarrete et al. 1990; Vejsada
et al. 1991, 1999). However, findings of facilitation of spinal
reflexes reported after neonatal nerve crush do not necessarily
infer that spinal reflexes are facilitated after injury to the adult
nerve in light of evidence of more extensive neuronal death in
newborn than in adult animals (Romanes 1946; Vejsada et al.
1999), more accurate distal reinnervation in postnatal animals
(Alsdkogius and Molander 1990), and the higher capability of
postnatal animals to reorganize central projections of misdirected collaterals, suggesting that functional recovery and spinal reorganization after nerve injury are age dependent (Holm-
Address for reprint requests: X. Navarro, Group of Neuroplasticity and
Regeneration, Faculty of Medicine, Universitat Autònoma de Barcelona,
E-08193 Bellaterra, Spain (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked ‘‘advertisement’’
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
INTRODUCTION
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Valero-Cabré, Antoni and Xavier Navarro. Changes in crossed
spinal reflexes after peripheral nerve injury and repair. J Neurophysiol
87: 1763–1771, 2002; 10.1152/jn.00305.2001. We investigated the
changes induced in crossed extensor reflex responses after peripheral
nerve injury and repair in the rat. Adults rats were submitted to non
repaired sciatic nerve crush (CRH, n ⫽ 9), section repaired by either
aligned epineurial suture (CS, n ⫽ 11) or silicone tube (SIL4, n ⫽ 13),
and 8 mm resection repaired by tubulization (SIL8, n ⫽ 12). To assess
reinnervation, the sciatic nerve was stimulated proximal to the injury
site, and the evoked compound muscle action potential (M and H
waves) from tibialis anterior and plantar muscles and nerve action
potential (CNAP) from the tibial nerve and the 4th digital nerve were
recorded at monthly intervals for 3 mo postoperation. Nociceptive
reinnervation to the hindpaw was also assessed by plantar algesimetry.
Crossed extensor reflexes were evoked by stimulation of the tibial
nerve at the ankle and recorded from the contralateral tibialis anterior
muscle. Reinnervation of the hindpaw increased progressively with
time during the 3 mo after lesion. The degree of muscle and sensory
target reinnervation was dependent on the severity of the injury and
the nerve gap created. The crossed extensor reflex consisted of three
bursts of activity (C1, C2, and C3) of gradually longer latency, lower
amplitude, and higher threshold in control rats. During follow-up after
sciatic nerve injury, all animals in the operated groups showed recovery of components C1 and C2 and of the reflex H wave, whereas
component C3 was detected in a significantly lower proportion of
animals in groups with tube repair. The maximal amplitude of components C1 and C2 recovered to values higher than preoperative
values, reaching final levels between 150 and 245% at the end of the
follow-up in groups CRH, CS, and SIL4. When reflex amplitude was
normalized by the CNAP amplitude of the regenerated tibial nerve,
components C1 (300 – 400%) and C2 (150 –350%) showed highly
increased responses, while C3 was similar to baseline levels. In
conclusion, reflexes mediated by myelinated sensory afferents
showed, after nerve injuries, a higher degree of facilitation than those
mediated by unmyelinated fibers. These changes tended to decline
toward baseline values with progressive reinnervation but still remained significant 3 mo after injury.
1764
A. VALERO-CABRÉ AND X. NAVARRO
berg and Schouenborg 1996a,b). The aims of this work were to
study the restitution of crossed extensor reflex responses conveyed by different types of sensory fibers after peripheral nerve
injuries in adult animals and to compare the effects of nerve
injuries of varying severity: nerve compression, section, and
resection, that result in different levels of peripheral reinnervation.
METHODS
Sixty-four adult female Sprague-Dawley rats [260 ⫾ 4 (SD) g]
were used in the study. All rats were kept on standard laboratory food
and tap water ad libitum with a light-dark cycle of 12 h. The experimental procedures were approved by the ethical committee of our
institution and were conducted in conformity with the Guiding Principles for Research Involving Animals and Human Beings, taking
adequate measures to minimize pain and animal discomfort during
surgery and testing procedures.
Animals were anesthetized with an intraperitoneal injection of
pentobarbital (50 mg/kg); the right sciatic nerve was then exposed at
the midthigh and injured at a constant point, 90 mm from the tip of the
third toe, well proximal to the sciatic trifurcation. Rats were divided
into five groups depending on the lesion made to the sciatic nerve. In
one group the right sciatic nerve was crushed during 30 s in three
different orientations by means of a fine forceps (CRH, n ⫽ 9). In two
other groups, the sciatic nerve was sharply transected and repaired
either by epineurial 10-0 sutures (CS, n ⫽ 11) or by tubulization with
a silicone guide (6 mm long, 2 mm ID, 0.5-mm wall thickness)
sutured between both nerve ends leaving a 4 mm gap (SIL4, n ⫽ 13).
In another group an 8-mm segment of the sciatic nerve was resected,
and the gap was bridged with a silicone tube (10 mm long; SIL8, n ⫽
12). The original alignment of the tibial and peroneal fascicles of the
sciatic nerve trunk was preserved. Immediately after injury, the distal
sciatic nerve was mechanically stimulated by gently pinching to
guarantee complete lesion, as the normal reflex response consisting in
contraction of muscles of the trunk was completely absent. To avoid
collateral reinnervation of the denervated sciatic territory by other
nerves supplying neighboring areas, the caudal cutaneous branch of
the sural nerve and the saphenous nerve were sectioned at the
midthigh and a long distal segment resected to prevent regeneration.
A control group of intact rats (CNT, n ⫽ 9) was submitted to sham
injury of the sciatic nerve plus resection of saphenous and caudal
cutaneous nerves and evaluated in parallel. All the animals were kept
during 90 days postoperation (dpo) to allow for nerve regeneration
and target reinnervation.
Electrophysiological evaluation
Before injury, 1 wk after operation, and thereafter on a monthly
basis, motor and nerve reinnervation and reflex recovery were assessed by means of nerve conduction tests. During the test, the rat
body temperature was kept constant between 34 and 36°C by means
of a thermostated flat coil, and anesthesia was maintained steady after
the initial induction (40 mg/kg ip) by injection of additional pentobarbital bolus (10 mg/kg ip) every 60 min. The right sciatic nerve was
stimulated with single electrical pulses (100-␮s duration and supramaximal intensity) delivered by monopolar needles percutaneously
placed proximal to the injury site, the cathode at the sciatic notch and
the anode one centimeter proximally. The compound muscle action
potentials (CMAPs) of the tibialis anterior and plantar muscles were
recorded during 20 ms by means of needle electrodes (27G), the active
electrode inserted on the muscle belly and the reference at the fourth
toe, and displayed in an oscilloscope (Sapphyre 4 M, Vickers). LikeJ Neurophysiol • VOL
Assessment of nociception recovery
Reinnervation of the plantar skin by C nociceptive fibers was
evaluated every month during 90 dpo by a heat-radiation method
using a modified plantar algesimeter (Hargreaves et al. 1988). Rats
were placed into a metacrylate box with an elevated glass floor.
The surface of the glass was kept at 30 ⫾ 1°C before starting each
test. From the bottom of the box, the light of a projection lamp was
focused directly onto the plantar surface of a hindfoot. The time
spent until raising the heated hindpaw was measured through a
time meter coupled with infrared (IR) detectors directed to the
plantar surface. The value for a test was the mean of three trials
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Surgical procedures
wise, the compound nerve action potential (CNAP) was recorded by
needle electrodes inserted near the tibial nerve at the ankle and the 4th
digital nerve. To ensure reproducibility, the recording needles were
placed at the microscopic magnification to secure the same placement
on all animals guided by anatomical landmarks. The active recording
electrode was placed subcutaneously at the first third of the distance
between knee and ankle on the belly of the tibialis anterior muscle and
at the third metatarsal space for plantar muscle recording, and the
maximal amplitude CMAP elicited was chosen for each animal at
each test day. The latency from the stimulus to the onset of the first
negative deflection and the maximal amplitude from baseline to the
negative peak of the evoked action potentials were measured (Navarro
et al. 1994). The stretch reflex, elicited by electrical stimulation of
large myelinated afferent fibers, was evaluated from the late H wave
recorded in the motor nerve conduction tests described in the preceding text. The latency from stimulus to the negative peak and the
amplitude from the onset to the peak of the H wave were measured
(see Navarro et al. 1996, 1999; Valero-Cabré and Navarro 2001 for
detailed traces of M and H waves).
The crossed extensor reflex was elicited by stimulating the right
tibial nerve with single electrical pulses (500-␮s duration and
supramaximal intensity for each component) with wide interstimuli
intervals (30 s or longer) through needles inserted the cathode at
the ankle under the Achilles tendon (distal to the injury) and the
anode 1 cm distally on the medial side of the paw. Crossed reflex
responses were recorded from the left tibialis anterior muscle by
means of monopolar needle electrodes. This muscle cooperates in
the fixation of the contralateral ankle joint when ipsilateral flexor
reflex is elicited. The reflex response was recorded up to 350 ms
after stimulation, rectified, and displayed in the oscilloscope at a
scale between 20 and 500 ␮V per division. The latency from the
stimulus to the onset of each burst of activity, the maximal amplitude from baseline to peak, and the number of peaks were measured (Navarro et al. 1996, 1999). To reduce variability, reflexes
were evoked eight times (with varying intervals of at least 30 s in
between), and the highest amplitude recorded for each reflex
component was measured.
At the time points during follow-up, the amplitude of each
response in all rats of each group, either with or without recorded
responses, was averaged to reflect the degree of recovery. To study
reflex facilitation, the amplitude of each reflex component was
normalized by the degree of peripheral regeneration. For the
stretch reflex, the H/M amplitude ratio was calculated for each
muscle tested, whereas for crossed extensor reflexes, the maximal
amplitude of each component was divided by the amplitude of the
CNAP of the tibial nerve at the ankle. As an estimate of the central
latency of the reflex responses, we subtracted the latency of the
peripheral afferent (from ipsilateral ankle to sciatic notch) and
efferent pathways (from contralateral sciatic notch to muscle) to
the total latency from stimulus to response. Thus the calculated
central latency is an estimate of the impulse conduction from
ipsilateral sciatic notch to contralateral sciatic notch, i.e., including
a segment of peripheral conduction at the dorsal ipsilateral and
ventral contralateral lumbar roots.
SPINAL REFLEXES AFTER PERIPHERAL NERVE INJURIES
1765
RESULTS
Functional reinnervation
separated by 30-min resting periods. The mean latency to withdrawal of the injured paw was expressed as percentage of that of
the contralateral intact paw. A maximum time of 40 s was allowed
for the algesimetry test before stimulation was automatically
stopped to avoid burns at the paw.
Data analysis
All data are presented as the group means ⫾ SE of percentages with
respect to presurgery values of each animal. The results have been
compared between groups by nonparametric Kruskall-Wallis test followed by Mann-Whitney U tests. ␹2 test has been used to compare
proportions of animals presenting a given response.
1. Percentages with respect to preoperative values of the amplitude and the latency of CMAPs of tibialis anterior and plantar
muscles and CNAPs of tibial and 4th digital nerves at 90 days postoperation
TABLE
Compound Muscle Action Potential (CMAP)
Percent amplitude
CNT
CRH
CS
SIL4
SIL8
Percent latency
CNT
CRH
CS
SIL4
SIL8
Compound Nerve Action Potential (CNAP)
Tibialis anterior muscle
Plantar muscle
Tibial nerve
113 ⫾ 4
97 ⫾ 1*
56 ⫾ 5*,†
51 ⫾ 3*,†
35 ⫾ 9*,†,‡,§
112 ⫾ 5
71 ⫾ 6*
35 ⫾ 5*,†
25 ⫾ 4*,†
14 ⫾ 2*,†,‡,§
106 ⫾
93 ⫾
80 ⫾
58 ⫾
43 ⫾
90 ⫾ 2
112 ⫾ 2*
135 ⫾ 9*
132 ⫾ 6*,†
149 ⫾ 8*,†
95 ⫾ 6
141 ⫾ 3*
156 ⫾ 8*
150 ⫾ 9*
164 ⫾ 6*
91 ⫾ 2
107 ⫾ 2*
115 ⫾ 4*
130 ⫾ 7*,†
146 ⫾ 12*,†,‡
1
1*
3*,†
7*,†,‡
9*,†,‡
4th digital nerve
110 ⫾
86 ⫾
37 ⫾
28 ⫾
3⫾
2
4*
6*,†
1*,†
3*,†,‡,§
93 ⫾ 3
159 ⫾ 8*
193 ⫾ 17*,†
190 ⫾ 11*,†
234 ⫾ 234*,†
CNT, control; CRH, group with sciatic nerve crushed; CS and SIL4, sciatic nerve sharply transected and repaired by sutures or by tubulization with a silicone
guide leaving a 4-mm gap, respectively; SIL8, 8-mm segment of sciatic nerve was resected with its gap bridged by a silicone tube. P ⬍ 0.05. *, vs. CNT; †,
vs. CRH; ‡, vs. CS; §, vs. SIL4.
J Neurophysiol • VOL
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FIG. 1. Percentage recovery with respect to preoperative values of compound muscle action potential (CMAP) amplitude recorded in tibialis anterior
(A) and plantar (B) muscles and compound nerve action potential (CNAP)
recorded at the tibial nerve (C) and the 4th digital nerve (D) along the 90 days
follow-up. Values are the mean for each group at each interval with SE
indicated as bars.
The results of electrophysiological tests for control rats
(group CNT) did not show significant changes over the 3 mo
follow-up after sham injury (Fig. 1, Table 1). There were
evidences of a slight and progressive increase in the amplitude
of CMAPs and CNAPs, attributed to an increase of muscle size
and nerve fiber size with age, and of a slight decrease in
latencies (indicating an increase in nerve conduction velocity),
which is attributable to mild increase in myelin thickness and
axonal caliber during adult age (see Verdú et al. 2000 for a
review). These findings indicate that adult rats 10 –12 wk old
have not reached yet a steady state of development with regard
to peripheral nerve function.
CMAPs and CNAPs were completely abolished after nerve
injury in all groups. The first evidence of motor reinnervation
appeared by 30 dpo in the proximal tibialis anterior muscle and
by 60 dpo in the distal plantar muscle with polyphasic CMAPs
of longer latency and lower amplitude than those recorded
prior to surgery (Fig. 1, A and B). CMAPs were recorded
during follow-up in all the rats except in the plantar muscle of
2 of the 12 animals of group SIL8. On the other hand, CNAPs
were recorded in the tibial nerve in all the rats from 60 dpo,
whereas recovery of digital CNAPs was more compromised
(Fig. 1, C and D). We were unable to record CNAPs in the
digital nerves in 1 rat of groups CS and SIL4 and in 11 of the
12 of group SIL8.
CMAPs and CNAPs reached significantly higher amplitude
values in group CRH than in all other injured groups. The
longer the gap between both nerve stumps, the lower amplitude
of compound action potentials; CS and SIL4 groups showed
significantly higher amplitudes of both CMAPs and CNAPs
than group SIL8. Notwithstanding, group CS had only significantly higher values for the tibial CNAP with respect to group
SIL4 (Table 1, Fig. 1). The latency of motor and nerve action
potentials decreased over follow-up, although in all groups it
remained longer than preoperative and CNT values at the end
of follow-up. CRH animals showed the best recovery, with
significant differences with respect to the other groups especially in the latency of CNAPs (Table 1).
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A. VALERO-CABRÉ AND X. NAVARRO
Withdrawal responses to plantar heat stimulation reappeared
by 60 dpo in all the rats, except in 5 of 12 of group SIL8,
which, however, had also a positive response by 90 dpo. Group
CRH had the best recovery of pain sensibility, with significantly lower withdrawal time to heat than groups CS and SIL4,
which in their turn showed shorter withdrawal time than group
SIL8 (Fig. 2).
Reflex responses
FIG. 2. Recovery of the latency until withdrawal in the operated right
hindpaw vs. the intact left paw found in the algesimetry test along the 90 days
follow-up. Final values were significantly (P ⬍ 0.05) increased in all injured
groups vs. controls, in group SIL4 vs. group CRH, and in group SIL8 vs. all
the other injured groups. Values are the mean for each group at each interval
with SE indicated as bars.
J Neurophysiol • VOL
FIG. 3. Single sample recordings of crossed extensor reflexes recorded at
the contralateral tibialis anterior muscle after stimulation of the right tibial
nerve in representative animals at preoperation (A), 30 (B), 60 (C), and 90 days
after sciatic nerve crush (D and E). C1 is the 1st component of the reflex
response to reappear, followed by C2, and later and in not all cases (see
example in D) by C3. Horizontal scale: 25 ms/div; vertical scale: 100 ␮V/div.
significantly lower than preoperative values (Table 3). In contrast, C1 and C2 components showed higher mean maximal
amplitude during follow-up than preoperative values in groups
CRH, CS and SIL4, but not in SIL8, in which amplitude
recovered similar to preoperative values. For C3, the maximal
amplitude achieved levels not different from those preoperation in groups CRH and CS but significantly lower in SIL4 and
SIL8. The number of peaks recorded in components C1 and C2
was by 90 dpo similar to the preoperative numbers in all the
injured groups, whereas there was a significant decrease to
about two-thirds in the number of peaks for C3 in groups SIL4
(65 ⫾ 11%) and SIL8 (66 ⫾ 16%) with respect to the controls.
The latency for all crossed extensor reflex components returned to normal levels with reinnervation in groups CRH, CS,
and SIL4 and remained significantly longer in SIL8. On the
contrary, the latency of the H wave was longer than in controls
for all experimental groups at the end of follow-up (Table 3).
The central conduction time, estimated for the different reflex
responses by subtracting the peripheral latencies of afferent
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In a large group of control rats (n ⫽ 205), stimulation of the
right tibial nerve at the ankle consistently yielded a contralateral crossed reflex response in the left tibialis anterior muscle,
recorded as bursts of motor unit action potentials grouped in
three components (Fig. 3, Table 2). The first component (C1)
appeared at a short latency (mean 12 ms), had short-duration (3
ms, 1–2 peaks), and had an average maximal amplitude of
around 650 ␮V. C1 was easily habituated with repeated stimulation. The second component (C2) appeared with longer
latency (about 23 ms) and had longer duration (20 ms, 5–7
peaks) but lower amplitude (300 ␮V). The third component
(C3) was detected starting 120 –135 ms after trigger, with long
duration (170 ms, around 30 peaks) and low amplitude (50 –
250 ␮V). The threshold and latencies measurements confirm
that each of the three components is likely mediated by peripheral fibers (A␤, A␦, and C fibers for C1, C2, and C3,
respectively) conveyed by different populations of sensory
dorsal root ganglion (DRG) neurons. In comparison, the ipsilateral H wave recorded in the right tibialis anterior muscle
showed shorter latency (6 ms) and duration (1 ms) but much
higher amplitude (mean 3.3 mV; Fig. 4, Table 2). When
thresholds were determined for each spinal reflex at different
stimulus duration, the H wave showed the lowest threshold
levels. For the crossed extensor reflex, C1 was evoked at
slightly lower stimulus intensity than C2, whereas C3 needed
about 15 times higher stimulation intensity (Table 2).
The three crossed extensor reflex components showed different patterns of recovery after nerve injury (see Figs. 3 and
4). C1 and C2, as well as the H wave, reappeared in all the rats
of the experimental groups during the 90 dpo. However, C3
failed to be evoked in one rat of groups CRH (10%) and CS
(10%), in two of SIL4 (15%), and in four of SIL8 (33%). In all
groups, the H wave reached values, between 72 and 88%,
SPINAL REFLEXES AFTER PERIPHERAL NERVE INJURIES
TABLE
1767
2. Characteristics of the three components (C1–C3) of the crossed extensor reflex
Crossed Extensor Reflex
Stretch Reflex H
Component
Latency, ms
Duration, ms
Peaks, number
Amplitude, ␮V
Central latency, ms
Threshold, mA
Pulse 500 ␮s
Pulse 200 ␮s
Pulse 100 ␮s
Pulse 50 ␮s
6.2
1.1
1.1
3260
4.9
⫾ 0.01
⫾ 0.04
⫾ 0.05
⫾ 90
⫾ 0.05
1.50 ⫾
2.17 ⫾
3.01 ⫾
4.88 ⫾
C1
C2
12.1 ⫾ 0.1
2.8 ⫾ 0.2
1.71 ⫾ 0.06
651
⫾ 60
9.2 ⫾ 0.2
1.63 ⫾
2.36 ⫾
3.21 ⫾
5.70 ⫾
0.07
0.12
0.13
0.23
23.5
19.9
6.4
305
21.2
⫾ 0.1
⫾ 1.1
⫾ 0.1
⫾ 15
⫾ 0.6
1.83 ⫾
2.38 ⫾
3.28 ⫾
5.81 ⫾
0.13
0.24
0.29
0.46
C3
0.14
0.21
0.23
0.42
129.4
472.6
28.9
214
126.6
⫾ 0.9
⫾ 48.6
⫾ 1.3
⫾ 11
⫾ 3.8
23.20 ⫾
40.14 ⫾
66.40 ⫾
86.50 ⫾
1.57
2.73
3.99
6.50
The crossed extensor reflex was recorded from the left tibialis anterior muscle after stimulation of the right tibial nerve, and the H wave reflex recorded from
the right tibialis anterior muscle after stimulation of the right sciatic nerve in a series of 205 control rats. Values are means ⫾ SE.
Spinal cord reflex facilitation
To analyze the functional performance of the crossed extensor reflex arch independently on the success of reinnervation of
the injured nerve, the amplitude of C1–C3 was normalized by
the amplitude of the tibial CNAP, whose regenerated sensory
afferents served to induce the reflex (Table 3). The reflex H
wave was also normalized by dividing its amplitude by that of
the M wave of the tibialis anterior CMAP. In group CNT, all
reflex responses remained stable over the 90 dpo. However, in
all injured groups, C1 reached values between 3.5 and 4.5
times its original values by 30 dpo (group CRH) or 60 dpo
(groups CS, SIL4 and SIL8; Fig. 5). Thereafter there was a
tendency to decline; final values were close to preoperative in
group CRH but remained higher than normal in groups CS,
SIL4, and SIL8. Similarly, the H/M ratio was largely increased
(2–7 times) 1 mo after injury and tended to return to near
normal values at the end of follow-up (Fig. 5). On the contrary,
for C2 and C3 components group CRH had only slight early
facilitation followed by return to preoperative levels. In groups
CS and SIL4, statistically significant increases were found for
C2 and C3 (2–3 times) with respect to preoperative levels.
Such facilitation was maintained for C2 in both groups still by
90 dpo, whereas final values of C3 were not different from
preoperation. In group SIL8, C2 increased 2.5 times and remained higher than normal over time, and C3 showed a mild,
not significant increase above controls (Fig. 5, Table 3).
DISCUSSION
FIG. 4. Single representative recordings of compound muscle action potentials in the tibialis anterior muscle of the operated hindlimb of 1 control rat (A),
1 rat subjected to section and suture repair (group CS) at 30 dpo (B) and at 90
dpo (C), and 1 rat subjected to section and tube repair (group SIL4) at 90 dpo
(D). Horizontal scale: 2 ms/square; vertical scale: 10 mV/div in A, C, and D
and 5 mV/div in B.
J Neurophysiol • VOL
Our results show that after peripheral nerve injuries not all
motor reflex responses show the same pattern of restitution.
Reflex arches dependent on myelinated afferent fibers, such as
the stretch reflex (H wave) and the components C1 and C2 of
the crossed extensor reflex, recovered in all the animals despite
the varying severity of the nerve injury induced. In contrast,
component C3 of the crossed extensor reflex, mediated by
unmyelinated afferents, reappeared in a lower proportion of
animals, especially if a gap was produced between the nerve
stumps. Furthermore, H wave, C1, and C2, but not C3, reflex
responses showed a marked increase in the amplitude of the
motor response during the first stages of regeneration, and, as
reinnervation progressed, tended to return down to normal
values. However, at the end of the 3 mo follow-up, the reflex
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sensory and efferent motor volleys, increased for all groups
and reflex components (C1: 105–130%, C2: 104 –144%, C3:
120 –150%, H: 104 –131%) after injury but tended to normalize
over time. However, for C1 (105–113%), C3 (106 –110%), and
H responses (105–113%), the final values remained significantly longer than preoperative values in all groups, whereas
for C2 significant differences were only found for groups SIL4
(104%) and SIL8 (108%).
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A. VALERO-CABRÉ AND X. NAVARRO
3. Percentage with respect to preoperative values of amplitude, latency, and normalized amplitude of the H wave and of C1–3
components of the crossed extensor reflex at 90 days follow-up
TABLE
Crossed Extensor Reflex
Stretch Reflex H
C2
C3
110 ⫾
88 ⫾
72 ⫾
79 ⫾
80 ⫾
5
5*
6*
6*
6*
99 ⫾ 7
151 ⫾ 36
243 ⫾ 24*
218 ⫾ 47*
104 ⫾ 18‡,§
98 ⫾ 4
103 ⫾ 15
183 ⫾ 34*
158 ⫾ 32
100 ⫾ 26
103 ⫾ 7
102 ⫾ 14
104 ⫾ 17
66 ⫾ 12*
48 ⫾ 14*,†,‡
97 ⫾
111 ⫾
126 ⫾
126 ⫾
123 ⫾
2
1*
4*
5*
3*
100 ⫾
105 ⫾
106 ⫾
105 ⫾
113 ⫾
99 ⫾
101 ⫾
101 ⫾
103 ⫾
108 ⫾
102 ⫾
109 ⫾
108 ⫾
108 ⫾
110 ⫾
108 ⫾ 6
92 ⫾ 7
140 ⫾ 13*,†
234 ⫾ 57*,†
216 ⫾ 15*,†,‡
3
3
2
5
3*
96 ⫾ 7
164 ⫾ 40
243 ⫾ 51*,†
345 ⫾ 74*
268 ⫾ 66*
5
3
2
2
3*
92 ⫾ 7
109 ⫾ 13
236 ⫾ 41*
331 ⫾ 91*
256 ⫾ 78*,†
1
4
3
3*
2*
99 ⫾ 6
111 ⫾ 17
140 ⫾ 27
197 ⫾ 51
121 ⫾ 16
P ⬍ 0.05. *, vs. CNT; †, vs. CRH; ‡, vs. CS; §, vs. SIL4.
amplitude remained steady at significantly higher levels than in
control animals, more in groups showing a lower degree of
sensory-motor peripheral reinnervation.
Peripheral reinnervation after injuries
After nerve injuries, the projection pathways of the spinal
reflex arches become disrupted and rewire with target receptors
and effectors only if axonal regeneration and target reinnervation take place. The amount of sensory and motor reinnervation
FIG. 5. Percentage with respect to preoperative values of the normalized
amplitude of C1–C3 components of the crossed extensor reflex and of the H/M
amplitude ratio during 3 mo postoperation in the groups of rats evaluated after
sciatic nerve crush (A), section and suture repair (B), or tube repair (C). Values
are the mean for each group at each interval.
J Neurophysiol • VOL
and the degree of accuracy of reinnervation of the originally
innervated dermatomes and myotomes are essential to achieve
the restitution of functional reflexes after nerve injuries. To
assess the effect of these factors on spinal reflex recovery, four
types of injury and repair, resulting in varying levels of reinnervation and of misdirected reinnervation (Evans et al. 1991;
Valero-Cabré et al. 2001), were applied to the rat sciatic nerve.
As expected, animals submitted to a crush injury showed the
fastest and highest recovery. A crush causes complete axotomy, but the endoneurial tubes remain intact, thus providing
the proximal axonal ends with immediate neurotrophic support
as well as a physical guide to their original targets (De Medinaceli 1988; Gutmann and Guttmann 1942). After nerve transection, epineurial suture repair allows the apposition of the
nerve stumps, with access of the transected axons to the degenerating distal stump, as a source of neurotrophic factors.
However, disruption of nerve continuity and misalignment of
endoneurial and perineurial sheaths lead some axons to misguidance toward territories different from the originals, resulting in poorer reinnervation (Brushart 1993; Navarro et al.
1994; Valero-Cabré et al. 2001). Tubulization of a gap between
nerve stumps allows for slower reinnervation timing because
extra time is needed to form an intratubular connective matrix
able to support the elongation of regenerating axons across the
tube (Butı́ et al. 1996; Williams et al. 1983). With a short
interstump gap, just produced by self-retraction of the nerve
stumps accounting for a 3– 4 mm gap, no significant differences in the speed and in the levels of reinnervation was found
with respect to a direct suture (Bodine-Fowler et al. 1997;
Navarro et al. 1994; Valero-Cabré and Navarro 2001). However, when a segment of the nerve is resected, resulting in a
longer gap, reinnervation is slower and achieves significantly
lower values at the end of the follow-up than with less severe
injuries (Butı́ et al. 1996; Valero-Cabré et al. 2001).
Our results also show that CMAPs and CNAPs recorded in
proximal targets recovered in all the animals used for the study,
whereas reinnervation of distal targets occurred in lower pro-
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Percent amplitude
CNT
CRH
CS
SIL4
SIL8
Percent latency
CNT
CRH
CS
SIL4
SIL8
Percent normalized amplitude
CNT
CRH
CS
SIL4
SIL8
C1
SPINAL REFLEXES AFTER PERIPHERAL NERVE INJURIES
portion and to lower levels at the same time points. Unpublished observations from our laboratory in rats subjected to
similar nerve lesions showed no significant changes of CMAPs
and CNAPs from 3 to 6 mo postoperation, assuring that in 90
days a steady state of reinnervation was achieved.
Spinal cord reflex recovery
J Neurophysiol • VOL
after nerve resection followed by short and long gap tubulization in comparison with crush and direct suture repair, and
although no histological confirmation is provided, a comparatively higher loss of small sensory neurons than of large
sensory neurons could partly explain the lower recovery of the
C3 component of the crossed extensor reflex with respect to
reflex components mediated by myelinated fibers. After unrepaired or repaired peripheral nerve injuries, myelinated A
primary afferents, shown by others to be less susceptible to
nerve injury-induced cell death, are able to extensively sprout
in the dorsal horn gray matter projecting to Rexed’s laminae I
and II previously occupied by unmyelinated nociceptive projections. In contrast, unmyelinated spinal projections are reduced, yielding to an empty space in the most superficial
laminae of dorsal horn that explains the extension of myelinated collaterals into these regions (Ma et al. 2000; Woolf et al.
1992, 1995). The decreased spinal projections of surviving
nociceptive cells would reduce reconnection with enough number of interneurons, thus diminishing the amplitude of reflex
responses evoked by peripheral afferent stimulation. On the
other hand, surviving nociceptive cells, probably selected for
their high resistance to neurotrophic factors deprivation, show
a high capability for peripheral sprouting and extensive reinnervation into a wider distal territory (Devor et al. 1979;
Navarro et al. 1994), thus compensating the loss of regenerative neurons for peripheral target innervation.
Spinal cord reflex facilitation
It is worthwhile to note that reflex components mediated by
thick myelinated afferent fibers showed higher amplitudes during the follow-up after the nerve injury than before the injury.
Despite the reduced number of regenerated myelinated fibers,
as indicated by the subnormal recovery of CNAP amplitudes,
components C1 and C2, although not C3, had similar to normal
latency and number of peaks but significantly higher levels of
maximal amplitude. Similarly, the ratio of H/M waves amplitude of ipsilateral muscles increases after repaired section and
resection injuries (Valero-Cabré and Navarro 2001). The facilitation of spinal reflexes was highly significant during the
initial stages of reinnervation and tended to decrease toward
preoperative values in a reinnervation-dependent manner.
After nerve lesions, spinal cord synapses are progressively
stripped by the action of astrocytes and microglia, thus impeding the transmission through reflex arches (Aldskogius and
Kozlova 1998; Brännström et al. 1992a; Mendell 1984). This
process reverses as soon as target reinnervation starts, and
motoneuron dendrites reextend and reestablish functional synapses with afferent inputs (Brännström 1992b). In agreement
with previous observations, our results indicate that after peripheral nerve injury functional or structural changes affect the
performance of spinal cord monosynaptic (Hellgren and Kellerth 1989; Valero-Cabré and Navarro 2001) and polysynaptic
(Navarrete et al. 1990; Vejsada et al. 1991), as well as brain
stem (Cossu et al. 1999) reflex circuits, so that they become
hyperresponsive to the stimulation of sensory terminals. Furthermore, after severe injuries, spinal reflexes remained facilitated at the end of the 3 mo follow-up, thus suggesting that
permanent rather than transitory new patterns of functional
connectivity in the spinal cord have been established (Koerber
et al. 1994).
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The stimulation of sciatic nerve skin sensory afferents in
control animals yield to ipsilateral flexion and crossed extension responses recordable at the biceps femoris (Navarro et al.
1996; Woolf and Sweet 1984) and at the tibialis anterior
muscle respectively (Clare and Landau 1975). Stimulation of a
limb induces an ipsilateral flexor reflex yielding to the withdrawal of the stimulated limb by inducing a flexion. Simultaneously, activation of several contralateral muscles is induced
by spinal crossed pathways to keep the whole limb in an
extended position with the aim to prevent loss of balance. This
crossed extensor reflex involves co-activation of extensor muscles of the knee (quadriceps muscle) to keep the limb extended
but also of dorsiflexors of the paw (tibialis anterior and peroneal muscles) and plantarflexor muscles (gastrocnemius and
soleus muscles) (Clare and Landau 1975), both contributing to
the fixation of the ankle joint. Although the contralateral response results in a general extension of the limb, we have
recorded it in a muscle (tibialis anterior) whose function is a
dorsal flexion of the ankle, contributing to the fixation of the
ankle to prevent loss of balance when the flexor withdrawal
reflex is produced. By stimulating with supramaximal pulses
the sensory fibers of the tibial nerve at the ankle, we recorded
a multiburst reflex signal with three differentiated components
of activity. Differences of latency and stimulus threshold indicated that they were compatible with being conveyed by A␤,
A␦, and C afferent fibers, respectively (Clare and Landau 1975;
Meyerson et al. 1995; Navarro et al. 1999; Woolf and Sweet
1984). We have previously proved that selective lumbar rhizotomy abolished the three components in ipsilateral and contralateral reflex responses and that their recovery was correlated with regeneration of sensory afferents onto spinal cord
dorsal laminae (Navarro et al. 1999).
The algesimetry test proved that thin nociceptive fibers
reinnervated the plantar skin in all the rats and reached higher
levels of recovery than that of CNAPs evoked by thick myelinated fibers at the paw, thus confirming the higher capabilities for sprouting and achieving target reinnervation of thin
than thick nerve fibers (Gutmann and Guttmann 1942; Navarro
et al. 1994). In contrast with these results, reflex components
mediated by thick sensory fibers recovered to higher amplitude
than those mediated by C fibers. Axotomized sensory neurons
are more likely to atrophy and die by apoptosis when access to
the distal stump as a source of trophic factors is not provided,
whereas surgical guidance of axons to the distal stump, by
interposing a nerve graft or an impermeable tube, prevents
neuronal death (Melville et al. 1989). However, according to
the literature, not all sensory neurons are equally affected by
ineffective or delayed regeneration (Fu and Gordon 1997;
Groves et al. 1997; Himes and Tessler 1989). Small sensory
neurons, such as nociceptive neurons projecting C fibers, seem
to be more sensitive than larger diameter neurons, such as
mechanoreceptive and propioceptive neurons, to cell death
(Tandrup et al. 2000). In our study, regeneration was delayed
1769
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A. VALERO-CABRÉ AND X. NAVARRO
The authors thank Drs. Anastas Propratiloff and Enrique Verdú for fruitful
discussions.
J Neurophysiol • VOL
This work was supported by grants from Comisión Interministerial de
Ciencia y Tecnologia (SAF97-0147) and Fondo de Investigaciones Sanitarias
(FIS00-0031-02), Spain, and ESPRIT programme (26322) of European Commission. A. Valero-Cabré was the recipient of a predoctoral research fellowship from Direcció General d’Universitats, Generalitat de Catalunya (Spain).
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