comparison of the fastest regenerating motor and sensory - brain

doi:10.1093/brain/awl184 Brain (2006), 129, 2471–2483

Comparison of the fastest regenerating motorand sensory myelinated axons in the sameperipheral nerve

Mihai Moldovan,1 Jesper Sørensen1,2 and Christian Krarup1,3

1Division of Neurophysiology, Institute of Medical Physiology, Panum Institute, University of Copenhagen, Departments of,2Plastic Surgery and 3Clinical Neurophysiology, The Neuroscience Center, Rigshospitalet, Copenhagen, Denmark

Correspondence to: Christian Krarup, MD, DMSc, FRCP, Department of Clinical Neurophysiology, The NeuroscienceCenter, NF3063, Rigshospitalet, 9 Blegdamsvej, 2100 Copenhagen, DenmarkE-mail: [email protected]

Functional outcome after peripheral nerve regeneration is often poor, particularly involving nerve injuries farfrom their targets. Comparison of sensory and motor axon regeneration before target reinnervation is notpossible in the clinical setting, and previous experimental studies addressing the question of differences ingrowth rates of different nerve fibre populations led to conflicting results. We developed an animal model tocompare growth and maturation of the fastest growing sensory and motor fibres within the same mixed nerveafter Wallerian degeneration. Regeneration of cat tibial nerve after crush (n = 13) and section (n = 7) wasmonitored for up to 140 days, using implanted cuff electrodes placed around the sciatic and tibial nerves andwire electrodes at plantar muscles. To distinguish between sensory and motor fibres, recordings were carriedout from L6–S2 spinal roots using cuff electrodes. The timing of laminectomy was based on the presence ofregenerating fibres along the nerve within the tibial cuff. Stimulation of unlesioned tibial nerves (n = 6) evokedthe largest motor response in S1 ventral root and the largest sensory response in L7 dorsal root. Growth rateswere compared by mapping the regenerating nerve fibres within the tibial nerve cuff to all ventral or dorsalroots and, regardless of the lesion type, the fastest growth was similar in sensory and motor fibres. Maturationwas assessed as recovery of themaximummotor and sensory conduction velocities (CVs) within the tibial nervecuff. Throughout the observation period the CV was �14% faster in regenerated sensory fibres than in motorfibres in accordancewith the difference observed in control nerves. Recovery of amplitude was only partial aftersection, whereas the root distribution pattern was restored. Our data suggest that the fastest growth andmaturation rates that can be achieved during regeneration are similar formotor and sensorymyelinated fibres.

Keywords: nerve; regeneration; laminectomy; cuff electrodes; cat

Abbreviations: CMAP = compound muscle action potential; CNAP = compound nerve action potentials; CRP = compoundroot action potential; CV = conduction velocity

Received March 28, 2006. Revised May 30, 2006. Accepted June 15, 2006. Advance Access publication August 10, 2006.

IntroductionAfter a nerve injury, both motor and sensory axons have the

ability to regenerate and, given a proper pathway, reconnect

with targets. Despite this capacity, the functional outcome of

peripheral nerve regeneration is often poor, particularly after

nerve injuries that sever peripheral nerves far from their

targets (Lundborg, 2003). In a long-term study of nerve

regeneration in non-human primates the lesion and repair

type, through the time to muscle reinnervation, influenced

recovery assessed by physiological methods (Krarup et al.,

2002). Thus, both the distance and rate of regeneration

probably play important roles in determining the outcome

of nerve regeneration.

Axonal transport responsible for nerve regeneration is slow

(Wujek and Lasek, 1983) for both motor and sensory axons

(Braendgaard and Sidenius, 1986). The fastest growth rates

approach 4 mm/day depending on the regeneration environ-

ment (Fugleholm et al., 1994, 1998, 2000; Sorensen et al.,

2001). In the mouse distal stump, Schwann cells previously

associated with motor axons retain specialized features

(Martini et al., 1992), and this was suggested to contribute

to preferential reinnervation of motor pathways by motor

axons (Brushart, 1988), which may improve functional out-

come of motor regeneration after section (Brushart, 1993). It

is unknown whether such environmental specializations

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accelerate motor axon growth rates compared with sensory

axon growth rates.

Comparison of sensory and motor axon regeneration

before target reinnervation is not possible in the clinical

setting, and previous experimental studies addressing the

question of differences in growth rates of different nerve

fibre populations led to conflicting results. In some studies,

axons in muscle or mixed nerves were found to regenerate

faster than fibres in pure sensory nerves (Jenq and Coggeshall,

1985; Chen and Bisby, 1993). A better motor than sensory

regeneration was also suggested in the same mixed nerve (da

Silva et al., 1985; Madison et al., 1988). Other studies, how-

ever, indicated that sensory fibres dominate in the early stages

of regeneration (Sanger et al., 1991; Madorsky et al., 1998;

Suzuki et al., 1998; Kawasaki et al., 2000). Nevertheless, the

fastest growing motor fibres were found to regenerate as fast

as the sensory fibres (Forman and Berenberg, 1978; Forman

et al., 1979). The reason for these controversies may be attrib-

uted to the technical difficulties of identifying the fastest

growing fibres at the front of regeneration.

The aim of this study was to compare, in a large animal

model, the fastest rates of regeneration that are achieved by

sensory and motor fibres within the same mixed nerve both

after crush and section. We previously developed an electro-

physiological method in cat to monitor growth of axons in

the tibial nerve through a series of electrodes implanted

around the nerves in a silicone cuff and confirmed by his-

tological studies (Fugleholm et al., 1994, 1998; Sorensen et al.,

2001). In this study, to distinguish between motor and sen-

sory fibres, we recorded evoked action potentials from all the

ventral and dorsal roots containing fibres projecting to

the tibial nerve. To compare rates of growth, we mapped

the distances reached within the tibial nerve cuff by fibres

belonging to different spinal roots. Furthermore, to compare

maturation, recovery of motor and sensory conduction velo-

cities (CVs) within the tibial nerve cuff was investigated for

up to 5 months of regeneration.

Material and methodsAnimals, experimental design and anaesthesiaElectrodes were implanted in both legs in the plantar muscles and

around the sciatic and tibial nerves of 15 adult female cats (2.5–

3.2 kg, Iffa-Credo, France). After implantation, all animals regained

normal movements and gait and stayed healthy during the experi-

ment. Data from four legs were excluded from the analysis because

the cat destroyed the cabling connecting the electrodes.

The study was performed in five consecutive steps: (i) surgery for

electrode implantation and nerve lesions; (ii) electrophysiological

validation of the nerve lesion; (iii) serial recordings to identify the

outgrowth of the tibial nerve fibres; (iv) terminal laminectomies to

compare growth of sensory and motor fibres in the tibial cuff after

crush (n = 5) and section (n = 4); (v) terminal laminectomies

to compare maturation of sensory and motor axons after target

reinnervation after crush (n = 8) and section (n = 3). Recordings

from roots were performed in six unlesioned tibial nerves for control

1 week after the electrode implantation.

Chronic serial investigations were carried out under anaesthesia

by a mixture of 0.7 ml Ketamine (10 mg/kg) and 0.4 ml Xylazine

(2 mg/kg) delivered by intramuscular injection and maintained by

subcutaneous injection as needed. Electrode implantations and

laminectomies were carried out during deep anaesthesia induced

with pentobarbital 40 mg/kg, 2.5 ml intraperitoneal, and maintained

by repeated intravenous injection of 1 ml from the 10% dilution.

During anaesthesia, the core temperature was controlled by placing

the animal on a thermostat-heated rubber pad (37�C) and the per-

ipheral temperature was maintained using a thermostat-controlled

heating lamp (37�C). At the completion of the investigations, the

cats were killed by an overdose of 250 mg pentobarbital i.v. The

experiments were performed with approval of the Danish National

Animal Experiment Committee.

Electrode implantations and nerve lesionsProcedures of nerve lesions and electrode implantation (Krarup

and Loeb, 1988) were described in detail previously (Fugleholm

et al., 1994, 1998, 2000; Sorensen et al., 2001). Briefly, after exposure

of the tibial nerves over �70 mm, crush lesions were carried out by

clamping a silicone coated forceps for 2 min, and complete transec-

tion lesions were immediately co-adapted using microsurgical tech-

niques by four epineural sutures (10-0 Taper Point, S&T Neuhausen,

Switzerland). Eight-lead semicircular cuff electrodes (Larsen et al.,

1998) with leads spaced 7.5 mm apart were implanted around the

tibial nerves (Fig. 1A) and positioned so that the first electrode (T1)

was 10 mm distal to the lesion site (marked with a 6-0 suture). Six-

lead cuff electrodes were implanted around the sciatic nerves (two

pairs of three leads, with 18 mm between the centre recording

leads). The electrode placed around the tibial nerve had an internal

diameter of 3 mm and the cuff around the sciatic nerve had an

internal diameter of 4 mm, which was at least 30% larger than

the diameter of the nerves to avoid compression. Wire electrodes

for recording of plantar muscle activity were sutured to the fascia

of the flexor digitorum brevis muscle and subcutaneously on the

dorsum of the paw. A ground wire electrode was implanted sub-

cutaneously. The connecting teflon-coated stainless steel wires (AS

631, Cooner WIRE Co., Chatsworth, CA, USA) connecting the elec-

trodes were passed subcutaneously to the back, resurfaced through a

skin excision and mounted on an integrated circuit board protected

by an aluminium shield.

The integrity of the implanted electrodes and electrical connec-

tions was tested in situ by measuring the impedance between the

leads and ground. Complete axonal loss after the nerve lesion was

ascertained electrophysiologically 1 week after the lesion.

Serial investigations to identify the growthfront of the tibial nerve fibresDuring the first month after the lesion, serial electrophysiological

studies were carried out every 4–6 days. An illustration of the

recording set-up is presented in Fig. 1A. Supramaximal stimulation

(10 mA at 0.5–3 Hz) was delivered consecutively to the T1–T7 leads

in the tibial cuff with the anode placed one lead distal to the cathode.

Biphasic rectangular negative-positive pulses (duration = 0.1 ms)

were generated from a photo-isolated battery-powered, constant

current stimulator. The ascending evoked compound nerve action

potentials (CNAPs) were recorded (10C02 Dantec, 0.2–6 kHz)

in two tripolar electrode configurations (R1 and R2) in the

sciatic cuff. The stimulus was first applied to T1, and if a clearly

2472 Brain (2006), 129, 2471–2483 M. Moldovan et al.

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identifiable CNAP (at least twice the background noise after

averaging of at least 200 repetitions on a Nicolet 20 Pro Digital

Oscilloscope) could be recorded at R1 and R2 the stimulus site

was stepwise moved distally. The front of growth was identified

at the most distal cathode where the CNAP had a longer latency

than the CNAP elicited at the next cathode in the proximal

direction (Fig. 1B and C). Our electrophysiological set-up

allowed identification of the growth front with single-fibre

resolution (Fig. 2).

Root recordings during terminallaminectomiesBefore the terminal laminectomy, the peroneal nerves were cut

bilaterally through a small skin incision below the knee to exclude

Fig. 1 Evaluation of the growth front in the tibial nerve, determined at 19 days after section. (A) The stimulus was first applied at T1–T2(cathode proximally) and then stepwise moved distally to T6–T7, at which no CNAPs could be recorded at R1 (B) and R2 (C) tripolarrecording sites in the sciatic cuff. The most distal fibres were stimulated at T5, which was therefore considered the growth front. Thelatencies recorded at R1 and R2 are marked with arrows (B and C). CV along the tibial nerve segment could be estimated through R1latency versus distance linear fit (D). CV of the parent fibres, whose sprouts were stimulated in the tibial cuff, was determined between R1and R2 in the sciatic cuff (E). The parent fibre CVs were constant between T1 and T3 (open bars and arrows). At T4 and T5, different fibres(illustrated in grey and black symbols) had lower CVs, indicating that the sprout contributing to the growth front did not originate from thefastest conducting parent fibre.

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erroneous conduction to the roots. The cat was placed in a stereo-

taxic frame and the vertebrae were exposed by stepwise removal

of fascia, muscles and ligaments adjacent to os sacrum and the

vertebrae. The L6–S2 (Highland et al., 1990; Bailey et al., 1992)

ventral and dorsal roots were separated (polished forceps, type

FRS-15 & RM-8; S&T Neuhausen, Switzerland) under optic mag-

nification and cut as close to the spinal cord as possible (�3 cm free

length). In order to ensure fixed geometry of the recording electro-

des, cuffs rather than hooks were used. Each ventral or dorsal root

was pulled gently through the tubular cuff (3 circular leads 7.5 mm

apart) by a thin thread (5–0) attached to its free tip. The internal cuff

diameter was 1.0 mm (around S1 and S2 roots) and 2.0 mm (around

the L7 and L6 roots), owing to the differences in root thickness. The

contact between the root and the leads in the cuff was ensured by

Fig. 2 The potential evoked at the growth front in Fig. 1 had triphasic morphology and amplitude <0.5 mV, suggesting thatstimulation at T5 evoked a response from a single fibre. (A) During double stimulation (s1 and s2) the unconditioned response r1 and thetest response r2 had similar shape and amplitude with occlusion at the absolute refractory period. (B) The difference between theinterresponse (DR) and the interstimulus (DS) interval (DR � DS) increased at smaller DS as a result of relative refractory period.Stippled line indicates theabsolute refractory period. (C) Amplitude of r2 relative to r1 (100%) as a function of DS. The sudden complete lossof the single-fibre response for DS < 0.3 ms may be noted.


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physiological saline solution maintained by the capillarity of the cuff.

In order to prevent tissue dry-out the remaining exposed spinal cord

was covered with gauze soaked in saline.

The ascending ventral and dorsal compound root action poten-

tials (CRPs) were recorded (10C02 Dantec, 0.2–6 kHz) in tripolar

electrode configurations identical to the R1 and R2 sciatic nerve

recording sites. The integrity of the root recordings was ascertained

by stimulation in the sciatic cuff with cathode placed at R2 and R1

electrode positions (Fig. 3). Sciatic stimulation evoked CRPs from all

ventral and dorsal L6, L7, S1 and S2, whereas no CRPs could be

Fig. 3 Comparison of distances attained by motor and sensory fibres for the tibial nerve in Fig. 1 was carried out during a terminallaminectomy (A). Tripolar recordings were carried out from cuffs placed around ventral (B) and dorsal (C) roots L6–S2. The integrityof root recordings was ascertained by stimulating in the sciatic cuff at R2 and R1 positions. Tibial nerve stimuli were firstapplied at T1–T2 (cathode proximally) and were then stepwise moved distally until no clearly identifiable CRPs—at least twice thebackground noise after averaging 500 repetitions–could be recorded at each of the investigated roots. The motor growth front wasidentified at T4 by S1V recording. The sensory growth front was identified at T5 by L6D recording. It may be noted that on thebasis of the refractory period measurements in Fig. 2 sensory growth front at T5 was represented by a single fibre that could be identifiedby CRP recordings.


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recorded from S2 dorsal root at tibial stimulation in any of the

investigated nerves. The largest CRP amplitudes were at least as

large as the corresponding R1 and R2 recordings and allowed

identification at the root level of the single fibre(s) at the front of

regeneration (Fig. 3).

Motor and sensory growth frontsLaminectomies were carried so that the growth front approached but

did not exceed the distal end of the tibial cuff. Stimuli were first

applied at T1–T2 (cathode proximally) and then stepwise moved

distally until no clearly identifiable CRP (at least twice the back-

ground noise after averaging 500 repetitions) could be recorded at

each of the investigated roots (Fig. 3). The motor growth front

was identified at the most distal cathode position where stimulation

evoked ventral root CRPs (Fig. 3B). Similarly, the sensory growth

front was identified at the most distal cathode position where

stimulation evoked dorsal root CRPs (Fig. 3C). The stability of

the growth front was ascertained after the 6–10 h of recordings

during laminectomy. The actual growth distance, and subsequently

the growth rate, could only be determined at the completion of the

study when the nerves were exposed and T1 position in respect to the

lesion mark was reassessed to allow corrections due to the sliding of

the cuff. At the completion of the experiment, the distances varied

from 8 to 14 mm, and the measured distance was used to calculate

growth rates. The slight displacement probably occurred during the

first week of the experiment when collagen formed around the

electrode (Fugleholm et al., 1994).

Motor and sensory CVsMaturation of the fastest axons was compared by investigating

recovery in motor and sensory CVs after target reinnervation.

Laminectomies were performed between 52 and 139 days after

the lesion, when compound muscle action potentials (CMAPs)

could be recorded (10C02 Dantec, 0.01–10 kHz) after tibial stimula-

tion. The latencies of the CNAPs and CRPs were measured to the

first positive peak and latencies of the CMAPs were measured to the

take-off of the negative phase to calculate the CVs of the fastest

fibres. Because of known distances between electrodes relative to

T1, the CV along the nerve segment in the tibial cuff could be

estimated from the slope of the latency versus distance linear fit

without measuring the distances to the recording sites. The

motor CV was estimated as the fastest CV among all ventral

roots. Similarly, the sensory CV was estimated as the fastest CV

to all dorsal roots. This method of CV calculation was validated

by comparing the motor CV with the plantar CV in the same nerve

both in regenerated (Fig. 4) and control nerves (data not shown).

Motor and sensory amplitudesThe amplitudes of CRPs and CNAPs were measured peak to

peak. Amplitude measurements depend on the number, distribution

and temporal dispersion of the different single-fibre potentials con-

tributing to the compound response. Since we could not compare

the ‘control’ and ‘regenerated’ amplitudes in the same nerve, we

limited our investigations to the recovery and distribution of the

maximum sensory and motor amplitude that could be attained

(among all stimulation sites and among all roots).

StatisticsResults are given as mean 6 standard error of measurement (SEM).

Owing to the small sample size, comparisons were performed using

non-parametric statistics (SPSS Inc. SPSS for Windows, release 13,

2005, Chicago). Individual tests are mentioned where used.

ResultsFastest rate of growth in the tibial nerveBy serial observations during the first month after the

lesion, the progression of the growth front without distinc-

tion between sensory and motor fibres could be followed

along the tibial nerve (Fig. 1). At the completion of the

experiments, the growth rate in the tibial cuff was calculated

from the slope of the linear fit of the growth distance versus

time after lesion plot (Fig. 5). The crushed fibres had a growth

rate through the cuff of �4 mm/day (Fig. 5A) and the sec-

tioned fibres slightly lower at 3.7 mm/day (Fig. 5B). The

intercept was�8 days both after crush and section, indicating

a similar delay between lesion and sprouting (Fig. 5A and B).

These values were in agreement with previous findings on the

same model (Krarup et al., 1989; Fugleholm et al., 1994).

Motor and sensory growth frontsTo compare the distances reached by sensory and motor

fibres within the tibial cuff a laminectomy was performed

between 18 and 21 days after the lesion (Fig. 3). The distances

reached by the motor and sensory fibres at the growth front

were similar both after crush (Fig. 5C) and section (Fig. 5D).

The differences between motor and sensory growth fronts

never exceeded one stimulation level (7.5 mm).

At the termination of the experiment after measurement

of the tibial cuff placement in respect to the lesion mark,

differences between the motor and sensory fibre growth

were compared with the variability of the overall fastest

growth rate in the tibial cuff (Fig. 5A and B). After crush,

the difference between the motor and the sensory growth

fronts remained within the 95% confidence limits

(67.3 mm, alpha = 0.05) of the growth front estimated

from serial observations. After section, the variability was

greater but still no significant differences between motor

and sensory fibres could be detected.

Parent fibre CVParent fibre CV of the axons stimulated in the tibial cuff could

be measured between the R1 and R2 sciatic recording sites

(Fig. 1E). Since only one recording site was feasible at the

root cuff, it was not possible to distinguish the parent fibre

CV of sensory and motor fibres in our study. In controls,

sciatic nerve CV was 85 6 1 m/s. During early outgrowth

(Fig. 1E) and during post-reinnervation maturation (data

not shown), parent fibre CVs at the sciatic nerve were in

the normal range. Nevertheless, when single fibres were acti-

vated at the tip of the regeneration front, their parent fibre CVs

were lower than the parent velocities of fibres activated at more

proximal stimulation sites with lower growth rates (Figs 1E

and 2). These observations indicated that it was not the fastest

fibres (parent fibre CV) that constituted the growth front.


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Recovery of CV along the tibial nervesIn control nerves, average motor CV (77 6 3 m/s; n = 6) was

lower than the sensory CV (956 4 m/s, P < 0.05, Wilcoxon).

During outgrowth through the tibial cuff the motor and

sensory CVs along the regenerating nerve segments were

<3 m/s both after crush and section (Fig. 1D). At this

early time point, latency changes were uneven as different

axons with different CVs reached different cathodes (Fig. 1A)

and no consistent differences were detected between the

sensory and motor mean conduction velocities estimated

from the linear slopes. After 7 weeks of regeneration, the

latency–distance relationships in the tibial cuff were linear

both after crush and section, and CVs could be accurately

estimated (Fig. 4). The recovery of CV from pooled observa-

tions collected at different laminectomies could best be

described by a logarithmic function (Fig. 6A) both for

motor CV (R2 = 0.94) and sensory CV (R2 = 0.98). The

slope seemed steeper for sensory (42 m/s/day) than for

motor (35 m/s/day), indicating that the absolute difference

between the motor and the sensory CVs increased with time.

Fig. 4 Calculation of motor CVs along the tibial nerve of a regenerated nerve, 96 days after crush. (A) Schematic representation ofthe recording set-up. (B) The integrity of root recordings was ascertained by stimulating in the sciatic cuff at R2 and R1 positions. The tibialnerve was stimulated at T1 through T7 (cathode proximally), and evoked responses were recorded from L6–S2 ventral roots. (C) Plantarmuscle responses (P) could be evoked by tibial nerve stimulated from T2 through T8 (cathode distally). (D) CV along the tibialnerve segment was calculated for the different recording sites from the latency versus distance linear fit. The fastest motor CV(recorded to L7V root cuff ) was similar to the CV calculated from plantar muscle recordings.


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For example, at 52 days after crush, motor CV was 13 m/s and

sensory CV was 15 m/s, whereas at 139 days after crush motor

CV was 49 m/s and sensory CV was 56 m/s.

During post-reinnervation maturation we found that the

sensory CV was faster at any time point (Fig. 6A). Never-

theless, the relative recovery in conduction was similar in

motor and sensory axons. For example, at 52 days conduction

recovered �16% and at 139 days conduction recovered

�60% relative to the respective motor and sensory controls.

In fact, in all nerves investigated after target reinnervation we

found a high correlation (R2 = 0.98) between motor CVs and

the corresponding sensory CVs (Fig. 7A), indicating that

sensory CVs were �14% faster than the corresponding

motor CVs. This finding was also consistent in the sectioned

Fig. 5 Growth rates of sensory and motor axons along the tibial nerve after crush and section. Growth distances of tibial nerveaxons at different time points after crush (A) and section (B) identified at sciatic nerve (star symbols). Mean growth rates (regression line)and 95% confidence intervals (stippled lines) are indicated. The corresponding motor (filled symbols) and sensory (open symbols)elongation fronts determined at laminectomy are presented with square symbols. The distances of corresponding motor and sensoryelongation fronts in the same nerves are shown after crush (C) and section (D).


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nerves and was similar to the findings in control nerves

(Fig. 7A). A likely explanation for these findings was that

at any time point CV recovery was proportional with that

of the parent fibre (Fig. 7B). Thus, with respect to the parent

fibre, the rate of maturation was similar in motor and sensory

sprouts.

Rate of amplitude recoveryIn control nerves, the largest ventral CRPs were 6416 116 mV

and the largest dorsal CRPs were 191 6 30 mV (P < 0.01,

Wilcoxon). During the third week of regeneration the CRPs

were dispersed and of low amplitude (<5 mV) both after

crush and section (Fig. 1A) and no differences could be

detected between sensory and motor fibres. The recovery

of motor and sensory amplitudes after target reinnervation

is presented in Fig. 6B. For example, at 52 days after crush, the

highest motor amplitude was 176 mV (27% of control) and

the highest sensory amplitude was 108 mV (56% of control).

At 139 days after crush the motor amplitude was 518 mV

(80% of control) and the sensory amplitude was 169 mV

(88% of control).

The amplitude recovery rate was 3.3 mV/day for motor

fibres, higher (Wilcoxon P < 0.05) than the 1.2 mV/day recov-

ery rate of the sensory fibres. Thus, both after crush and

section we found that most of the recovery in motor ampli-

tude along the tibial nerve occurred at later stages during

maturation (Fig. 6B). While we cannot exclude differences

in CRP dispersion and distribution between motor and sen-

sory fibres, it is possible that growth of motor axons was more

abundant at later stages during regeneration, as seen in rat

(Kawasaki et al., 2000). Our electrophysiological comparison

Fig. 6 Recovery of CV along the tibial nerve (A) and largestamplitude—among all roots and stimulation sites (B). Data werepooled from different nerves at various times after targetreinnervation.

Fig. 7 Relationship between motor CV and sensory CV aftertarget reinnervation. (A) Data were pooled from different nerves:unlesioned controls (filled symbols), regenerated nerves aftercrush (open circles) and regenerated nerves after section (opentriangles). Regression line was calculated on the basis ofregenerated nerves. The high correlation between motor CV andsensory CV during regeneration that approaches the measure-ments in control nerves may be noted. (B) A cartoon indicating apossible explanation for the observed correlation where the CV ofthe sprout (distal to the lesion) remains proportional with CV ofthe parent fibre (proximal to the lesion).


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was, however, aimed at the fastest regenerating motor and

sensory axons that were not influenced by such regenerative

differences within the motor and sensory fibre populations.

Recovery of fibre distribution betweendifferent rootsIn control nerves, the tibial nerve CVs of fibres projecting to

different ventral roots were similar. However, the sensory

CVs to the L7 root (956 1 m/s) were significantly (Wilcoxon,

P < 0.05) faster than sensory CVs to the S1 root (846 5 m/s)

(Fig. 8A). Distances from T1 to the root recordings (calcu-

lated from the intercept of the latency versus distance linear

fit) were �187 mm to all dorsal roots. It was therefore

possible that the different sensory fibre populations (with

different CVs) projected to different roots.

In spite of a large variation of absolute amplitudes between

the different nerves, the relative amplitude distribution

between the different roots in control nerves was, however,

consistent: L7 dorsal CRPs contained 61 6 8% of the total

sensory amplitude (P < 0.05, Wilcoxon), whereas S1 ventral

CRPs contained 88 6 2% of the total motor amplitude

(P < 0.05, Wilcoxon) (Fig. 8B). Thus, myelinated alpha-

motor fibres in the tibial nerve originated mostly from S1

root and large sensory fibres mainly from L7 root. This

distribution was maintained during outgrowth through the

tibial cuff in all investigated nerves, both after crush and

section. The motor elongation front contained fibres from

S1 in four of five crushed nerves and in two of four sectioned

nerves, and the sensory elongation front contained fibres

from L7. After target reinnervation (Fig. 8C), the S1 and

L7 regained the largest relative amplitude both after crush

and after section. Thus, it is likely that for sensory fibres, as

for motor fibres, the regeneration pattern for a root was

dictated by the number of fibres before the lesion and not

by specialized fibre-type projection between roots.

DiscussionIn humans the rate of regeneration after peripheral nerve

lesions cannot be directly measured. The delay between

nerve lesion or repair and functional recovery of target

organs, which has been converted to a growth rate of 1–

2 mm/day (Seddon et al., 1943; Hodes et al., 1948), consists

of the delay before fibre growth, the time used to cross the

lesion site, the rate of growth through the distal nerve stump,

the reinnervation of target organs and the outcome measure

to ascertain reinnervation. The time to reinnervation was

the single-most important factor to influence the level of

outcome after different types of nerve lesions in monkeys

(Krarup et al., 2002), and in humans there is a general

agreement on the improved functional recovery when time

to reinnervation is short and that surgical treatment when

needed should be carried out as quickly as possible after a

nerve lesion (Lundborg, 2000). Functional recovery after

axonal degeneration is probably dependent on the number,

accuracy and growth rate of axons and reinnervation of

target organs, and these factors may be linked to the pro-

gressive atrophy and loss of Schwann cell tubes in the distal

nerve stump (Weinberg and Spencer, 1978) and target organs

(Gutmann and Young, 1944). Hence, the rate of growth of

Fig. 8 Distribution of CRP amplitude and CVs between differentventral (filled symbols) and dorsal (open symbols) roots. (A) Meandistribution of motor and sensory CRPs and CVs in control nerves.(B) Distribution of mean motor and sensory CRP amplitudes incontrols from each root in per cent of the total motor and sensorycumulated responses, Error bars indicate SEM. (C) Relativerecovery of S1V and L7D CRP amplitudes (largest CRPs incontrols). Data from crushed nerves are presented with circles andthose from sectioned nerves, with triangles. The mean relativeamplitudes in control nerves are presented with continuous (S1V)and dashed (L6D) horizontal lines. It may be noted that relativedistribution of CRP amplitudes from ventral and dorsal rootsrecovered after both crushing and sectioning.


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motor and sensory axons may have direct implications

on functional recovery. Since the reinnervation of sensory

receptors is considerably more difficult to ascertain than

muscle fibre reinnervation in humans and non-human

primates owing to the small sensory nerve and receptor

responses (Buchthal and Kuhl, 1979; Krarup et al., 1990,

2002), direct comparison of motor and sensory regeneration

rates is inaccurate. We therefore extended a method to

monitor growth of axons during regeneration after Wallerian

degeneration to directly compare elongation and maturation

of motor and sensory fibres within the same mixed tibial

nerve. Both after crush and section we found that similar

growth and maturation rates were attained by myelinated

motor and sensory fibres.

Growth of sensory and motor axonsWe could not directly measure the rate of growth of sensory

and motor axons in serial studies. We compared instead

the distances reached by the motor and sensory fibres at

specified times during regeneration in different nerves by

stimulating in the tibial cuff and recording from the L6–S2

dorsal and ventral roots. The spatial and temporal resolutions

of our method were determined by the length of the tibial

cuff, the inter-electrode spacing and the intervals between

studies. During serial studies we found that fastest outgrowth

started after �8 days and progressed at a rate of �4 mm/day,

which was largely in agreement with our previous observa-

tions (Fugleholm et al., 1994). Under these circumstances, to

discriminate a distance difference of one stimulation level

(7.5 mm) after 20 days of regeneration, the difference in

growth rates should have been >0.5 mm/day (4 versus

3.5 mm/day).

We found that the fastest growth rates achieved by

myelinated fibres were similar in motor and sensory fibres

populations and fell within the confidence intervals of the

mixed nerve growth rates (Fig. 5). This indicated that

growth rates of motor and sensory fibres were similar within

0.5 mm/day accuracy. While it was not possible to increase

the length of the cuff owing to anatomical constraints, one

would expect that an even greater accuracy could have been

obtained by reducing the inter-electrode distance. We

previously verified by electron microscopy that the 10 mA

stimulation current required to excite the immature axons

could accurately predict the growth front within 7.5 mm

resolution (Fugleholm et al., 1994). By reducing inter-

electrode distance, however, stimulus spread could have

been a source of error.

Axons contributing to the growth frontWe stimulated the immature axons at the growth front in

the tibial and recorded the evoked responses in cuff electro-

des. Our electrophysiological approach was constrained by

the methodological limitation that action potentials could be

recorded from single myelinated fibres with diameters larger

than 5–7 mm (Rindos et al., 1984; Krarup and Loeb, 1988).

Thus, our method allowed us to investigate alpha-

motor fibres but was limited to only the larger sensory fibres.

A functional study in rat showed that responses from

nociceptive and sudomotor fibres occur slightly earlier and

to a greater extent than recordings of compound muscle and

nerve action potentials from large myelinated fibres (Navarro

et al., 1994). It is, however, unlikely that small sensory fibres

regenerate strikingly faster than large sensory fibres since a

recent retrograde tracer study showed that regeneration of

unmyelinated sensory fibres and large sensory fibres in rat

had similar rates (Lozeron et al., 2004).

However contraintuitive, our study did not support

the assumption that the largest fibres have the fastest growth

rate. While our method favoured detection of regeneration of

the largest fibres with the largest action potential amplitudes,

we found evidence that within the respective sensory and

motor fibre populations it was not the largest and fastest

conducting fibres that grew first (Figs 1E, 2 and 8A), and

that motor and sensory fibres made similar contributions to

the growth front (Fig. 5). It may therefore be speculated that

growth rates were maximized for an ‘optimal’ relationship

between radial growth and longitudinal growth, which is

similar in sensory and motor axons.

Maturation of sensory and motor axonsMaturation (the increase in axonal calibre and myelination)

occurs slower (Wujek et al., 1986; Zhu et al., 1997) and

independently (Fried et al., 1991; Macias et al., 1998) of

growth. In regenerating cat axons (Fugleholm et al., 2000)

CV remains proportional with the fibre diameter and we

could compare maturation of sensory and motor axons

by investigating recovery in CV. After target reinnervation,

the change in latency along the tibial cuff was linear and we

could calculate the average CV along the tibial nerve as the

latency versus distance linear fit and then determine the

fastest motor and sensory CV among the different roots.

This method of calculation was validated by comparing

the motor CV estimated to the roots with the CV to the

plantar muscles. Furthermore, this method of calculation

was able to detect the slightly faster sensory than motor

CV corresponding to the differences in largest diameters of

sensory and motor fibres (Hursh, 1939).

We found that at various times during maturation, the

sensory CV was slightly faster than the motor CV (Fig. 6A).

This did not indicate that sensory maturation occurred at a

faster rate, because a similar difference in CV was also seen in

control nerves, and the ratio between sensory and motor CV

in control nerves was preserved during regeneration. It was

more likely that maturation occurred at a rate proportional to

the diameter of the parent fibre (Wujek et al., 1986). Further-

more, early studies in rat sensory fibres showed that CV of the

sprout was proportional with CV of its parent fibre (Devor

and Govrin-Lippmann, 1979). Thus, in respect to the parent

fibre diameters, maturation rate was similar between sensory

and motor fibres.


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Crush versus sectionWhile the theoretical rate-limiting factor of axonal regenera-

tion may be the speed of up to 6.5 mm/day at which slow

axonal transport moves cytoskeletal elements involved in the

motility of the growth cone and elongation of the axon

(Wujek and Lasek, 1983), the linear growth rate is much

slower, dependent on the growth cone environment

(Fugleholm et al., 1994, 1998, 2000; Sorensen et al., 2001).

The fastest rates of regeneration could be attained after crush,

where growth cones move along their original Schwann

cell tubes. We found that in these ideal conditions the

growth rates were similar between motor and sensory fibres.

In the clinical situation of a co-adapted nerve section, regen-

erating fibres are more likely to grow through unfamiliar

pathways that may impair the growth rates. In the mouse

motor axons preferentially reinnervated motor pathways

(Brushart, 1988) after section. It was therefore possible

that such environmental specialization may favour the

growth of motor fibres. Our data suggests that after section,

as after crush, the fastest growth rates that could be attained

by motor and sensory fibres were similar. Thus, even in the

clinically relevant regenerative environment some motor

and sensory axons retain similar growth capacity and will

simultaneously compete for target selection.

AcknowledgementsThe project was supported by Lundbeck Foundation, the

Novo Nordisk Foundation, the Danish Medical Research

Council, the Ludvig and Sarah Elsass Foundation and the

Foundation for Research in Neurology.

ReferencesBailey CS, Kitchell RL, Guinan MJ, Sharp JW. Dorsal nerve root origins of

the cutaneous nerves of the feline pelvic limb. Anat Histol Embryol 1992;

21: 23–31.

Braendgaard H, Sidenius P. Anterograde components of axonal transport in

motor and sensory nerves in experimental 2,5–hexanedione neuropathy.

J Neurochem 1986; 47: 31–7.

Brushart TM. Preferential reinnervation of motor nerves by regenerating

motor axons. J Neurosci 1988; 8: 1026–31.

Brushart TM. Motor axons preferentially reinnervate motor pathways.

J Neurosci 1993; 13: 2730–8.

Buchthal F, Kuhl V. Nerve conduction, tactile sensibility, and the

electromyogram after suture or compression of peripheral nerve: a

longitudinal study in man. J Neurol Neurosurg Psychiatry 1979; 42:

436–51.

Chen S, Bisby MA. Impaired motor axon regeneration in the C57BL/Ola

mouse. J Comp Neurol 1993; 333: 449–54.

da Silva CF, Madison R, Dikkes P, Chiu TH, Sidman RL. An in vivo model

to quantify motor and sensory peripheral nerve regeneration using

bioresorbable nerve guide tubes. Brain Res 1985; 342: 307–15.

Devor M, Govrin-Lippmann R. Maturation of axonal sprouts after nerve

crush. Exp Neurol 1979; 64: 260–70.

Forman DS, Berenberg RA. Regeneration of motor axons in the rat sciatic

nerve studied by labeling with axonally transported radioactive proteins.

Brain Res 1978; 156: 213–25.

Forman DS, Wood DK, DeSilva S. Rate of regeneration of sensory

axons in transected rat sciatic nerve repaired with epineurial sutures.

J Neurol Sci 1979; 44: 55–9.

Fried K, Govrin-Lippmann R, Rosenthal F, Ellisman MH, Devor M.

Ultrastructure of afferent axon endings in a neuroma. J Neurocytol 1991;

20: 682–701.

Fugleholm K, Schmalbruch H, Krarup C. Early peripheral nerve regenera-

tion after crushing, sectioning, and freeze studied by implanted electrodes

in the cat. J Neurosci 1994; 14: 2659–73.

Fugleholm K, Sorensen J, Schmalbruch H, Krarup C. Axonal elongation

through acellular nerve segments of the cat tibial nerve: importance of

the near-nerve environment. Brain Res 1998; 792: 309–18.

Fugleholm K, Schmalbruch H, Krarup C. Post reinnervation maturation of

myelinated nerve fibers in the cat tibial nerve: chronic electrophysiological

and morphometric studies. J Peripher Nerv Syst 2000; 5: 82–95.

Gutmann E, Young JZ. The re-innervation of muscle after various periods

of atrophy. J Anat 1944; 78: 15–44.

Highland TR, Ottoni L, Lamont RL, Goshgarian HG. The contribution of

the dorsal and ventral roots of the tibial nerve in the propagation of the

spinal somatosensory evoked potential in cats. Spine 1990; 15: 560–6.

Hodes R, Larrabee MG, German W. The human electromyogram in

response to nerve stimulation and the conduction velocity of motor axons.

Arch Neurol Psychiat 1948; 60: 340–65.

Hursh JB. Conduction velocity and diameter of nerve fibers. Am J Physiol

1939; 127: 131–9.

Jenq CB, Coggeshall RE. Numbers of regenerated axons in tributary

nerves following neonatal sciatic nerve crush in rat. Neurosci Lett 1985;

61: 43–8.

Kawasaki Y, Yoshimura K, Harii K, Park S. Identification of myelinatedmotor

and sensory axons in a regenerating mixed nerve. J Hand Surg [Am] 2000;

25: 104–11.

Krarup C, Loeb GE. Conduction studies in peripheral cat nerve using

implanted electrodes: I. Methods and findings in controls. Muscle Nerve

1988; 11: 922–32.

Krarup C, Loeb GE, Pezeshkpour GH. Conduction studies in peripheral cat

nerve using implanted electrodes: III. The effects of prolonged constriction

on the distal nerve segment. Muscle Nerve 1989; 12: 915–28.

Krarup C, Upton J, Creager MA. Nerve regeneration and reinnervation after

limb amputation and replantation: clinical and physiological findings.

Muscle Nerve 1990; 13: 291–304.

Krarup C, Archibald SJ, Madison RD. Factors that influence peripheral nerve

regeneration: an electrophysiological study of the monkey median nerve.

Ann Neurol 2002; 51: 69–81.

Larsen JO, Thomsen M, Haugland M, Sinkjaer T. Degeneration and

regeneration in rabbit peripheral nerve with long-term nerve cuff electrode

implant: a stereological study of myelinated and unmyelinated axons. Acta

Neuropathol (Berl) 1998; 96: 365–78.

Lozeron P, Krarup C, Schmalbruch H. Regeneration of unmyelinated and

myelinated sensory nerve fibres studied by a retrograde tracer method.

J Neurosci Methods 2004; 138: 225–32.

Lundborg G. A 25-year perspective of peripheral nerve surgery: evolving

neuroscientific concepts and clinical significance. J Hand Surg [Am] 2000;

25: 391–414.

Lundborg G, Richard P. Bunge memorial lecture. Nerve injury and repair—a

challenge to the plastic brain. J Peripher Nerv Syst 2003; 8: 209–226.

Macias MY, Lehman CT, Sanger JR, Riley DA. Myelinated sensory and alpha

motor axon regeneration in peripheral nerve neuromas. Muscle Nerve

1998; 21: 1748–58.

Madison RD, da Silva CF, Dikkes P. Entubulation repair with protein

additives increases the maximum nerve gap distance successfully bridged

with tubular prostheses. Brain Res 1988; 447: 325–34.

Madorsky SJ, Swett JE, Crumley RL. Motor versus sensory neuron

regeneration through collagen tubules. Plast Reconstr Surg 1998; 102:

430–36.

Martini R, Xin Y, Schmitz B, Schachner M. The L2/HNK-1 carbohydrate

epitope is involved in the preferential outgrowth of motor neurons on

ventral roots and motor nerves. Eur J Neurosci 1992; 4: 628–39.

Navarro X, Verdu E, Buti M. Comparison of regenerative and reinnervating

capabilities of different functional types of nerve fibers. Exp Neurol 1994;

129: 217–24.


Dow



ovember 2018

Rindos AJ, Loeb GE, Levitan H. Conduction velocity changes along lumbar

primary afferent fibers in cats. Exp Neurol 1984; 86: 208–26.

Sanger JR, Riley DA, Matloub HS, Yousif NJ, Bain JL, Moore GH. Effects

of axotomy on the cholinesterase and carbonic anhydrase activities of

axons in the proximal and distal stumps of rabbit sciatic nerves: a temporal

study. Plast Reconstr Surg 1991; 87: 726–38.

Seddon HJ, Medawar PB, Smith H. Rate of regeneration of peripheral

nerves in man. J Physiol 1943; 102: 191–215.

Sorensen J, Fugleholm K, Moldovan M, Schmalbruch H, Krarup C.

Axona elongation through long acellular nerve segments depends on

recruitment of phagocytic cells from the near-nerve environment.

Electrophysiological and morphological studies in the cat. Brain Res

2001; 903: 185–97.

Suzuki G, Ochi M, Shu N, Uchio Y, Matsuura Y. Sensory neurons regenerate

more dominantly than motoneurons during the initial stage of the reg-

enerating process after peripheral axotomy. Neuroreport 1998; 9: 3487–92.

Weinberg HJ, Spencer PS. The fate of Schwann cells isolated from axonal

contact. J Neurocytol 1978; 7: 555–69.

Wujek JR, Lasek RJ. Correlation of axonal regeneration and slow

component B in two branches of a single axon. J Neurosci 1983; 3: 243–51.

Wujek JR, Lasek RJ, Gambetti P. The amount of slow axonal transport is

proportional to the radial dimensions of the axon. J Neurocytol 1986;

15: 75–83.

Zhu Q, Couillard-Despres S, Julien JP. Delayed maturation of regenerating

myelinated axons in mice lacking neurofilaments. Exp Neurol 1997;

148: 299–316.


Dow



ovember 2018

comparison of the fastest regenerating motor and sensory - brain

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