Brain Research, 333 (1985) 131-138 13 l Elsevier

BRE 10700

Sensory Reinnervation of Cat Peroneus Brevis Muscle Spindles After Nerve Crush

D. BARKER, J. J. A. SCOTT and M. J. STACEY

Departmemt of Zoology, University of Durham, Durham, DH1 3LE (U. K.)

(Accepted July 31st, 1984)

Key words: cat - - muscle spindles - - sensory reinnervation - - nerve-crush injury

Results are presented of examining the postcrush sensory reinnervation of cat peroneus brevis muscle spindles previously investi- gated physiologically by Hyde and Scott 11. It is shown that primary and secondary endings were successfully restored in their final form in the early stages of recovery. The primary endings were shorter than normal and had fewer transverse bands; 12% were judged to be hyperinnervated. Some secondary endings showed signs of growth through the primary region apparently designed to establish sec- ondary terminals in the opposite pole. This is compared with the collateral regeneration of intact motor axons in partially denervated muscle. It is concluded that the defects observed in the regenerated sensory endings had no effect on their functional recovery.

INTRODUCTION

The re innervat ion of skeletal muscle that follows

nerve-crush injury results in res tora t ion of function

to the muscle spindles6, H and complete recovery of

stretch reflexes, such as the knee jerk 2. Recordings

of the responses of regenera ted spindle afferents to

ramp-and-hold stretch, and to fusimotor st imulation,

show that they eventual ly re turn to normal , though

during the early stages of recovery many of the after-

ents respond only phasically6,11. The t ime course for

the restorat ion of normal function is to some extent

dependent on the per iod of denervation~5.

Histological observat ions have establ ished that

both pr imary and secondary endings are res tored to

their normal sites in the spindles, though their axons

regenerate more slowly than those involved with the

motor reinnervation4,12,13. Ip et al. t3 describe the ap-

pearance of the re innervated spindles as 'normal , or

nearly normal ' , but more recent studies4,15 have re-

vealed a number of abnormali t ies . For example , hy-

per innervat ion occurs, and the regenera ted pr imary

endings are shorter than normal with fewer trans-

verse terminal bands. At the ul t ras t ructural level

Schr6de¢ 4 has descr ibed abnormal i t ies in the senso-

ry re innervat ion of rat spindles after nerve crush;

some of the regenera ted terminals were associated

with Schwann cells, others were only part ial ly in con-

tact with the underlying muscle fibre.

This paper is part of a histophysiological investiga-

tion of the recovery of muscle spindles after nerve

crush, and presents the results of examining the sen-

sory reinnervat ion of those spindles investigated

physiologically by Hyde and Scott H.

MATERIALS AND METHODS

Peroneus brevis muscles were removed from 26

adult cats (average weight 2.2 kg) at the end of re-

cording exper iments carried out by Hyde and Scott 11

under sodium pen tobarb i tone anaesthesia (Sagatal;

May and Baker , 45 mg/kg i .p.) 20-140 days after the

common peroneal nerve to the left hindl imb had

been crushed. Detai ls of the surgery employed to ef-

fect the crush injury are given by Hyde and Scott 11.

After administering a lethal dose of Sagatal, the re-

innervated peroneus brevis was removed a n d pro-

cessed according to Barker and Ip 's 3 technique for

producing teased, silver prepara t ions . Modif icat ions

to this technique introduced by A. Boddy and F. Di-

wan (personal communicat ion) were employed as

follows. (i) Af te r fixation, the muscle was washed at

Correspondence: D. Barker, Department of Zoology, University of Durham, South Road, Durham DH1 3LE, U.K.

0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

132

a flow rate of i litre/h for 24 h in dilute aluminium sul-

phate (l g AI 2 (SO4) 3" 16H20 per litre distilled water) made up to pH 9 with a saturated solution of NaOH.

(ii) After 30 h in ammoniacal alcohol the muscle was

dipped repeatedly into a 1% agar solution. The agar coating was then left to gel. (iii) The muscle was incu-

bated in freshly prepared 1.5% silver nitrate in the

dark at 37 °C in a shaking water bath. The agar coat-

ing was then removed before placing the muscle in

the reducer for 24 h.

Barker and Boddy 4 described their results in terms

of reinnervation time (RT), which they defined as be-

ing 'the number of days that the fastest-growing ax-

ons are calculated to have entered and been reinner-

vating the muscle'. They calculated that, after a reor-

ganization time of 5.8 days at the site of the crush in-

jury, the fastest-growing axons in the common pero-

neal nerve advance at a rate of 3.2 mm/day. Since the

average distance from the crush site to the entry of the nerve into peroneus brevis was 51 mm, the first

axons would have reached the muscle approximately

22 days post-crush, which is therefore day 0 RT. In the Results all recovery times are given in terms of

days RT, observations on reinnervation being made

at intervals o f - 2 , 4, 11, 18, 25, 39, 53, 74 and 118

days RT. A nerve-crush injury leaves most of the endoneuri-

al and basal lamina tubes intact so that there is a high- ly accurate return of the regenerating axons to their

original target sitesS,9.16 In muscle spindles this re-

sults in the restoration of clearly recognizable senso- ry and motor endings in their normal locations 4, Nev-

ertheless the fact that some endoneurial tubes are in- evitably cut rather than crushed by the injury, nec- essarily introduces an element of doubt into the iden-

tification of regenerated endings and their axons. Thus, for example, it is conceivable that the axon ter-

minating in what appears to be a primary ending is not a regenerated crushed la axon, but a regenerated cut Ib axon that has grown down a cut Ia endoneurial tube. Such connexions may be possible, but if they do occur in the reinnervation that follows nerve crush, they are unlikely to do so with any significant fre- quency. We have therefore identified regenerated endings and their axons on the basis of the appear- ance of the endings and their intrafusal location, as is done with normal cat spindles (see Barker and BanksS). Unless otherwise specified the results refer

to the reinnervation of spindles composed <~ all thiee

types of intrafusal muscle fibre.

All means are given with their standard errors

(S.E.).

RESULTS

The process of reinnervation was generally very

successful. Once the regenerating axons established

contact with the intrafusal muscle fibres there was rapid restoration of both motor and sensory innerva-

tion. Sensory endings of normal configuration were re-established in their normal positions. Termi-

nations resembling typical primary endings reinner-

vated both types of nuclear-bag fibre and the nucle-

ar-chain fibres in the equatorial region; characteristic

secondary endings reformed in the juxta-equatorial

$1, $2 and, rarely, S 3 positions; and motor axons ter- minated in the polar regions. Nevertheless. a number

of features, such as hyperinnervation and growth by some secondary endings through the primary region,

distinguished the material as reinnervated, and the

full restoration of normal sensory innervation was

never achieved.

Primary endings At -2 days RT no primary endings were present in

24 spindles teased from two peroneus brevis muscles,

though some thin regenerating axons were visible in

the intramuscular nerve trunks. The first reinnerva- tion by presumed primary (Ia) axons was observed at

4 days RT, but at this stage the endings were very ru-

dimentary. One week later 78% (n = 18) of spindles had received primary endings; after 25 clays RT the

proportion had increased to 89% (n = 27), and there- after (39-118 days RT) only 1.5% (n = 132) lacked

primary endings.

Regeneration of primary endings was well ad- vanced after 11 days RT and complete by 18 days RT. Among 153 primary endings sampled from t l t o 118 days RT only 5% were incomplete in that termi- nals were not supplied to all three types of intrafusal muscle fibre, i.e. bag 1, bag 2 and chain. In these pri-

maries one of the two types of bag fibre lacked termi- nals; this proved to be the bag i fibre in those in- stances (3 of 8) where bag-fibre type could be identi- fied with certainty. We could not determine whether each chain fibre received terminals in every primary

133

Fig. 1. Photographs of teased, silver preparations of muscle spindles from cat peroneus brevis, a: Primary (P) and S 1 secondary (St) endings in a normal spindle, b: Afferent reinnervation of a spindle 7 weeks postcrush (25 days RT). The primary ending is shorter than normal and has fewer transverse bands. A branch (arrowed) from the regenerated S 1 secondary ending has grown through the primary region to terminate in the S 1 secondary region in the opposite pole (compare with Fig. 3b). Scale as shown in (a).

134

ending, but there were always at least two that did so.

The regenera ted pr imary endings were shor ter

than normal and among the annulospiral terminals

suppl ied to their bag fibres there were fewer trans-

verse bands (Figs. lb and 2). The mean length of

33 pr imary endings in normal peroneus brevis was

314.8 _+ l l . 6 /~m, and the mean number of t ransverse

bands per bag fibre among 45 bag 1 and 42 bag 2 fibres

was 14 + 0.44 (range 5 - 2 7 ) By contrast , 149 regen-

e ra ted pr imary endings sampled from 11 to 118 days

Fig. 2. Photographs of teased, silver preparations showing regenerated primary endings supplied to cat peroneus brevis spindles re- innervated 6 weeks postcrush (18 days RT). Both endings are shorter than normal (compare with Fig. la); scale in (b) as shown in (a). Though moderately well regenerated, the transverse bands are fewer than normal and more widely spaced. In (a) the bag~ (b I) fibre has been very sparsely reinnervated, b~, bag 2 fibre: cap., capillary.

135

RT had a mean length of 269.7 + 14.1 gm, and the

mean number of bands per bag fibre among 204 re- innervated bag fibres was 7.2 + 0.25 (range 0-20). The regenerated endings are thus significantly short- er than normal (P < 0.05: Student's t-test), and have significantly fewer bands per bag fibre (P < 0.01). There was no obvious tendency for the endings to get

longer, or to have more transverse bands, as RT in- creased from 11 to 118 days (see Table I).

Some regenerated primary endings were reinner- vated by two or three axons that travelled separately within the original Ia endoneurial tube for as far back as could be traced. We regarded such axons as en- gaged in hyperinnervation only if they remained sep- arate when traced back from the spindle for at least 1000 gm. This virtually excluded the possibility that they were branches of Ia axons cut distal to their first branching node during teasing. In normal peroneus brevis spindles the mean distance between this node and the centre of the primary ending in 120 Ia axons was 321.8 _ 17.0/~m; in only 0.8% was it more than 1000 urn. In reinnervated spindles the situation was very similar, the mean distance between the first branching node of 152 regenerated Ia axons and the centre of the primary ending being 382.7 + 19.4 Bm; in only 2.3% was it more than 1000 gm. Among 171 reinnervated spindles sampled from 11 to 118 days RT only 12% were judged on this basis to have hy- perinnervated primary endings. Most of these were supplied by two axons contained within the original Ia endoneurial tube, one of which innervated a single bag fibre only. A few primaries were hyperinner- vated by three axons, and one ending was supplied by

five. There was no evidence of any trend in the occur- rence of hyperinnervation that could be associated with period of recovery.

T A B L E I

In each recovery stage there were a few markedly

irregular primary endings with terminals more dis-

persed than normal and with very little banding around the bag fibres. These endings were distinct from the irregular primary endings supplied to spin- dle capsules that are part of tandem spindles and lack

a bag 1 fibre. We encountered 13 of these reinner- vated 'bzc spindle units'a; apart from one instance of hyperinnervation, the primary endings appeared normal.

In four reinnervated spindles a myelinated branch of the la axon had grown away from the primary re- gion into the adjacent part of the intrafusal bundle

where it ended in one or two simple terminals. In two of the spindles these lay among the terminals of a re- generated $1 secondary ending. Such overspill does not occur in normal spindles.

Secondary endings In normal peroneus brevis muscles most of the

spindles receive secondary endings (Fig. la). In 5 muscles from 5 cats the mean number of secondaries per spindle among 152 spindles was 1.28 + 0.08; only 34 (22.4%) lacked them (Table II). In most of the re- covery stages (i.e. except 18 and 118 days RT) the proportion of spindles without secondaries was about normal (Table IIA), but the mean number of second- aries per spindle was reduced to 1.05 + 0.06. Com- pared with normal spindles there was thus a signifi- cant shortfall of secondary endings (P < 0.05: x2-test)

amounting to 18.3%. We suspect that most of this may be accounted for by our failing to detect the presence of regenerated S 2 and $3 secondaries in the densely reinnervated polar regions (Table IIB).

Secondary endings first appeared in the spindles together with primary endings at 11 days RT, but

Regenerated primarv endings: length o fending and number of transverse terminal bands after various perio& of reinnervation time (R T)

Time (days RT) No. primary endings Mean ending length (~um) with S.E. No. bag fibres Mean no. transverse bands with S. E.

l l 12 248.6__+ 17.3 8 6.9 + 1.3 18 24 258.6 + 14.9 28 7.6 + 0.82 25 23 288.7 + 15.6 32 7.5 __+ 0.58 39 18 269.9 + 10.1 18 7.9 __+ 0.86 53 26 276.8 __+ 13.3 87 6.2 + 0.28 74 20 256.3 __+ 12.3 26 10.6 __+ 0.75

118 6 297.5 __+ 21.2 5 6.0 +__ 1.3

N o r m a l 33 314.8 + 11.6 87 14.0 + 0.44

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TABLE II

Regenerated secondary endings (A) Mean number per spindle and percentage of spindles without secondaries after various periods of reinnervation time fR [~

e / ~ • Number spindles Mean number secondaries/spindle with S.E. ~ spmdles wtthout.~e~ondaries Time (days RT)

11 16 1.00 +___ 0.18 25.0 18 30 0.83 __+ 0.15 40.0 25 25 1.08 -+ 0.14 20.0 39 26 1.08 -+ 0.16 26.9 53 60 1.00 -+ 0.10 26.6 74 27 1.11 __+ 0.15 22.2

118 18 0.61 __+ 0.18 55.5

Normal 152 1.28 + 0.08 ,~..4

(B) Number of secondary endings in reinnervated spindles (excluding 18 and 118 days RT samples) compared with number in normal spindles

Number secondaries~spindle Reinnervated spindles Normal spindles

Number % Number %

0 38 24.7 34 22.4 1 74 48.0 60 39.5 2 39 25.3 42 27.6 3 3 2.0 13 8.5 4 - - 3 2.0

were not fully established until 39-53 days RT. In the

later stages of recovery it was evident that the major-

ity of Sl secondary endings were distributed to all

three types of intrafusal muscle fibre, as in our sam- ple of normal spindles. Owing to the irregular nature

of secondaries it was not possible to make a quantita-

tive assessment of the quality of their restoration, but

most appeared to be less elaborate than those sup-

plied to normal spindles.

At each recovery stage, but especially from 53 days RT onwards, there were some S1 secondary

endings that showed signs of growth through the pri- mary region. This took the form of a single pretermi-

nal axon branch that left the site of an $1 secondary ending to thread its way through the primary termi- nals (Figs. lb and 3). In most instances the branch ended as a small bulb (growth cone?) in the primary region (Fig. 3a), but in some spindles it traversed this

region to form a few simple terminals in the $1 region of the opposite pole (Figs. lb and 3). Among the to- tal sample of 202 reinnervated spindles there were 31 (15.3%) in which such growth occurred. In most of these the opposite pole did not have an $1 secondary ending. When one was present, the invading branch either contributed a few terminals (Fig. 3d), or

turned to grow back to the site of its own ending

(Fig. 3e). In 3 spindles $1 secondary endings on both

sides of the primary region had preterminal axons

growing through the primary ending (Fig. 3f, g and

h). In normal spindles there is never any growth through the primary region by preterminal axons

originating from S 1 secondary endings.

Anomalies

In seven of the spindles that lacked a regenerated

primary ending, the primary region had been in- vaded by foreign axons that had grown down the

spindle nerve and entered the capsule in place of the Ia axon. In five spindles (from the 11.74 and 118 days RT stages) the thickness and branching pattern of the axons suggested that they were sensory. Four of

them ended in short simple tapers in the primary re- gion and could not be identified, but the fifth axon produced terminal ramifications that suggested it was a free-ending afferent. The remaining two spindles (from the 11 and 25 days RT stages) were invaded by numerous thin axons which each formed a bulbous swelling in the primary region. In one spindle the ax- ons were all derived from a single medium-sized axon which first branched in a large intramuscular nerve

137

S 1 P S t $1 P $I

a e

11 - l l8days RT |18days RT

b f

18, 25, 53, 74 days RT 53days RT

c g

11, 53days RT 74days RT

d h

53days RT 74days RT

Fig. 3. Schematic representation of instances of growth by S 1 secondary endings (S 0 through the region of the primary ending (P) in spindles reinnervated 5-20 weeks postcrush (11-118 days RT).

trunk 2.2 mm from capsule entry. In the other spin- dle, the axons, at least seven in number, formed a distinct bundle within the original Ia endoneurial tube and remained separate as far as could be traced. It is possible that both spindles provide examples of motor axons misguidedly entering the primary re- gions whilst growing through the equatorial region towards the poles.

DISCUSSION

Hyde and Scott's 11 study of the recovery of re- sponses from spindle afferents after nerve crush showed that there was a continuous process of im- provement so that by 118 days RT the proportion of those responding abnormally had decreased to 11%. By contrast the process of afferent reinnervation is completed in the early stages of recovery, and, once restored, the endings (except for a few secondaries) appear to be established in their final form. Compari-

son of these findings suggests two conclusions. First, that the gradual improvement of spindle afferent re- sponses during recovery does not have any morpho- logical basis. Hyde and Scott n arrive at the same con- clusion and suggest that the return to normality is probably due to the pacemaker thresholds of the af- ferents gradually regaining normal levels. And sec- ond, that such defects as occur in the regenerated endings (e.g. fewer transverse terminals bands than normal in the primaries) apparently do not impair their responsiveness.

A few S 1 secondary endings showed signs of continuing regeneration in the form of growth through the primary region, which is some spindles led to terminals being established in the $1 region of the opposite pole. We suggest that such growth only occurs in spindles originally supplied with an Sa sec- ondary ending on each side of the primary. In our normal sample of 152 peroneus brevis spindles 28.3% had this type of afferent innervation. During

138

the postcrush reinnervat ion of such spindles there

will inevitably be several occasions when the Si sec-

ondary on one side of the primary will be restored be-

fore the other. The situation that then obtains would

be comparable to that existing in partially dener-

vated muscle when intact motor axons sprout to

make new connexions with the end-plates that have

been denervated 7.t0. If S 1 secondary afferents have a

similar capacity for collateral regeneration, then we

would expect that in most instances the sprout would

be growing towards the site of an Sl secondary ending

that had not been reinnervated. We believe that this

was so since in 74% of the spindles in which such

sprouting occurred there was no S~ secondary ending

present in the opposite pole, though the equatorial

length of the periaxial space indicated that a site for

one was present. The arrival of the original Si sec-

ondary afferent to reinnervate this site, as has oc-

curred in the instances represented in Fig. 3c-h , pre-

sumably ultimately leads to the suppression of the

collateral regeneration, but we have insufficient data

to speculate about this.

The muscles in Hyde and Scott's 1~ experiments

were denervated by the nerve-crush injury for 22

days before the regenerating axons arrived back to

begin reinnervation. Under these circumstances, as

we have seen, the spindle afferents ultimately recov-

er to respond normally despite having regenerated

endings with some morphological defects. By short-

ening the denervation time to 10 days Scott 15 has

shown that the functional recovery of the spindle af-

ferents is improved, but not the quality of their re-

generated endings. Progressively longer denervation

times presumably lead to the regeneration of increas-

ingly abnormal endings, and a stage should be

reached when these morphological abnormalit ies im-

pair functional recovery. We are at present investi-

gating whether this is so.

ACKNOWLEDGEMENTS

We wish to express out thanks for financial support

from the University of Durham Special Projects

Fund; an S.E.R.C. Research Studentship for

J .J .A.S.; technical assistance from Heather Young;

and photographic assistance from David Hutchinson.

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