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
136
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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3 Barker, D. and Ip, M. C., A silver method for demonstra- ting the innervation of mammalian muscle in teased prepa- rations, J. Physiol. (Lond.), 69 (1963) 73P-74P.
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