hypertrophy of rat extensor digitorum longus muscle injected with

J. Anal. (1992) 181, pp. 11-27, with 16 figures Printed in Great Britain

Hypertrophy of rat extensor digitorum longus muscle injectedwith bupivacaine. A sequential histochemical,immunohistochemical, histological and morphometric study

J. D. ROSENBLATT AND R. I. WOODS

Physiological Laboratory, University of Cambridge

(Accepted 11 April 1992)

ABSTRACT

Histochemical, immunohistochemical, histological and morphometric properties of bupivacaine-injected ratskeletal muscle were studied at times spanning the complete course of degeneration and regeneration toestablish when, if ever, 'normality' is reached. This was achieved in a sequence of measurements made onthe same series of rat fast-twitch extensor digitorum longus muscle (EDL), of the fibre type composition,myosin heavy chain content, fibre size, connective tissue content and myofibril size at 1-2 h and 2, 4, 8, 11,21, 40, 60, 80 and 180 d after treatment. By 2 d after injection 86% of the fibres had undergone necrosis. Arapid restoration of histochemical, immunohistochemical and morphometric properties then occurred, beingapparently complete by 21 d after injection. A pattern of ongoing changes recognised when regeneration wasessentially 'complete' are reminiscent of changes that occur in muscles following compensatory hypertrophyproduced by synergist ablation. These changes included an increase in muscle weight, a decline in normalisedpeak twitch and tetanic tensions, and normalised force in response to different stimulation frequencies(Rosenblatt, 1992), an increase in the relative number of type I fibres and of fibres reacting with the slowmyosin heavy chain antibody, an increase in whole muscle cross-sectional area, an increase in type I andtype II fibre cross-sectional area and diameter, an increase in myofibril cross-sectional area, density, number,and area fraction, and an increase in the relative proportion of intramuscular connective tissue collagen.This suggests that the EDL muscle is being made to do more active work and is being influenced by passiveforces (stretch) imposed on it. These changes appeared permanent: they stabilised at about 60 d afterinjection and were maintained for at least the next 120 d.

INTRODUCTION

The local anaesthetic bupivacaine is a potent andspecific myotoxic agent (Hall-Craggs, 1974; Foster &Carlson, 1980). Injection of the drug into smallskeletal muscles of rat or mouse precipitates animmediate and massive myonecrosis (Bradley, 1979)followed by phagocytosis of necrotic debris (Orimo etal. 1991) and a rapid and apparently completeregeneration of muscle fibres 3-4 wk after injection(Hall-Craggs, 1974). Recently, it was reported (Rosen-blatt, 1992) that the peak isometric twitch and tetanictensions produced by rat fast-twitch extensor digi-torum longus muscle injected with bupivacaine returns

to normal values by 21 d after injection. Thereafterthe injected muscle grows at an accelerated rate andby 60 d after injection, when the growth rate hasslowed to, and stabilised at, that of control, the muscleis 70% heavier and produces 30% less normalised(N/g) peak isometric twitch and tetanic tensions thancontrol. Although this was the first study of thecontractile properties of bupivacaine-injected muscle,descriptive accounts of the morphological (Bradley,1979), enzyme histochemical (Hall-Craggs & Seyan,1975; Abe et al. 1987; Sadeh, 1988), myosin immuno-histochemical (Abe et al. 1987) and morphometricproperties (Hall-Craggs, 1974; Sadeh, 1988) havebeen made, and none reported muscle hypertrophy.

Correspondence to Dr J. D. Rosenblatt, Department of Physiology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa,Ontario KIH 8M5, Canada.

I I

12 J. D. Rosenblatt and R. I. Woods

However, quantitative techniques were not used inthese studies and the muscles were examined up to,but not beyond, 3-4 wk recovery, when the propertiesof the regenerated muscle fibres are virtually in-distinguishable from control. There are no quan-titative studies over a wide range of recovery times toestablish the changes that occur in muscle duringdegeneration, through early regeneration, and to wellbeyond the times at which regeneration was previouslybelieved to be complete.

In the light of findings of muscle hypertrophypreviously reported (Rosenblatt, 1992) and because ofthe apparent paucity of quantitative data to sub-stantiate the descriptive claims, the object of thepresent study was to examine further the effect ofbupivacaine on skeletal muscle at times spanning thecomplete course of degeneration and regeneration andto establish when, if ever, 'normality' is reached.

MATERIALS AND METHODS

A total of 256 male CHFB Wistar rats (120-150 gbody weight at the start of the experiments), obtainedfrom Interfauna UK Ltd (Huntingdon, UK), wereused. The care and treatment of the animals were inaccordance with the Animals (Scientific Procedures)Act, 1986.Animals were anaesthetised with pentobarbitone

sodium (60 mg/kg body weight, i.p.; Sagatal, Mayand Baker Ltd, Dagenham, UK) and the rightextensor digitorum longus (EDL) muscle was exposedsurgically. A 0.6 ml injection of either 0.5 % bupi-vacaine HCI (Marcain, Astra Pharmaceuticals Ltd,Hertfordshire, UK) or isotonic saline was made intothe muscle: a 26 gauge needle was introduced into thedistal myotendinous junction and advanced prox-imally along the longitudinal axis of the muscle untilit reached the tendon. The needle was then slowlywithdrawn as the muscle swelled during the injection.A third group of animals received no treatment. Theconcentration of bupivacaine used in the presentstudy was the same as that most frequently used forthe study of bupivacaine myotoxicity and has beenrepeatedly shown to cause massive whole muscledamage (e.g. Hall-Craggs, 1974).At 1-2 h and 2, 4, 8, 11, 21, 40, 60, 80, and 180 d

after injection in situ recordings of isometric con-tractile properties were made from the bupivacaine-injected (BI) and noninjected (NI) EDL muscle whilethe rat was anaesthetised with pentobarbitone sodium(Rosenblatt, 1992); saline injected (SI) muscles werestudied at 1-2 h and 2 d only. The number of animals

12, with the exception of the 180 d recovery timegroups, in which there were 8 animals. At thecompletion of the tension measurements the rightEDL was removed and weighed and a cross-section ofEDL muscle was taken from the midbelly region,covered in cryopreservative (Tissue-Tek II O.C.T.Compound), quick-frozen by immersion in 2-methylbutane that had been cooled to a slush in liquidnitrogen, and stored in a sealed plastic tube main-tained at -70 °C until further processing.

Transverse serial 8 gm sections were cut from eachfrozen muscle specimen on a cryostat microtome at-20 °C and mounted on warm uncoated slides. Twoserial slices were stained to demonstrate, respectively,NADH-tetrazolium reductase (NADH-TR) activity(Novikoff et al. 1961) and myofibrillar ATPaseactivity (Padykula & Herman, 1955) after pre-

incubation at pH 10.3 (Brooke & Kaiser, 1969). Fibresthat were unstained with the ATPase method (type I)and uniformly dark with the NADH-TR method were

labelled slow-oxidative (SO). Fibres that stained darkwith the ATPase method (type II) and either centri-petally light or uniformly light were labelled as fast-oxidative-glycolytic (FOG) and fast-glycolytic (FG),respectively (Peter et al. 1972).

Three serial slices were used to demonstrate thepresence of different myosin heavy chain (MHC)isoforms. Adult fast skeletal muscle MHC (WB/MHCf) and slow skeletal muscle MHC (WB/MHCs)monoclonal antibodies were produced by Dr WendyE. Brown (Department of Surgery, Jefferson MedicalCollege, PA, USA) and obtained through ProfessorJohn B. Harris (Muscular Dystrophy Group ResearchLaboratories, Newcastle General Hospital, Newcastleupon Tyne, UK). The antibodies were raised inBalb/c mice against native fast MHC (taken fromadult rabbit psoas muscle) and native slow MHC(taken from adult rabbit soleus muscle). The specifi-city of the antibodies against adult rat fast and slowMHC was verified on Western blots by ProfessorHarris (personal communication). In the present studyWB/MHCf stained 4 d regenerating fibres in additionto the adult fast (type II) fibres and thus it appearedthat WB/MHCf recognised an antigenic site on an

immature form of rat MHC in addition to adult ratfast myosin. Because regenerating muscle fibres reactwith embryonic and neonatal MHC antibodies (Sar-tore et al. 1982), a third antibody to the MHC foundin developing muscle, kindly donated by ProfessorHarris, was used. The antibody was raised in miceafter they were immunised with myosin from neonatalrat muscle. Its specificity was checked against em-

in the BI, SI and NI groups at each recovery time was bryonic and neonatal MHCs in hindlimb muscle of

Morphology of regenerating rat muscle

7-d-old rats and the MHCs of cultured rat L6myotubes, and was verified by Western blots. Theantibody did not distinguish between the embryonicand neonatal forms of MHC and will therefore bereferred to as the perinatal MHC. The MHCantibodies were adsorbed to the muscle sections andthen stained by the indirect immunoperoxidasemethod as described by Rowlerson et al. (1981). Pre-liminary testing demonstrated good agreement be-tween the fibres that stained positively with theWB/MHCs antibody and fibres that stained nega-

tively with the WB/MHCf antibody. Furthermore,the 2 antibodies were type specific; type I fibresreacted with WB/MHCs antibody only and the typeII fibres reacted with the WB/MHCf antibody only.To identify myonuclei another section of muscle was

stained with Mayer's haematoxylin (Mayer, 1903) andeosin.A comparator projecting microscope was used to

view stained EDL muscle cross-sections. The numberof fibres in a cross-section of EDL muscle was

counted on sections stained for NADH-TR. Type Iand type II fibres, SO, FOG and FG fibres, fibresreacting with WB/MHCs, WB/MHCf and perinatalMHC antibodies, and fibres exhibiting central myo-

nuclei were counted in cross-sections of muscleappropriately stained, and the numerical ratio of eachvariable was calculated as a percentage of the totalfibre number. A preliminary test-retest analysis was

done to estimate the reliability with which each ofthese numerical ratio measurements could be made.For all variables, the coefficient of variation was lessthan 3 %.Morphometric measurements were made with a

microscope with a camera lucida attachment, a

Houston Instruments Hipad Plus 9012 digitisingtablet (Austin, Texas, USA) connected to a CompaqPortable III computer (Houston, TX, USA) and a

software applications program (SigmaScan, JandelScientific, CA, USA). Muscle midbelly cross-sectionalarea (CSA) was measured on NADH-TR-stainedsections. Type I and type II fibre CSA and diameter(Brooke & Engel, 1969) were measured on ATPase-stained sections. In regenerating muscle, fibre CSAand diameter measurements were also made on fibreswhich reacted with the perinatal MHC antibody. Therelative fibre area fraction was calculated for type Iand type II fibres and for fibres reacting with theperinatal MHC antibody. The number of fibresneeded to make an accurate estimate (chosen as 5 %relative S.E.M.) of the fibre CSA and diameter was

determined using the method described by Aherne

fibres and 100-130 type II fibres were digitised from atleast 5 different regions within a section of muscle. Apreliminary test-retest analysis was used to estimatethe reliability with which muscle and fibre CSAmeasurements could be made. The coefficient ofvariation between repeated CSA measurements of agiven section of muscle was always less than 3 %.Means and S.E.M. were calculated for each continuousvariable measured. Differences between groups foreach variable quantified were analysed in a 2-wayANOVA (type of injection by duration of recovery)and, when warranted, a post hoc t' test comparison ofleast-squares means.

After examining some of the results obtained fromthe BI muscles during the course of the study it wasconsidered necessary to know the intramuscularconnective tissue content and myofibril size. Con-nective tissue was examined in muscles that hadrecovered for 21 d and longer. For this analysis, aserial section of muscle was stained with picro-anilineblue (0.1 % aniline blue, C.I. 42755) in saturated picricacid (Lillie, 1945). An ordinal rating scale of integersfrom 1-5 was adopted to express the relative amountof perimysial and endomysial connective tissue ineach section of muscle. Untreated EDL muscles foreach of the recovery times were examined and thenassigned a reference value of 3. Bupivacaine-treatedmuscles were related to this on the 1-5 scale: a sectionof muscle was rated at, respectively less than 3 or morethan 3 when judged to have a smaller connective tissuearea fraction or larger connective tissue area fractionthan the control muscle. The ordinal data wereassessed using the Mann-Whitney U test.

Heidenhain's iron haematoxylin (Heidenhain, 1896)was used to stain myofibrils. This was done only forsamples of muscle from animals which had recoveredfor 180 d, a time when hypertrophy was clear in theBI muscles. For this stain the EDL muscle specimenswere fixed in 2.5 % glutaraldehyde, postfixed inosmium tetroxide and embedded in epoxy resin(Araldite); 1-2 gm sections were cut on an ultra-microtome and stained. Myofibrils in fibres ofdifferent sizes were photographed at a magnificationof x 625, and the negatives enlarged to give a finalmagnification of x 3906. Myofibril density was de-termined by counting the number of myofibrils in adefined area on the photographs. Mean myofibrilCSA was measured directly on the photographs usingthe computerised planimetry system described above;100 myofibrils were digitised from each fibre photo-graphed. The relative myofibril area fraction was thencalculated. Means and S.E.M. were calculated for themeasured variables and differences between BI and NI

13

(1968); this was usually achieved when 50-75 type I


muscles were assessed using a t test for independentsamples. For all statistical analyses, a level ofsignificance of P < 0.05 was used.

RESULTS

None of the SI muscles underwent necrosis and exceptfor a transiently elevated relative water content at1-2 h (Rosenblatt, 1992), they were not different thanNI muscle for any of the variables quantified. Since BImuscles showed significant changes at both theserecovery times it was deemed unnecessary to examineSI muscles at later recovery times.

General light microscopic observations

Fibres from NI muscles appeared normal with allstains at all recovery times. Normal fibre growth wasobserved during the course of the study. Bupivacaine-treated EDL muscles exhibited a sequence of de-generative and regenerative changes. At 1-2 h afterbupivacaine injection, most of the muscle fibres werecircular (Fig. 1) with pyknotic nuclei and hyalinecytoplasm. The ATPase staining reaction was reducedbut the NADH-TR staining reaction was normal. Thefibres reacted with either the fast or the slowWB/MHC antibody. A minority of fibres appearednormal. Two days after the injection of bupivacaine,most of the fibres had degenerated completely.Macrophages filled the necrotic fibres and ghost-likeremnants of the original fibres could occasionally beseen (Fig. 2). The remaining fibres displayed featuresindicative of partial damage: circular shape and'moth-eaten' appearance, hyaline cytoplasm andpyknotic (but peripheral) myonuclei, and reducedATPase and NADH-TR staining reactions. The fibresstill reacted with either the fast or the slow WB/MHCantibody. On the fourth day, many small colonies ofmyotubes with central myonuclei were observed. Thestaining reaction of these fibres for ATPase was weakto moderate but was weak and ambiguous for NADH-TR. The fibres reacted strongly with both theWB/MHCf and perinatal MHC antibodies (Fig. 3),but not the WB/MHCs antibody. At 8 d, theregenerating myotubes had grown larger and many ofthe myonuclei were still central. All fibres exhibited astrong reaction for ATPase activity and a weak andindistinct reaction for NADH-TR (Fig. 4). Fewerfibres reacted with the perinatal MHC antibody thanat 4 d (Fig. 3), but all still reacted with the WB/MHCfantibody. On d 11, the regenerating fibres were larger.The myonuclei of many of the fibres were peripheral.

Fig. 1. Transverse sections of rat EDL muscle 1-2 h after injection:(a) bupivacaine injection (BI), (b) no injection (NI), (c) salineinjection (SI). Many of the fibres in the BI muscle are circular orhave a rounded contour and there are large extracellular spacesseparating fibres. The fibres in the NI and SI muscles are closelyapposed and polygonal. ATPase. Bar, 50 gm.

) S %.~~~~IF~~ r <R

X6 f LO r..,0

itwf

Fig. 2. Transverse sections of rat EDL muscle after 2 d recovery: (a)no injection (NI), (b) saline injection (SI), (c, d) bupivacaineinjection (BI). The fibres in the NI and SI muscles were closelyapposed, polygonally shaped and had peripherally located myo-nuclei. Bupivacaine caused massive muscle fibre degeneration andthe necrotic debris was removed by macrophages (c). A smallpercentage of fibres survived the bupivacaine insult (asterisk in (d)).Mayer's haematoxylin-eosin. Bar, 50 gm.


Fig. 3. Reaction of transverse sections of bupivacaine-injected ratEDL muscle with perinatal MHC antibodies: (a) 4 d recovery, (b)8 d recovery, (c) 11I d recovery, (d) 21 d recovery. At 4 d all theregenerating myotubes reacted with the perinatal MHC antibodies;thereafter the number gradually diminished (b, c) and by 21 d noneof the fibres reacted with the antibody (d). Immunoperoxidase. Bar,50 gim.

Fig. 4. Transverse sections of bupivacaine-injected rat EDL muscle

stained for NADH-TR activity: (a) 4 d recovery, (b) 8 d recovery,

(c) 11I d recovery, (d) 21 d recovery. Although the regenerating

fibres demonstrated NADH-TR activity, division of the fibres into

distinct fibre types was not possible until 21 d after injection. Bar,

50 gm.

Fig. 5. Abnormal morphological features exhibited by bupivacaine-injected (BI) rat EDL muscle after 40 d recovery. (a) Section stainedfor ATPase activity demonstrating type I fibre grouping, (b) sectionstained with Mayer's haematoxylin-eosin demonstrating centrallyor eccentrically located myonuclei; the central myonuclei werefound in large, normal sized and atrophic fibres (arrows), (c) sectionstained for ATPase activity demonstrating an atrophic type II fibre(arrow). These were characteristic features of all hypertrophic BImuscles. All bars, 50 gim.

Division of the fibres into distinct fibre types waspossible with the ATPase reaction, but not with theNADH-TR reaction (Fig. 4). The reaction with theWB/MHCf antibody was strong and the number offibres that reacted with the perinatal MHC antibodyhad further declined (Fig. 3). On d 21, the fibredifferentiation and morphology appeared normal:differentiation of the fibre types with both the ATPaseand NADH-TR reaction was possible (Fig. 4), fibresreacted with either the WB/MHCf or WB/MHCs butnot with the perinatal MHC antibody (Fig. 3), andmost of the myonuclei were now located at theperiphery of the fibre. From 40 to 180 d, the BImuscles appeared to be much larger than the NImuscles. Both the type I and type II fibres of the BImuscles were generally larger than the equivalentfibres of the NI muscles and many of the type II fibreswere grossly hypertrophic (Fig. S b, c), although somewere extremely atrophic (Fig. S c). Infrequently, smallclusters of type I fibres (Fig. 5 a) were observed. Somefibres still had central myonuclei (Fig. Sb).

15


c.o 3500

T

0

, 2500

a2000-VD

1500 Bupivacaine_' 1 000 - Control

1000

E* 500-0

O-_

E 0 4 8 20 60 100 140 180

z (1-2h)' 2 6 10 40 80 120 160Recovery time (days)

Fig. 6. Mean number of muscle fibres in a midbelly cross-section ofbupivacaine-injected and noninjected control EDL muscles.

Slow- oxidative4- m Fast-oxidative-glycolytic 4g Fast-glycolytic

0

0

C

-2i~~~~~~~~<11 ¢ Lib

21 40 60 80 180Recovery time (days)

Fig. 7. Absolute change in the relative number of slow-oxidative,fast-oxidative-glycolytic and fast-glycolytic fibres in the bupi-vacaine-injected muscles from 21 to 180 d recovery.

Fibre number

The mean number of fibres in NI muscles did notchange during the course of the study (combinedmean, 3160+ 30). A small but significant drop of themean fibre number in BI muscles occurred by 1-2 hafter injection; after 48 h the number of fibres in theBI muscles was 14% of control (Fig. 6). By 4 d,when many small colonies of myotubes were observed,the fibre number had returned to normal, andremained normal thereafter as the myotubes de-veloped into mature fibres.

Histochemical fibre type

The mean percentage of type I and II fibres in both BIand NI muscles is shown in Table 1. The percentage oftype I and II fibres in the NI muscles was constant forthe duration of the study (4.4+ 0.1 for type I and II

fibres, respectively). At 1-2 h after injection, thepercentage of type I fibres in BI muscles was

significantly, but only slightly, less than that in controlmuscles. At 2 d recovery, the type I fibres represented0.8% of the total fibre number (or 19% of control)and the figure was only slightly higher at 4 and 8 drecovery (26 % and 24% of control, respectively). By11 d, the relative number of type I fibres had risen to57% of that in normal muscle. At 21 d recovery, therelative number of type I fibres in treated musclesexceeded that in controls. This condition continued to180 d.The relative number of SO, FOG and FG fibres in

NI muscles did not change during the course of thestudy (combined mean, 4.4 + 0. 1, 58.3 + 0.2 and

Table 1. Percentage of type I (I), type II (II), slow-oxidative (SO), fast-oxidative-glycolytic (FOG) andfast-glycolytic (FG)fibres in bupivacaine-injected (BI) and noninjected (NI) control EDL muscles

Fibre type

n I II SO FOG FGRecoverytime (d) BI NI BI NI BI NI BI NI BI NI BI NI

0 7 8 3.2+0.5 4.4+0.4* 96.8+0.5 95.6+0.4* 3.2+0.5 4.4+0.4* 60.3+2.0 58.8+ 1.3 36.5+2.0 36.8+ 1.22 7 8 0.8+0.3 4.3+0.3* 99.2+0.3 95.7+0.3* 4.3+0.3 59.3+0.7 36.4+0.74 6 8 1.1+0.1 4.3+0.4* 98.8+0.1 95.7+0.4* 4.3+0.4 58.6+0.7 37.2+0.58 6 8 1.1 +0.3 4.5+0.3* 98.9+0.3 95.5+0.3* 4.5+0.3 57.3+0.8 38.3+0.6

11 8 8 2.4 +0.7 4.2+ 0.3* 97.6 +0.7 95.8 + 0.3* 4.2+0.3 58.8 +0.9 37.1+0.921 8 8 6.8+0.5 4.2+0.3* 93.2+0.5 95.8+0.3* 6.8+0.5 4.2+0.3* 55.2+ 1.6 57.2+0.6 38.0+ 1.3 38.6+0.740 7 8 6.7+0.6 4.4+ 0.6* 93.3 +0.6 95.6+ 0.6* 6.7+0.6 4.4+ 0.6* 56.6+1.2 59.4+1.7 36.7+1.8 36.3 +1.360 7 8 6.9 + 0.3 4.7 + 0.3* 93.1 +0.3 95.3 + 0.3* 6.9+0.3 4.7+ 0.3* 56.4 + 3.0 58.1 +0.6 36.7 + 2.9 37.2 +0.680 7 7 7.6 +0.8 4.7 + 0.4* 92.4+0.3 95.3 + 0.4* 7.6 +0.8 4.7+ 0.4* 54.7 + 1.2 57.7 + 1.0 37.7 + 1.1 37.6 +0.8180 8 7 5.6+0.6 4.3+0.2* 94.4+0.6 95.7+0.2* 5.6+0.6 4.3+0.2* 56.7+0.9 58.6+ 1.1 37.7+ 1.1 37.1 + 1.0

Values are expressed as means + S.E.M. * Significant difference between BI and NI at a given time, P < 0.05, 2-way ANOVA., Differentiation not possible.


Table 2. Percentage offibres expressing slow ( WB/MHCs), fast (WB/MHCJ) andperinatal myosin heavy chain in bupivacaine-injected (B!) and noninjected (NI) control EDL muscles

Myosin heavy chain composition (%)

n WB/MHCs WB/MHCf PerinatalRecoverytime (d) BI NI BI NI BI NI BI NI

0 7 8 5.4+0.4 4.6+0.6 96.7+0.4 95.9+0.8 0.0+0.0 0.0+0.02 7 8 9.8+1.0 4.5+0.5* 98.3+0.5 96.6+0.6* 0.0+0.0 0.0+0.04 6 8 2.9+0.2 5.0+0.6* 98.9+0.1 96.3+0.5* 80.0+6.7 0.0+0.0*8 6 8 4.3 +0.6 4.7+0.3 98.8 +0.3 95.3 + 0.3* 26.9+4.5 0.0 + 0.0*

11 8 8 4.3+0.9 4.2+0.3 97.7+0.6 95.8+0.3* 12.3+2.6 0.0+0.0*21 8 8 7.4+0.6 4.1 +0.3* 94.3+0.5 95.9+0.3* 0.1+0.0 0.0+0.040 7 8 7.3+0.6 4.6+0.6* 93.8+0.6 95.4+0.6* 0.2+0.1 0.0+0.060 7 8 8.2+0.6 4.8+0.4* 93.6+0.3 95.2+0.4* 0.0+0.0 0.0+0.080 7 7 7.7+0.9 4.7+0.4* 92.5 +0.8 95.3 +0.4* 0.0+0.0 0.0+0.0180 8 7 - - -

Values are expressed as means +S.E.M. * Significant difference between BI and NI at a given time, P < 0.05, 2-way ANOVA. WB/MHCs,Wendy Brown slow myosin heavy chain antibody; WB/MHCf, Wendy Brown fast myosin heavy chain antibody.

37.3 +0.2, respectively). Differentiation of fibre typesfrom 2 to 11 d after injection of bupivacaine was notpossible (Fig. 4). At all other recovery times thepercentages of SO fibres and type I fibres were equalboth in the BI and NI muscles (Table 1). The increasein the percentage of SO fibres observed at laterrecovery times (21-180 d) was paralleled by a reduc-tion in the percentage of FOG fibres. Although thechange in the percentage of FOG fibres in the BImuscle was not significantly different from thecontrols, the direction of change was consistent at allof the recovery times (Fig. 7). The percentage of FGfibres in the BI muscles changed very little.

Myosin heavy chains

Table 2 gives the mean percentage of fibres in both BIand NI muscles that reacted with the WB/MHCs,WB/MHCf and perinatal MHC antibodies. For theNI muscles, the percentage of fibres reacting withWB/MHCs and WB/MHCf did not change duringthe course of the study (4.6 + 0.1 and 95.7 + 0.2 forfibres reacting with MHCs and MHCf antibodies,respectively); fibres in these muscles did not react withthe perinatal MHC antibody. There were no differ-ences between BI and NI muscles 1-2 h after injection.At 2 d, the percentage of fibres reacting with WB/MHCs in the BI muscles more than doubled. Thepercentage of fibres reacting with WB/MHCf wasalso significantly greater than in NI muscles. This wasthe only recovery time showing marked overlapbetween WB/MHCs and WB/MHCf. At 4 d, thepercentage of fibres reacting with WB/MHCs in BImuscle was less than that in NI muscles, but by 8 d

had returned to normal. At 21 d, the percentage offibres reacting with WB/MHCs in BI muscles in-creased to 180% of that in NI muscles and remainedelevated thereafter. Fibres were first observed to reactwith the perinatal MHC antibody 4 d after injectionwhen 80% of the fibres reacted with antibody. Thenumber of fibres reacting with perinatal MHCgradually fell and by 21 d was not different fromcontrol (Fig. 3). With the exception of a few fibres at40 and 60 d (0.1 % and 0.2 %, respectively), there wasno further expression of perinatal MHC in the BImuscles.

Central myonuclei

Virtually all the fibres in all the NI muscles examinedexhibited peripheral myonuclei. In BI muscles, centralmyonuclei were present in 74% of the fibres after 4 drecovery (Fig. 8). Thereafter, the number graduallyfell and by 21 d only 6% of the fibres exhibited anycentral myonuclei. Subsequently, a gradual increase incentral myonuclei occurred and by 60 d they werefound in 14% of fibres. No further change wasobserved during the remainder of the study.

Muscle andfibre morphometry

From 1-2 h to 21 d, the CSA of the BI and NI muscleswas not significantly different. Subsequently, the BImuscles grew at an accelerated rate and by 40 d were18% larger, and at 60 d 52 % larger, than the controls(Fig. 9). After further growth of animals to 180 d, theywere still about 50% larger.

Fibre CSA and diameter were highly correlated(r = 0.97, Pearson product-moment correlation,

ANA 181

17


100 -

_ Bupivacain

: 80

0(o.

U) >~

LE

* _X

40-lCDC

LL 200 |FF

0 2 4 8 11 21 406080180(1-2 h) Recovery time (days)

Fig. 8. Percentage of fibres in bupivacaine-injectedwith centrally located myonuclei.

e 4000 -

_- 3000 -

< 2000 -U)

1000-_. IE

a)

E0

Control line

(BI) muscles

I--V 30-E

,25 T T.X~~~~~~'i

C 20-0

or~~~~~~~~~~~~~~~._

c' 15- O

0

nnjt10- oControl

2 5 -.'

> 0 2 4 6 810 20 40 60 80100120140160180( /-2h Recovery time (days)

Fig. 9. Midbelly cross-sectional area of bupivacaine-injected andnoninjected control rat EDL muscle. Plotted values are means+S.E.M. The asterisks indicate significant differences between BI andNI muscle at a given recovery time (P < 0.05, 2-way ANOVA).

1400 -

¢s^ 1 200 -

E-=- 10001-

< 800-U) -U 600-

400 -

40 -

30 -

20 -

10 -

* * *

-i.ii--

,~~~~~~~~~~~~~~~

,.

. * *

- .

* * *

0° -- . Bupivacaineo Control

0 I, I, II, I, I , ,-" ,

0 2 4 6 810 20 40 60 80 100120140160180

(1-2 h) Recovery time (days)Fig. 10. Cross-sectional area (CSA) and diameter of type I fibres inbupivacaine-injected and noninjected control rat EDL muscle.Plotted values are means+S.E.M. The asterisks indicate significantdifferences between BI and NI muscles at a given recovery time(P < 0.05, 2-way ANOVA).

60 -

40 -

20 -

0-

* * *

U .a'

s ,..U

* * *

m Bupivacaineo Control

0 2 4 6 810 20 60 100 140 180(1-2 h) 40 80 120 160

Recovery time (days)Fig. 11. Cross-sectional area (CSA) and diameter of type II fibres inbupivacaine-injected and noninjected control rat EDL muscle.Plotted values are means+S.E.M. The asterisks indicate significantdifferences between BI and NI muscles at a given recovery time(P < 0.05, 2-way ANOVA).

P < 0.0001), hence, the results of fibre size deter-minations are reported for CSA only (Fig. 10). Thetype I fibres in the BI muscles were, respectively, 17%and 18 % smaller than those in controls at 2 and 4 drecovery. The fibres in the BI muscles measured atthese recovery times were intact surviving fibres orpartially damaged fibres. From 8 to 40 d there were nodifferences between the type I fibre CSAs in BI and NImuscles, but at 60 d the fibres in the BI muscles were26% larger.At 2 d recovery, the surviving type II fibres in the BI

muscles had atrophied 33 % (Fig. 11). AT 4 d, when apreponderance of myotubes was observed, the meanfibre CSA was 82% smaller than in control muscles.Fibres in the BI muscles grew faster than in thecontrols from 8 to 40 d; at 21 d fibres were the samesize in both groups and by 40 d those in BI muscleswere 33 % larger. From 40 to 60 d the growth rate ofthe type II fibres in the BI muscles decreased and wassimilar to that of controls (Fig. 11). Thereafter therewas little growth in type II fibres in either BI or NImuscle. From 21 d onwards, the BI muscles had1-3% frankly atrophic type II fibres (< 600 iim2),which were not seen in NI muscles.

Fibres reacting with the perinatal MHC antibodywere observed in BI muscles from 4 to 11 d recoveryin decreasing numbers. A comparison of the sizes ofthe fibres that survived intact or were only slightlydamaged by bupivacaine, and the sizes of the fibresregenerating after total dissolution by bupivacaine isshown in Figure 12. The surviving fibres were onlyslightly smaller than the control fibres, whereas theregenerating fibres were substantially smaller. Withsubsequent growth of the regenerating fibres, and

a)a)CDE0


1600 -

1400 -

1200 -

1000 -

800 -

600 -

400

200

Control~-----T Surviving fibres

= Surviving andregeneratingfibres

..-------------------- . R g n rt g.. Regeneratingfibres

Q Control35 i Surviving fibres

30 T s-nn

25- urviving ana

regenerating

fibres20

.----------------.-.--... Rege1 5 -... fibre

,. :.

10 *Tl5 * Bupivacaine

o Control

4 8 11

eneratingII

Recovery time (days)Fig. 12. Cross-sectional area (CSA) and diameter of type II fibres inbupivacaine-injected and noninjected control rat EDL muscle at 4,8 and 11 d recovery. The CSAs and diameters of the type II fibresin the BI muscles are plotted separately for regenerating fibres only,surviving fibres only, and surviving and regenerating fibres. Plottedvalues are means +S.E.M.

discontinued reactivity with the perinatal MHCantibody, surviving fibres could not be distinguishedfrom regenerating fibres.

Relative fibre area fraction

The total area of the muscle cross-section occupied bytype II fibres was significantly larger in BI muscles at

Surviving fibres

90Control.....-Surviving and-90- e ---------.. regeneratingc

80 l fibres

70-

40a, 60

' 40-

30

=20-a) 20 - *Bupivacaine-- .F 10- OControl

Regenerating fibres° I -I ~ ~~~I

4 8 11Recovery time (days)

Fig. 13. Relative area fractions of type II muscle fibres inbupivacaine-injected and noninjected control rat EDL muscle at 4,8 and 11 days recovery. The relative area fractions of the type IIfibres in the BI muscles were plotted separately for regeneratingfibres only, and surviving and regenerating fibres. Plotted values aremeans + S.E.M.

2 d recovery and from 8 to 21 d recovery than incontrols. At all recovery times later than 40 d, the areafraction was smaller in the BI muscles; the differenceat 180 days, however, was not statistically significant(Table 3).From 4 to 11 d the area fraction of all the type II

fibres (regenerating + surviving fibres) and the re-generating fibres only was significantly smaller thanthe type II area fraction of the NI muscles (Fig. 13).However, the area fraction of the surviving fibresalone was not significantly different from that of theNI muscles. At 4 d recovery, the area fraction of theregenerating fibres were not significantly differentfrom that of all the type II fibres in the BI muscles, butfrom 8 to 11 d the area fraction of the regenerating

Table 3. Relative areafractions oftype I and type II musclefibres ofbupivacaine-injected (BI) and noninjected (NI) control EDLmuscles

Relative area fraction (%)

n Type I fibres Type II fibresRecoverytime (d) BI NI BI NI BI NI

0 8 8 1.6+0.3 2.1+0.3 98.4+0.3 97.9+0.32 8 8 0.4+0.1 1.9+0.1* 99.6+0.3 98.1 +0.1*4 5 8 1.5+0.3 2.0+0.2 98.5+0.3 98.0+0.18 6 8 0.5+0.2 1.9+0.2* 99.5+0.2 98.1 +0.1*

11 8 8 1.1+0.4 1.8+0.2* 98.9+0.4 98.2+0.2*21 7 8 2.4+0.3 1.6+0.2* 97.6+0.3 98.4+0.2*40 8 8 1.8+0.3 1.9+0.4 98.2+0.3 98.1+0.460 7 8 2.2+0.1 1.6+0.2* 97.8+0.1 98.4+0.2*80 7 7 2.4+0.3 1.6+0.1* 97.6+0.3 98.4+0.1*180 8 7 1.8+0.2 1.5+0.1 98.2+0.2 98.5+0.1

Values are expressed as means +S.E.M. * Significant difference between BI and NI at a given time, P < 0.05, 2-way ANOVA.

2-2

N

1-

-.0.O00

I-

Ea)

00.E'a)Q

19


F

/A

I' /

Fig. 15. Myofibrils in transverse sections of rat EDL muscle after180 d recovery: (a) no injection, (b) bupivacaine injection. Circles in(a) enclose areas which contain myofibrils with a punctateFibrillenstruktur appearance (lower circle) and banded or Felder-struktur appearance (upper circle). A third fibre (indicated by blackarrow) contains distinct regions of punctate and banded myofibrils.Note the central myonuclei in the BI muscle fibres (white arrow).Heidenhain's haematoxylin. Bar, 50 pm.

t<) / 'CAFig. 14. Intramuscular connective tissue collagen in transversesections of noninjected (left column) and bupivacaine-injected(right column) rat EDL muscle at 21 d (a), 40 d (b), 60 d (c), 80 d (d)and 180 d (e) after injection. The BI muscles were judged as havinga greater relative connective tissue collagen content than control at40, 60, 80, and 180 d recovery. The difference was most apparent forthe perimysium (arrows). Picro-aniline blue. Bar, 50 gim.

fibres became progressively smaller with a concurrentincrease in the total type II area fraction.

Intramuscular connective tissue

At all recovery times after 21 d, the rating given toexpress the amount of perimysial and endomysialconnective tissue was higher for sections of BI musclesthan NI muscles, indicating a greater area fraction of

-- Mww -

Fig. 16. Myofibrils in transverse sections of rat EDL muscle after180 days recovery: (a) no injection, (b) bupivacaine injection. Themyofibrils in the BI muscles occupy a larger area fraction of musclefibre than the myofibrils in the NI muscles. Heidenhain's haema-toxylin. Bar, 10 gm.


Table 4. Myofibril density, myofibril area and myofibril relative area fraction of bupivacaine-injected (BI) and noninjected (NI)control EDL muscles

Number Density Cross-sectional area Area fractionof fibres (myofibrils/tm2) (pm2) (%)

Recoverytime (d) BI NI BI NI BI NI BI NI

180 25 9 1.05 +0.02 1.46+0.06* 0.88+0.02 0.54+0.01* 92.4+ 1.3 78.9+2.7*

Values are expressed as means + S.E.M. * Significant difference between BI and NI, P < 0.05, t test.

connective tissue in the BI muscles (Fig. 14). Differ-ences in the relative perimysial connective tissuecontent were more apparent than differences in therelative endomysial connective tissue content.

Myofibrils

In transverse section, 2 different shapes of myofibrilwere observed (Fig. 15 a, b); the shape appeared to bedependent on the size of the fibre but independent ofthe muscle from which it was taken. The myofibrils inthe relatively large fibres had a punctate or Fibrill-enstruktur appearance (Kruger, 1929). The smallfibres contained predominantly banded myofibrils(Felderstruktur appearance; Kruger, 1929) that wereoften arranged in parallel strips or in a rosette,although there were usually small, scattered regions ofpunctate myofibrils. The medium-size fibres containedregions which generally approximated the bandedmyofibrils or punctate myofibrils but with definiteintermediate zones with mixed characteristics. TheCSA of the banded myofibrils was not measured. TheBI muscles had a 28 % lower myofibril density (Fig.16), a 63 % larger myofibril CSA and a 17% largermyofibril area fraction than control (Table 4).

DISCUSSION

Intramuscular injections of bupivacaine cause wide-spread fibre degeneration (Hall-Craggs, 1974; Foster& Carlson, 1980) and both the massive dissolution ofmuscle fibres, measured 2 d after bupivacaine in-jection, and the large number of regenerating, cen-trally-nucleated fibres reacting with the perinatalMHC antibody, measured 4 d after injection, confirmthese observations. The rate and extent of regen-eration of the BI muscles during the first 3 wk wasessentially as expected from previous qualitativeaccounts of muscle regeneration following bupi-vacaine injection (e.g. Hall-Craggs & Seyan, 1975).Necrosis was followed by a rapid period of re-generation during which time muscle fibre numberwas restored, histochemical and immunohisto-

chemical fibre type differentiation occurred, andmuscle force, dry mass (Rosenblatt, 1992) and fibreCSA and diameter gradually improved to matchcontrol values.

Preservation of a muscle's nerve and vascularsupply, basal membranes and satellite cells signifi-cantly contributes to the rate and extent of musclefibre regeneration (Grounds, 1991). Several experi-mental models of muscle degeneration and regen-eration have been designed which selectively damageone or more of these elements. Three of the mostcommonly used models involve grafting, which alwaysproduces massive whole muscle damage (see Carlson& Faulkner, 1983). Minced muscle grafting damagesall of the elements. Free muscle grafting damages themuscle's nerve and blood supply. Nerve-intact graft-ing damages the muscle's blood supply but leaves themotor nerve intact. In each of these models, the degreeof satellite cell proliferation and of vascular andneural reintegration influences the extent of musclefibre regeneration; accordingly, the most severedeficits following regeneration are exhibited by mincedmuscle graft regenerates. Free muscle graft regeneratesand nerve-intact muscle graft regenerates are pro-gressively more complete. In contrast to the damagecaused by the various grafting procedures, bupi-vacaine does not damage the muscle's intramuscularnerves (Tomas i Ferre et al. 1989), vasculature (Grimet al. 1983), basal laminae (Hall-Craggs, 1980a) orsatellite cells (Hall-Craggs, 1980b).

Fibre type and central myonuclei

The differentiation of the regenerating fibres intotypes I and II fibres was not observed until 11 daysafter injection, a finding that is already well docu-mented for rat fast-twitch muscles injected withbupivacaine (Hall-Craggs & Seyan, 1975; Abe et al.1987). At 21 days after injection the percentage of typeI and WB/MHCs fibres in BI muscle was significantlygreater than that in controls, a difference thatremained for the duration of the study. Histo-

21

22 J. D. Rosenblatt and R. L Woods

chemically, it appeared that the increase in thepercentage of type I or SO fibres occurred at theexpense of the FOG fibres. This finding is at odds withthe earlier descriptions of a normal distribution ofslow and fast histochemical fibre types (Hall-Craggs &Seyan, 1975) and of fibres expressing slow and fastMHCs (Abe et al. 1987) in bupivacaine-treated fast-twitch muscle, but this discordance can probably beexplained in the following way. The relative increasein the number of type I and WB/MHCs fibres wassubstantial (approximately 48% and 67%, respect-ively), but since type I fibres (Eddinger et al. 1985) orfibres containing the slow MHC (Thomason et al.1986) normally exist in relatively small numbers(4.4 + 0.1 % for NI muscles in the present study) theabsolute magnitude of the increase was actually small(approximately 2.2% and 3.1 %, respectively, for typeI fibres and fibres reacting with WB/MHCs). Todetect such a difference in a cross-section of musclecontaining 3000-3500 fibres by mere observationwould be extremely difficult.

There were infrequent instances of type I fibreclustering (type grouping) in some sections of muscleat all recovery times after 21 d. This is likely to be dueto the splitting or 'forking' of fibres, though it is alsopossible that there was some irreparable damage tosome of the intramuscular nerve axons followinginjection and that the affected fibres became re-innervated by collateral sprouts from nearby slowmotor neurons (Banker & Engel, 1986). The incidenceof group typing was too low to affect the fibre typemeasurements significantly.Another abnormal feature in hypertrophic BI

muscle was the significant proportion of the fibrescontaining central or eccentric myonuclei. The num-ber of fibres with central myonuclei was highest 4 dafter injection (74 %) when the muscle was composedprimarily of regenerating myotubes, and the numbergradually fell to reach a minimum of 6% by 21 d.Thus the migration of the myonuclei from the fibreaxis to the periphery remained incomplete even afterregeneration appeared to have stabilised. However,the proportion of central myonuclei then graduallyincreased and by 60 d had settled at 14%. Persistingcentral myonuclei is a consistent finding in bupi-vacaine-treated muscles (Hall-Craggs, 1974; 0klandet al. 1989), and its significance has never beenaddressed. It is difficult to interpret the persistenceand subsequent rise of central myonuclei in thepresent study as virtually nothing is known about thekinetics of myonuclei during development or re-generation. Migration of myonuclei to a central or

myonuclei is considered to be an aberrant, butnonspecific, response because it is a characteristicfeature of many neuromuscular diseases (Banker &Engel, 1986) and, as such, the prevalence of centralmyonuclei is used as a measure of fibre damage sincethe myonuclei often fail to marginate during re-

generation. If the central myonuclei can be used as a

marker of fibre damage, then the increase seen in thepresent study suggests that some of the fibres suffereddamage during the latter part of the study, althoughother evidence of this was not apparent by microscopicexamination of any of the stained sections.

Fibre size and relative fibre area fraction

In the only previous quantitative study, Yoshimura &Schotland (1987) measured the perimeters of fibresfrom rat EDL muscles at 8, 10, 15, 30 and 60 d afterinjection with bupivacaine. At 30 d the perimeterswere 88% of control and at 60 days they were 98% ofcontrol. In the present work, the CSA of the fibres inthe treated muscles were 81 % of control at 11 daysand 95 % of control at 21 days, ratios that are similarto, but a time course that is different from, thatreported by Yoshimura & Scotland (1987). Hall-Craggs (1974) and Sadeh (1988) observed normal fibresizes by 3 to 4 wk after injection. It is possible thenthat the rate of muscle regeneration in Yoshimura &Schotland's study was slower than that in the presentwork and in other studies of muscle fibre size.Yoshimura & Schotland (1987) dissolved bupivacainein 0.1 % methylparaben, which may have slowed therate of regeneration. Since the regenerating muscleswere still growing between 30 and 60 d, it is notknown whether growth had stabilized at 60 d. Furtherrecovery may have been necessary to reveal fibrehypertrophy.The absolute magnitude ofchange in the percentage

of type I or WB/MHCs fibres is probably too small toexert a significant effect on the contractile properties,including contraction times. Indeed, no change was

found for the twitch time, time to peak tension, or

half-relaxation time (Rosenblatt, 1992), even thoughmuscles with a high proportion of type I fibres displaylonger isometric twitch times than muscles with a highproportion of type II fibres (Bairainy, 1967). Egginton(1990) has shown that the relative area fraction ratherthan the numerical ratio of each fibre type might be a

better indicator of the relative contribution that eachfibre type has on contractile function, especially whenchanges in fibre type are accompanied by changes infibre size. In the present study, at some of the later

eccentric position or incomplete margination of recovery times, the hypertrophy of type I and type II


fibres negated the changes in numerical ratio. Anincrease in the relative number of type I fibres wasobserved from 21-180 d recovery, but the relative areafraction was only significantly larger at 60 and 80 dand was actually lower at 21 d. In addition, therelative area fractions at all recovery times weresmaller than the numerical proportions suggestingthat the type I fibres have less influence on overallmuscle function than would be indicated by numericalratio alone.

Fibre number

There was no apparent difference in the number offibres in midbelly transverse sections of BI and NImuscle at any of the recovery times longer than 2 d.However, the reliability with which muscle fibres canbe counted is influenced by the geometric design of themuscle. The rat EDL muscle is a pinnate, fusiformmuscle (Balice-Gordon & Thompson, 1988) composedof approximately 5000-5200 fibres. This value hasbeen obtained by direct enumeration of individualfibres dissected from the muscle (Timson et al. 1989),and by indirect enumeration from sections throughthe midbelly of the muscle at an angle to its long axisso that all the fibres are included in the section (Balice-Gordon & Thompson, 1988). Because of its archi-tecture, it is not possible to obtain a single cross-section ofEDL muscle which contains all the fibres inthe muscle. In the present study, fibres were countedon a single cross-section of muscle that was cut fromthe region of greatest diameter, perpendicular to thelong axis of the fibres. This region of muscle wasselected because it presumably contains the largestnumber of fibres obtainable in a transverse sectionand was therefore thought to be 'most representative'of the muscle as a whole. Although the mean numberof fibres in this section (3160+30) is similar to thatreported by others using the same sampling andcounting technique (e.g. Eddinger et al. 1985), it isconsiderably less than the total fibre number. Since, inthe present study, only a portion of the fibres in themuscle was counted it is possible that hyperplasia didoccur but was missed.

Alterations in the geometry of a muscle and itsfibres will affect the proportion of fibres contained ina transverse section of the belly of a muscle. Increasesin muscle mass can produce changes in the angle ofpinnation of the fibres. If a cross-section of muscle isobtained by cutting perpendicular to the long axis ofthe fibres, then the number of fibres in the section ofa hypertrophied muscle is likely to be either greater

degree of fibre hypertrophy and angle of pinnation; ifmost of the muscle enlargement was produced by fibrehypertrophy, then there will be more fibres in thehypertrophied muscle than in the control muscle. Ifthe cut is made perpendicular to the long axis of themuscle, then the number of fibres in a section of a

hypertrophied muscle will be the same as or less thanthat in control, depending on the amount by whichthe angle of fibre pinnation increases; however in thisinstance fibre CSA will be overestimated (Gollnick etal., 1981).The angle of pinnation of normal EDL muscle

fibres is approximately 3.5° (Close, 1964). A directmeasurement of angle of pinnation in the BI muscleswas not made in the present study. However, if therewas a change, then it was probably small because thefibres in the hypertrophic BI muscles were almostregular polygons when viewed from sections cut at thesame angle as those cut from control muscles. Had theangle changed significantly, then the fibres would haveappeared as elongated polygons. Because the fibres ofnormal rat EDL muscle run almost parallel to thelong axis of the muscle, it is unlikely that any

significant change in the number of fibres in themidbelly of the BI muscle had occurred.

Connective tissue

The magnitude of the increase in fibre CSA (31 %)was not commensurate with the increase in muscleweight and muscle midbelly (70% and 52 %, re-

spectively). It seems unlikely that fibre hyperplasiacontributed significantly, if at all, to the increased sizeof the muscle. In addition, the relative water contentof hypertrophic BI muscle is not different than control(Rosenblatt, 1992). The discrepancy was furtherinvestigated by assessing the relative amount ofintramuscular connective tissue in each muscle thatrecovered for 21 d and longer. Sections of BI musclewere rated as having a larger area fraction ofintramuscular connective tissue than NI muscle from40 d onwards, although the method of assessmentused in this study did not allow the determination ofthe amount by which it was greater nor the extra-cellular fluid volume contained in it. Thus it seems

that the incompatibility between muscle size and fibresize can be explained, at least in part, by a larger area

fraction of intramuscular connective tissue.

Myofibrils

The myofibril density was significantly lower in thefibres in the BI muscles at 180 d recovery and to a

than or less than that in control, depending on the

23

large extent this was attributable to a larger myofibril


Table 5. Estimate of the number ofmyofibrils per musclefibrebased on musclefibre cross-sectional area (CSA) and myofibrildensity of bupivacaine-injected (BI) and noninjected (NI)control EDL muscles

Number ofFibre CSA Myofibril myofibrils(IrM2) density (gm-2) per fibre

Recoverytime (d) BI NI BI NI BI NI A%

180 3730 3180 1.05 1.46 3552 2178 63

CSA in these muscles. The total number of myofibrilsin a muscle fibre was not measured directly. Aprovisional comparison between the relative numberof myofibrils per fibre in BI and NI muscles can bemade from the myofibril densities and the mean fibreCSAs of the muscles from which the myofibrildensities were measured (Table 5). Estimated in thisway there are 63 % more myofibrils per fibre in BImuscles than in NI muscles at 180 d recovery. Thelarger relative mass of myofibrils in the BI muscles isthus due not only to myofibril hypertrophy but also to

myofibril proliferation; consequently, the hypertro-phied muscle fibres exhibit both an increase in size andin the number of myofibrils. This indicates that thetotal myofibril CSA of BI muscle is greater than NImuscle. Thus not only do the BI muscles produce lessforce per unit mass (Rosenblatt, 1992), but they alsoproduce significantly less force per myofibrillar CSA.

There are no studies on the myofibrils in musclesinjected with bupivacaine nor in muscles damaged byother methods. Studies on myofibrils of hypertrophicmuscles have yielded conflicting results. Rowe (1969)reported a disproportionate increase in myofibril massrelative to muscle weight or fibre CSA for mouse

soleus muscles which were enlarged by the ablation oftheir synergist muscles, although it was not indicatedwhether this was due to myofibril proliferation and/orhypertrophy. The muscles also produced 21-42% lesspeak tetanic tension per unit mass and 46% less peaktetanic tension per unit total myofibril area. Usingbiochemical methods, Tsika et al. (1987b) found thatthe myofibrillar protein content of rat plantaris musclesubjected to chronic overload by ablation of thesynergists increased in proportion to the increase inmuscle mass. Goldspink & Howells (1974) foundincreases in the total myofibril content, but no changein the myofibril density, of hamster biceps brachii,soleus and EDL muscles which had been subjected toa 5 wk weight lifting programme. The reason for thediscrepancies are unclear but may be due to thedifferent stimuli produced by the different methods

used for invoking hypertrophy. Synergist ablationsubjects the overloaded muscle to increased active andpassive tensions, whereas weight training producesintermittent increases in the work done by the musclebut not passive load.Growth of muscle fibres is associated with an

increase in both the number and the CSA of myofibrils(Goldspink, 1970, 1971). When myofibrils reach acritical size they proliferate by splitting longitudinally;as the filament mass of the growing myofibrilincreases, the tension produced by 2 adjoiningsarcomeres and the rate at which the tension isdeveloped produces a mechanical stress at the mid-point of each Z-disc causing it to rupture (Goldspink,1971). The proliferation of myofibrils during fibregrowth is thought to be important because it permitsthe developing sarcoplasmic reticulum and transversetubular system to penetrate and ensheath theincreased myofibrillar mass (Goldspink, 1971).

Relationship to force deficit

Between 40 and 80 d after injection the stimulationfrequency at which the BI muscles produced asignificantly larger absolute isometric force than theNI muscles was equal to or greater than the fusionfrequency (- 40-50 Hz) (Rosenblatt, 1992). Thehypertrophy of the myofibrils in BI muscle suggeststhat this may be due to impairment in muscleactivation. Since the myofibril CSA increases duringhypertrophy, it is possible that the larger diffusiondistances that must be traversed by the releasedcalcium during excitation-contraction coupling mayprevent complete activation of the myofibril at lowstimulation frequencies. At the higher stimulationfrequencies the calcium, which would normally havebeen sequestered by the sarcoplasmic reticulum atlower frequencies of stimulation, remains in thesarcoplasm, and with the addition of more calciumfrom successive stimuli, there is adequate time for thecalcium to pervade and activate the whole myofibril.

However, at all stimulation frequencies the nor-malised (N/g) isometric tension produced by thehypertrophic BI muscles was significantly less thancontrol (Rosenblatt, 1992). This can be ascribed, atleast in part, to the larger relative mass of noncon-tractile connective tissue in the BI muscles.

Trigger and stimulus for hypertrophy

During regeneration following massive damage pro-duced by other methods, the structure and function ofthe muscles gradually improve but never reach nor-mality (see Carlson & Faulkner, 1983). When the

Morphology of regenerating rat muscle 25

changes which follow damage have finally stabilisedregeneration is said to be complete. However, duringregeneration in the present study, muscle structureand function appeared to have returned to normal by21 d, but continued changes were observed until 60 dat which time a plateau period was finally reached.The changes which occurred in the muscle during thefirst 21 d can be described as muscle regeneration, butwhat of the changes that ensued?The trigger for muscle degeneration and regen-

eration was bupivacaine; by 2 d after injection 86%of the fibres had degenerated, and during the sameperiod neither the SI nor the NI muscles showed anysign of necrosis. Less clear, however, is whether thechanges revealed at the later recovery times were alsotriggered by the surgery and/or the injection orwhether they were completely independent of bupi-vacaine and triggered by some other agent activeduring the first 21 d following injection or which wasactive at or after the first 21 d. With hindsight, SImuscles should have been examined at all recoverytimes, or at least at recovery times representative ofthe time course of all changes, and not just at 1-2 hand 2 d after injection. It seems unlikely, however,that the injection vehicle exerted an effect as there areno reports of fibre damage, except to a small numberof fibres along the needle track (Svendsen, 1983), or ofmuscle hypertrophy following saline injection norfollowing the injection of any other drug vehicle. Ifthis is true then the changes that occurred from 21 donwards were also triggered by bupivacaine.

If the ongoing changes were triggered by bupi-vacaine then it is important to determine whether thestimulus for the ongoing changes exerts its effectduring the re-established use of the EDL or after theEDL has reached a point of near normality (21 d). Ifthe changes observed after 21 d were stimulated duringthe first 21 d after injection, then these changes beganat some time during the re-established use of the EDLmuscle. If the changes observed after 21 d werestimulated after the muscle reached normality thenthe two events are sequential. Thus the questionremains, is the stimulus for hypertrophy present andactive during the course of fibre regeneration or afterthe muscle has reached normality? The answers tothese questions are not given by the present data.Another important issue is the nature of the

stimulus for hypertrophy. The present results indicatethat the stimulus may be increased active and passivetension. The similarity between the properties of thehypertrophic BI muscles and fast-twitch muscles withhypertrophy induced by the ablation of their synergistmuscles is remarkable: like BI muscles these exhibit

larger absolute, but smaller normalised, isometrictwitch and tetanic tensions (Olha et al. 1988); agreater proportion of SO (type I) muscle fibres(lanuzzo et al. 1976) and slow myosin isoenzymes(Gregory et al. 1986); type I and type II fibrehypertrophy (lanuzzo et al. 1976); an accumulation ofintramuscular connective tissue collagen (Williams &Goldspink, 1981); and a greater myofibrillar areafraction (Rowe, 1969). Synergist ablation subjects theoverloaded muscle to both an increased passivetension, resulting from stretch imposed by antag-onistic muscle activity (Gutmann et al. 1971), andincreased active tension, resulting from a greaterweight bearing and work requirement (Tsika et al.1987 a). Since a primary function of the intramuscularconnective tissues is to provide an infrastructure formuscle by binding together and maintaining musclefibres and fascicles in an orderly and contiguousarrangement (Borg & Caulfield, 1980), part of thegreater workload may be the resistance imposed bythe extra collagen, which could only be overcome byadditional (active) force. Michel et al. (1989) demon-strated that both increased active and passive tensionare required to produce simultaneous increases inmuscle mass, fibre size, absolute isometric force andthe ratio of type I to type II fibres. Given thesimilarities between the hypertrophic BI muscles andmuscles made hypertrophic by the ablation of theirfunctional synergists, it is not unreasonable to suggestthat BI muscles have undergone a compensatoryadaptation, presumably as a consequence of anincreased active and passive tension. Future studiesshould be directed towards establishing this inferenceby examining the changes occurring in BI EDLmuscles under different conditions affecting the degreeof stretch (passive tension) alone or in combinationwith different levels of activity (active tension).

ACKNOWLEDGEMENTS

We thank Mrs A. Shelly for preparing Araldite-embedded sections and Dr A. Silver for her helpfulcomments and suggestions. J. D. R. is grateful to theCambridge Commonwealth (Canadian) Trust (Tid-marsh Scholarship), the Committee of Vice-Chan-cellors and Principals of the Universities of the UnitedKingdom (Overseas Research Student Award), theCouncil of the Canadian Centennial Scholarship Fundand the Wellcome Trust for their financial support.

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