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Regenerative response of fast and slow twitch skeletal muscle to denervation and devascularization Bockhold, Kathy 1988

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REGENERATIVE RESPONSE OF FAST AND SLOW TWITCH SKELETAL MUSCLE TO DENERVATION AND DEVASCULARIZATION  By Kathy Bockhold B . S c , The University of B r i t i s h Columbia, 1986 A THESIS IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES (Anatomy)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA January 1989 Q Kathy Bockhold, 1988  In  presenting this  degree at the  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department  of  Anatomy  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  j k ^ i i m :  ii ABSTRACT The  contractile  properties  of  fast-twitch extensor digitorum longus were  denervated/devascularized  (EDL) and slow-twitch soleus (SOL)  studied at 3, 6, 9, and 12 weeks post-surgery.  examination establish  of these a  mouse  physiological model  of  mouse  parameters  A comprehensive  i s desired  regeneration.  The  in order to  surgical  technique  involved shimmying a piece of s i l k thread along the b e l l y of the muscle thus severing the nervous and the vascular supply to the individual EDL or SOL muscles.  The denervated/devascularized muscles were divided  two groups, reinnervated and non-reinnervated tetanic  tensions.  During  showed a gradual tensions. twitch  By  and tetanic  the SOL  tensions.  12  reinnervated  increase toward control  12 weeks  based on t h e i r twitch and  the  post-denervation/devascularization,  reached  into  values  week  EDL  and  period  SOL  muscles  in twitch and tetanic  107% and 98% of the control  In contrast, the reinnervated  EDL  only  recovered 52% of the twitch tension and 64% of the tetanic tension by 12 weeks  post-denervation/devascularization.  twitch  and  tetanic  tensions  were  The  non-reinnervated  significantly  less  than  control  (p<0.05) and reinnervated values at 6 and 9 weeks but by 12 weeks were  not d i f f e r e n t  produced periods  from  significantly studied.  controls  less  (p<0.05).  twitch  and  The non-reinnervated  tetanic  At 3 and 6 weeks post-surgery  tension  SOL  they EDL  at a l l time  the reinnervated EDL  contracted very slowly, but the speed of contraction gradually increased to  control  significantly reinnervated  values  by  slower  than  12  weeks.  the control  and non-reinnervated  The  non-reinnervated  EDL  was  and reinnervated muscles.  The  SOL were slower  than  control muscles  at 9 weeks but they recovered to control values by 12 weeks.  The  iii  post-tetanic potentiation (PTP) of reinnervated and non-reinnervated  EDL  was  The  20%  by  12 weeks and  there was  maximum v e l o c i t y of shortening at a l l ages.  The  (Vo)  non-reinnervated  no  PTP  for EDL and  for reinnervated and SOL  SOL.  remained unchanged  reinnervated  EDL  muscles were  more fatigue resistant than the controls at 3 weeks post-surgery but the reinnervated EDL weeks  eventually returned to control values  post-surgery).  The  reinnervated  and  non-reinnervated  s i g n i f i c a n t l y less fatigable than controls at 3, 6, and which there was  and  SOL  12  were  9 weeks, after  no difference in f a t i g a b i l i t y between the three groups.  Both the  reinnervated  measured  by  their  classically  reveals  PTP  and  EDL  and  contractile  resembled a  (at 6,9,  SOL  muscles successfully  properties.  denervated  muscle.  The The  a fatigue pattern suggestive  branch of a fast nerve.  regenerated  as  non-reinnervated  EDL  non-reinnervated  SOL  of reinnervation by  a  IV  TABLE OF CONTENTS  Abstract  11  L i s t of Tables  vl  L i s t of Figures  .  vii  Acknowledgements  viii  Key  1x  Introduction  x  Literature Review Development of Motor Nerves -Wallerian Degeneration -Regeneration of the Nerve  2 3 5  Muscle Development and Growth -Muscle Fiber Type Development -Denervation and Muscle Development Denervation -Morphological Changes -Metabolic Changes -Changes i n E l e c t r i c a l Properties -Changes in ACh S e n s i t i v i t y -Changes i n the Motor End Plate -Changes in Contractile Properties  6 8 10 .  12 12 12 13 13 .•.. 14 14  Regeneration  15  -The Regenerating Muscle Fiber  17  Satellite Cells  22  Muscle Blood Flow  23  Methods Denervation/devascularization Muscle Dissection Experimental Apparatus Experimental Procedures H i s t o l o g i c a l Preparation Analysis of Data Controls Results P i l o t Study The Sham Experiments Clinical  26 27 27 28 31 34 34 36 37 38 43 Observations 43  V  Discussion  70  Conclusion  80  References  81  Appendices A. Control and Sham Experiments B. S c i a t i c Neurectomy  85 86 91  vi L i s t of Tables  1.  Twitch tension and twitch tension normalized to muscle weight from Control ( C ) , Reinnervated (R), and Non-reinnervated (NR) mouse EDL.  2.  Twitch tension and twitch tension normalized to muscle weight from Control ( C ) , Reinnervated (R), and Non-reinnervated (NR) mouse SOL.  3.  The tetanic tension and tetanic tension normalized to muscle weight from Control ( C ) , Reinnervated (R), and Non-reinnervated (NR) mouse EDL.  4.  The tetanic tension and tetanic tension normalized to muscle weight from Control ( C ) , Reinnervated (R), and Non-reinnervated (NR) mouse SOL.  5.  The maximum v e l o c i t y of shortening in Control ( C ) , Reinnervated (R), and Non-reinnervated (NR) mouse EDL.  6.  The maximum v e l o c i t y of shortening i n Control ( C ) , Reinnervated (R), and Non-reinnervated (NR) mouse SOL.  7.  The post-tetanic potentiation in Control (C), Reinnervated (R), and Non-reinnervated (NR) mouse EDL.  8.  The post-tetanic potentiation in Control ( C ) , Reinnervated (R), and Non-reinnervated (NR) mouse SOL.  9.  The c o n t r a c t i l e properties and muscle weight of Control and Sham EDL muscles.  10.  The c o n t r a c t i l e properties and muscle weight of Control and Sham SOL muscles.  11.  The absolute and normalized twitch and tetanic tensions of Control, (C), Reinnervated (R), Non-reinnervated (NR), and Denervated (D) EDL muscles.  12  The remaining c o n t r a c t i l e properties and muscle weight of C o n t r o l , ( C ) , Reinnervated (R), Non-reinnervated (NR), and Denervated (D) EDL muscles.  13.  The absolute and normalized twitch and tetanic tensions of Control, (C), Reinnervated (R), Non-reinnervated (NR), and Denervated (D) SOL muscles.  14.  The remaining c o n t r a c t i l e properties and muscle weight of Control, (C), Reinnervated (R), Non-reinnervated (NR), and Denervated (D) SOL muscles.  vi i L i s t of Figures  1.  Experimental apparatus.  2.  Determination  of the maximum v e l o c i t y of unloaded shortening.  3.  A longitudinal section of normal 21 day mouse EDL muscle. s t a i n . 650X.  4.  Longitudinal sections of denervated/devascularized H&E s t a i n . 700X. A. B. C.  H&E  mouse EDL muscle.  4 days post-denervation/devascularization. 7 days post-denervation/devascularization. 21 days post-denervation/devascularization.  5.  Weights of control and denervated/devascularized  EDL muscles.  6.  Weights of control and denervated/devascularized  SOL muscles.  7.  The time-to-peak twitch tension and the half-relaxation time of c o n t r o l , reinnervated, and non-reinnervated mouse EDL muscles.  8.  The time-to-peak twitch tension and the half relaxation time of c o n t r o l , reinnervated, and non-reinnervated mouse SOL muscles.  9.  The fatigue regime of c o n t r o l , reinnervated, and nonreinnervated mouse EDL muscle. The values are expressed as a proportion of the i n i t i a l Po value.  10.  The fatigue pattern of c o n t r o l , reinnervated, and nonreinnervated mouse SOL muscles.  11.  The fatigue regime f o r control and sham EDL and SOL.  12.  The fatigue regime f o r c o n t r o l , reinnervated, non-reinnervated and denervated EDL and SOL.  vi i i  ACKNOWLEDGEMENTS  The  author  experience  would  like  and knowledge  to thank as well  Dr. B.H. Bressler as his guidance  f o r sharing his  i n supervising this  research p r o j e c t . A very special and  help.  My  thanks are due to Dr. J . Anderson f o r a l l of her advice  appreciation i s also  extended  to Jean  McLeod  f o r her  technical assistance, to Darlene Redenbach for permission to use figures 1 and  3, and to Dr. W.K. Ovalle and Corinne Reimer f o r t h e i r help with the  photography. manuscript.  Teresa  Johnson  also  was a t e r r i f i c  help i n preparing the  ix KEY  ACh=  Acetylcholine  AChE=  Acetylcholinesterase  EDL=  extensor digitorum longus (fast twitch muscle)  S0L=  soleus (slow twitch muscle)  NCAM's=  neural c e l l adhesion molecules  L C l f , LC2f, LC3f= MHC=  myosin heavy chain  MHCemb=  myosin heavy chain, embryonic  f l - f 4 , FM1-FM3= SMI, SM2= FDL= SR=  l i g h t chain fast muscle  fast myosin isozymes.  slow myosin isozymes.  f l e x o r digitorum longus sarcoplasmic  reticulum  SER=  smooth endoplasmic reticulum  rER=  rough endoplasmic reticulum  LG= SDH=  l a t e r a l gastrocnemius succinic dehydrogenase  C=  control  R=  reinnervated  NR=  non-reinnervated  D=  s c i a t i c neurectomy  CT=  contraction time  Pt=  Twitch tension  Po=  Tetanic tension  Pt or Po /MW= 1/2RT= TTP= Vo= PTP=  Twitch or tetanic tensions normalized to muscle weight.  Half-relaxation time  Time-to-peak twitch tension. Maximum v e l o c i t y of unloaded Post-tetanic potentiation.  shortening.  -X-  INTRODUCTION  The physiological widely The  analyzed  properties of denervated  using  a  number of  skeletal  techniques  muscle have been  (nerve crush, cordotomy).  early phases of denervation, characterized by a c e n t r a l i z a t i o n of the  nuclei and a progressive increase in the disorganization of the components of  a muscle c e l l ,  are b a s i c a l l y  consistent between fast and  muscle regardless of the technique used. changes  include  time-to-peak  (TTP)  fatigability has  also  a  decreased and  tetanic  half-relaxation  of the muscle f i b r e s .  been  examined although  regeneration  has  denervation  have  The corresponding physiological tension time  (Po),  (1/2RT),  a and  regeneration of  a complete analysis  yet to be done. involved  The  limb  slowing an  of  increased  skeletal  muscle  of many aspects of  Thus f a r , most of the  whole  slow-twitch  techniques  immobilization in young  for  animals.  Whole limb immobilization causes atrophy of a l l of the muscles of the limb thus  creating  muscles. the  a  very  unstable  environment  In p a r t i c u l a r , the importance  normal  functioning  (Allbrook,  1962).  structural  support.  For  of  an  Regeneration  the  regrowth  of  the  of the surrounding environment to  individual  example,  for  muscle  neighbouring  is  muscle  well  documented  fibres  provide  studies have yet to look at the regrowth  of i n d i v i d u a l l y denervated muscles, i . e . , ones whose external environment remains normal. Vrbova and co-workers have studied the regeneration of skeletal muscle in young and  adult rats  using s c i a t i c  1984; Navarrete and Vrbova, 1984). response of a muscle denervated conducted completely  on adult animals. recovered  their  nerve  section  They observed  (Lowrie and Vrbova,  a d i f f e r e n t regenerative  at an early age as opposed to denervations  The fast-twitch twitch  or  muscles of young rats  tetanic  never  tensions; however, these  parameters f u l l y recovered in adult denervated muscle.  xi  A  thorough  muscle  examination  after  denervating  and Vrbova, 1984). on  of  regenerating  other  contractile  young animals  However, a d i s t i n c t  fast  and  slow-twitch  adult muscles.  A d d i t i o n a l l y , to our  denervating  devascularizing an  and  has  properties of also  regenerating  been undertaken  (Lowrie  lack of information i s available skeletal  muscle  after  denervating  knowledge the combined technique  individual  of  muscle has never been done  before. This project i s an attempt to provide a more complete analysis of the  physiological  devascularizing technique  used  properties of regenerating muscle a f t e r denervating  adult  fast  immobilized  namely the fast-twitch EDL A  and  slow-twitch  only the and  muscle.  individual  i s that i t severs the nervous and  chances of reinnervation by  vascular stump attached to the muscle. correlation  between the  Burnstock  short nerve and  et al (1983) reported a  length of time for reinnervation with the length They discovered that the  shorter the stump l e f t behind, the faster the reinnervation process. technique used had one other potential advantage. of the whole muscle thus  immature state from which the new because  any  parameters;  surviving  the  This procedure could  leaving a very  of the nerve stump l e f t attached to the muscle.  complete degeneration  the  muscles of the mouse.  blood supplies immediately as they enter the muscle. maximize the  addition,  muscles desired for study  the slow-twitch SOL  unique feature of this technique  In  and  muscle  therefore,  muscle can  fibres  masking  denervated/devascularized muscles.  may the  The  This procedure causes a taking i t back to a very  develop.  alter true  This  the measured recovery  is  important  contractile of  the  LITERATURE REVIEW  -2DEVELOPMENT OF MOTOR NERVES  Nerve but  and  muscle  eventually they  development  initially  are  independent  processes  become highly dependent upon each other once  they  have acquired the properties that allow them to interact with each other -  i e : release of  Acetylcholine (ACh)(nerve) and  the  response  neural  tube  to  ACh  (muscle). The  neuroepithelium  germinal  cells  (Burnstock,  lining  undergoing  O'Brien,  and  the  rapid  Vrbova  embryonic  proliferation  1983).  A l l of  to  contains  produce  the  cells  neurons  within  the  neuroepithelium undergo mitosis with one of the resulting daughter c e l l s eventually developing into a neuroblast. of  further  division,  migrates  away from the germinal  layer just underneath the mantle. matter  of  the  spinal  cord.  This neuroblast, now  It w i l l  From  their  The  formation  differentiation running  the  of  the  length  of  these  neuroblasts  position  of  the  nerve to  around  the  embryonic  cord and  paths  life,  the developing  the  nerves  by  these  traced  elongate. first  the muscle causes a rapid  motility  of  the  chemosensitivity, desensitization  of  animal  elaboration the  the  motor  cells  Outgrowths  from  these  Later, other During  axons normal  The  development of the  increases the  with  muscle mass extends further  nerves.  of  nerve  of  most  of  subneural  extrajunctional membrane  from  grow along  the  development  the  contact of the nerve subneural  (postsynaptic membrane and associated AChE) (Burnstock the  central  As the animal grows during  nerves reach the muscle at the myotube stage. with  begins  column  cord.  neuroblasts form the axons of motor neurons. early  a  the  be the f i r s t  fibres  form  spinal  to form a  eventually become the white  canal, the neuroblasts send out processes which w i l l fibres.  layer  incapable  the  et a l , 1983).  As  localization  of  apparatus are  apparatus  related  and to  the the  -3activity  of the neuromuscular j u n c t i o n .  visible  on  the  innervation  has  embryonic been  muscle,  completed.  As soon as the end plates are  the  characteristic  Vrbova,  Gordon,  and  pattern Jones  of  (1978)  suggest that the a c t i v i t y of the muscle desensitizes the extrajunctional muscle membrane to ACh. the  nerve  synaptic  stimulates cleft.  Within  the end plate region, ACh released from  the release  These  enzymes  of proteolytic  then  digest  enzymes  into  the superfluous  the nerve  terminals causing them to retreat from the end plate region.  Wallerian Degeneration The  cell  synthesis  body of a neuron  i s the major  site  and i t i s the transportation of material  of macromolecular produced  in this  region and sent to the nerve terminal that i s c r i t i c a l f o r the synthesis of  various  substances,  transmission. Wallerian  like  neurotransmitters,  and  finally  The separation of the axon from the c e l l  Degeneration.  This obviously  neural  body results in  removes the n u t r i t i o n a l  supply  to the nerve terminal, a job normally accomplished by a slow transport system subunits shipping  (1-5 mm/day), which  also  of neurofilaments, membranous  transmitter Consequently, transmission  and a fast  organelles  synthesis  and  without continues  tubulin  and  and the  system (400 m/day),  membrane  transmitter  as long  actin,  transport  f o r normal  secretion,  further only  transports  turnover,  f o r axonal synthesis,  for  metabolism. neuromuscular  as the remaining transmitter lasts  (Burnstock et a l , 1983). Microscopically, r e s u l t of axotomy.  changes  can be seen  within  There i s a swelling and a migration  to an eccentric position and an increased  nuclear  Also apparent i s the disappearance of the Nissl constitutes  the c e l l  clusters  of  ribosomes  and  body as a  of the nucleus  and nucleolar  size.  substance which normally  ordered  arrays  of  rough  -4endoplasmic indicates  reticulum (rER).  that  a  change  chromatolysis may final  stages  of  disorganization final  be  a response  the  altered  in protein  Wallerian of  The  appearance of the ribosomes  synthesis may  be  occurring, thus  to help regenerate the lost axon.  Degeneration  neurotubules  involves  and  the  the  dissolution  neurofilaments  disintegration of the axons and t h e i r phagocytosis.  proliferation nutritional maintain  of  Schwann  supply and  cells  is  also  apparent.  and  with  the  An  increased  a  decreased  With  the loss of mitochondria the axon can  i t s necessary structural  The  no  longer  c h a r a c t e r i s t i c s in order to carry out  i t s function (Burnstock et a l , 1983). Reinnervation  and  regeneration are f e a s i b l e  can i n i t i a t e s t r u c t u r a l , metabolic and the  repair  of  cellular  damage and  because the  functional  the  cell  soma  changes necessary for  regeneration  of the  lost axon  (Vrbova et a l , 1978). In addition to be  an  altered  to the changes with the ribosomes, there also appears content of neurofilaments.  neurofilaments, neurotubules, triplet  protein) and  proportions  than  neurofilament microtubules  and  neurofilaments. state  similar  growth cone and microtubules  and  microtubules  protein  the  is a  with  When  microfilaments This  to  actin-containing  neurotubules.  triplet  The  sign  embryonic  the  an  neurofilaments  axon  is  that  increases  relative  where  the  in  the  to  number  the to  greater  amount  the  dedifferentiation  the newly formed axon i s composed  cut axon becomes a growth cone.  cut  so  of  axon contains  filaments (neurofilament  decreases  neuron  microfilaments.  normal  In a sense, the proximal  of  number of  an  immature  cytoskeleton of almost  of  entirely end  Burnstock et al (1983) suggest  the of  of the that the  growth cone in regenerating axons arises s p e c i f i c a l l y as a result of the reorganization  of f i b r i l l a r  proteins which  ends of cut axons during the  accumulate  latent period before  in the  proximal  regenerative axonal  -5elongation  (See  reasonable mammals  to  would  necessary tubulin  section  on  suggest  be equal  that  the  to the rate  to make actin  and actin  regeneration).  of  i t seems  regeneration  in  of transportation of the proteins  and t u b u l i n ,  i s appropriate  rate  Therefore,  or 3-4 mm/day.  f o r growth since actin  The increase in i s required f o r  the m o t i l i t y of the growth cone and for axonal elongation.  Regeneration of the Nerve The  turning point  contacts the muscle. of  i n the regenerative  process  begins when the nerve  As mentioned, axotomy promotes the d e d i f f e r e n t i a t i o n  the neurons resulting i n the formation  of a growth cone.  The contact  of nerve with muscle halts that growth and promotes the maturation  of the  nerve which leads to synaptic transmission. In  embryogenesis,  property  the growth  of the neurons and elongation  supply of material from the c e l l for  a guiding  part  of  this  accumulate.  body.  the Schwann  I t i s these  cells  i s an  inherent  proceeds as a consequence of the The branching  nerve fibres  search  motor end plate and synaptic is critical  divide  and  fibroblasts  begin  to  structures that help bridge the gap between the Their mechanical guidance causes the nerve  grow i n the proper d i r e c t i o n .  contact  initially  structure to enable them to find the peripheral stump. As  central and peripheral stumps. to  potential  The motor axons then reach the o r i g i n a l  transmission  begins.  The nerve  to muscle  to regeneration, without i t the diameter of the axon  remains small and therefore conduction  velocity  i s reduced  (Burnstock et  a l , 1983). Covault, Cunningham, and Sanes special  matrix-bound  the synapse. denervated  (1987) have suggested  the presence of  factors which a i d i n stimulating axonal growth near  Their results were derived from the study of innervated and  muscles  with  the l a t t e r  releasing large  quantities of these  -6factors  that  stimulate neurite outgrowth from cultured neurones.  u n l i k e l y that pools of these factors can be maintained;  It is  therefore, i t most  l i k e l y i s the changes in the surface of the denervated muscle fibres that is  recognized  molecules  by  the growing axons.  (NCAM) and  Increases  in neural  sulfate  proteoglycan  laminin-heparin  been noted i n denervated muscles (which also have an of n e u r i t e s ) .  The  site.  extensive  studies involving  Buller,  Eccles,  to  fast  reinnervation; contractile  and  Eccles  complex  have  increased outgrowth  and  slow-twitch  of  denervation/regeneration  have  conducted  are  altered  discovery  according  to  the  In a d d i t i o n , they found that a f t e r axotomy the  consequently, speeds  (1960b)  cross-reinnervation leading to the  properties of the muscle  nerve that supplies them. motoneuron  adhesion  result i s that 80% of the neurites contact the original  synaptic  that c o n t r a c t i l e  cell  as  the  muscles  the  axon  muscles.  returned size  to  normal  changed  Further  so  detail  after  did on  the these  studies i s provided in a subsequent section.  MUSCLE DEVELOPMENT AND GROWTH  C e l l s within the embryonic mesoderm represent the pool of c e l l s which proliferate The  to form the most primitive  distinguishing  of  muscle  features of a myoblast c e l l  the a b i l i t y to synthesize and  cells,  are:  another  cell  division,  1978).  Upon  completion  many  proteins  synthesized  is  and  specific creatine  of for  they  fuse  division, striated  with the  each  phosphokinase,  other  followed  The by  first the  of molecules of a c t i n , myosin  At  development, ACh  this  same  point  in  receptors  are  never enter  (Vrbova  begin  assembly into myofibrils  with  tropomyosin into  cycle and  myoblasts  muscle.  myoblasts.  they are c e l l s  assemble myosin, actin and  m y o f i b r i l s , they eventually withdraw from the c e l l  the  to  et a l ,  synthesize  such  enzyme  synthesis  and  and  tropomyosin.  synthesized  and  -7incorporated  into  the membrane.  The  myoblasts then elongate and fuse to  form myotubes (Vrbova et a l , 1978). Unique to myoblasts  and  myotubes are special c e l l  (NCAM's), the  modulation  of which  muscle  modelling  (Kelly  tissue  matures, the a b i l i t y NCAM's,  declines  contributes  and  they  to adhere  are  d i f f e r e n t i a t i o n of secondary c e l l s . formation  of  additional  fibres.  in a  significant  Rubinstein, 1986).  of the fibres  until  adhesion molecules  no  to  each  longer  As  way  the  to  animal  other, v i a these  able  to  guide  the  This results in the cessation of the The  remaining  myoblasts  then are  left  only with the option of fusing with other primary or secondary c e l l s . Just before f u s i o n , a number of metabolic changes occurs including an increase  in the  utilizing  number of mitochondria, the  glucose and  glycogen  changes are made in preparation synthesis myotube  further at  increases out  of  increases  this  phase  has  and  development of  for contractile  when a  the  appearance  the  low  the  cell  improves,  and  the  begin  membrane  with time as the a b i l i t y of the c e l l myotube  enzymes for  rER.  A l l these  a c t i v i t y , however, their  myotubes resting  of  contracting. potential,  The which  membrane to transport can  produce  and  Na  +  conduct  e l e c t r i c a l a c t i v i t y along i t s entire length. The muscle i s able to develop only to a certain innervated.  It i s at the myotube phase in normal  point without being embryonic development  and in regeneration that the nerve seeks and attempts to connect with the myotube. of  Motor axons are f i r s t  gestation.  between  The  days  recognizable gestation.  14  first  and  synapses By  the  seen in rat trunk muscle by the 12th day  physiological  15 seen  (Vrbova by  eighteenth  et the  day,  signs  of  innervation  a l , 1978), fifteenth  polyneuronal  are  seen  with morphologically  or  sixteenth  innervation  day  of  reaches  a  maximum a f t e r which the number of nerve terminals decreases until weeks a f t e r birth only 1 nerve terminal i s l e f t at each end p l a t e .  2 or 3  -8Muscle Fibre Type Development At  birth  reticulum the  very  (SR)  simple  and  resting  the  membrane  contraction  times  are  moderate oxidative These  neonatal  (Burnstock  et  complexes  are  t-tubules.  At  potential,  the  a l l quite  enzyme  muscles  a l , 1983).  this  With  a  between  sarcoplasmic  stage in normal  development,  of  shortening  In a d d i t i o n , there  and  show  the  speeds  slow.  activity  also  formed  a  low  very  further  myosin  high  ATPase  the  the  i s a uniform  resistance  maturation  and  activity. to  fatigue  "basic"  muscle  begins to develop s p e c i f i c q u a l i t i e s enabling one to d i f f e r e n t i a t e between fast and  slow muscle responses.  have a well developed SR t-tubules  at  the  triads  permits  times.  Other  Z-line and labile  A-I  the  The  f u l l y mature fast-twitch white fibres  network which in turn forms many triads with the  junctions  rapid  of  sarcomeres.  sequestering  characteristics  of  a  of  The  calcium  fast-twitch  abundance  for short muscle  of  these  contraction  are:  a  narrow  a high myosin ATPase a c t i v i t y (which i s stable in a l k a l i n e but  in  acid  conditions,  shortening).  It  a c t i v i t y , and  is fatigable.  potentiation,  a  large-diameter velocity.  also contains  shorter  low  with  its  oxidative and  high  axon which enables them to maintain  Conversely  rapid  rate  glycolytic  of  enzyme  Fast twitch fibres also exhibit post-tetanic  after-hyperpolarization and  slow-twitch  small-diameter axons which long periods  corresponding  are  fibres  r e a d i l y excited  of time but with low  muscle very fatigue r e s i s t a n t .  red  A  f i r i n g rates.  are a  innervated  greater  are  and  which  by  a  conduction  innervated  by  discharge  for  These q u a l i t i e s make the  slow contracting muscle, l i k e  soleus,  produces a smaller maximal tension as predicted from the r e l a t i v e l y fewer number of t r i a d s , the high oxidative capacity and  the lower myosin ATPase  a c t i v i t y as compared with fast-twitch muscle (Vrbova et a l , 1978). As  mentioned,  complete  maturation  into  fast-twitch  or  slow-twitch  muscle f i b r e s i s c r i t i c a l l y dependent on the success of innervation at the  -9myotube stage. glycolytic clear  At b i r t h , EDL and SOL are composed of Type I I 2 oxidative  fibres;  difference  variety  of  however, by two weeks of age between  enzymatic  levels  fast-oxidative-glycolytic (SO), depending fibres  on  two  also  fibre  types  evident.  (slow  They  (FOG), f a s t - g l y c o l y t i c  their  differentiate  the  in the  from  metabolic  and are predominate in the SOL.  and  have  fast)  been  (FG) and  properties.  the Type  rat there  By  with  labelled  a as  slow-oxidative  adulthood,  I I ^ oxidative-glycolytic  In EDL, the Type  is a  Type  fibre  1^ pool  I I 2 glycolytic  fibres  d i f f e r e n t i a t e from this same pool (Kelly and Rubenstein, 1986). The myosin component of muscle i s composed of fast and slow isoforms. The myosin molecule consists of six subunits. myosin  heavy  chains  plus  3 pairs  of  light  Fast muscle contains  chains  (LClf,  LC2f,  two  LC3f).  While slow muscle contains 2 heavy chains plus 2 pairs of LCls and LC2s. During the development of fast-twitch muscle, the type of myosin changes. 7-11  Fetal  days  Finally  of  by 21  muscle age  contains  the  muscle  MHC-emb, LClemb-LClf, and contains  MHC-neo, L C l f ,  isoform  LC2f, but by  LC2f  and  LC3f.  days of age, or adulthood, the fast-twitch muscle contains  MHCF, L C l f , LC2f, and LC3f (Redenbach, 1985).  Studies by Dhoot and Perry  (1982) have revealed slow and fast forms of tropomyosin and troponin I, T and C.  At b i r t h , the fast form prevails  but during d i f f e r e n t i a t i o n  some  of the f i b r e s change, switching to the synthesis of the slow forms. It i s apparent that in the early muscles  develop  differentiation we SOL  call  as  certain  "fast-twitch"  slow-twitch  stages of embryological muscles  but  muscles acquire q u a l i t i e s muscle.  On  that  with  development time  characteristic  of what  the other hand, the slow muscles  of the rabbit begin as slow contracting muscles and  then with  they become even slower (Gutmann, Melichna, and Sycovy, 1974).  and  like time  -10Denervation and Muscle Development Buller et al (1960a) conducted studies on,the d i f f e r e n t i a t i o n of fast and  slow muscle  of the cat hind-limb.  Their  results  muscles in the newborn kitten were slow (contraction 1/2RT=80  msec)  but  that  within  a  d i f f e r e n t i a t e d into the f a s t form. muscle  of 4 week old kittens was  few  The  the  muscles  contraction  times  were 80 msec, at 8-16 weeks 25 msec with 1/2RT=18 msec, and by  old k i t t e n s ,  weeks  (fast)  of  fast muscle  whose muscles  Buller et al then transected  should have been well  that age, at the f i r s t or second lumbar segment. 13  some  faster than the slow muscle of 28 week  adulthood CT=27 msec and 1/2RT=18 msec. day  time (CT): 80 msec,  In addition, they found that the slow  old cats (CT=70 msec, 1/2RT=75 msec). at birth  days  indicated that a l l  post-surgery  and  SOL  revealed  (slow)  that  were  33  the and  differentiated  at  Study of the muscles at  contraction 32  1-4  msec  time  for crureus  respectively.  The  half-relaxation time for the two were 24 and 25 msec respectively (Buller et a l , 1960a). fast  and  These results as compared to values from normal  slow  differentiation weeks.  muscle of  suggest  slow  muscle  that  there  is  a  failure  with  normally occurs  between  5  the  and  16  In fact the slow muscles resembled normal fast muscles much closer  in terms of t h e i r physiology. fast  that  developing  muscles  seemed  to  be  evidence f o r the suggestion largely affected  by neural  On the other hand, the d i f f e r e n t i a t i o n unaffected  by  transection.  that the d i f f e r e n t i a t i o n influences  emanating  of  This slow  of  provides muscle i s  from the spinal  cord.  A  follow-up study conducted by Buller et al (1960b) looked at the effect of neural  influence  They conducted longus  (FDD  on  the speeds of contraction  cross-reinnervation as  well  as  doing  studies  of slow and  with SOL  self-reinnervation  and  fast  flexor  muscle.  digitorum  experiments.  Their  results indicated that the speed of contraction of the SOL i s accelerated when reinnervated by FDL nerve f i b r e s , whereas there was  a slowing of the  -11contraction  speed in FDL muscle reinnervated by SOL nerve f i b r e s .  transformations  in  twitch  contraction  characteristics  also  These  held  for  tetanic contraction. Cord i s o l a t i o n studies (L2-S2) provided additional et a l , 1960b).  from  together  normal.  provide  influence  (Buller  Here i t was found that the late phases of d i f f e r e n t i a t i o n  of slow muscle were altered differ  information  ( i n a young kitten) yet fast muscles did not  The cord  evidence  i s required  isolation  that  some  studies type  and  of  transection  neural  for the proper development  studies  differentiating  of slow muscle.  This  substance (nerve trophic factor) travels down the axon of the motoneuron, traverses the neuromuscular junction and then enters the muscle fibres to change t h e i r c o n t r a c t i l e c h a r a c t e r i s t i c s (Davis and Kiernan, 1980). Hatano, observed  a  Suge, Ikuta, Miyamoto, Yoshioka, and change  denervation.  in the  shape  of the  in size  more than Type  denervation.  dehydrogenase checkerboard approximately  fibres  Type II f i b r e s were also  have after  (SDH)  The  muscle  a c t i v i t y which  appearance the  red  third  of  the  week).  seen to  I f i b r e s , weighing 50% of the control  weight by the second week, 30% by the fourth after  muscle  (1981)  Instead of being a regular spindle-shape, the fibres assumed  more of a rod shape a f t e r denervation. decrease  individual  Hiramatsu  fibres  can  be  also  above  types  15-20% 3 months  lost  visualized  different The  week and  of  changes  as  their a  succinic  loss  muscle  of the  fibres  became  even  (by more  extensive by one month post-neurectomy but with a peak at approximately five  months where the muscle fibres  fragments (Hatano et a l , 1981).  appeared merely as  debris  and  tiny  DENERVATION  Denervation  of skeletal muscle by cordotomy, nerve section, and  nerve  crush results in s t r u c t u r a l , metabolic and functional changes.  a) Morphological Changes: One' of the e a r l i e s t  changes to be  appearance of the n u c l e i . rounded and  swollen  and  Two  seen i s the altered  their  chromatin  dispersed.  position in the centre of the muscle f i b r e  The  number of s a t e l l i t e As  atrophy, f i r s t interior  of  cells  denervation beginning the  They also  (Burnstock  take up a  et a l , 1983).  increase (Schultz, 1984), as does the  time i s lengthened, the muscle f i b r e s at the periphery and  muscle  and  weeks after denervation, the nuclei become  new  content.  position  fibre.  then  Alterations  mitochondria and SR can also be seen.  finally  in  begin  reaching  the  DNA to the  myofilaments,  The SR and the mitochondria follow  the same pattern of change, f i r s t they increase in size or volume and  then  they both shrink.  b)  Metabolic Changes Generally speaking, substances used for energy production decrease  the enzymes to breakdown these substances  also change thus resulting in a  greatly altered metabolic state of the muscle (Leung, J e f f r e y , and 1984), after  Specifically,  glycogen  content  within  denervation whereas within the rabbit  decreases. content  Several of  sialyl-galactosyl of  the  +  +  Na /K -ATPase  inositol  substances  hexosamine,  are  known  hexose,  and  rat muscle  i t first to  Rostas,  decreases  increases and  increase,  membrane-bound  including sialic  then the acid,  and  N-acetylglucosaminyl  transferase and the a c t i v i t i e s  and  guanylate  The  cyclase.  turnover  of  phosphatidyl  i s also increased but only small changes are seen in phospholipid  -13composition;  however,  the  membrane  content  fucosylglycoprotein transferase are known to decrease.  of  fucose  and  Leung et al (1984)  suggest that the changes in the glycoprotein component of membranes after denervation  are to enable the muscle to respond to i t s new physiological  environment. denervation  The major  changes  i n these  glycoproteins  i s that  after  they contain more carbohydrates or they have more sugars added  to the same carbohydrate chain. c)  Changes i n E l e c t r i c a l Properties There are several  and  muscle a f t e r  permeability  changes i n the e l e c t r i c a l  denervation.  of the membrane  There  appears  properties of the nerve  to be an increase  in the  to Na , manifested as a decreased  resting  +  membrane potential and therefore, an altered membrane e x c i t a b i l i t y . result  of these  membrane  changes  i s a slower rate of r i s e  The  and smaller  overshoot of the action potential as well as the appearance of spontaneous asynchronous contractions  (Hatano et a l , 1981).  action potential i s lower as well et al (1983) have also found that constants and  (Kirsch and Anderson, 1986).  of the  Burnstock  the resistance and the time and space  of the membrane just outside  SOL of the cat were increased  The peak height  the nerve/muscle junction of EDL  in the f i r s t few hours a f t e r sectioning  but l a t e r t h i s spread to the whole muscle f i b r e .  d)  Changes i n Acetylcholine S e n s i t i v i t y Denervation  in  produces an increased  sensitivity  the number of cholinergic receptors.  This  to ACh v i a an increase  sensitivity  also tends to  spread over the entire length of the muscle as opposed to being to a small junctional region.  localized  -14e)  Changes in the Motor End Hatano  effects  et  of  inhibited. slightly  al  Plate  (1981) conducted  degeneration  on  the  After 1 week, the end  decreased  histological  i n t e n s i t y as  end  in s i z e .  plates  to  when  look  at  the  regeneration  was  plates were c l e a r l y outlined but with a compared to the  week post-denervation, the i n t e n s i t y was reduction  studies  controls.  moderate and  At  the  there was  third  a distinct  By the fourth week, the end plate had changed from i t s  c h a r a c t e r i s t i c round shape to a rather longer the outline of the end  configuration.  Thereafter,  plate became increasingly d i f f i c u l t to see until 3  months post-denervation, at the peak of the changes, when the outline not  visible  at a l l . The  disorganized studies well  and  there  internal structure of the end  was  a  at 5 months revealed  as a population  however,  the  of end  internal  poor an  of  increase  folds and  of  in the number of end  these  "new"  they s t i l l  totally  enzyme a c t i v i t y .  plates which reacted  structure  normal internal synaptic  return  plate was  with  was  Further plates as  normal i n t e n s i t y ;  end  plates  lacked  maintained t h e i r  the  lengthened  appearance.  f)  Changes in C o n t r a c t i l e Properties B r i e f l y , there appears to be an increased  tetanus  tension,  post-tetanic  a  prolongation  potentiation  of  TTP  (PTP)(Davis,  Lieber, Friden, Hargens, and  Feringa  year  transection  after  thoracic  force-generating  capacity  this length of time. of contraction and the of  increased SOL  and  and  1/2RT  Bressler,  and and  (1986) looked at  contraction  (T9).  They  a  loss  of  Jasch, rat SOL  found  and  EDL 1  that  EDL s  by  50%  but  I n t e r e s t i n g l y , the  i t was  able  1  speed remained unchanged a f t e r  Soleus, on the other hand, showed an  frequency.  the  1988).  increased  relaxation times, as r e f l e c t e d in the decreased TTP  fusion  decreased  spinal  twitch tension, a decreased  cross-sectional  to generate the  same  rate and area  absolute  tension; area.  therefore, the SOL was able to produce a greater force per unit  I t i s also apparent that a f t e r cordotomy (and a year latent period)  the c o n t r a c t i l e  properties of slow muscle fibres  fast muscle f i b r e s .  Morphometric analysis  become more similar to  supports  this  finding  (Kelly  and Rubenstein, 1986). Morphometric analysis reveals that one year a f t e r almost a complete important  cordotomy there i s  conversion from Type I to Type II muscle f i b r e s .  to note  muscle  fibre  atrophy  occurred  i n both  It i s  EDL and SOL;  however, Type II atrophy reached the same extent in both muscles but Type I atrophy  i n SOL was much more  severe.  One must remember that  these  results were gathered 1 year a f t e r cordotomy whereas most of the previous information has focussed on the f i r s t month or two a f t e r denervation.  REGENERATION  Experimental  procedures  such as cordotomy, nerve section  cause degeneration changes i n fast and slow muscle.  and tenotomy  With time however, a  process takes over which serves to regenerate the damaged muscle and bring it  back to normal  functioning.  Carlson (1973) has described 2 modes of  muscle regeneration. They are the epimorphic mode of regeneration and the tissue mode of regeneration. The  epimorphic  differentiations muscle f i b r e s  mode  of  regeneration  involves  a  series  of  leading to blastemal formation before the regeneration of  occurs.  In this  case, development  closely  resembles the  phases of embryonic development, with the blastema being the key factor in the morphogenetic c o n t r o l . The muscle  tissue  mode  and follows  of regeneration which the degeneration  occurs  i n damaged mammalian  of the sarcoplasm  i n the area of  injury.  Subsequent  surface  of the basement membrane of the o r i g i n a l  myoblasts muscle  to t h i s , myoblasts appear associated  then  fibres  the o r i g i n a l Allbrook  differentiate  into  myotubes.  with  muscle  Most  of  the inner  fibre.  These  the regenerated  eventually become arranged i n a fashion similar to that of muscle  (1962)  by following  the long  conducted an interesting  axis  of the o r i g i n a l  series  fibre.  of experiments where he  rotated a minced muscle so i t was oriented at right angles to i t s original axis. and  When the myotube lengthened past i t s basement membrane i t turned  continued  provided factor  to grow p a r a l l e l  with  the long axis  of the muscle.  evidence that tension along the length of the muscle i s a major in  reorganizing  surrounding  the  environment  also  minced poses  muscle  for  limitations  regeneration.  because  dense  the new  fibres  to cross.  As mentioned,  the blastema  morphogenetic control  i n epimorphic regeneration;  mode of regeneration  the physiological  the muscle  has with  The  connective  tissue present i n ,the regenerating area proves to be a d i f f i c u l t for  This  boundary  i s the key  however, i n the tissue  relationship or constraints  i t s surrounding tissue  that  i s presumed to be the major  morphogenetic f a c t o r . Ali  (1979) examined the way myotubes are formed  evidence  f o r 2 modes of myotube  theory, i s based on the finding damaged muscle f i b r e s . the surviving form  of  disrupted. stems  from  sarcolemmal  tube.  of sarcoplasmic  healthy portion  predominates  The second the  The f i r s t , buds  the continuous  growing  from the  The nuclei of these "buds" possibly originate from  and presumably  regeneration  formation.  and his work reveals  form  discovery These  when  of the muscle  the sarcolemmal  of regeneration, of  mononucleated  mononucleated  cells  inside cells  This  sheaths are  the "discontinuous  or s a t e l l i t e  into myoblasts which then fuse to form myotubes.  fibre.  the  form" intact  differentiate  -17The Regenerating Muscle Fibre The  process  of regeneration  f i b r e s into an injured area. must  begins  with  the sprouting of the new  In order f o r this to occur, the damaged area  be cleared of necrotic  sarcoplasm, myofibrils  and other debris by  phagocytic c e l l s (Hudson and F i e l d , 1973). The one,  success  the  of the regeneration  degree  regeneration.  of  injury  i s dependent on many f a c t o r s .  poses  a  serious  critical  to  complete  I f the muscle fibres are in an advanced stage of atrophy,  the chances of complete recovery are not as good. satellite  threat  For  cells  or myoblasts  i n regeneration.  from  Also  Second, the presence of  which the muscle f i b r e s  of importance  originate i s  i s the i n t e g r i t y  of the  sarcolemmal sheath and the endomysial framework of the necrotic segment so that the growth of the new f i b r e s pathways.  However, the most c r i t i c a l  capacity of each individual response,  can be directed  both  cells  a c t i v a t e d , the former to give r i s e  hours  of  the  During the regenerative  and the muscle  fibre  nuceli  to myoblasts and the l a t t e r  development of the sarcoplasmic buds. ninety-six  the anatomical  factor in muscle regeneration i s the  f i b r e to regenerate.  the s a t e l l i t e  along  become  to start  Muir (1970) has divided the i n i t i a l  degenerative/regenerative  process  into  three  phases:  Stage 1:  24-72 hours a f t e r injury:  At this p o i n t , one can see a pool between occurs  the undamaged first  myoblasts mitosis.  with  myofibres  the formation  and subsequently  these  of mononucleated myoblasts  and the necrotic of a new  new  tissue.  generation  myoblasts  become  This  forming process  of mononucleated more abundant by  -18Stage 2: The cells  72-96 hours after injury: new  myoblasts then fuse with  (myotubes).  chromatin  contains  to  form  Carlson (1973), using electron and  at the myotubes and discovered  scattered  other  multinucleated  It i s only in the myotube phase that myofibrillogenesis  becomes detectable. looked  each  numerous  and free  a  very  a very large nucleus with loosely  prominent  ribosomes  l i g h t microscopy,  and  nucleolus.  consequently  The  stains  cytoplasm basophilic.  These ribosomes are typical of active secretory c e l l s and in regenerating muscle  they  production. individual  can  be  seen  during  They therefore myofilament  the most active  seem to be involved  proteins.  The  rER  phases  of  myofilament  i n the formation of the  and Golgi  may  be  involved in  protein synthesis and transport of materials used in building basement membrane which surrounds the myotube. muscle,  the  Golgi  apparatus  i s quite  up the  new  In normal mature striated  small.  Mitochondria with  well  defined c r i s t a e are also evident in the cytoplasm. Stage 3.: This  96 hours + stage  myofibres.  involves  the  gradual  Here myofibrillogenesis  cross-striations  of  skeletal  previously, the complete motor innervation.  i s more  muscle  development  Axonal  maturation  prominent  become  of  regeneration  and  muscle  occurring  in  the  myotube  stage.  the As  depends  into  typical mentioned  upon proper  proceeds at a rate of 3.2  mm/day (Barker, Scott and Stacey, 1986) with the actual to  myotubes  visible.  the muscle itself  of  Lowrie  contact of nerve and  Vrbova  (1984)  suggest that the functional recovery of the muscle occurs by approximately 11 days a f t e r injury (with s c i a t i c crush).  The myofilaments f i r s t  appear  in the subsarcolemmal cytoplasm.  Even at this early stage, Z-, A-, I-, H-  and M-bands are c l e a r l y v i s i b l e  and the sarcomeres have already attained  the  lengths  increase,  seen i n adult muscle. new  filaments  are  In order f o r the mass of muscle  added  to  the  periphery  of  the  to  young  myofibrils;  consequently,  there  i s a gradient of m y o f i b r i l l a r s i z e , with  the thickest and oldest fibres located c e n t r a l l y and newly formed ones  located at the periphery (Allbrook, 1962).  in embryogenesis, vesicular with maturation  one  elements and  nucleolus  has  the SR  begin  As i s seen  to take  shape  and  sees a migration of the c e n t r a l l y placed nuclei to the  periphery of the muscle f i b r e . the  the thinnest and most  faded  has completed maturation  and  Once the  nucleus  has  in  size,  the nuclear chromatin condenses, the myotube  into a young muscle f i b r e .  is the fact that this new  decreased  Most important though  f i b r e closely resembles the o r i g i n a l f i b r e which  was damaged. According  to A l l brook (1962), 3 weeks a f t e r injury muscle regeneration  is complete as suggested by the normal appearance of the muscle excluding a  slightly  increased  possible to s t i l l The  content  of  interstitial  see macrophages and  collagen.  I t may  also be  fibroblasts.  actual return of function to the muscle a f t e r nerve crush appears  to occur 10-12  days a f t e r the operation (on 5 to 6 day old rats) (Lowrie,  Krishnan,  Vrbova, 1982).  months  and  post-operation  tension  production  developed  only  yet  about  examination, the  the  SOL  the 50%  This  same study  recovered  also  almost  revealed  completely  that by 2  in  terms  fast muscles, t i b i a l i s anterior (TA) and of  the  tension  fast muscles contained  of  the  controls.  Upon  fewer muscle fibres and  of EDL,  close  the ones  there were, were ones with high levels of oxidative enzymes; however, the quantity  of  Therefore,  motoneurons  nerve  crush  muscles which are  remained  in very  young  similar  to  rats  causes  pre-operative  not caused by motor neuron death.  changes  in  counts. the  fast  When nerve crush i s  conducted on adult r a t s , there i s a complete recovery of muscle function. Several  theories  have  been  postulated  to  explain muscle f i b r e  loss  a f t e r regeneration has begun with many people supporting the concept that the  new  fibres  are  smaller  in  stature because of the  inability  of  the  -20motoneuron  to  support  a l l of  i t s branches.  This  would  result in  incomplete reinnervation and a decreased muscle f i b r e weight or on actual loss  of  some  hypothesis muscle.  fibres.  Lowrie  to explain  Initial  et  the results  al  (1982)  of degeneration  presented  and  another  regeneration of  neural development produces motor units that f i r e at low  r a t e s , but with time some of these motor units firing.  have  increase t h e i r  rates of  The muscle then develops special c h a r a c t e r i s t i c s to suit the mode  of stimulation i e : SOL-slow contracting and f a t i g u e - r e s i s t a n t . Upon exposed be  reinnervation,  the new  to the high f i r i n g  the cause  of t h e i r  immature  fibres  of EDL (fast) would be  rates of the old fast motoneuron.  degeneration.  Obviously this  This  would be not be a  problem f o r SOL which i s always innervated by a slow motor u n i t . to  this  i s the finding  could  Contrary  by Davis, Bressler, and Jasch (1988) where the  addition of a nerve extract to a denervated muscle aids i n the development of the p a r t i c u l a r muscle f i b e r type. Lowrie after  and Vrbova  sciatic  nerve  (1984) have crush  looked  at the regenerative  in 5-6 day old r a t s .  They found  recovery i n SOL and EDL by the 18th day a f t e r surgery; SOL  and EDL only reached  respectively.  By 21 days, EDL produced  tension which remained the  at this level  other hand, the tetanic  normal  values  contractile  two  remained  with  denervation and  contraction  which  affected Vrbova  after  changed  so f o r several  speed  Lowrie  the  tension  months  properties  reinnervation  the  55% and 70% of normal  Ca  ++  this  produced  tetanic  Other During  parameters the early  times  Prolonged relaxation  system also  tension values tetanic  by the SR.  so as to slow reported  On  by SOL reached 75% of  relaxation  i s sequestered  (1984)  however, at day 18  f o r the rest of the experiment.  as w e l l .  months.  functional  only 40% of i t s normal  surgery.  and  response  were time  and  depends upon  I t may  in  1  EDL s  stages of slower  the uptake  changes  of  its  be that of  the  Ca  ++  .  fatigue  c h a r a c t e r i s t i c s of EDL.  At day 18, the muscle was as fatigable as normal  muscle, but with age normal muscle becomes more f a t i g a b l e .  The  opposite  happens with reinnervated EDL, i t becomes more f a t i g u e - r e s i s t a n t . these physiological and  reinnervation  fibres. after its  in young  fairly  possibly  lost  loses  a  with  large  two-thirds of t h e i r  characteristics.  to account f o r the altered  stained  because  denervation  number of muscle fibres  by 1 month  This huge loss i s not seen in SOL which accounts for  consistent  tetrazolium  change  animals EDL  In f a c t , they have  nerve crush.  helped  properties  A l l of  reductase  stain  both  Muscle  fatigue EDL  type  distribution  characteristics.  and  with only patches of denervation  fibre  SOL  appeared  at day  18.  With a NADH  more  uniformly  Not present in the  reinnervated EDL but which normally makes up 35% of the f i b r e  content of  normal fast muscle were the pale-staining g l y c o l y t i c f i b r e s . These  same nerve crush  experiments conducted in adult  d i f f e r e n t results (Navarrete and Vrbova, 1984) induces only temporary changes in motoneuron  .  rats  produced  Nerve crush in an adult  activity.  G i l l e s p i e , Gordon,  and Murphy (1986) using r a t s , sewed the nerve to the l a t e r a l gastrocnemius (LG) muscle and then looked at the physiology of the muscle 4 to 14 months later.  The contraction speed of reinnervated LG was similar to normal but  the relaxation SOL. LG.  This  rate declined  toward  suggests an increased  rates  of relaxation  slow muscle f i b r e  in the  control  content in the fast  In contrast, the reinnervated SOL had an increased relaxation speed,  suggesting a larger proportion of fast muscle fibres than normal.  Also,  both muscles exerted less tetanic force than the controls (LG 45% and SOL 61%  of  normal  values).  Thus,  the  reinnervated  muscles  revealed  c h a r a c t e r i s t i c s intermediate between the normal fast LG and the slow SOL. Albani old r a t s . in  minced  and Vrbova (1985) conducted a series of experiments on 4 week They studied the physiology and the development EDL  and SOL  which  had been  re-introduced  into  of end  plates  i t s own  bed or  placed  into  the other muscles' bed  into SOL bed or EDL bed. twitch  and  tetanic  became more also  Their  tension  results  after injury).  with  that  were similar  to others' in that  values decreased and the regenerated muscles  fatigue-resistant.  correlated  i e : EDL into EDL bed or SOL bed, SOL  The  timing  of Lowrie and  of muscle  Vrbova  function  (1984)(i.e.  recovery  10-12  days  The unique part of this experiment were the results on end  plate development.  The regenerated fibres had more than one end plate and  some end plates had more than one axon terminal.  On EDL, the end  plates  are normally found as a continuous band which stretches diagonally across the muscle when the new muscle develops. end plates  are found.  original  end  plate  running  longitudinally  From EDL fragments, 2 groups of  These are a proximal group, part of the remaining  zone, and along  a  distal  the  group,  regenerated  long  thin  muscle.  nerve If  fibres  no  muscle  fragments are l e f t behind then the regenerated muscle fibres develop their own  end plate zone  located  in the most proximal and d i s t a l  ends of the  muscle. SATELLITE CELLS  S a t e l l i t e c e l l s are mononucleated c e l l s which l i e between the basement membrane  and  the  sarcolemma  of  fibre.  They  Their numbers have been found to increase during the f i r s t  level.  parallel  are  muscle f i b r e .  weeks after which  long axes oriented  normally  structures  postnatal  their  muscle  spindle-shaped  3  with  the  they decrease in number to reach a basal  Evidence has been collected showing the incorporation of  satellite  cell  nuclei  into  (Carlson, 1973), suggesting myoblasts.  Schultz  to the  the  growing  (regenerating)  the d i f f e r e n t i a t i o n  of  labelled  muscle  satellite  cells  fibre into  (1984) conducted cross-reinnervation studies with SOL  and EDL and looked at the effects of cross-reinnervation on the s a t e l l i t e cell  population.  His results indicate that a minimum number of s a t e l l i t e  -23c e l l s are required f o r a successful reinnervation but that increasing the quantity of these stem c e l l s does not necessarily mean that will as  be improved.  nerve supply  mass  In f a c t , i t was apparent that e x t r i n s i c influences such  and stretch are more  in regenerating  population.  regeneration  Schultz  muscle  than  influential  i s the size  (1984) further  suggested  i n determining  muscle  of the s a t e l l i t e  that  cell  the s a t e l l i t e  cells  could be the controllers of the actual rate of muscle regeneration.  MUSCLE BLOOD FLOW  In general, blood vessels run p a r a l l e l  to the muscle f i b r e s ; however,  there i s a difference in t h e i r organization between red and white f i b r e s . The  capillaries  form elongated  loops p a r a l l e l  to white muscle f i b r e s  the transverse branches actually e n c i r c l i n g the individual  fibres.  with  In red  muscle, the loops are of equal length but the longitudinal branches of the network  form  dilations.  sinusoids  the  transverse  branches  have  varicose  Red muscle has a greater c a p i l l a r y network (Hudlicka, 1973).  Long-term Within  and  ischaemia  produces  drastic  effects  two hours a f t e r the onset of ischaemia,  on  skeletal  muscle.  neuromuscular transmission  i s impeded and one can see a disruption of the c r o s s - s t r i a t i o n s within the muscle  fibre.  After  four  hours  of ischaemia,  10% of the f i b r e s are  damaged and by 8 hours 60% of the fibres are damaged (Hudlicka, 1973). rat muscle g r a f t s , the c a p i l l a r i e s In  large  vessels  degenerates. the  details  endothelium  disappears  to find  and  They may use the p e r s i s t i n g t h e i r way to the muscle.  basal  the  24 hours.  smooth  muscle  lamina of the original  Very l i t t l e  of r e v a s c u l a r i z a t i o n ; however, suggestions  interaction  the f i r s t  By two days new blood vessels are seen at the periphery of  "muscle".  vessel  the  breakdown within  In  i s known about the  have  focussed  on an  between the ends of the vascular network and the macrophages  -24that  exist  in  this  area.  This  macrophage-induced angiogenesis  hypothesis  stems  from  evidence  of  (Carlson, 1986).  Hudlicka and Tyler (1986) have summarized blood vessel growth during denervation  and  vascularization Poorly  regeneration.  They  described  how  the  lack  may actually help i n the regeneration of skeletal  vascularized  grafts  grow  faster  than  well  of  muscle.  vascularized  ones.  Before v a s c u l a r i z a t i o n , the Krebs cycle operates through the pentose shunt and  therefore, hypoxia may a f f e c t vascular growth.  regenerated grow  2-3  complete.  Carlson  (1973) using  minced muscle transplants found that blood vessels started to days He  after found  implantation myoblasts  previous to the c a p i l l a r i e s .  and  to be  by  9 days  present  This suggests  vascularization  i n a l l of the sections  that v a s c u l a r i z a t i o n , as long  as i t occurs, i s not the l i m i t i n g factor in muscle regeneration. support f o r this stems from the work of Carlson et al (1981). compared  standard  standard  graft  placing one.  this  grafts  Further  Here they  and nerve-intact grafts of EDL i n the r a t .  involves severing a l l connections  muscle  was  back  A nerve-intact graft  into  A  of the muscle and then  the same muscle bed or into a d i f f e r e n t  involves the same procedure but i n this  the nerve supply to the muscle i s not severed. same sequence of muscle f i b r e degeneration  case  Both groups underwent the  and regeneration but the return  of function to the muscle was faster and greater i n the nerve-intact graft as compared to the standard  graft.  Apparently  i t i s the presence of an  intact nerve supply that can bring a muscle graft i n a r a t almost back to control condition. It  i s apparent  response.  that  age plays  Both the morphological  a  large  role  i n the regenerative  parameters of regenerating  EDL and SOL  in young animals have been closely examined with f a i r l y consistent results produced.  However, detailed physiological  c h a r a c t e r i s t i c s over the course  of the regeneration of EDL and SOL i n the adult mouse remain to be f u l l y  -25described.  Also,  most  of  the  denervation  experiments  muscle consist of immobilizing the whole hind limb. will  make use  only  EDL  will  of  or SOL.  then  undergo  a  unique technique  The an  physiological  whole  following project  that denervates and  muscles, removed at various extensive  The  involving  devascularizes  times  after  analysis  in  surgery, order  to  establish the physiological basis for a mouse model of muscle regeneration.  -26-  METHODS  -27All strain.  experiments  were carried  Denervation  and  out on male mice of the C57BL/6J  devascularization  of  the  fast-twitch  +/+  extensor  digitorum longus (EDL) muscle and the slow-twitch soleus (SOL) muscle were carried out in mice at 4 weeks of age and c o n t r a c t i l e properties measured at  3,6,9, and  12 weeks following  surgery.  In a d d i t i o n , a sham-operated  group was studied at 6 weeks post denervation/devascularization.  DENERVATION/DEVASCULARIZATION  Mice  were  anesthetized  anesthesia was  with  sodium  pentobarbitol  maintained with an ether c u f f .  (50mg./kg.)  and  Each animal was then placed  on a dissecting board and the right hindlimb supported on a perspex block and  secured with surgical  tape.  A small i n c i s i o n  was  made in the skin  overlying the approximate position of the respective muscle (either EDL or SOL).  With  the  aid of a dissecting  planes between t i b i a l i s cut,  anterior and  microscope, the  connective tissue  EDL, or gastrocnemius  thus exposing the desired muscle.  A 5 cm.  and  SOL  piece of unbraided  were 5-0  surgical s i l k was then s l i d under the d i s t a l tendon of EDL or the proximal tendon of SOL opposite  and was  tendon.  gently shimmied along the b e l l y of the muscle to i t s  This  procedure  supply to the individual The  animals  surgery and  removed  both  the  nerve  and  the blood  minutes  following  muscle.  regained  were observed  consciousness  within  30  to run about the cage quite e a s i l y immediately  a f t e r awakening.  MUSCLE DISSECTION  At 3,6,9, and 12 weeks post-surgery, animals were k i l l e d dislocation  and  their  body weights were recorded.  by cervical  The right leg of each  -28animal was removed and pinned to a cork board and bathed i n buffered Krebs solution  to maintain v i a b i l i t y  dehydration.  For EDL, the f a s c i a  anterior muscle was removed. excised  o f the tissue  to expose the SOL.  preparation  and to prevent  was cut and the overlying  S i m i l a r l y , the overlying  tibialis  gastrocnemius was  Subsequently, the tendons of EDL or SOL were  freed and tied with short segments of surgical  silk  (5-0).  The ties were  made as close as possible to the myotendinous junction i n order to avoid stray series compliance i n the preparation.  The muscle was then removed  by gentle dissection  with fine  the  o f the adhering f a s c i a  scissors.  SOL or EDL was used from a single animal f o r measuring  Either  the c o n t r a c t i l e  properties.  EXPERIMENTAL APPARATUS  The muscle was transferred to an experimental chamber ( F i g 1.). The. chamber consisted o f a 1.43 cm. wide perspex bath embedded i n a plate of hardened was  aluminum.  With  the a i d o f a dissecting  microscope, the muscle  t i e d at one end to a stainless steel wire secured to a perspex block  (dummy force-transducer) and the other end to a galvanometer (Cambridge, model 300H). as  a  length  The galvanometer was used  servo-system  which  was extremely  examination of isometric c o n t r a c t i l e properties.  torque motor  to measure force and  stiff  to allow f o r  the  Both the motor and dummy  force-transducers were secured to 3-way micropositioners which allowed for critical  alignment of the muscle and adjustment o f length.  Throughout the  experiment, the muscle was immersed i n Krebs solution which contained NaCl 115mM;  KC1  MgS0^7H20  5.0  1.2mM  mM;  CaCl2  3.1mM;  NaHC03  and 2gm. g l u c o s e / l i t r e ,  25mM;  gassed  NaH2P04H20  with  95% 0 2  C0 9 and maintained at pH 7.4 and a temperature of 2 T C ± T C .  1.2mM; and 5%  -29-  Fig 1.  Experimental apparatus:  The muscle i s immersed i n Krebs  solution in bath chamber, tied at one end to the lever arm of the  motor  and at the other  extension of the dummy force bubbled  with  95%  end to a stainless transducer.  and 5% C02-  Length  by means of a 3-way micro-positioner.  steel  wire  It i s continuously adjustment  i s made  I Lb'.  -31EXPERIMENTAL PROCEDURES  For  purposes  of comparison, muscles were mounted at the length at  which the maximum isometric twitch was recorded.  Length could be adjusted  in  assembly  lOum.  motor.  increments  by means of a micrometer  held the  Stimulation consisted of supramaximal square wave pulses of 1 ms.  duration.  For tetanic contraction, the stimulation frequency and duration  were adjusted to produce a fused tetanus. by  which  a tetanus,  with  A regime of 3 twitches  a contraction once  every  fatigue of the muscles due to repeated t e t a n i .  followed  90 s was used to prevent For each muscle, a minimum  of 12 twitches and 4 tetani were recorded. The using  maximum v e l o c i t y of unloaded  the slack  test  method  shortening  (Edman, 1979)(Fig  (Vo) was then measured 2.). This  consisted of  giving the muscle ramp length changes of 100 Hz. during the plateau of an isometric tetanus, which were s u f f i c i e n t to reduce the tension to zero and remain there u n t i l  the muscle contracted to take up the slack.  of 4 or 5 d i f f e r e n t the  amplitudes were used.  slack i s proportional  to the amplitude  Releases  The time required to take up of the length  change.  The  slope of this r e l a t i o n s h i p , determined by linear regression a n a l y s i s , was used as a measure of the maximum v e l o c i t y of unloaded Following  Vo determination,  was allowed measured  the stimulator was turned o f f and the muscle  to rest f o r 20 min.  by the following  shortening (Vo).  Post-tetanic potentiation (PTP) was then  procedure.  a  single  stimulus (pre-twitch) and was followed 90 s later by a 1 s tetanus.  After  20 seconds, the muscle was given twitches  were  stored  The muscle  a second  on a Nicolet  Digital  oscilloscope was used to record the tetanus.  stimulus  was  given  (post-twitch).  O s c i l l o s c o p e , and a  Both second  -32-  Fig 2. A.  Determination of the maximum v e l o c i t y of unloaded  shortening.  Four d i f f e r e n t length changes and the resultant drop and then recovery of the tension.  Change in length i s the amplitude of  the release and change in time i s the time required to take up the slack.  B.  Shows the l i n e representing the least squares regression of change in length upon change in time.  The slope i s then div-  ided by the muscle length to express Vo in Lo/sec.  -33-  -34Following t h i s , the fatigue p r o f i l e of the muscle was measured. consisted  of giving  This  the muscle a 1 s tetanus at a rate of 12/min. for 5  min. At the conclusion of each experiment, the length of the muscle was measured  with  fine  calipers,  and  the  muscle  removed,  blotted  dry  and  weighed.  Histological Preparation  The animal was was  killed  by cervical  dislocation and the right hind leg  removed and pinned to a cork board.  the muscle was  The  cut along the length of the muscle.  the EDL, the overlying t i b i a l i s anterior was SOL  was  tendons  exposed were  between two then  was  removing  removed to expose  gastrocnemius.  the muscle  was  removed  The from  on  embedded a  placed  cork  in mouse chuck.  had been in  a  The  cooled  cryostat  liver  which  sample  was  was  proximal the  at  frozen  -20'C  EDL.  The  and  distal  leg and  placed  The muscle was  then mounted  to -160'C in l i q u i d cabinet  overlying  During dissection of  layers of gauze moistened with 0.9 M s a l i n e .  isopentane which it  by  cut and  carefully  tragacanth  connective tissue  for 30  gum  sec. in  nitrogen  f o r 30  in  and  min.  then  Serial  sections of 10 urn thickness were taken from the midbelly of the muscle and collected  on glass  cover  slips.  The  sections were dried for 1 hour and  stained with Haematoxylin and Eosin as previously described by  Redenbach,  O v a l l e , and Bressler (1988).  ANALYSIS OF DATA  All Asahi  the c o n t r a c t i l e  Pentax  camera  which  responses were recorded on 35 mm. was  mounted  on  the oscilloscope  f i l m with an frame.  The  -35analog  signal  Apple H E  from the tension transducer  by the use of Scopedriver software  and the data stored on d i s c . analog-digital records.  was  converter  recorded  on the  (RC E l e c t r o n i c s , C a l i f o r n i a )  A 12 ms. delay between triggering of the  and the stimulator provided  Custom software  directly  written f o r the analysis  a baseline on the  of tension data  was  used to calculate the twitch parameters of Pt, Pt/MN, TTP, 1/2RT, and the tetanic parameters Po and Po/MW. Maximum v e l o c i t y of unloaded shortening was determined  from measurements of the length and tension signals which  were collected in the Nicolet D i g i t a l Oscilloscope and plotted plotter.  Least  squares  linear  regression analysis  by an x-y  was used to calculate  the slope of the relationship of the amplitude of the length change to the duration of time taken by the muscle to take up the slack. were expressed were measured standard projected  i n muscle lengths per second directly  photographic images.  the f i l m .  The negatives  enlarger and the records  PTP was  post-twitch tension. expressed  from  (Lo/sec).  expressed  were  as a r a t i o  A l l Vo values  PTP and fatigue were placed  analyzed  in a  from the  of the pre-twitch to  For the fatigue p r o f i l e , each tetanic contraction i s  as a f r a c t i o n  of the i n i t i a l  tetanic  tension of the fatigue  regime and plotted over time. Data f o r normal and denervated/devascularized groups at 3,6,9,and 12 weeks post surgery were analysed using a two-way analysis order  to assess  changes  with  time  and  changes  due  to regeneration.  Pairwise comparison at each time was done using Tukey's comparisons, a p r o b a b i l i t y  of p<0.05 was used.  of variance in  test.  For  all  The twitch and tetanic  tension values f o r the denervated/devascularized muscles at 6, 9, and 12 weeks post-surgery f e l l  into 2 s t a t i s t i c a l l y  distinct  groups; therefore,  we divided the denervated/devascularized data into 2 groups, reinnervated and  non-reinnervated.  week post-surgery  The d i v i s i o n  animals  of the data was not done f o r the 3  since the differences  i n twitch  and  tetanic  -36tensions  were  not  as  denervated/devascularized conducted  again  in  non-reinnervated time.  The  After  into  groups  data  order  data.  apparent.  to  condition  12, representing  compare  factor  was  the groups  again used to detect s t a t i s t i c a l  divided  ANOVA  intervals  into  statistics  were  reinnervated  and  3  levels,  data  control,  as was the time factor with levels 6, 9, studied.  A probability  significance.  contained  of p<0.05 was  The analysis of the 3 week  two-way ANOVA since this group was  into reinnervated and non-reinnervated.  of the fatigue  interval.  the  the  control,  divided  post-surgery data involved the i n i t i a l not  of  An ANOVA was used with two f a c t o r s , condition and  reinnervated and non-reinnervated and  two  division  The set-up  one additional  factor,  f o r the fatigue  This factor constitutes the mean+SD which i s determined at 30 s ( f o r up to 320 s) f o r each group studied.  Only the mean f o r  each 5 s interval was p l o t t e d .  CONTROLS  Control  experiments consisted of unoperated age-matched animals.  In  addition a series of sham experiments, 6 EDL and 6 SOL, were conducted in 4-week  o l d male  animals.  The surgical  procedure  followed  that  just  described except that only the connective tissue planes were cut to expose either  the EDL or SOL muscle.  These animals  were only  characteristics control  animals.  studied  The i n c i s i o n  was then  6 weeks post-surgery  sutured  closed.  and physiological  were compared v i a t - t e s t s , to the age-matched unoperated  -37-  RESULTS  -38PILOT STUDY A p i l o t study was conducted to determine the time course of the f i r s t appearance  of  regenerating  muscle  fibres  reinnervation of the muscle had occurred.  which  would  indicate  that  For this study, the surgery was  performed at 4 weeks of age and then the EDL was removed f o r h i s t o l o g i c a l examination at 4, 7, and example of a  normal  21  days post-denervation/devascularization.  mouse EDL  skeletal  muscle  An  i s shown in Fig 3.  The  multiple peripheral nuclei within each d i s t i n c t muscle f i b r e i s obvious as is the close association of neighboring muscle f i b r e s . be  seen that 4 days  connective could  be  tissue  after  and  white  EDL,  connective  with  central  myoblasts.  tissue  nuclei  post-surgery  myoblasts  cells,  containing  of the  EDL  including  was  invaded  neutrophils,  staining.  of  the  also  but  seen  round  eosin  (Fig 4B.). fibres  (Fig 4C.)  stained  These  with  this  at 21 would  days be  an  muscle  to a  nuclei.  structures  presumably  peripheral  in addition  centrally-located  muscle  post-denervation/devascularization fibres  which  Remnants of  was SOL.  seen The  post-surgery in both the EDL appropriate  time  period  in  and to  were  preponderance Connective  presence  were  nuclei  was also evident i n greater quantities as compared to c o n t r o l s . appearance  by  In the 7 day post-denervation/devascularization  predominated  were  site  Eosin  F i n a l l y , rows of muscle  21-days  fused  blood  seen with Hemotoxylin and  fibres were also evident.  seen  surgery the  In Fig 4A. i t may  of  of  tissue  A similar  the  21-day  mature  muscle  SOL,  suggested  that  begin  assessing  the  c o n t r a c t i l e properties of these reinnervating muscles.  -39-  Fig 3.  A longitudinal old mouse.  section of EDL muscle from a normal 21 day  H&E s t a i n .  650X.  -41-  Fig 4.  A section of a denervated/devascularized muscle.  H&E s t a i n .  mouse EDL  700X.  A.  4 days posf-denervation/devascularization.  B.  7 days post-denervation/devascularization.  C.  21 days post-denervation/devascularization.  Following denervation/devascularization connective tissue c e l l s invade the muscle. arrow) undergo degeneration.  Muscle fibers  Seven days  (large  post-surgery  myoblasts (my) appear and by 21 days post-surgery  dis-  t i n c t mature muscle f i b r e s can be seen (small arrow).  -42-  -43The Sham Experiments The data from the sham experiments were compared to the data from the control  experiments  normalized  no s i g n i f i c a n t  difference  in absolute and  twitch and tetanic tensions, TTP, 1/2RT, Vo, PTP, or in their  fatigability for  revealing  (Appendix A ) . As the sham experiments were only carried out  one post-operative period  the results  were  not  included  with  the  results of the unoperated animals.  Clinical  Observations  Upon i n i t i a l  removal of the p a r t i c u l a r muscle from the leg several  features  were observed.  compared  to  their  Some of the EDL or SOL muscles were very  respective  control  muscles.  In  other  thin  cases, the  respective muscle appeared to be as large as the control muscles but with a  l o t of connective  Subsequently  tissue  surrounding  and  clinging  the weights of the muscles were found  (see Fig 5. and Fig 6.). reinnervated,  and  therefore  physiology yet were found muscles i n this  On  to  to f a l l  several occasions, muscles had  not  to produce  regenerated,  no tension.  group that were observed  the  muscle.  into 2 groups that  were  had not  prepared  The total  for  number of  during the study was 5:  1 in  each of the 6, 9, and 12 week post-surgery SOL muscles and 2, 12 week post-surgery EDL muscles. It tetanus  became  apparent  tensionsell  in assessing  into  two  groups  the results  were designated  muscles were designated properties  were placed  the twitch and  f o r the denervated/devascularized  muscles at 6,9, and 12 weeks post-surgery. muscles  that  The greater tension-producing  as reinnervated and the lower tension-producing as non-reinnervated. into  the two groups  The remaining with  contractile  the tension being the  determining f a c t o r . The muscle weights from the control and the denervated/devascularized  -44-  Fig 5.  Weights of control and  surgical  (All values are means±SD).  EDL  muscles.  Weights of control and surgical EDL muscles  Weeks post-surgery  -46-  Fig 6.  Weights of control and  surgical  ( A l l values are means±SD).  SOL  muscles.  Weights of control and surgical SOL muscles Q •  control den/devasc.  Weeks post-surgery  -48muscles f o r the 4 post-operative periods are presented i n Fig 5. (EDL) in  Fig 6.  between  (SOL). There was  the  control  post-surgery  and  (p<0.05).  At  could  divided  be  tension.  At  6,9,  and  12  difference in muscle  denervated/devascularized  EDL  into  these  significant  In  denervated/devascularized controls.  no  contrast,  this  distinct  time periods  surgery,  groups  on  post-surgery  EDL  the  the  3  same  the  weights  at  muscles weighed s i g n i f i c a n t l y  weeks a f t e r  2  at  SOL  weeks  time  the  less than the muscle  basis  muscle  and  of  weights  isometric  weights  of  the  reinnervated muscles were not s i g n i f i c a n t l y d i f f e r e n t from the unoperated EDL.  The  non-reinnervated  the controls and SOL  Similar  significant weights than the  divided into 2 groups based on EDL,  at  6  difference between  the  control  and  to  the  the  reinnervated  non-reinnnervated  groups studied (9 and was  no  significantly  the reinnervated muscles (p<0.05).  could also be  values.  muscles were  difference  non-reinnervated  muscle  muscle  weeks  and  weights  the  muscles  the  weights.  the  were  than  both  The muscle weights of isometric tension  post-surgery,  there  was  reinnervated significantly  However, in  the  no  muscle greater  oldest  age  12 weeks post-denervation/ devascularization), there in  muscle  weights  between  reinnervated  and  SOL muscles.  Table 1 i s a summary of the absolute and of  lighter  fast-twitch produced  EDL  muscle.  significantly  Both less  normalized  reinnervated twitch  and  tension  twitch  tensions  non-reinnervated than  the  control  muscles at a l l time periods (weeks post-surgery); however, at 6,9, and  12  weeks post-surgery the reinnervated muscles produced s i g n i f i c a n t l y greater tension  than  the  non-reinnervated  muscles.  post-surgery, the reinnervated muscles had of wet  control muscle tension. weight,  the  a d d i t i o n , by  recovered  When twitch tension was  normalized  denervation/devascularization  In  twitch did  not  tension differ  at  12  weeks  to greater than  50%  normalized  to muscle  3  following  weeks  significantly  from  the  Table 1: Twitch tension and twitch tension normalized to muscle weight from Control (C), Reinnervated (R), and Non-reinnervated (NR) mouse EDL. Twitch Tension (g) a  Time  3  8.80 ± 1.3 (6)  R (n)  NR (n)  3.39 ± .42C (6) +A  6  8.56 ± 1.2 (6)  3.09 ± .12* 1.85 ± .46 (2) (4)  9  9.38 ± .80 (6)  4.43 ± .81* 1.29 ± .71 (3) (6)  12  a: b:  b  C (n)  Twitch Tension/ Muscle Height (g/mg)  10.12 ± 1.0 5.28 ±1.2* (6) (3)  1.55±1.1 (4)  +A  +A  C (n)  R (n)  NR (n)  0.83 ± .08 (6)  0.69 ± .19 (6)  0.70+ .13 (6)  0.28 ± .01* 0.30 ± .06 (2) (4)  A  A  0.72 ± .09 0.39 ± .04* (6) (3)  0.31 ± .17 (6)  0.78 ±.11 (6)  0.70 ± .17+ (4)  0.33 ± .09* (3)  Weeks post-surgery This data was not separable into reinnervated and non-reinnervated groups, based on twitch and tetanus tensions, c: All values are means±SD and the level of significant difference is p<0.05 *: A significant difference exists between the reinnervated and the control muscles A: A significant difference exists between the non-reinnervated and the control muscles +: A significant difference exists between the reinnervated and the non-reinnervated muscles  Table 2: The twitch tension and the twitch tension normalized to muscle weight from Control (C), Reinnervated (R), and Non-reinnervated (NR) mouse SOL Twitch Tension (g) a  Time  Twitch Tension/Muscle Weight (g/mg)  R (n)  C (n) c  NR (n)  C (n)  3b  3.38+.85 (6)  6  3.29 + .53 (6)  2.51 + .35* (5)  1.50 + .31** (2)  9  3.83 + .40 (6)  2.70 + .20* (3)  1.32 + .32 (4)  12  3.44 + .80 (6)  3.68 + 1.1 (5)  1.93 + .85 (2)  a: b:  3..13 + .92 (8)  0.34 + .09 (6)  +A  R (n)  NR (n) 0.24 + .09 (8)  0.23 + .04 (6)  0.19 + .03 (5)  0.27 + .08 (2)  0.28 + .06 (6)  0.20 + .05* (3)  0.16 + .05+ (4)  0.24 + .07 (6)  0.19 + .06 (5)  0.26 + .17 (2)  weeks post-surgery data for reinnervated and non-reinnervated muscles could not be divided based on twitch or tetanic tension, c: A l l values are means±SD and the level of significant difference is p<0.05 *•: A significant difference exists between the reinnervated and the control muscles +: A significant difference exists between the non-reinnervated and the control muscles A: A significant difference exists between the reinnervated and the non-reinnervated muscles  -51respective  age-matched  controls;  subsequently,  at  6,9,  and  12  weeks  post-surgery a s i g n i f i c a n t decrease compared to controls was seen in the reinnervated  muscles.  reinnervated  Moreover,  and non-reinnervated  weeks following  difference  muscles  was  at these  observed  time  between  periods.  At 3  denervation and devascularization of the slow-twitch SOL,  there was no difference and  no  in absolute twitch tension between the controls  the operated muscles (Table 2 ) . However the denervated/devascularized  SOL, both  reinnervated and non-reinnervated, produced  significantly  less  twitch tension than controls at 6 and 9 weeks of age. The non-reinnervated SOL produced s i g n i f i c a n t l y less twitch tension at 6 and 9 weeks than both the reinnervated and the control muscles. weeks there was no s i g n i f i c a n t  difference  in twitch tension between the  c o n t r o l , reinnervated and non-reinnervated muscles. tensions  f o r SOL  follow  a  different  pattern  The normalized twitch  than  the EDL.  s i g n i f i c a n t differences were seen at 9 weeks where the control twitch  tension  exceeded  both  By 12  reinnervated  and  The  only  normalized  non-reinnervated  values  although no difference existed between the l a t t e r two. Table 3 i s a summary of the isometric tetanic tension values of the fast-twitch  EDL.  muscles  significantly  was  post-surgery.  By  The  tetanic less  tension of the denervated/devascularized than  the controls  12 weeks the tetanic  at 3,6, and  tension produced  9 weeks  by reinnervated  muscles was not s i g n i f i c a n t l y d i f f e r e n t from the tetanic tension produced by  the control  muscles.  Moreover, the non-reinnervated  s i g n i f i c a n t l y less tetanic tension than  the control  muscles at 6,9, and 12 weeks post-surgery. was  normalized  to muscle  wet weight,  a  muscles produced  and the reinnervated  When the EDL tetanic tension  significant  decrease  was  seen  between controls and the denervated/devascularized muscles at 3,6, and 12 weeks. more  However, at these  tension  per muscle  age groups weight  than  the reinnervated muscles did the non-reinnervated  produced muscles.  Table 3: The tetanic tension and the tetanic tension normalized to muscle weight from Control (C), Reinnerrvated (R), and Non-reinnervated (NR) mouse EDL. Tetanic Tension (g) a  Time ~P  12  a: b:  C (n) 31.42±3.1C (6)  R (n)  Tetanic Tension/Muscle Weight (g/mg) NR (n)  C (n)  8.72±4.5* (6)  2.95±.39 (6) +A  36.56±3.1 (6)  15.39±.06* (2)  3.30±.53 (4)  34.48±5.7 (6)  22.64±5.8* (3)  7.64+10 (6)  38.80±1.5 (6)  25.02±5.0 (3)  3.87±3.73 (4)  +A  +A  NR (n)  R (n) 1.62±.49* (6)  +A  3.00+.63 (6)  1.41+.10* (2)  0.54±.11 (4)  2.64±.52 (6)  1.98±.35 (3)  1.60+1.9 (6)  2.95±.26 (6)  1.54±.25* (3)  0.759+.93 (4)  +A  weeks post-surgery This data was not separable into reinnervated and non-reinnervated based on twitch and tetanic tensions, c: All values are means+SD and the level of significant difference is p<0.05 *: A significant difference exists between the reinnervated and the control muscles A: A significant difference exists between the non-reinnervated and the control muscles +: A significant difference exists between the reinnervated and the non-reinnervated muscles  Table 4: The tetanic tension and the tetanic tension normalized to muscle weight from Control (C), Reinnerrvated (R), and Non-reinnervated (NR) mouse SOL. Tetanic Tension (g) Time*  Tetanic Tension/Muscle Height (g/mg)  R (n)  C (n) C  NR (n)  C (n)  3»>  15.81+2.7 (6)  6  19.46+ 2.4 (6)  12.89+1.5* (5)  2.56+.64+ (2)  9  24.33+3.1 (6)  15.81+1.9* (3)  12  19.78+1.6 (6)  19.32+4.1 (5)  a: b:  12.98+7.6 (8)  1.59+.21 (6) A  R (n)  NR (n)  1.07+.45 (8) +A  1.34+.19 (6)  1.00+.23* (5)  0.45+.07 (2)  5.58+2.7 (4)  1.75+.24 (6)  1.19+.30* (3)  0.70+.41 (4)  10.89+6.6 (2)  1.36+.15 (6)  1.04+.36 (5)  1.349+.63 (2)  +A  A  weeks post-surgery This data was not separable into reinnervated and non-reinnervated based on twitch and tetanic tensions, c: A l l values are means±SD and the level of significant difference i s p<0.05 *: A significant difference exists between the reinnervated and the control muscles A: A significant difference exists between the non-reinnervated and the control muscles +: A significant difference exists between the reinnervated and the non-reinnervated muscles  -54Finally,  at  9  reinnervated  weeks  and  no  difference  non-reinnervated  was  muscles  seen in  between  terms  of  the  control,  the  normalized  tetanic tension. Table the  4 summarizes the absolute  SOL  muscle.  and  Three  normalized  weeks  tetanic  following  tensions  surgery,  for the  denervated/devascularized  muscles s t i l l  produced 82% of the control twitch  tension.  reinnervated  and  However,  the  the  non-reinnervated  muscles  produced s i g n i f i c a n t l y less tetanic tension than the controls at 6 and weeks.  In  produced  a d d i t i o n , at  significantly  muscles.  these  age  groups, the  greater tetanic  tension than  F i n a l l y , at 12 weeks there was  between  the  control  and  the  was  no  significant  produced by SOL produced  the  reinnervated  muscles  non-reinnervated  In  particular,  the  up to 98% of the control tetanic tension.  difference in the  greatest tetanic and  the  muscles.  at 3 weeks post-surgery.  muscles  reinnervated  no difference in tetanic tension  operated  reinnervated muscles recovered There  2  9  normalized  tetanic  tension  At 6 weeks, the control muscles  tension per muscle weight followed  finally  the  non-reinnervated  by  muscles.  the The  difference in the normalized  tetanic tension produced between reinnervated  and  muscles  non-reinnervated  post-denervation/devascularization;  disappeared however,  by  both  muscle  9  weeks  groups  still  produced s i g n i f i c a n t l y less tetanic tension per wet muscle weight than the age-matched  controls.  Finally,  by  12  weeks  there  was  no  significant  difference in this parameter between any of the three experimental A  comparison  of  the  denervated/devascularized denervated/devascularized than  the  slowing  of  age-matched the  TTP  devascularization.  changes EDL  EDL  are had  control. and  at by  TTP  and  shown  a  in  1/2RT Fig  significantly  The  1/2RT  However,  in  reinnervated 6 12  and  of 7.  slower EDL  9  weeks  weeks,  there  groups.  control The  TTP  3 and  initially  no  week 1/2RT  showed a  post-denervation was  and  and  significant  -55-  Fig 7.  The time-to-peak, twitch tensions and the half-relaxation times of c o n t r o l , reinnervated, and non-reinnervated mouse EDL muscles.  ( A l l values are means±SD).  -56-  EDLTTP • • - •  control reinnerv. non-reinnerv.  i  i  i  EDL • " • • •  i  i  i  L  1/2RT  control reinnerv. non-reinnerv.  I  2  .  I  .  4 6 8 10 12 Age (weeks post-surgery)  14  -57-  Fig 8.  The time-to-peak twitch tension and the half-relaxation time of c o n t r o l , reinnervated, and non-reinnervated mouse SOL muscle.  ( A l l values are means+SD).  -58-  SOLTTP  140 o a>  E, ^.  CO  a • •  120  control reinnerv. non-reinnerv.  100  a>  80 CD  E  60  -  it J  40  i  L  SOL ^ CD V)  ~  J  i  L  1/2RT  400 r Q control 300  • •  reinnerv. non-reinnerv.  CD  E  "r-  200  CO X _fO 12  100  CO X  0  a>  -i  i_  4  j  i  i_  6 8 10 12 Age (weeks post-surgery)  14  difference observed reinnervated  muscles.  significant muscle  and  The  and  TTP and 1/2RT  1/2RT  at 3 and 6 weeks  non-reinnervated  existing  non-reinnervated  slowing of both  the TTP  controls  i n either of these parameters between the control and  were  not  muscles  between the former two.  at 6  and  a  For the SOL  different  from  the  ( F i g 8.). By 9 weeks the TTP of reinnervated the controls  with  no difference  However, by 12 weeks post-surgery the  TTP of the reinnervated and non-reinnervated  slowing  exhibited  up to 12 weeks.  significantly  SOL was slower than  from the c o n t r o l s .  still  muscles  was  not d i f f e r e n t  The 1/2RT followed the same pattern as f o r TTP i e . a 9  weeks.  By  12  weeks  however, the  1/2RT  of  both  reinnervated and non-reinnervated SOL was not s i g n i f i c a n t l y d i f f e r e n t from control muscles. Tables  5 and  6 are summaries of the mean values  v e l o c i t y of unloaded  shortening i n denervated/devascularized  EDL and SOL muscles respectively. EDL  were  not  of the maximum  significantly  denervated/devascularized  control  S t r i k i n g l y , Vo values f o r both SOL and  different  muscles  and  between  the  at any age group.  controls  and  This  true f o r  was  the  both the reinnervated and the non-reinnervated muscles. Values  of post-tetanic twitch potentiation, a c h a r a c t e r i s t i c  typical  of fast-twitch skeletal muscle but not slow-twitch muscle, are contained in Tables 7 and 8.  The control  and by 6, 9, and 12 weeks reinnervated  showed  difference disappeared.  i t has reached  EDL followed a similar  between i t and the controls only  EDL exhibits a 6% potentiation at 3 weeks  a  5%  between  an average value of 20%.  trend with no s i g n i f i c a n t difference  at any age group.  potentiation  The  at 6 weeks  the non-reinnervated  PTP  The non-reinnervated  but by 9 and and  the  EDL  12 weeks the  control  PTP  The slow-twitch SOL does not normally exhibit any PTP.  had  Table 5: The maximum velocity of shortening in Control(C), Reinnervated (R) and Non-reinnervated (NR) mouse EDL. Maximum Velocity of Unloaded Shortening (Lo/sec) a  Time  a: b: c:  R (n)  C (n) C  NR (n)  3"  8.15+1.2 (5)  7.08+1.7 (6)  6  6.26+1.9 (6)  5.16+.24 (2)  5.86+2.5 (4)  9  5.37+.91 (5)  5.16+1.4 (3)  3.06+1.3 (5)  12  7.57+3.0 (5)  9.10+1.7 (3)  10.5+11 (2)  weeks post-surgery This data was not separable into reinnervated and non-reinnervated based on twitch.and tetanic tensions, A l l values are means+SD and the level of significant difference is p<0.05  Table 6: The maximum velocity of shortening in Control(C), Reinnervated (R) and Non-reinnervated (NR) mouse SOL. Maximum Velocity of Unloaded Shortening (Lo/sec) Time*  3  a: b: c:  b  R (n)  C (n) C  6.07+1.0 (6)  NR (n) 6.39+2.0 (8)  6  4.95+1.2 (6)  7.86+3.6 (5)  5.73+1.6 (2)  9  5.82+2.4 (6)  10.6+7.7 (3)  2.73+.66 (3)  12  5.58+1.3 (5)  5.46+2.0 (5)  5.05+2.0 (2)  weeks post-surgery These data were not separable into reinnervated and non-reinnervated based on twitch and tetanic tensions, A l l values are means±SD and the level of significant difference is p<0.05  Table 7: The post-tetanic potentiation in Control (C), Reinnervated (R), and Non-reinnervated (NR) mouse EDL. Post-Tetanic Potentiation a  Time (n)  a: b:  C (n)  R (n)  NR (n)  3b  1.06+.07C (6)  6  1.18+.07 (6)  1.23+.01 (2)  1.05+.04*+ (4)  9  1.20+.09 (6)  1.19+.04 (3)  1.04+.14 (6)  12  1.21+.05 (5)  1.14+.09 (3)  1.08+.08 (4)  1.03+.05 (6)  weeks post-surgery These data were not separable into reinnervated and non-reinnervated based on twitch and tetanic tensions, c: All values are means±SD and the level of significant difference is p<0.05 *: A significant difference exists between the non-reinnervated and the control muscles +: A significant difference exists between the reinnervated and the non-reinnervated muscles  Table 8: The post-tetanic potentiation in Control (C), Reinnervated (R), and Non-reinnervated (NR) mouse SOL. Post-Tetanic Potentiation Time* (n)  a: b: c:  NR (n)  R (n)  C (n) c  0.99+.03 (8)  3»>  1.04+.05 (6)  6  0.97+.04 (6)  1.09+.23 (5)  1.02+.03 (2)  9  0.99+.02 (6)  0.98+.03 (3)  0.95+.04 (4)  12  0.98+.02 (6)  0.98+.03 (5)  1.12+.16 (2)  weeks post-surgery This data was not separable into reinnervated and non-reinnervated based on twitch and tetanic tensions, All values are means±SD and the level of significant difference is p<0.05  -64There  was  no  s i g n i f i c a n t difference  non-reinnervated periods  between  c o n t r o l , reinnervated and  SOL muscles i n terms of t h e i r  PTP at any of the time  studied.  The  fatigue  patterns  derived  from  pooled  data  of  denervated and devascularized  muscles are shown in Figs  fatigue  based  regime  statistically (control,  was  analysed  s i g n i f i c a n t difference  reinnervated  or  was  included  in  the  trends  persisted  and  9. and 10. The  in f a t i g a b i l i t y .  If a  between any of the groups  non-reinnervated)  approximately 40 s and continuing this  on  control  i.e.  beginning  at  f o r the remainder of the stimulus, then  description  of  the  fatigue  data  as  a  s t a t i s t i c a l l y significant difference. At  3  significantly  weeks  post-surgery,  (p<0.05  age-matched c o n t r o l s . resemble the pattern non-reinnervated  after  the  35  s)  denervated/devascularized more  fatigue-resistant  By 6 weeks, the reinnervated of fatigue  muscles  exhibited  more  up to 12 week time  exhibiting  greater  period  with  fatigue-resistance  than  the  However, the  fatigue-resistant  both the controls and the reinnervated muscles at this time. continued  was  muscles more closely  by the c o n t r o l s .  were s i g n i f i c a n t l y  EDL  than  This  pattern  the non-reinnervated  muscles  than  the  control  and  the  reinnervated muscles. At 3 weeks, the operated SOL was s i g n i f i c a n t l y more fatigue-resistant than the control  SOL.  The reinnnervated 6 week SOL i s s i g n i f i c a n t l y more  fatigue r e s i s t a n t than the control; however, no difference existed between the  non-reinnervated  reinnervated  SOL i n terms of t h e i r fatigue  there was no s i g n i f i c a n t difference the  The 9 week  and non-reinnervated muscles fatigue s i m i l a r l y although they  were both more fatigue-resistant than the c o n t r o l s .  and  regime.  reinnervated  muscles.  in the f a t i g a b i l i t y  The  s i m i l a r l y to the control muscle until  F i n a l l y , by 12 weeks  non-reinnervated  of the controls muscles  fatigued  the l a t t e r half of stimulation when  -65the  non-reinnervated muscles  were  significantly  more fatigue  resistant  than the c o n t r o l s . The and any  statistics  treatment. of  the  used also accounted for any interaction between time  There was parameters  no interaction studied  between time and  i.e.  the  control  treatment for  muscles  show  a  developmental increase in twitch and tetanic tensions and a basic maturity of  the  muscle  (contractile  times  achieve  those  of  a  mature  fast  or  slow-twitch skeletal muscle) as does the denervated/devascularized muscle; however, the l a t t e r  muscles do not show an increase  above and beyond the control  level.  in these parameters  -66-  Fig 9.  The fatigue regime of c o n t r o l , denervated/devascularized, reinnervated, and non-reinnervated mouse EDL muscle.  The  values are expressed as a proportion of the i n i t i a l Po value.  -67-  Fatigue: 1.0  EDL 3 weeks post-surgery  EDL 6 weeks post-surgery control reinnerv. non-reinnerv.  EDL 9 weeks post-surgery control reinnerv non-reinnerv.  EDL 12 weeks post-surgery control reinnerv. non-reinnerv.  100  200 Time(sec)  300  400,:  -68-  Fig 10.  The fatigue pattern of c o n t r o l , denervated/devascularized, reinnervated, and non-reinnervated mouse SOL muscle.  -69-  Fatigue: SOL 3 weeks post-surgery  c g •</> c CD  H  CD >  IS CD  DC  SOL 6 weeks post-surgery  c g "w c CD  ICD > control reinnerv. non-reinnerv.  CD  DC  SOL 9 weeks post-surgery  c g 'w c: CD  H  CD > CD  DC  0.2  SOL 12 weeks post-surgery  CD > CD  control reinnerv. non-reinnerv  DC  0.2  i  100  L_  200 Time (sec)  300  400  -70-  DISCUSSION  -71In  this  contractile  project  a  properties  slow-twitch  comparison  of  the  oxidative  mice  technique  and  very  of  denervation  immature  state,  from  made  fast-twitch  SOL  denervated/devascularized  was  of  the  the  isometric  glycolytic  EDL  and  muscles  of  the  from  C57BL/6J  devascularization  which  the  normal  strain. brought  regenerative  the  The  the  and surgical  muscle to a  responses  of  these  a  strong  muscles at 3, 6, 9, and 12 weeks post-surgery were studied. During  the  relationship tetanic that  recovery,  between  tensions.  the  tetanic  the  appropriate  were  muscle  which the  contraction  (reinnervated).  and  twitch  and  Bressler  (TTP  Conversely,  1/2RT  and  less  EDL,  greater that  with  twitch  i t was  absolute  had 12  twitch  weeks  and  their  post-surgery  muscle  and  were generally slower than the control muscles or  the  subsequently grafted  and  by  rat triceps  40%  decline in tetanic tension  The  actin  of  the  as reinnervated.  coincided with Carlson  tetanus  weights  tension  (1985)  and  and  apparent  recovered  smaller  twitch  was  absolute  1/2RT) by  absolute  there  the with  muscles  muscles  tetanic tension  content  and  produced the  group of muscles which were designated in  process,  weights  same  times  (non-reinnervated) produced t h e i r TTP and  regenerative  Furthermore, p a r t i c u l a r l y  larger muscles tensions  or  similar  (1973).  surae and  In  The early decline reports  a d d i t i o n , minced  plantaris  muscles  (Bertrand, Plaghki, and  muscles  increased  i n d i c a t i n g that this reduced tension was  as  by Webster  the  and  revealed  a  Marechal, 1981)  .  tension  increased  due to a poor regeneration of the  m y o f i b r i l l a r apparatus. It i s i n t e r e s t i n g to note the d i f f e r e n t regenerative responses of the SOL and the EDL in our study.  The 3, 6, 9, and  12 week post-operative  muscles produced less twitch tension than the c o n t r o l s . biphasic response in twitch tension.  The  SOL  twitch  produced  93%  of  the  control  3 week  The  SOL  EDL  showed a  denervated/devascularized  tension  before  dropping  to  -72approximately 75% and then r i s i n g again at 12 weeks to 107% of the control values.  Millar  and Das  (1981) reported  a biphasic  response  i n twitch  tension a f t e r orthotopic grafting of f l e x o r digitorum s u p e r f i c i a l i s rabbit muscle. weeks  They attributed the i n i t i a l  post-grafting  second change Although twitch  we  Carlson,  observed  and  conditions  cause.  this  differed Irwin,  from  their  response study.  the timing  et a l , 1978)  revascularization  takes  approximately  14-21  days  after  injury to mouse  supply  and the  of the g r a f t . of the r i s e in  I t i s agreed  Vrbova  (Hansen-Smith,  that  under  7-10  muscle;  optimal  days  transplant  studies, including Carlson  the recovery  maturation of the muscle.  rise  i n twitch  tension  and  therefore, the  which we saw at 3 and 12 weeks must be due to some  Other  while  to the reinnervation  biphasic  have attributed the i n i t i a l fibres  later  between 1 and 2  blood  (1980);  reinnervation alterations  to the a c q u i s i t i o n of a new  1 or 2 months  tension  change which occurred  and Gutmann  other  (1975),  to surviving muscle  seen at 12 weeks may be accounted f o r by the While the EDL exhibits a steady  recovery in  twitch tension with time post-injury i t never reaches a level greater than 58% of the control value. of  EDL although again  A sharper r i s e was found i n the tetanic tension  the SOL was much more successful  in i t s recovery  (Tables 3 and 4 ) . The  recovery  of the tetanic tension of the reinnervated muscles by 12  weeks coincides with the work of Beranek et al (1957) who used adult mouse muscle  and  observed  that  following  They found a complete recovery Niemeyer, Maxwell, and White recovery  crush  the nerve  in a l l c o n t r a c t i l e (1980) working  of the tetanic tension  nerve anastomoses.  nerve  properties.  on adult  120 days a f t e r  regenerates.  cats  Faulkner,  found a 50%  EDL transplantation with  The f a t i g a b i l i t y of these muscles only reached 40% of  the controls and the muscle mass reached 80% of the controls; therefore, the decreased tetanic tension was accounted f o r by the lower muscle mass.  -73In  our experiments, the muscle  comparable  weights  to that of the controls.  of the reinnervated  observation), the decreased absolute tetanic  (Carlson, 1973; personal  tension  could  to  a genuine  et  al (1987) speculated that a large part of the loss  reinnervation.  can be accounted  They  suggest  f o r by muscle  that  an  immature  some muscle f i b r e death. slow  Lowrie  i n muscle mass of  fibre  atrophy  muscle  traumatized by the reinnervation by a mature fast nerve.  the  be attributed  decrease i n the number of muscle fibres per muscle.  muscle  was  I f the content of connective tissue  was quite high as observed in denervated muscles  fast-twitch  muscles  is  after  actually  This results in  This of course would not apply to SOL because of  discharge frequency of i t s nerve.  A d d i t i o n a l l y , the possible  increase i n connective tissue makes the analysis of the normalized twitch and  tetanic  decreased controls  tensions  very d i f f i c u l t  f o r the reinnervated  to i n t e r p r e t .  and  This  non-reinnervated  parameter  EDL  compared  was to  at a l l ages, but i t had recovered to control values by 12 weeks  post-surgery normalized  in the SOL.  tetanic  Faulkner  and  tension of grafted  Cote  (1986)  found  that  the  EDL decreased when compared to the  control but when the tetanic tension was measured per square centimeter of viable muscle fibres the  then there was no difference between the grafts and  controls. In  addition  to the reduced muscle mass accounting f o r the decreased  tetanic  tension,  Faulkner and Cote  motor unit functions.  (1986) reported  deficits  in single  Again, using grafts made with r a t EDL, they found a  small but s i g n i f i c a n t decrease in the average tension developed of single motor  units.  ability  This would imply that there i s a genuine  of the muscle fibres  to generate  tension  decrease in the  at this  point  in the  Carlson, Hnik, Tucek, Vejsada, Bader, and Faulkner, (1981)  studied  regenerative response.  the  problem  of reinnervation  and revascularization  i n muscle  grafting.  -74Th ey  found  postulated was  that  80%  of  original  muscle  the  lack  of  successful  is  the  injured s i t e .  He  had  mass  as i t occurs, i s not  reinnervation. connective  of  regeneration  to  produce  a  The  major  muscle  blockade  to  long  factor of the  the  blood  vessels,  occur  without  mature  muscle  a  that  the  evidence to support that r e v a s c u l a r i z a t i o n , as limiting  tissue  of  and  the  myoblasts appear before  course,  degenerated  infiltrates  the  of  stems from the finding that in the i n i t i a l  phases  fibres  that the major l i m i t i n g factor for regeneration  reinnervation  This  the  regeneration  phases of regeneration  i n d i c a t i n g that  fully  fibre  of muscle.  the  i n t a c t blood  both  the  initial  supply.  Of  revascularization  and  reinnervation would seem mandatory. The  denervated/devascularized  experiments severing  was  designed  the nerve and  procedure used  to maximize the  in  the  present  chances for reinnervation.  blood vessels at their entrance to the  expected the reinnervation and  indicated  that  week post-surgery and myotubes  would  muscle.  Our  achieved.  results  The  group consisted and  another  latter  the  reinnervation would soon follow. and  of  eventually  indicate  muscles where  muscle, we  minimized.  revascularization would occur within  that  data seemed to f a l l  group  develop  a  into  complete  into two  this  apparently  recovery  that  r e v a s c u l a r i z a t i o n , or  not  connective both.  was  groups for EDL  was  The  The  the  first  Subsequently, the  a normal  successfully reinnervated  muscles, i t i s possible  reinnervation, of EDL  mature  By  revascularization to take place quickly and  e a s i l y since the distance for these processes to occur was literature  series  and  the  and  not  always  SOL.  One  revascularized  case.  tissue  functioning  For  these  prevented  their  non-reinnervated muscles  were quite c l e a r l y representative of a denervated muscle; however,  non-reinnervated  tetanic tensions  SOL  exhibited  to control values.  fatigue regime suggested that the SOL  an  eventual  return  In addition the TTP,  of  twitch  and  1/2RT, PTP,  and  resembled fast-twitch muscle in i t s  -75contractile may  properties.  be accounted  for by  from i t s lack of the muscle may thread  was  muscle.  failure  an  reflect  a  of a complete return of the tension  incomplete recovery  reinnervation.  The  problem  shimmied along  the nerve was the  The  the  of the muscle, resulting  f a i l u r e of the nerve to reinnervate  with  the  actual  technique.  b e l l y of the muscle, i t was  pulled away or pushed further from the This  increased  site  When  the  possible that of entry  into  distance over which reinnervation must occur  would slow the regenerative process of the muscle or completely prevent i t . The of the have  faster c o n t r a c t i l e speeds, the PTP and the increased f a t i g a b i l i t y non-reinnervated  been  SOL  reinnervated  i n d i c a t e , moreover, that  by  fast-twitch gastrocnemius.  a  fast  nerve,  muscles  branch  from  may the  nerves in the  not apparent.  conclusion we can draw regarding the above f a c t o r s , which heavily  influence  the  regenerative  ability  of  the  subgroups, reinnervated and non-reinnervated It  i s apparent  in our  study  that  regenerative capacity of EDL and SOL. twitch  a  A similar sprouting by adjacent  region of the fast-twitch EDL muscle was One  possibly  these  and  tetanic tensions.  The  muscles, i s that  there  was  a  difference in  a larger percent of i t s  EDL.  In the early phases of EDL and SOL regeneration, there was slowing  of  the  non-reinnervated reinnervated  contraction muscles.  EDL  non-reinnervated  had  times  By  12  for  the  weeks, the  recovered  to  muscles were s t i l l  the  noticable in both absolute  recovered  twitch and tetanic tensions compared to  have 2  muscles.  This was  SOL  we  reinnervated  contractile  control  values;  an i n i t i a l and  times  the  of  however,  the the  s i g n i f i c a n t l y slower contracting than  t h e i r control or reinnervated counterparts. The  contractile  difference in the TTP muscles  and  the  times and  of  SOL  the 1/2RT  controls  at  were  more . puzzling.  between the 3  and  6  There  was  no  denervated/devascularized weeks  (reinnervated  and  -76non-reinnervated);  however,  by  9  weeks  the  reinnervated  and  the  non-reinnervated muscles both contracted slower than the controls with no difference  between  significant  the  former  difference  in  two.  TTP  Finally  and  by  1/2RT  non-reinnervated and control muscles.  12  weeks, there was  between  response  of  the  EDL  and  SOL  control  exhibited This was  values  by  60  days  slower c o n t r a c t i l e  and  not the case in our study.  of TTP  and  1/2RT.  slowed and then sped up to  post-surgery.  speeds  found a d i f f e r e n t i a l  in terms  Similar to our experiments, the EDL i n i t i a l l y reach  reinnervated,  Using EDL and SOL muscle grafts in  one month old r a t s , Carlson and Gutmann (1975) also regenerative  the  no  The  grafted  SOL  never did reach control  The TTP and 1/2RT  also  values.  of reinnervated SOL  were slower than the controls early on but by 12 weeks they had recovered. The normal  slowing and  values  development.  was  a pattern  A l l early  produce an embryonic The  fast-twitch  isozymes of  f2  f2,  and  eventual return  f3  contain  the  isozymes  contracting.  The  embryonic  neonatal  and  EDL seen  during  which  are  heavy chain  a l , 1983).  isozymes  MHC  The  SOL  development;  f4  FM1-FM3 appear.  follows  the a  however, the  slow  isozyme  forms  that  reinnervated  embryological development.  appear and  contracting  fraction The  and  rest  parts of  Between  f3, 10-15  At this point, the muscle  similar loss  of  becomes course  fast  during  eMHC i s much  contains 2 isozymes, si and s2, which are  precursors to the adult slow isozymes SMI and SM2. slow adult  embryological  (MHC)(Close, 1964).  (eMHC).  begin to disappear and  slow-twitch  speeds to  4 weeks of age, contains 4 myosin  embryonic  A d d i t i o n a l l y , the SOL  the  contractile  represent isozymes with neonatal MHC.  the adult fast  developmental  muscles  the f i r s t et  the  to that  form of the myosin  EDL, during  a l l of f 1  days of age  slower.  developing  (f1-f4)(Rubenstein and  similar  of  By 15 days of age, the  (Rubenstein et al,1983). regenerated  SOL  and  EDL  It i s apparent recapitulate  Young EDL muscle produces a slower acting form  -77of  myosin but with  manifested times.  in  The  shortened  development, the mature fast form predominates as i s  our  data  by  reinnervated  the  SOL,  gradual  on  the  shortening  other  speeds are greatly affected by  1983).  indicates  The  final  that  therefore, the  the  return  muscle  nerve has  to  the  cold-injured pectoralis  has  been  transformed  adult chicken  major  fibres  and  the  contained  characteristic  addition, weeks,  slow  the  adult  the  reinnervation (Rubenstein  et  isoform  phase of  and  the  embryonic  and  development; synthesis  of  neonatal  however, the  and SOL  the  normal SOL  and  the  and type.  were  initial  quantity of  reduced.  expressed.  Marechal  et  In  By  al  3-4  (1984)  with similar r e s u l t s . regeneration  that the  closely  is  the  of  the  synthesis  that  of  characterized  does not.  They  as opposed to  time course  parallels  stage, which  adult forms of isomyosins,  The  greatly  i s characterized by  isomyosins second  1/2RT  regeneration of the fast  was  appeared.  They discovered that  and  Strohman and Matsuda (1983), using  B-tropomyosin  study on rat EDL  regeneration  TTP  reinnervated  the muscle to the  muscle,  pattern  development.  of  appropriately  studied the time course of development during embryological  values  embryonic forms of MHC  chains  However,  Unlike the EDL,  anterior latissimus d o r s i .  embryonic  a  SOL  muscle, followed slow  of  light  conducted a s i m i l a r  early  exhibits  the  control  Others have reported similar f i n d i n g s .  FLC3,  contraction  contraction time because of the embryonic form of MHC.  contractile  muscle  the  hand, i n i t i a l l y  with time the c o n t r a c t i l e speeds gradually slow.  al,  of  of  early by  It appears  the that  this second stage takes longer in regenerating muscle. The  maximum v e l o c i t y  unchanged Studies  throughout  conducted  by  the  of  unloaded  regenerative  Faulkner  and  agreed that a f t e r muscle grafting control  value.  Unfortunately  shortening response  in  Cote (1986) and the Vo  in  values  neither  of  for the  EDL  present  Faulkner  returned these  and  SOL  was  study.  et al (1980)  to 100% studies  of did  the the  -78investigators  report  Vo  during  the  early phases of regeneration.  (1964) studied force-velocity properties of EDL days. two  He  discovered  muscles  are  increases but  that at birth  similar.  With  that of SOL  the  aging  from that found by Close  but  muscles  i n Close's  mature  d i f f e r s from embryological the muscle.  in  length  had  a  Our Vo was  over  change  f o r c e - v e l o c i t y properties of  the  one  speed  SOL  of  constant.  shortening  EDL  Our Vo data for EDL  must r e a l i z e nerve  of  that the  immature  reinnervating them.  This  development where the nerve matures along  Another possible reason  data a n a l y s i s .  100  remains f a i r l y  differs  study  from birth to  the  and  Close  in  for the  discrepancy  determined from the time, whereas  i s the mode of  slope derived  Close  with  determined  from change  the  speeds of  shortening from a force-velocity r e l a t i o n s h i p . The that  20%  PTP  some of  for EDL by 12 weeks post-surgery was  the  EDL  had  reinnervated.  should  not  show any  typical  slow-twitch  SOL  not.  However, the  which  may  do  tension  SOL  muscles  be  attributed  to  On  PTP  non-reinnervated  the  other  because the  SOL  some of  a further indication  revealed  the  slow  hand, a  reinnervated post-tetanic  SOL  fibres  being  reinnervated by a fast nerve. The and  fatigue data gathered for EDL  t-tests.  A  significant  and SOL  difference was  were analyzed  found  using ANOVA  i f p<0.05.  We  have  interpreted the data with the understanding that we are looking for trends in the f a t i g a b i l i t y of the denervated/devascularized pattern  of the  early regenerating  EDL  was  muscles. The  fatigue  similar to the early stages  of  development of muscle which were more fatigue-resistant than the controls (personal  communication  with  D.  Redenbach).  By  6  weeks, the  pattern of the reinnervated muscles resembled that of the reinnervated weeks.  muscles  This may  be  remain  slightly  accounted  for by  greater oxidative capacity of the  more  regenerated  controls.  fatigue-resistant  a greater  capillary  fatigue  up  to  density and  muscle f i b r e s , as  The 12 a  reported  -79by  Faulkner  muscles  and  Cote (1986) in a study on rat EDL.  remain  reinnervated  more  fatigue-resistant  than  The  the  non-reinnervated  controls  muscles which i s consistent with denervated  and  the  muscle (Davis et  a l , 1988).  the  The denervated/devascularized SOL  i s also more fatigue-resistant than  controls  the  contrasts  at  3  that  of  in  the  difference  weeks;  EDL  however,  between  6-12  fatigability  weeks.  between  non-reinnervated muscles unlike what we for this a r i s e . the slow SOL account  fatigability By  the  12  pattern  weeks, there  control,  SOL  was  no  reinnervated,  and  saw in the EDL.  One, which i s supported  of  Two  possibilities  by the PTP data, i s that some of  muscles have been reinnervated by a fast nerve and this would  for  the  Alternatively,  increased  the  non-reinnervated  fatigability  non-reinnervated  but  rather,  as  of  the  older  muscles  may  not  suggested  SOL  muscles.  be  above,  completely  have  delayed  reinnervation. It i s apparent that under certain chance  f o r recovery, i . e .  fast-twitch  and  reinnervation and  slow-twitch  skeletal  regeneration  differs  slightly  between  muscles  through  stages  similar  go  conditions and  given  revascularization  muscle  do  occur.  these  two  types  to  the  those  optimal of  both  Moreover,  this  of muscle.  during  Both  embryological  development with the f i n a l product a fast-twitch or a slow-twitch skeletal muscle very muscles  that  denervated fatigue  similar did  muscle.  pattern  to not The  i t s age-matched reinnervate  maintain  non-reinnervated  suggesting  that  control.  it  SOL too  reinnervated by the sprouting of a fast nerve.  Those  fast-twitch  characteristics  typical  muscles reveal a PTP may  have  been  and  EDL of a  aberrantly  -80Conclusions  The basis of this project was to investigate the usefulness of a new preparation and  i . e . denervated/devascularized  to characterize  during  regeneration.  the fast-twitch and were found  the mechanical  properties of these  mouse  After such surgery, the physiological  EDL and the slow-twitch  SOL were  muscle, muscles  properties of  statistically  analysed  to recover in those muscles that had reinnervated. Another  group of muscles designated same extent.  slow and fast-twitch  as non-reinnervated  did not recover  to the  In p a r t i c u l a r , the non-reinnervated SOL appeared to receive  delayed  reinnervation or they  results  of this  study  were  were  reinnervated  to provide  by a fast  a mouse model  nerve.  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(1986) Reinnervation of the l a t e r a l gastrocnemius and soleus muscles i n the r a t by t h e i r common nerve. J . P h y s i o l . 372: 485-500.  Gutmann, E., Melichna, J . , and Sycovy, I. (1974) Developmental changes in contraction time, myosin properties and f i b r e pattern of fast and slow skeletal muscles. Physiol. Bohem. 23: 19-27.  Hansen-Smith, F.M., Carlson, B.M., and Irwin, K.I. (1980) Revascularization of the f r e e l y grafted extensor digitorum longus muscle i n the r a t . Am. J . Anat. 158: 65-82.  -83Hatano, E., Suge, T., Ikuta, Y., Miyamoto, Y., Yoshioka, K., and Hiramatsu, N. (1981) Denervated changes i n muscle fibers and motor end p l a t e s . Hiroshima J . M. S c i . 30(4): 293-299.  Hudgson, P. and F i e l d , (1973) THE STRUCTURE AND FUNCTION OF MUSCLE Academic Press, NY, V o l . 2.  Hudlicka, 0. (1973) B.V.-Amsterdam.  MUSCLE BLOOD FLOW. Swets and Z e i t l i n g e r  Hudlicka, 0. and T y l e r , K.R. (1986) Academic Press, NY.  THE GROWTH OF THE VASCULAR SYSTEM.  K e l l y , A.M. and Rubinstein, N.A. (1986) Development of neuromuscular s p e c i a l i z a t i o n . Med. S c i . Sports Exerc. 18(3): 292-298.  K e l l y , A.M. (1979) Variations i n s a t e l l i t e c e l l d i s t r i b u t i o n i n developing and mature muscles of the r a t . Muscle Regeneration. Mauro. Raven Press, New York, pp.167-177.  A.  K e l l y , A.M. and Rubenstein, N.A. (1986) Muscle h i s t i o g e n i s i s and muscle d i v e r s i t y . Mol. B i o l , of Muscle Dev. 29: 77-84.  K i r s c h , G.E. and Anderson, M.F. (1986) Sodium channel kinetics i n normal and denervated rabbit muscle membrane. Muscle Nerve 9: 738-747.  Leung, W.N., J e f f r e y , P., and Rostas, J.A. (1984) The e f f e c t of denervation on mammalian sarcolemmal proteins and glycoproteins. Muscle Nerve 7: 35-49.  Lieber, R.L., Friden, J . , Hargens, A., and Feringa, E. (1986) Long-term effects of spinal cord transection on fast and slow r a t skeletal muscle. Exp. Neurol. 91: 423-434.  Lowrie, M.B. and Vrbova, G. (1984) Different pattern of recovery of fast and slow muscles following nerve injury i n the r a t . J . P h y s i o l . 349: 397-410.  Lowrie, M.B., Krishnan, S., and Vrbova, G. (1982) Recovery of slow and fast muscles following nerve injury during early post-natal development i n the r a t . J . Physiol. 331: 51-66.  Lowrie, M.B., Krishnan, S., and Vrbova, G. (1987) Permanent changes i n muscle and motoneurones induced by nerve injury during a c r i t i c a l period of development of the r a t . Dev. Brain Res. 31: 91-101.  -84Marechal, G., Schwartz, K., Beckers-Bleukx, G., and Ghins, E. (1984) Isozymes of mysosin in growing and regenerating rat muscles. Eur. Biochem. 138: 421-428.  M i l l a r , T.A. and Das, S.K. (1981) An early report of free muscle grafts in r a b b i t s . MUSCLE TRANSPLANTATION: 83-89.  Muir, A.R. (1970) REGENERATION OF STRIATED MUSCLE AND MYOGENESIS. Excerpta Medica, Amsterdam.  Navarrete, R. and Vrbova, G. (1984) D i f f e r e n t i a l e f f e c t of nerve injury at b i r t h on the a c t i v i t y pattern of reinnervated slow and fast muscles of the r a t . J . Physiol. 351: 675-685.  Redenbach, D. (1985) Histochemical and c o n t r a c t i l e properties following neonatal denervation i n the fast-twitch extensor digitorum longus muscle of the mouse. Thesis: University of B r i t i s h Columbia.  Redenbach, D.R., Ovalle, U.K., and Bressler, B.H. (1988) Effect of neonatal denervation on the d i s t r i b u t i o n of f i b e r types i n a mouse fast-twitch skeletal muscle. Histochem. 89: 333-342.  Rubenstein, N.A., Lyons, G., Gambke, B., and K e l l y , A. (1983) Control of myosin isozymes in the r a t . Adv. Exp. Med. B i o l . 182: 141-154.  Schultz, E. (1984) A quantitative study of s a t e l l i t e c e l l s i n regenerated SOL and EDL. Anat. Rec. 208: 501-506.  Strohman, R.C, and Matsuda, R. (1983) Myosin expression during regeneration and in denervated skeletal muscle. Adv. Exp. Med. B i o l . 182: 259-264.  Vrbova, G., Gordon, J . , and Jones, R. (1978) Chapman and Hall L t d . , London.  NERVE MUSCLE INTERACTION.  Webster, D. and Bressler, B.H. (1985) Changes in isometric c o n t r a c t i l e properties of extensor digitorum longus and soleus muscles of C57BL/6J mice following denervation. Can J . P h y s i o l . Pharm. 63(6): 681-686.  -85-  APPENDICES  -86APPENDIX A-Control and Sham Experiments  A  set of  comparison  to  determine  sham experiments the  normal  was  unoperated  done on  EDL  C57BL/6J  and  SOL  mouse.  muscles for This  was  to  the e f f e c t , i f any, of surgery on the c o n t r a c t i l e properties  of the respective muscles. The results of these experiments can be seen in Tables 9 (EDL) and 10 (SOL), and in Fig 11. there was  no difference  SOL and t h e i r respective  Statistical  analysis  ( t - t e s t ) determined that  in any of these parameters between the EDL and controls.  DISCUSSION  This  set of  controls.  The  experiments  technique  used  denervation/devascularization nerves  were  anesthesia cutting  not  and  initial  connective tissue  conducted  mimicked  surgery but  severed.  the  was  We  hoped  phases  of  to  to determine  the  technique  here  used  blood  determine  surgery  planes, had on  the  ie.  our  what  set of f o r the  vessels  and  effect  the  opening  the l e g ,  the c o n t r a c t i l e parameters of  the EDL or SOL muscles. The effect  on  muscles. our l a b .  results the  consistently  contractile  suggested that the sham operation had no  parameters  of  either  the  EDL  or  the  SOL  This i s consistent with the results found by other members of  Table 9: The contractile properties and muscle weight of Control and Sham EDL Muscles C-EDL (n)  Sham (n)  a  Twitch tension (g)  8.560+1.2 (6)  8.330+1.1 (5)  Twitch tension/muscle weight (g/mg)  0.698±.13  0.662±.05  (6)  (5)  Tetanus tension (g)  36.56+3.1 (6)  34.38+2.7 (5)  Tetanus tension/muscle weight (g/mg)  3.011+.63 (6)  2.742+.26 (5)  Time -to-peak twitch (ms)  24.01+1.4 (6)  24.17+3.4 (5)  Half-relaxation time (ms)  40.06+3.9 (6)  37.89+5.3 (5)  Shortening velocity (Lo/s)  6.26+1.9 (6)  5.00+.89 (5)  Twitch potentiation  1.18+.07 (6)  1.24+.20 (5)  EDL muscle weight (g)  12.48+2.4 (6)  12.56+1.1 (5)  a:  All values are means+SD and the level of significant difference is p<0.05  Table 10: The contractile properties and muscle weight of Control and Sham SOL Muscles C-SOL (n)  Sham (n)  a  Twitch tension (g)  3.288+.53 (6)  3.197+.51 (5)  Twitch tension/muscle weight (g/mg)  0.246+.05 (6)  0.227+.04 (5)  Tetanus tension (g)  19.46+2.4 (6)  18.08+1.9 (5)  Tetanus tension/muscle weight (g/mg)  1.340+.19 (6)  1.412+.36 (5)  Time -to-peak twitch (ms)  60.62+4.1 (6)  67.48+11 (5)  Half-relaxation time (ms)  155.9+22 (6)  137.3+14 (5)  Shortening velocity (Lo/s)  4.95+1.2 (6)  5.40+1.3 (5)  Twitch potentiation  0.974+.04 (6)  1.01+.07 (5)  SOL muscle weigh (g)  14.57+.80 (6)  13.36+1.9 (5)  a:  A l l values are means±SD and the level of significant difference i s p<0.05  -89-  F i g . 11.  The fatigue regime for control and  sham EDL and  SOL.  -90-  Fatigue: Sham EDL o control * sham  0  100  200  300  400  Time (sec)  Fatigue: Sham SOL  0.2 H 0  i  r -  100  1  1  200  Time (sec)  1  300  •  ,  400  -91Appendix B-Sciatic Neurectomy  In  order  to  assess  devascularization, sciatic  a  the  group  neurectomy was  of  effects  of  denervation  experiments  performed  on  was  5 mice  alone  carried  at  4  out  without  on  weeks of  which a  age.  The  r e s u l t i n g data were then compared to the age-matched control data and to the data from the denervated/devascularized studied TTP,  included  1/2RT, Vo,  absolute PTP,  and  and  normalized  fatigue.  A  experiments. twitch and  significance  The  parameters  tetanic  level  tensions,  of p<0.05  was  used in the t - t e s t s . A set of animals, in which a s c i a t i c neurectomy was of age  and  studied at 12 weeks post-denervation  for both EDL the  and  SOL  The  produced muscle weights  that were s i g n i f i c a n t l y less than the controls and  reinnervated muscle weights, but  non-reinnervated  done at 4 weeks  they were not  different  from  the  (Table 11)  was  muscle weights (p<0.05)  twitch tension produced by  the denervated  EDL  s i g n i f i c a n t l y less than that produced by  the  not  reinnervated muscles.  different  other  from that produced by  hand, the  denervated  than the non-reinnervated muscle  weight  control  no  muscles.  produced by from  the  however,  significantly  twitch muscles.  control,  tension In  between  significantly  greater  (Table  non-reinnervated  values.  twitch than  than  both  contrast,  reinnervated  normalized  however, i t was On  greater  the  denervated  the  reinnervated  absolute  13)  was  or  non-reinnervated  the  and  to the  produced a s i g n i f i c a n t l y  the  tension  the  tension  When the twitch tension i s normalized  Moreover, the denervated EDL  denervated SOL  the  EDL.  produced  difference exists  greater normalized non-reinnervated  EDL  the  controls;  not  twitch  significantly  for  control,  and  tension different  muscle  denervated  the  values; SOL  reinnervated  is and  Table 11: The Absolute and Normalized Twitch and Tetanic Tensions of Control, Reinnervated, Non-reinnervated and Denervated EDL Muscles Pt (g) (n)  Pt/MW (g/mg) (n)  a  Po (g) (n)  Po/MH (g/mg) (n)  Control  10.12+1.0 (6)  0.784±.l (6)  38.81+1.5 (6)  2.95±.26 (6)  Reinnervated  5.28±1.2 (3)  0.33W.09 (3)  25.00±5.0 (3)  1.54±.25 (3)  Non-reinnervated  1.55+1.1  0.287±.27 (4)  3.87+3.7 (4)  0.759±.93 (4)  0.704±.17ta (5)  25.14+11 ^ (5)  (4)  Denervated  a: •: f: a:  6.30+1.8°* (5)  1  2.59±.74* (5)  All values are means+SD and the level of significant difference i s p<0.05 A significant difference exists between denervated and control muscles, A significant difference exists between denervated and reinnervated muscles A significant difference exists between denervated and non-reinnervated muscles  Table 12: The Remaining Contractile Properties and Muscle Height of Control.Reinnervated, Non-reinnervated and Denervated EDL Muscles TTP (ms) (n)  Control  22.42±1.7 (6)  Reinnervated  a  1/2RT (ms) Vo (Lo/sec) (n) (n)  PTP (n)  Muscle Weight (g/mg) (n)  54.7U4.6 (6)  7.57±3.0 (6)  1.21±.05 (6)  13.00±1.1 (6)  24.23±1.6 (3)  52.95+11 (3)  9.10±1.7 (3)  1.14±.09 (3)  14.97±2.3 (3)  Non-reinnervated  42.34±6.5 (4)  84.52±19 (4)  10.51+11 (4)  1.08U.08 (4)  6.325±1.4 (4)  Denervated  25.46±ll (5)  a: f: a:  a  41.46±15 (5)  a  5.92±1.4t (5)  1.116+.10 (5)  9.3±2.9 (5)  All values are means±SD and the level of significant difference i s p<0.05 A significant difference exists between denervated and reinnervated muscles A significant difference exists between denervated and non-reinnervated muscles  Table 13: The Absolute and Normalized Twitch and Tetanic Tensions of Control, Reinnervated, Non-reinnervated and Denervated SOL Muscles Pt (g) (n)  Control  3.44±.80 (6)  Rei nnervated  Pt/MW (g/mg) Po (g) (n) (n)  a  Po/MW (g/mg) (n)  0.24±.07 (6)  19.78+1.6 (6)  1.36+.15 (6)  3.68+1.1 (5)  0.19±.06 (5)  19.32±5.0 (5)  1.54±.25 (5)  Non-rei nnervated  1.55+1.1 (2)  0.287±.27 (2)  3.87+3.7 (2)  0.759±.93 (2)  Denervated  6.30+1.8 (5)  0.704±.17 t« (5)  25.14+11 (5)  2.59±.74 (5)  a: •: f: a:  D  A l l values are means±SD and the level of significant difference i s p<0.05 A significant difference exists between denervated and control muscles A significant difference exists between denervated and reinnervated muscles A significant difference exists between denervated and non-reinnervated muscles  Table 14: The Remaining Contractile Properties and Muscle Weight of Control, Reinnervated, Non-reinnervated and Denervated SOL Muscles. TTP (ms) (n)  Control  70.49±5.1 (6)  Reinnervated  1/2 RT (ms) Vo (Lo/sec) (n) (n)  a  PTP (n)  Muscle Weight (g/mg) (n)  184.U35 (6)  5.58±1.3 (6)  0.980±.02 (6)  14.66+1.5 (6)  73.43±23 (5)  211.7+104 (5)  5.46±2.0 (5)  0.980±.03 (5)  19.54±3.0 (5)  Non-reinnervated  50.07±3.6 (2)  108.3±37 (2)  5.05±2.2 (2)  1.120+.16 (2)  10.45±9.8 (2)  Denervated  42.23±18°t 75.25133°* (5) (5)  8.24±2.lQf (5)  1.0501.06°* (5)  10.3813.7°* (5)  a: •: a:  A l l values are means±SD and the level of significant difference is p<0.05 A significant difference exists between denervated and control muscles A significant difference exists between denervated and non-reinnervated muscles  -96The denervated EDL produced less tetanic tension than the controls; however, i t produced muscles.  greater tetanic  In a d d i t i o n , there was  tension  than  no difference  in the absolute tension  produced  by the denervated and reinnervated EDL.  tension  of  denervated  reinnervated  values  non-reinnervated from  the  EDL but  tetanic  control  or  did it  not  was  tension.  The normalized tetanic  differ  from  significantly The  the non-reinnervated  denervated  the  control  greater SOL  did  denervated/devascularized muscles  or  than not  in  the  differ  terms  of  were  not  reinnervated muscles  but  than the non-reinnervated muscles.  The  absolute and normalized tetanic tension. The  TTP  significantly  and  1/2RT  different  from  they were s i g n i f i c a n t l y denervated SOL both  the  faster  from  Vo  denervated  the  faster  control  EDL or  (Table  (Table 14) on the other hand was  control  different  of  and  the  for  the  reinnervated  denervated  SOL  versus  faster contracting than  muscles  non-reinnervated SOL.  12)  but  Correlated  control  they with  muscles. EDL  was  were this  The  not is a  maximum  v e l o c i t y of unloaded  shortening f o r denervated  slower than  the  reinnervated muscles  but not s i g n i f i c a n t l y d i f f e r e n t from the control or  non-reinnervated muscles. As  mentioned,  approximately a 20% which  is  not  reinnervated produces  a  12  week  potentiation.  considered and  5%  the  no  non-reinnervated  potentiation  significant  non-reinnervated The  The  significantly PTP  control  denervated different values.  EDL  difference  EDL  produces  produces  from The  which i s uncharacteristic  muscle including the control muscles is  age-matched  the  a  PTP  control,  denervated  SOL  of a slow-twitch  in this experiment; however, there  in PTP between the denervated SOL and the  SOL.  fatigue  examined (Fig 12.).  pattern The  for  the  denervated  denervated EDL  was  EDL  and  SOL  was  also  s i g n i f i c a n t l y more fatigue  -97-  F i g . 14.  The fatigue regime for c o n t r o l , reinnervated, non-reinnervated and denervated EDL AND  SOL.  -98-  Fatigue: EDL Denervation  T  0  100  200  300  400  Time (sec)  Fatigue: SOL Denervation  0  100  200  Time (sec)  300  400  -99resistant sciatic  than  the controls.  neurectomy  pattern;  muscle  however,  resistant  than  There  was no difference  fatigue pattern  the non-reinnervated  the denervated  between the  and the reinnervated  fatigue  muscles  fatigue  EDL. In contrast,  were  more  the denervated SOL  produced a fatigue pattern that was riot s i g n i f i c a n t l y d i f f e r e n t from the controls,  reinnervated,  or non-reinnervated fatigue  patterns  at  any  point during the t r a i n of tetanic stimulation.  DISCUSSION  These experiments involved the study o f the c o n t r a c t i l e of  denervated  neurectomy The  EDL and SOL.  The muscles  were denervated  and then removed f o r physiological  results  were used  parameters  analysis  by s c i a t i c  12 weeks  later.  to compare to the previous experiments involving  denervation and devascularization o f EDL and SOL muscles. The  denervated  reinnervated  EDL  EDL muscles  closely  resembled  o f the previous  1/2RT, Vo, and PTP of the denervated d i f f e r e n t from the controls. and  fatigue  The  only  muscles  regime  similarity was  and the  study.  The Po/MW, Pt/MW,  EDL were  not s i g n i f i c a n t l y  A l s o , the Pt, Po, Po/MW, TTP, 1/2RT, PTP,  were a l l similar between  the muscle  the control  to the reinnervated  the denervated  weight.  We  muscles (Webster and Bressler, 1985).  and the non-reinnervated  have  resemblance o f the non-reinnervated muscles  EDL muscles.  already  discussed the  to c l a s s i c a l l y  denervated  The denervated EDL i n this study  did not follow the pattern of a denervated muscle, rather i t appeared to have  become  reinnervated.  This  could  explain  the s i m i l a r i t y  to  the  control and in p a r t i c u l a r to the reinnervated muscles. The  denervated  SOL muscles  appeared  to be "truely"  denervated.  They resembled the non-reinnervated SOL muscles o f the previous study i n  -100terms of t h e i r muscle pattern.  At  12  weights, weeks  P t , Po, TTP, 1/2RT, PTP, and fatigue post-denervation/devascularization,  reinnervated and non-reinnervated muscles closely muscles  but a  difference  was  still  seen  muscle weight, TTP, 1/2RT, and PTP. the denervated but  SOL differed  resembled  the  interpretation  of  non-reinnervated  from the control  data  muscles with  muscles,  respect to  I t was these four parameters where  non-reinnervated  these  resembled the control  among the control  reinnervated muscles, and the non-reinnervated  the  was  SOL. that  and reinnervated muscles One  problem  with  the  i f we  assumed  that  the  SOL had become reinnervated but by a fast nerve or they  had acquired delayed innervation, then the TTP, 1/2RT, and PTP data are somewhat  misleading.  Consequently,  denervated SOL i s a c t u a l l y completely  denervated  responses  muscle.  reinnervated,  thus  The  cannot  conducted  their  sure  between denervated/devascularized EDL  and  excluding  sciatic  possibly  them  from  neurectomy  l a t e r a l p o p l i t e a l ) on adult r a t s .  that  the  to provide a basis f o r comparison  comparison, the denervation data from Finol they  be  denervated.  These experiments were designed of physiological  we  muscle and  the SOL  appear  to have  further  analysis.  For  et al (1981) can be used as  experiments  ( s p e c i f i c a l l y the  

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