THEANATOMICALRECORD263:139–154(2001) ReparativeMyogenesisinLong-Term DenervatedSkeletalMusclesofAdult RatsResultsinaReductionofthe SatelliteCellPopulation EDUARDI.DEDKOV,1*TATIANAY.KOSTROMINOVA,1 1 1,2 ANDREIB.BORISOV, ANDBRUCEM.CARLSON 1DepartmentofCellandDevelopmentalBiology,UniversityofMichigan, AnnArbor,Michigan 2InstituteofGerontology,UniversityofMichigan,AnnArbor,Michigan ABSTRACT Thisstudy,conductedon25-monthdenervatedrathindlimbmuscles,wasdirected towardelucidatingthebasisforthepoorregenerationthatisobservedinlong-termdener- vatedmuscles.Despiteaϳ97.6%lossinmeancross-sectionalareaofmusclefibers,the musclesretainedtheirfasciculararrangement,withthefasciclescontainingϳ1.5timesmore fibersthanage-matchedcontrolmuscles.Atleastthreedistincttypesofmusclefiberswere observed:degenerating,persisting(original),andnewlyformed(regenerated)fibers.Ama- jorityofnewlyformedfibersdidnotappeartoundergocompletematuration,andmorpho- logicallytheyresembledmyotubes.Sitesofformermotorend-platesremainedidentifiablein persistingmusclefibers.Nucleardeathwasseeninalltypesofmusclefibers,especiallyin degeneratingfibers.Nevertheless,theseverelyatrophicskeletalmusclescontinuedtoex- pressdevelopmentallyandfunctionallyimportantproteins,suchasMyoD,myogenin,adult andembryonicsubunitsofthenicotinicacetylcholinereceptor,andneural-celladhesion molecule.Despitetheprolongedperiodofdenervation,slowandfasttypesofmyosinwere foundinsurvivingmusclefibers.Thenumberofsatellitecellswassignificantlyreducedin long-termdenervatedmuscles,ascomparedwithage-matchedcontrolmuscles.In25-month denervatedmuscle,satellitecellswereonlyattachedtopersistingmusclefibers,butwere neverseenonnewlyformedfibers.Ourdatasuggestthattheabsenceofsatellitecellsina populationofimmaturenewlyformedmusclefibersthathasarisenasaresultofcontinuous reparativemyogenesismaybeacrucial,althoughnotnecessarilytheonly,factorunderlying thepoorregenerativeabilityoflong-termdenervatedmuscle.AnatRec263:139–154,2001. ©2001Wiley-Liss,Inc. Keywords:prolongeddenervation;skeletalmusclefibers;satellitecells;elec- tronmicroscopy;immunohistochemistry;RT-PCR;Western-blot analysis;myogenin;MyoD;nAChR;N-CAM Prolongeddenervationcausesmultiplefunctionaland functionalstateevenafternerveregenerationintothe morphologicalchangesinskeletalmuscleduetotheab- muscle(CarlsonandFaulkner,1988;Carlsonetal.,1996; senceofmotorandtrophicregulatorycontrolbythenerve FuandGordon,1995;Gulati,1988,1990;Gutmann,1948; (Gutmann,1962).Themostprominentfeaturesofdener- vatedskeletalmusclesaretherapidatrophyofmuscle fibersandadecreaseinthenumberofbothmyonucleiand satellitecells(AnzilandWernig,1989;BorisovandCarl- Grantsponsor:NIH;Grantnumber:PO1-AG10821. son,2000;GutmannandZelena´,1962;Rodriguesand *Correspondenceto:Dr.EduardI.Dedkov,M.D.,Ph.D.,De- Schmalbruch,1995;SchmalbruchandLewis,2000; partmentofCellandDevelopmentalBiology,4643MedicalSci- Schmalbruchetal.,1991;Viguieetal.,1997).Amajor encesIIBuilding,UniversityofMichigan,AnnArbor,MI48109. questionthatcontinuestobepoorlyunderstoodiswhy, E-mail:[email protected] afterseveralmonthsofdenervation,skeletalmuscleloses Received2August2000;Accepted8February2001 theabilitytobecomerestoredtoafullstructuraland Publishedonline00Month2001 ©2001WILEY-LISS,INC. 140 DEDKOV ET AL. Gutmann and Young, 1944; Irintchev et al., 1990). Among Light and Transmission Electron Microscopy the possible explanations for the incomplete restoration of Small pieces of EDL muscles from the midbelly area very long-term denervated muscle are a failure of regen- were fixed with a mixture of 4% paraformaldehyde and erating nerves to reach all of the atrophic muscle fibers 2.5% glutaraldehyde in 0.1M phosphate buffer saline and establish mature muscle-nerve contacts (Fu and Gor- (PBS) at pH 7.4, washed in PBS and post-fixed in 1% don, 1995; Gutmann and Young, 1944; Irintchev et al., OsO4. Samples were further dehydrated in a graded series 1990) and/or a steady decline in the number of satellite of ethanol and absolute acetone and then embedded in cells (Rodrigues and Schmalbruch, 1995; Schmalbruch mixture of Epon/Araldite using an Eponate 12™-Araldite and Lewis, 2000; Viguie et al., 1997). However, there are 502 Kit (Ted Pella, Inc., Redding, CA). Transverse semi- observations that during muscle denervation, from sev- thin and ultrathin sections were cut with an ultramic- eral weeks to a few months, the regeneration of new fibers rotome. The 1.0-␮m sections were mounted on glass slides from satellite cells is often sufficient for maintaining a and stained with toluidine blue for general observation. normal or even increased number of viable muscle fibers Ultrathin sections were collected on formvar-coated slot- (Bittner et al., 1995; Mussini et al., 1987; Schmalbruch et ted grids, stained with uranyl acetate and lead citrate, al., 1991; Schmalbruch and Lewis, 2000; Viguie et al., and examined with a Philips CM-100 transmission elec- 1997). tron microscope. Development of a number of new methods during the last several years has allowed better evaluation of the Quantitative and Morphometric Analysis changes in muscle fibers under normal and pathological Quantitative analyses. Forty-eight and 90 muscle conditions at both molecular and structural levels. For fascicles, from control and denervated EDL muscles, re- example, it has been shown that denervation of skeletal spectively, were examined on ultrathin sections by trans- muscle fibers results in reactivation of the myogenic basic mission electron microscopy. Before any calculation was helix-loop-helix transcription factors, MyoD and myogenin performed, the phenotype of each cell in the fascicles was (Adams et al., 1995; Eftimie et al., 1991; Merlie et al., recognized under the high magnification. The criteria 1994; Weis, 1994), as well as elevation of the expression used to identify the muscle fibers were the presence of levels of some embryonic muscle-specific proteins, such as basal lamina and myofibrils. The numbers of muscle fiber N-CAM (Covault and Sanes, 1985, 1986; Moore and profiles, myonuclei and satellite cell nuclei on cross-sec- Walsh, 1986; Sanes et al., 1986; Walsh and Moore, 1985) tions of muscle fascicles were recorded as follows: 1.) the and the ␥-subunit of nAChR (Adams et al., 1995; Witze- distribution of fascicles according to the number of muscle mann et al., 1987). fibers per fascicle was laid out in histograms; 2.) the mean The current study is a continuation of research that has number of myonuclei per fascicle plotted against the mean been done in our laboratory (Lu et al., 1997; Viguie et al., number of muscle fibers in the same fascicle was pre- 1997) designed to elucidate the status of skeletal muscles sented as a histogram; 3.) the mean numbers of muscle after long-term denervation. The specific question in this fibers and myonuclei per fascicle were also calculated; 4.) study was whether the changes in extremely long-term the myonuclei/muscle fiber (Mn/F) ratio was calculated; 5.) satellite cell numbers were expressed in two ways: denervated skeletal muscles represent only a steady dete- first, as a percentage of the total number of nuclei counted rioration of muscle tissue or if concurrent active cellular beneath the basement membranes in cross-sections, and compensatory processes accompany them. second, as ratio of the total number of satellite cells to the total number of muscle fibers calculated in all cross-sec- MATERIALS AND METHODS tioned fascicles that were examined. Animals and Denervation Morphometric analyses. Measurements of cross-sec- This study was conducted on 4-month-old male rats of tional area (CSA) of the muscle fibers were made on trans- the WI/HicksCar strain. After ether anesthesia, the right verse semi-thin sections from the same samples that were sciatic nerve was tightly ligated with silk in two places used for electron microscopy. Images of the tissues were and the nerve was cut between the sutures. Both proximal captured from the sections onto a DELL Precision 410 and distal nerve stumps were implanted into muscular computer by using a Zeiss Axiophot-2 Universal Micro- tissue as far away from each other as possible. This scope with a Zeiss Axiocam digital camera (Carl Zeiss Inc., method is routinely used for long-term denervation of the Germany). The images were converted to grayscale on a hind limb (Viguie et al., 1997). All operations and subse- Power Macintosh 7300/200 computer by using NIH Image quent animal care were carried out in accordance with the 1.62. The circumferences of muscle fibers were electroni- guidelines of the Unit for Laboratory Animal Medicine at cally traced by using an ArtPad II and a graphics tablet the University of Michigan. After operations, the rats with an Erasing UltraPen, and the CSAs were calculated were treated with oral terramycin for 5 days. At 29 in square micrometers (mm2) with the help of NIH Image months of age the extensor digitorum longus (EDL), tibi- 1.62. The distribution of muscle fibers according to CSA in alis anterior (TA), and gastrocnemius muscles were re- control and denervated EDL muscles was laid out in his- moved from both denervated and normal contralateral tograms. legs from each of four rats, and the animals were eutha- nized by an overdose of anesthetic. Age-matched muscles Immunohistochemistry from both non-operated rats (two rats) and contralateral Technique. TA muscles were fixed in freshly prepared legs of denervated animals (four rats), along with normal 2% paraformaldehyde in PBS at 4°C and were then trans- and 1-month denervated muscles from three 5-month-old versely cut in the midbelly area. The