Nerve Growth in Cardiac Muscle - Europe PMC

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Addressfor reprints: M.A. Glasby, B.M., B.Ch., M.A., M.Sc., F.R.C.S., The Royal College. ofSurgeons ofEngland, 35-43 Lincoln's Inn Fields, London WC2A 3PN, ...
Nerve Growth in Cardiac Muscle A.H. Davies, M.A., B.M., B.Ch., B.A. De Souza, B.Sc., M.A. Glasby, B.M., B.Ch., M.A., M.Sc., F.R.C.S., S.E. Gschmeissner, B.Sc., and C.L.-H. Huang, Ph.D., M.D., D.M. The failure of reinnervation after cardiac transplantation is probably of scar formation at the suture lines. However, it must be established whether there are any intrinsic properties of the muscle that prevent reinnervation. This is examined in experimental peripheral nerve implants using cardiac muscle isografts. The results show extensive growth of regenerating axons into the implanted cardiac muscle. It is therefore unlikely that the failure of reinnervation depends upon the physical or chemical properties of the muscle itself. (Texas Heart Institute Journal 1986; 13:447452) a consequence

Key words: Cardiac transplant reinnervation; cardiac isographs GENERALLY ACCEPTED observations following orthotopic cardiac transplantation in human beings suggest that the heart is never reinnervated by either its adrenergic or cholinergic extrinsic neural pathways. All subsequent electrical activity, and thus neural regulation, is of an intrinsic form. 1-3 Work in our laboratory has been centered around the use of fresh and degenerated rat and primate skeletal muscle as a medium for repair in both the central and peripheral nervous systems. We have found that the tubular matrix of basement membrane formed when muscle degenerates is able to support and direct axonal regeneration4-7 in the manner of a bioprosthesis. Three views pertain to the failure of reinnervation of the transplanted heart. First, and recognized as most likely, it is thought that scarring at the suture lines acts as a mechanical barrier to the progress of pioneering axons into the atria. Second, it has been shown in skeletal muscle8'9 that where electrical activity persists, new neuromuscular connections are not

formed when an additional pathway of innervation is established. Third, it may be that intrinsic physical or chemical differences between skeletal and cardiac muscle will prevent reinnervation of the latter in whatever circumstances. The object of the present experiments is to test, specifically, the third of these options, using cardiac muscle isografts implanted into peripheral nerves. MATERIALS AND METHODS

Inbred male rates of the Fisher strain were used in the experiments. Anesthesia was established using intramuscular Hypnorm (Jansen Pharmaceuticals, U.K., 0.5 ml/kg-'). Donor rats were injected intravenously with 1,000 units of heparin; then a median sternotomy was performed to remove the heart, from which strips of left ventricular muscle were cut. The donor muscle was immersed in normal saline at 37°C while the graft site was prepared. The recipient rats underwent surgical exposure of the sciatic nerve through a skin

From the Department of Anatomy, The Royal College of Surgeons of England, London. Address for reprints: M.A. Glasby, B.M., B.Ch., M.A., M.Sc., F.R.C.S., The Royal College of Surgeons of England, 35-43 Lincoln's Inn Fields, London WC2A 3PN, England. Texas Heart Institute Journal

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incision over the iliac crest and retraction of gluteus maximus to expose the greater sciatic foramen, through which the sciatic nerve emerges. Using a Zeiss Jena operating microscope, we excised a 10 mm nerve segment, taking care to preserve the adventitial sheath of the nerve and its vascular supply. Cardiac muscle was first shaped to fit the dimensions of the excised nerve and then interposed into the nerve gap and sutured to the proximal and distal stumps with approximately six 10/0 monofilament nylon sutures at each junction. Only the epineurium was sutured to the periphery of the cardiac muscle graft. The lumbar fascia and skin were closed with 5/0 Dexon. In order to compare the results of nerve growth into cardiac muscle with those of growth into skeletal muscle, we prepared a second population of rats, using grafts made of

autogenous gluteus maximus according to the technique described by Glasby et al.4 At fifty days, following administration of a similar anesthetic, we reopened the original incision and removed the graft en bloc with the proximal and distal nerve stumps. Serial sections of the sample were cut with a razor blade after the specimen had been immersed in cacodylate-buffered 2% glutaraldehyde. Each sample was then transferred to fresh cacodylate-buffered 2% glutaraldehyde for complete fixation. This was followed by postfixation in 1% osmium tetroxide and dehydration in graded alcohols. The segments were embedded in Araldite, and 1 ,um transverse sections were cut on an ultramicrotome. Individual sections were stained with toluidine blue and mounted in DPX. We counted the axons in these sections, using a Nomarski

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RESULTS Nerve growth into cardiac muscle grafts was observed in all cases studied (n = 12), at 50 days after implantation. Figure 1 shows mean numbers of myelinated axons observed at 50 days in whole sections of proximal nerve, cardiac muscle graft, and distal nerve. Several observations may be made. There is a gradient of axon numbers decreasing from proximal Texas Heart Institute Journal

nerve, through cardiac muscle graft, to distal nerve segment; this suggests that longitudinal

growth is not yet complete. A similar observation in both fresh and denatured skeletal muscle grafts was made by Glasby et al,5 who showed that equality of myelinated axon numbers in these three categories was not achieved until 100 days in the case of denatured muscle, and still later in the case of fresh muscle grafts. The present experiments also show that when fresh cardiac and skeletal muscle grafts are compared at 50 days, there are significantly (p < 0.001) more myelinated axons to be found per unit area in the cardiac muscle grafts than in skeletal muscle grafts (Fig. 2). In all of the grafts studied, a finite but variable number of small blood vessels was seen through the microscope. The presence of Nerve Growth in Cardiac Muscek 449

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these is taken to imply that some degree of revascularization of the graft had taken place and that these vessels likely had played some supporting role for the axons contained in the graft, since no axon appeared to be undergoing visible degeneration. Figure 3 is an electron micrograph (x 3,000) of a transverse section through the center of a cardiac muscle graft, 50 days after implantation into a rat sciatic nerve. Numerous myelinated (m) and unmyelinated (u) axons are seen enclosed within their Schwann cell (s) sheaths. Figure 4 is an electron micrograph (x 3,000) of the distal nerve segment to which the graft seen in Fig. 3 was attached. Note the presence of both myelinated and unmyelinated axons, their Schwann-cell endoneurial collagen, and perineurial cells. Figure 5 (x 3,000) shows a cross-section of fresh skeletal muscle that had been implanted as a sciatic nerve graft 50 days before. Note the persistence of muscle fibers and myelinated and unmyelinated axons. Texas Heart Institute Journal

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DISCUSSION The results obtained in this study indicate that when denervated and implanted as an isograft into transected peripheral nerve, devascularized cardiac muscle is able to support axonal growth and maturation in a manner similar to that seen in conventional nerve grafts and in fresh and denatured muscle autografts.5 However, cardiac muscle differs from fresh skeletal muscle in so far as it seems to support more rapid growth. Furthermore, at 50 days after implantation, there seems to be far less morphologically identifiable residual cardiac muscle than is the case with skeletal muscle when skeletal muscle grafts are used. The reasons for this are largely speculative. Glasby et a15 found that at a similar time after implantation of fresh skeletal muscle grafts, there were relatively few axons traversing the graft, and it was supposed that those that had found their way through might have done so by taking advantage of routes opened up by the Nerve Growth in Cardiac Musck 451

spontaneous degeneration of muscle fibers. At 50 days, explanted cardiac muscle resembles muscle that has been "predegenerated" by thermal or osmotic insults, but since it has not been subjected to these stresses, we must suppose that under the same conditions it is more prone to degenerate than is skeletal muscle, and this may be no more than a reflection of its greater metabolic requirements and consequent greater vulnerability in unfavorable environments. In any case, it seems that this more rapid degeneration, without becoming frankly fibrotic, is of advantage to the pioneering axons growing out of the proximal nerve segment. While it is hardly likely that cardiac muscle can serve any useful purpose in peripheral nerve repair, the observation made here-that there seem to be no direct physical or chemical impediments to nerve regrowth into the muscle-raises a number of interesting points. Although nerve fibers may grow into the denervated myocardium, it does not necessarily follow that an electrically active myocardium would support reinnervation. The analogy with skeletal muscle8'9 goes only so far, because cardiac muscle denervated by transplantation has lost its extrinsic drive, and such muscle, relying only on an intrinsic mechanism, will not of necessity behave in the way that other muscle does. Work is continuing in our laboratory to investigate this point, and should there prove to be a situation in cardiac muscle parallel to that in skeletal muscle, it would then seem most likely that the failure of reinnervation seen after transplantation is the consequence of simple mechanical interference with nerve elongation at the site of the suture scar.

REFERENCES 1. Stinson EB, Griepp RB, Schroder JS, Dong E, Shumway NE. Hemodynamic observations one and two years after cardiac transplantation in man. Circulation 1972; 45:1183. 2. Mason JW, Stinson EB, Harrison DC. The autonomic nervous system and arrhythmias: Studies in the transplanted, denervated human heart. Cardiology 1976; 61:75. 3. Bexton RS, Hellestrand KJ, Cory-Pearce R, Spurrell RAJ, English TAH, Camm AJ. Unusual atrial potentials in a cardiac transplant recipient. Possible synchronization between donor and recipient atria. J Electrocardiol 1983; 16(3):313. 4. Glasby MA, Gschmeissner SE, Hitchcock RJI, Huang CL-H. Regeneration of the sciatic nerve in rats. J Bone Joint Surg [Br] 1986; 68(5):829-833. 5. Glasby MA, Gschmeissner SE, Hitchcock RJI, Huang CL-H. The dependence of nerve regeneration through muscle grafts in the rat on the availability and orientation of basement membrane. J Neurocytol 1986; 15(4):497-510. 6. Glasby MA, Gschmeissner SE, Hitchcock RJI, Huang CL-H, de Souza BA. A comparison of nerve regeneration through nerve and muscle grafts in rat sciatic nerve. Neuro-Orthopedics 1986; 2(l):21-28. 7. Glasby MA, Gschmeissner SE, Huang CL-H, de Souza BA. Degenerated muscle grafts used for peripheral nerve repair in primates. J Hand Surg [Br] 1986 (In press). 8. Brown MC, Holland RC, Hopkins WG. Motor nerve sprouting. Annu Rev Neurosci 1981; 4:17. 9. Lomo T. The role of activity in the control of membrane and contracile properties of skeletal muscle. In Thesleff S (ed): Motor Innervation of Muscle. New York, Academic Press, 1976, pp 289-321.

ACKNOWLEDGMENTS The authors thank Miss A.C. Culverhouse for skilled technical assistance and the National Fund for Research into Crippling Diseases (Action Research for the Crippled Child) for a research grant for the study of nerve growth through muscle grafts.

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