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In vivo guidance of regenerating nerve by laminin-coated filaments
EXPERIMENTAL
NEUROLOGY
96,469-473
(1987)
RESEARCH
NOTE
In Vivo Guidance of Regenerating Nerve by Laminin-Coated Filaments SATORU
YOSHII,* TAKAO YAMAMURO,* AND MASAO HAYASHI$?
SEIJI ITO,?
*Department of Orthopedic Surgery, Faculty ofMedicine, Kyoto University, Sakyo-ku. Kyoto 606; tHayaishi Bioinformation Transfer Project, Research Development Corporation of Japan, Minami-ku, Kyoto 601; and *Department of Biology, Ochanomizu University, Bunkyo-ku, Tokyo 112, Japan Received November 3,1986 Laminin is reportedly an extremely potent neurite-promoting agent in vitro. We investigated the effect of laminin on regeneration of axons in vivo, using a cord of polyester filaments coated with laminin as a guide, replacing a 1O-mm segment of the rat sciatic nerve. After 4 weeks the cross section of the nerve guide at its midportion was examined under an electron microscope. Many regenerated axons were seen in the laminin-coated group, whereas no axon was observed in the controls. We conclude that laminin guides the regeneration of axons in vivo. 0 1987 Academic Press, Inc.
During regeneration, nerve fibers may be guided by specific neurite-promoting macromolecules within the extracellular matrix. Interactions between microspikes and the substratum adjacent to the growth cone were found to be important determinants of the directions and pathways of axonal elongation (7). It is generally accepted that the basement membrane provides a scaffolding along which regenerating cells migrate during tissue regeneration after injury. Macromolecules, including laminin, fibronectin, and heparan sulfate proteoglycan, are major components ofthe basement membrane (6, 15). The large glycoprotein, laminin, is a major component of all basement membranes and promotes epithelial cell attachment to the substrata in i We thank M. Kanato for skillful technical assistance, and M. Harada for typing the manuscript. Please address correspondence to Dr. Satoru Yoshii. 469 0014-4886187 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
YOSHII ET AL.
470 0)
:. G
b)
LAMININ
GUIDES REGENERATING
NERVE IN VZVO
471
vitro whereas fibroblasts attach to fibronectin (14). Laminin is an extremely potent net&e-promoting agent in vitro ( 10, 13). Neurite outgrowth is guided by laminin in culture medium (3). In vivo experiments revealed that laminincontaining gel in a tube increased the rate of peripheral nerve regeneration (9). However, tube-shaped nerve guides usually induce good regeneration of nerve fibers in a short distance without laminin ( 1, 2, 8, 11, 12, 17). In this study, we developed a new type of nerve guide coated with laminin and assessed its effect on in vivo nerve guidance. Nerve guides were made of polyester filaments. Two thousand polyethylene telephtalate filaments ( 10 pm in diameter, Toyobo, Osaka, Japan) were wound together with 10-O monofilament nylon thread (S&T, West Germany) to form a cord 10 mm in length and 1 mm in diameter (Fig. la). All nerve guides were immersed 24 h in poly-DL-omithine (Sigma) solution (0.1 mg/ml in 15 mM borate buffer, pH 8.4) at room temperature (4). Then, the guides were washed four times with sterile double-distilled water. These guides were used as controls, or were subsequently coated with laminin. Laminin was purified from mouse EHS sarcoma as reported elsewhere ( 15). Polyornithine-treated nerve guides were incubated 2 h at room temperature in a solution consisting of 1 mg laminin and 20 ml phosphate-buffered saline (PH 7.4). Before grafting, the nerve guides were rinsed three times with phosphate-buffered saline. Ten 3-month-old Wistar rats weighing about 250 g were used, 4 in the control group and 6 in the laminin-coated nerve guide group. All operative procedures were carried out under pentobarbital anesthesia (40 mg/kg). The right sciatic nerve was exposed at the buttock and a IO-mm segment of the nerve was removed; the nerve guide was inserted and sutured to the proximal and distal stumps of the nerve under an operating microscope, using 10-O monofilament nylon epineurial sutures (Fig. 1b). The wound was closed in two layers. At 4 weeks after surgery, the animals were killed with a lethal dose of pentobarbital. The sciatic nerve was reexposed and the nerve guide was removed. At the midportion of the nerve guide, a l-mm long specimen was taken and its ultrathin sections were examined with a Nihon Denshi JEM200cx electron microscope at 100 kV. In five of the six nerve guides coated with laminin, numerous myelinated and unmyelinated axons were seen among the polyester filaments (Fig. 1c). FIG. 1. a-A scheme of our nerve guide (G). b-Epineurial suture of the nerve guide to the stumps of rat sciatic nerve (N). c-A transverse section of the laminin-coated nerve guide. Myelinated (M) and unmyelinated (U) axons were seen among polyester filaments(P). d-A transverse section in a control nerve guide.Cross-sectioned collagen fibrils (C), fibroblast (F), and macrophage (M) were seen among polyester filaments (P) but no regenerating axons were present.
472
YOSHII ET AL.
Myelin sheaths of regenerating nerve fibers were thinner than the myelin sheaths of the normal sciatic nerve of the rat. In the control group, no axon was seen in any of the four nerve guides and many collagen fib&, fibroblasts, and macrophages were found instead of nerve fibers (Fig. Id). Polyornithine coating has been reported to have no inhibitory effect on nerve regeneration (4, 7). These results demonstrate that laminin had a remarkable effect on regeneration of axons along polyester filaments in viva Fibers of severed peripheral nerves are known to require the presence of segments of nerve bundles for regeneration. Whereas the dead Schwann cells are phagocytosed by macrophages, the basement membranes of the Schwann cells remain as tubular scaffolds and regenerating axons grow through the scaffolds (5). It appears important that axons always attach to the inner side (cellular surface) of the basement membrane scaffolds. It has been reported that laminin is localized mainly on the cellular surface of the basement membrane of Schwann cells ( 16). Many tube-shaped nerve guides usually induce good regeneration of nerve fibers in a short distance without laminin (1, 2, 8, 11, 12). Therefore, tube-shaped nerve guides are not suitable to assessthe effect of laminin on in vivo nerve guidance. Weiss and Taylor used fiber cores with collagen tubes or with sleeves of arteries ( 17). However, it remains undecided whether the addition of a fiber core was of any real advantage. Their nerve guide should be regarded as one of the tube-shaped nerve guides. Our nerve guide is made of filaments that are opened to the surrounding extracellular matrix. Therefore, regenerating nerve fibers can extend in any direction without being confined among filaments. Poor axonal regeneration was seen in the control group. The finding that rat peripheral nerve was evidently guided by mouse laminin suggests that laminin derived from one species may guide regenerating nerve fibers of another species in viva as observed for neurites in vitro (3, 10, 13). We demonstrated that laminin guided the regenerating fibers of a peripheral nerve in vivo using a new type of nerve guide coated with laminin. This approach may be applicable to nerve guide bridging for a lengthy defect of a peripheral nerve. REFERENCES 1. CHIU, D.T.W., LJANECKA, T.J. KRIZED, M. WOLFF,ANDR.E.L~VELACE. 1982.Autogenous vein graft as a conduit for nerve regeneration. Surgery91: 226-233. 2. DUCKER, T. B., AND G. J. HAYES. 1968. Peripheral nerve injuries: a comparative study of the anatomical and functional results following primary nerve repair in chimpanzees. Milit. Med. 133: 298-305. 3. HAMMARBACK,J.A.,S.L.PALM,L.T.FURCHT,ANDP.C.LETOURNEAU. 198.5.Guidance of neurite outgrowth by pathways of substratum-adsorbed laminin. J. Neurosci. Rex 13: 213-220.
LAMININ
GUIDES
REGENERATING
NERVE
IN
VW0
473
4. HELFAND, S. L., G. A. SMITH, AND N. K. WESSELLS. 1976. Survival and development in culture of dissociated parasympathetic neurons from ciliaty ganglia. Dev. Biol. 50: 54 l547. 5. IDE, C., K. TOHYAMA, R. YOKOTA, T. NITATORI, AND S. ONODERA. 1983. Schwann cell basal lamina and nerve regeneration. Brain Res. 288: 6 l-75. 6. KEFALIDES, N. A., R. ALPER, AND C. C. CLARK. 1979. Biochemistry and metabolism of basement membranes. Int. Rev. Cytol. 61: 167-228. 7. LETOURNEAU, P. C. 1975. Cell-to-substratum adhesion and guidance of axonal elongation. Dev. Biol.44: 92-101. 8. LUNDBORG, G., L. B. DAHLIN, N. DANIELSEN, H. A. HANSSON, A. JOHANNESSON, F. M. LONGO, AND S. VARON. 1982. Nerve regeneration across an extended gap: a neurobiological view of nerve repair and the possible involvement of neurotrophic factors. J. Hand
Surg. 7: 580-587. 9. MADISON, R., C. F. DA SILVA, P. DIKKES, T. CHIU, AND R. L. SIDMAN. 1985. Increased rate of peripheral nerve regeneration using bioresorbable nerve guides and a laminincontaining gel. Exp. Neural. 88: 767-772. 10. MANTHORPE, M., E. ENGVALL, E. RLJOSLAHTI, F. M. LONGO, G. E. DAVIS, AND S. VARON. 1983. Laminin promotes neuritic regeneration from cultured peripheral and central neurons. J. Cell Biol. 97: 1882- 1890. 11. MIDGLEY, R. D., AND F. M. WOOLHOUSE. 1968. Silicone rubber sheathing as an adjunct to neural anastomosis. Surg. Clin. North. Am. 48: 1149-l 157. 12. RICE, D. H., F. D. BURSTEIN, AND A. NEWMAN. 1985. Use of polytetrnthtorinated ethylene compound in peripheral nerve grafting. Arch. Otolaryngol. 111: 259-26 1. 13. ROGERS, S. L., P. C. LETOURNEAU, S. L. PALM, J. MCCARTHY, AND L. T. FURCHT. 1983. Neurite extension by peripheral and central nervous system neurons in response to subtratum-bound fibronectin and laminin. Dev. Biol. 98: 2 12-220. 14. TERRANOVA, V. P., D. H. ROHRBACH, AND G. R. MARTIN. 1980. Role of laminin in the attachment of PAM 2 12 (epithelial) cells to basement membrane collagen. Cell 22: 7 19726. 15. TIMPL, R., H. ROHDE, P. G. ROBEY, S. I. RENNARD, J. M. FOIDART, AND G. R. MARTIN. 1979. Laminin-a glycoprotein from basement membranes. J. Biol. Chem. 254: 99339937. 16. TOHYAMA, K., AND C. IDE. 1984. The localization of laminin and fibronectin on the Schwann cell basal lamina. Arch. Histol. Jap. 47: 5 19-532. 17. WEISS, P., AND A. C. TAYLOR. 1946. Guides for nerve regeneration across gaps. J. Neuro-
surg. 3: 375-389.
NEUROLOGY
96,469-473
(1987)
RESEARCH
NOTE
In Vivo Guidance of Regenerating Nerve by Laminin-Coated Filaments SATORU
YOSHII,* TAKAO YAMAMURO,* AND MASAO HAYASHI$?
SEIJI ITO,?
*Department of Orthopedic Surgery, Faculty ofMedicine, Kyoto University, Sakyo-ku. Kyoto 606; tHayaishi Bioinformation Transfer Project, Research Development Corporation of Japan, Minami-ku, Kyoto 601; and *Department of Biology, Ochanomizu University, Bunkyo-ku, Tokyo 112, Japan Received November 3,1986 Laminin is reportedly an extremely potent neurite-promoting agent in vitro. We investigated the effect of laminin on regeneration of axons in vivo, using a cord of polyester filaments coated with laminin as a guide, replacing a 1O-mm segment of the rat sciatic nerve. After 4 weeks the cross section of the nerve guide at its midportion was examined under an electron microscope. Many regenerated axons were seen in the laminin-coated group, whereas no axon was observed in the controls. We conclude that laminin guides the regeneration of axons in vivo. 0 1987 Academic Press, Inc.
During regeneration, nerve fibers may be guided by specific neurite-promoting macromolecules within the extracellular matrix. Interactions between microspikes and the substratum adjacent to the growth cone were found to be important determinants of the directions and pathways of axonal elongation (7). It is generally accepted that the basement membrane provides a scaffolding along which regenerating cells migrate during tissue regeneration after injury. Macromolecules, including laminin, fibronectin, and heparan sulfate proteoglycan, are major components ofthe basement membrane (6, 15). The large glycoprotein, laminin, is a major component of all basement membranes and promotes epithelial cell attachment to the substrata in i We thank M. Kanato for skillful technical assistance, and M. Harada for typing the manuscript. Please address correspondence to Dr. Satoru Yoshii. 469 0014-4886187 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
YOSHII ET AL.
470 0)
:. G
b)
LAMININ
GUIDES REGENERATING
NERVE IN VZVO
471
vitro whereas fibroblasts attach to fibronectin (14). Laminin is an extremely potent net&e-promoting agent in vitro ( 10, 13). Neurite outgrowth is guided by laminin in culture medium (3). In vivo experiments revealed that laminincontaining gel in a tube increased the rate of peripheral nerve regeneration (9). However, tube-shaped nerve guides usually induce good regeneration of nerve fibers in a short distance without laminin ( 1, 2, 8, 11, 12, 17). In this study, we developed a new type of nerve guide coated with laminin and assessed its effect on in vivo nerve guidance. Nerve guides were made of polyester filaments. Two thousand polyethylene telephtalate filaments ( 10 pm in diameter, Toyobo, Osaka, Japan) were wound together with 10-O monofilament nylon thread (S&T, West Germany) to form a cord 10 mm in length and 1 mm in diameter (Fig. la). All nerve guides were immersed 24 h in poly-DL-omithine (Sigma) solution (0.1 mg/ml in 15 mM borate buffer, pH 8.4) at room temperature (4). Then, the guides were washed four times with sterile double-distilled water. These guides were used as controls, or were subsequently coated with laminin. Laminin was purified from mouse EHS sarcoma as reported elsewhere ( 15). Polyornithine-treated nerve guides were incubated 2 h at room temperature in a solution consisting of 1 mg laminin and 20 ml phosphate-buffered saline (PH 7.4). Before grafting, the nerve guides were rinsed three times with phosphate-buffered saline. Ten 3-month-old Wistar rats weighing about 250 g were used, 4 in the control group and 6 in the laminin-coated nerve guide group. All operative procedures were carried out under pentobarbital anesthesia (40 mg/kg). The right sciatic nerve was exposed at the buttock and a IO-mm segment of the nerve was removed; the nerve guide was inserted and sutured to the proximal and distal stumps of the nerve under an operating microscope, using 10-O monofilament nylon epineurial sutures (Fig. 1b). The wound was closed in two layers. At 4 weeks after surgery, the animals were killed with a lethal dose of pentobarbital. The sciatic nerve was reexposed and the nerve guide was removed. At the midportion of the nerve guide, a l-mm long specimen was taken and its ultrathin sections were examined with a Nihon Denshi JEM200cx electron microscope at 100 kV. In five of the six nerve guides coated with laminin, numerous myelinated and unmyelinated axons were seen among the polyester filaments (Fig. 1c). FIG. 1. a-A scheme of our nerve guide (G). b-Epineurial suture of the nerve guide to the stumps of rat sciatic nerve (N). c-A transverse section of the laminin-coated nerve guide. Myelinated (M) and unmyelinated (U) axons were seen among polyester filaments(P). d-A transverse section in a control nerve guide.Cross-sectioned collagen fibrils (C), fibroblast (F), and macrophage (M) were seen among polyester filaments (P) but no regenerating axons were present.
472
YOSHII ET AL.
Myelin sheaths of regenerating nerve fibers were thinner than the myelin sheaths of the normal sciatic nerve of the rat. In the control group, no axon was seen in any of the four nerve guides and many collagen fib&, fibroblasts, and macrophages were found instead of nerve fibers (Fig. Id). Polyornithine coating has been reported to have no inhibitory effect on nerve regeneration (4, 7). These results demonstrate that laminin had a remarkable effect on regeneration of axons along polyester filaments in viva Fibers of severed peripheral nerves are known to require the presence of segments of nerve bundles for regeneration. Whereas the dead Schwann cells are phagocytosed by macrophages, the basement membranes of the Schwann cells remain as tubular scaffolds and regenerating axons grow through the scaffolds (5). It appears important that axons always attach to the inner side (cellular surface) of the basement membrane scaffolds. It has been reported that laminin is localized mainly on the cellular surface of the basement membrane of Schwann cells ( 16). Many tube-shaped nerve guides usually induce good regeneration of nerve fibers in a short distance without laminin (1, 2, 8, 11, 12). Therefore, tube-shaped nerve guides are not suitable to assessthe effect of laminin on in vivo nerve guidance. Weiss and Taylor used fiber cores with collagen tubes or with sleeves of arteries ( 17). However, it remains undecided whether the addition of a fiber core was of any real advantage. Their nerve guide should be regarded as one of the tube-shaped nerve guides. Our nerve guide is made of filaments that are opened to the surrounding extracellular matrix. Therefore, regenerating nerve fibers can extend in any direction without being confined among filaments. Poor axonal regeneration was seen in the control group. The finding that rat peripheral nerve was evidently guided by mouse laminin suggests that laminin derived from one species may guide regenerating nerve fibers of another species in viva as observed for neurites in vitro (3, 10, 13). We demonstrated that laminin guided the regenerating fibers of a peripheral nerve in vivo using a new type of nerve guide coated with laminin. This approach may be applicable to nerve guide bridging for a lengthy defect of a peripheral nerve. REFERENCES 1. CHIU, D.T.W., LJANECKA, T.J. KRIZED, M. WOLFF,ANDR.E.L~VELACE. 1982.Autogenous vein graft as a conduit for nerve regeneration. Surgery91: 226-233. 2. DUCKER, T. B., AND G. J. HAYES. 1968. Peripheral nerve injuries: a comparative study of the anatomical and functional results following primary nerve repair in chimpanzees. Milit. Med. 133: 298-305. 3. HAMMARBACK,J.A.,S.L.PALM,L.T.FURCHT,ANDP.C.LETOURNEAU. 198.5.Guidance of neurite outgrowth by pathways of substratum-adsorbed laminin. J. Neurosci. Rex 13: 213-220.
LAMININ
GUIDES
REGENERATING
NERVE
IN
VW0
473
4. HELFAND, S. L., G. A. SMITH, AND N. K. WESSELLS. 1976. Survival and development in culture of dissociated parasympathetic neurons from ciliaty ganglia. Dev. Biol. 50: 54 l547. 5. IDE, C., K. TOHYAMA, R. YOKOTA, T. NITATORI, AND S. ONODERA. 1983. Schwann cell basal lamina and nerve regeneration. Brain Res. 288: 6 l-75. 6. KEFALIDES, N. A., R. ALPER, AND C. C. CLARK. 1979. Biochemistry and metabolism of basement membranes. Int. Rev. Cytol. 61: 167-228. 7. LETOURNEAU, P. C. 1975. Cell-to-substratum adhesion and guidance of axonal elongation. Dev. Biol.44: 92-101. 8. LUNDBORG, G., L. B. DAHLIN, N. DANIELSEN, H. A. HANSSON, A. JOHANNESSON, F. M. LONGO, AND S. VARON. 1982. Nerve regeneration across an extended gap: a neurobiological view of nerve repair and the possible involvement of neurotrophic factors. J. Hand
Surg. 7: 580-587. 9. MADISON, R., C. F. DA SILVA, P. DIKKES, T. CHIU, AND R. L. SIDMAN. 1985. Increased rate of peripheral nerve regeneration using bioresorbable nerve guides and a laminincontaining gel. Exp. Neural. 88: 767-772. 10. MANTHORPE, M., E. ENGVALL, E. RLJOSLAHTI, F. M. LONGO, G. E. DAVIS, AND S. VARON. 1983. Laminin promotes neuritic regeneration from cultured peripheral and central neurons. J. Cell Biol. 97: 1882- 1890. 11. MIDGLEY, R. D., AND F. M. WOOLHOUSE. 1968. Silicone rubber sheathing as an adjunct to neural anastomosis. Surg. Clin. North. Am. 48: 1149-l 157. 12. RICE, D. H., F. D. BURSTEIN, AND A. NEWMAN. 1985. Use of polytetrnthtorinated ethylene compound in peripheral nerve grafting. Arch. Otolaryngol. 111: 259-26 1. 13. ROGERS, S. L., P. C. LETOURNEAU, S. L. PALM, J. MCCARTHY, AND L. T. FURCHT. 1983. Neurite extension by peripheral and central nervous system neurons in response to subtratum-bound fibronectin and laminin. Dev. Biol. 98: 2 12-220. 14. TERRANOVA, V. P., D. H. ROHRBACH, AND G. R. MARTIN. 1980. Role of laminin in the attachment of PAM 2 12 (epithelial) cells to basement membrane collagen. Cell 22: 7 19726. 15. TIMPL, R., H. ROHDE, P. G. ROBEY, S. I. RENNARD, J. M. FOIDART, AND G. R. MARTIN. 1979. Laminin-a glycoprotein from basement membranes. J. Biol. Chem. 254: 99339937. 16. TOHYAMA, K., AND C. IDE. 1984. The localization of laminin and fibronectin on the Schwann cell basal lamina. Arch. Histol. Jap. 47: 5 19-532. 17. WEISS, P., AND A. C. TAYLOR. 1946. Guides for nerve regeneration across gaps. J. Neuro-
surg. 3: 375-389.