Nerve regeneration through allogeneic nerve grafts, with special reference to the role of the schwann cell basal lamina

Nerve regeneration through allogeneic nerve grafts, with special reference to the role of the schwann cell basal lamina

Progress in Neurobwlogy Vol. 34, pp. 1 to 38, 1990 Printed in Great Britain. All rights reserved 0301-0082/90/$0.00+ 0.50 © 1990PergamonPress plc NE...

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Progress in Neurobwlogy Vol. 34, pp. 1 to 38, 1990 Printed in Great Britain. All rights reserved

0301-0082/90/$0.00+ 0.50 © 1990PergamonPress plc

NERVE REGENERATION THROUGH ALLOGENEIC NERVE GRAFTS, WITH SPECIAL REFERENCE TO THE ROLE OF THE SCHWANN CELL BASAL LAMINA C. IDE,* T. OSAWA~ a n d K. TOHYAMA* *Department of Anatomy School of Medicine and "~Department of Oral Anatomy School of Dentistry, lwate Medical University, Morioka 020, Japan (Received 13 July 1989)

CONTENTS 1. 2. 3. 4.

Introduction Structure of the peripheral nerve Principles of nerve degeneration and regeneration Allografts 4.1. Fresh grafts 4.2. Alcohol treatment 4.3. Storage 4.4. Freezing 4.5. Freeze-drying (lyophilization) 4.6. Irradiation 4.7. Immunosuppression of the host 4.8. Other treatments 4.8.1. Cialit treatment 4.8.2. Ensheathment 4.8.3. Predegeneration 4.9. Blood supply 5. Electron microscopic studies of nerve regeneration in the auto- and allograft 5.1. Autograft in mice 5.2. Allograft in mice 5.3. Allograft in rats 5.4. Allograft in rabbits 5.5. Allograft in monkeys 6. Considerations on the main components of the graft 6.1. Schwann cells 6.2. Basal laminae 7. Concluding remarks Acknowledgements References

1. INTRODUCTION Allogeneic (homogeneic) nerve grafts of the periplaeral nerves have been studied for almost 100 years since the first attempt by Albert (1885). The application of the nerve allograft for the clinical use still continues to be the subject of extensive study, but without any definitely affirmative results. At present, the defect of the injured nerve is operated on clinically by grafting an autogeneic nerve segment obtained mainly from the sural nerve. However, the graft obtainable from the sural nerve is not always large enough for filling the gap of injured nerves. In addition, the excision of the sural nerve results in sensory deficits in its innervation area at the lateral surface of the ankle and foot. Owing to such a limited availability of the allograft, the possibility of using allogeneic nerves has been actively investigated. Even heterogeneic nerves have been tried as grafts (Gluck, 1880; Sencert, 1918; Marmor et al., 1966; Hirasawa JPN

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and Marmor, 1967; Hirasawa et al., 1968; Marmor and Hirasawa, 1968; Fujii, 1972; Osawa et al., 1987). If the allograft can be generally used with successful results, clinicians can select any nerve from the tissue bank that is suitable in thickness as well as in its internal funicular structures for the defective part of the injured nerve. The main concern of the nerve allograft studies is how to reduce the immune reactions which would lead to the necrosis or fibrosis of the graft. For this purpose, various kinds of attempts have been made, which include pretreatment of the graft by alcohol, freezing, freeze-drying, etc. and suppression of the immunoreactive ability of the host animal by drugs. Recently the value of immunosuppression of the host has been emphasized (Zalewski and Silvers, 1980; Zalewski et al., 1981; Zalewski and Gulati, 1981; Mackinnon et al., 1987; Bain et al., 1988; Gulati, 1989). l

( IDl~et at. Almost all the studies of nerve allograft conducted so far have been clinically oriented studies, in which the detailed morphological analysis of the graft has been lacking or almost completely neglected: in some studies the histology of the grafted nerve was never examined, and in others it was presented only briefly with a few light microscopic micrographs. Accordingly, histological changes including the outgrowth of regenerating nerves through the graft have not been demonstrated precisely. Before our electron microscopic studies, there were only a few studies on nerve allografts by electron microscopy (Schrfder and Seiffert, 1970; Pollard and Fitzpatrick, 1973a, b). By studying the nerve auto- and allografi by electron microscopy, we found that Schwann cell basal laminae play an important role in nerve regeneration by serving as conduits for growing axons (Ide, 1983; Ide et al., 1983; Osawa et al., 1986, 1989). It appeared that basal laminae remain t0r a relatively long time without undergoing immunological rejection in the allograft. This fact indicates the possibility of acellular grafts of Schwann cell basal laminae, contrary to the generally held concept of using cellular grafts containing viable Schwann cells (Gulati and Zalewski, 1985; Gulati, 1988) In this article, we review the nerve allograft studies from the purely morphological point of view.

2. STRUCTURE OF THE PERIPHERAL NERVE

The peripheral nerve fiber consists of axons and Schwann cells. In the myelinated fibers Schwann cells form the myelin sheath around the axon, while in the unmyelinated fibers Schwann cells surround the naked axons directly. In the former, a nerve fiber contains one axon, but in the latter several axons are enclosed by a common Schwann cell. Neighboring Schwann cells are separated from each other at the node of Ranvier in the myelinated fibers, while they are directly contiguous with each other in the unmyelinated fibers. Schwann cells of both myelinated and unmyelinated fibers are invested by the basal lamina on their external (ad-connective tissue) surface (Fig. 1). Because the basal lamina is not interrupted even at the Schwann cell junctions of both myelinated and unmyelinated fibers, it can be said that each nerve fiber resides within its proper basal lamina tube which is continuous with its entire length from its exit at the spinal cord or brain to its end at the targets in the periphery. Outside of the Schwann cell basal lamina is the endoneurial space, which is filled with bundles of collagen fibrils running longitudinally. The endoneurial space is, in turn, separated by the perineurial sheath from the surrounding connective tissue. Contiguous with the external surface of Schwann cell basal lamina is a thin layer of fine collagen fibrils in very loose networks which is called the sheath of Plenk-Laidlaw (Plenk, 1934; Laidlaw, 1930). More external to the sheath of Plenk-Laidlaw, is a layer of collagen bundles running closely associated with individual nerve fibers. This layer of collagen bundles is known as the sheath of Key-Retzius (Key and

Retzius, 1873). Thomas (1963) observed by electron microscopy the connective tissue of peripheral nerves, noting the sheaths of Plenk-Laidlav~' and ~t~ Key-Retzius. Similarly Gamble (1964) and Gamble and Eames (1964) studied by electron microscopy the connective tissue sheaths in rat and human peripheral nerves. The fine structure of these "classical" sheaths defined by light microscopy was beautifully demonstrated by scanning electron microscopy (Ushiki and Ide, 1986). The "Schwann tube" (Hohnes and Young, 1942) and "endoneurial sheath or ~ubc" (Sunderland and Bradley, 1950) in the old literature are considered to include the Schwann celt basal lamina, the sheath of Plenk-Laidlaw, and part ,:~t'the sheath of Key-Retzius. The nerve bundle surro~mded by each perineurial sheath is called the funiculu:~, ~md several nerve funiculi are surrounded by the c(~r~mon epineurium to form a thick nerve trunk (Sunclcrland, 1978).

3. PRINCIPLES OF NERVE DEGENERATION AND REGENERATION

The degeneration of the axon in the distal stump following the transection of the nerve fibers was first described correctly by Waller (1850), who observed granular degeneration of myelin sheaths along with disappearance of axons in the distal portion 1-5 months after transection of glossopharyngeal and hypoglossal nerves of the frog. The degeneration of axon is caused by the interruption of the continuity of the axon with its trophic center, the cell body. This axonal degeneration is called secondary degeneration, or more commonly Wallerian degeneration. The process of changes of cellular components in Wallerian degeneration was extensively studied by light (Cajal, 1928; Sunderland and Bradley, 1950), and electron microscopy (Vial, 1958; Ohmi, 1961; Webster, 1962; Lee, 1963; Nathaniel and Pease, 1963a; Thomas, 1964; Thomas and Jones, 1967; Williams and Hall, 1971; Calabretta et al., 1973). The myelin sheaths of transected fibers are degraded into "myelin balls" of various sizes probably under the influence of surrounding Schwann cells. Although it appeared that Schwann cells phagocytose myelin sheath debris (Wechsler and Hager, t962a; Fisher and Taruno, 1963; Satinsky et al., 1964), Schwann cells are probably not scavengers of myelin sheath debris. Macrophages invading from the outside, i.e. from blood vessels, vigorously phagocytose myelin sheath debris as scavengers (Holmes and Young, 1942). On the other hand, the degeneration of the axons themselves is considered to occur in an autolytic fashion. Schwann cells which are deprived of axons remain alive, becoming elongated with slender processes which are interconnected with one another to tbrm a cell column called the Schwann cell column or band of Biingner (1891). In the old literature, Schwann cell columns were named Schwann cell tubes (Young, 1942) or neurilemma bands (Guth, 1956). Neurilemma means the Schwann cells ensheathing the axons. Schwann cell columns are formed in the same fashion in both myelinated and unmyelinated fibers. It should be noted that the Schwann cell basal

NERVE ALI.OGRAFT

laminae remain almost unaffected during the process of Wallerian degeneration, followed by the formation of Schwann cell columns. Therefore, every Schwann cell column is located within its own basal lamina tube. Growth of axonal sprouts takes place from the proximal stump after certain periods (1-7days) following the transection of the nerve. Holmes and Young (1942) made correct observations on the processes of axonal growth and maturation in Schwann cell tubes, which are relevant to the present electron microscopic findings. They described that all the regenerating axons always grew through the Schwann cell columns for a long distance, and that axons extended over the surface of the Schwann cells in the space between the Schwann cells and the Schwann tube (endoneuriai sheath). At that time, many other investigator.s believed that regenerating axons extended within vacuoles inside the Schwann cell cytoplasm (Bielschowsky and Unger, 1917). Although Holmes and Young (1942) were mistaken in explaining that regenerating axons later came to lie within the Schwann cell cytoplasm, their observations on the processes of separation (sorting out) and remyelination of regenerating axons within the individual Schwann.cell columns are precise in view of the present electron microscopic findings. The fact that several axons would grow down through an individual Schwann cell column had already been demonstrated (Cajal, 1928; Nageotte, 1932). Early electron microscopic studies clearly revealed that regenerating axons grow down through intercellular spaces of the Schwann cell column (Wechsler and Hager, 1962b; Barton, 1962; Thomas, 1964a; Dyck and Hopkins, 1972; Morris et al., 1972; Fig. 2). Inasmuch as Schwann cell columns are invested by the basal laminae, regenerating axons growing through Schwann cell columns are regarded as being located within the basal lamina tubes. However, since the basal laminae are difficult to identify in ordinary light microscopic preparations, no light microscopic studies have inferred the role of basal laminae in nerve regeneration. By observing regenerating axons which grew in the spaces between the basal laminae and Schwann cells in the crushed lumbosacral dorsal roots of the rat, Nathaniel and Pease (1963b) suggested a role of the Schwann cell basal laminae in guiding and limiting the regenerative processes by providing scaffolds for regenerating axons. In our electron microscopic studies, the basal lamina tubes of Schwann cells were shown to play an important role as effective conduits for the growing axons in the auto- (Ide, 1983; Ide et al., 1983), allo- (Osawa et al., 1986, 1989), and heterograft (Osawa et al., 1987). 4. ALLOGRAFTS Various attempts have been made to reduce the immune reactions to allogeneic nerve graft: allograft research is a history of endeavours to find a way of coping with the immune reaction. We will first survey the studies of nerve allograft using fresh nerve segments, and then proceed to examine allograft studies according to the various treatments employed in each study to suppress immunological reactions. We will then discuss the effects and significance of such

treatments from a morphological point of view based on our recent studies. 4.1. FP~SH GR~TS In the literature, the allograft (homograft) of the peripheral nerve was reported for the first time by Albert 0885), who transplanted a 3 cm long nerve segment obtained from an amputated human leg into the gap of the right median nerve made after the resection of sarcoma: the final outcome of this first nerve ailograft was not known. In a second case, a 10 cm long nerve segment was similarly transplanted, which became necrotic 1 week after the surgery. He also made experiments using autografts, in which a nerve segment was exchanged between the right and left sciatic nerve in the dog. His main concern in these studies was whether the graft could reunite with the host nerve stumps. Since the principles of the nerve regeneration were not yet clearly understood, observations and explanations of the results were inadequate. Kilvington (1908) reported 4 experiments, in which 1 in long ulnar nerve segments of the dog were grafted autologously into another dog. Fair motor responses were obtained in these cases 15 days after grafting: this unusually early functional recovery was probably due to inadequate experiments. Maccabruni (1911) performed allograft of sciatic nerves in rabbit, but he observed only degenerative processes of the nerve in the homograft. Eden (1919) reported that fresh grafts were most desirable in autoas well as ailograft using 2-4 cm long nerves in dog and human: stored or heterogeneic grafts had no value. Duel (1934) reported aUografts of the facial nerve in clinical cases with a certain amount of SUCCESS.

Before allogeneic grafts, the first attempt to bridge a gap with an autogeneic nerve graft was performed by Phillipeaux and Vulpian (1870), who transplanted a 2 cm long segment of the lingual nerve into the hypoglossal nerve in the dog: 2 out of 5 dogs showed some functional recovery 70 days after grafting. Convincing results of nerve autograft were obtained in the treatment of facial nerve palsy by Ballance and Duel (1932), who performed transplantation of 0.5-1 cm long segments of Bell's nerve (thoracicus longus nerve) into the injured facial nerves at the facial canal (fallopian canal). The autograft which has proven to be as successful as the end-to-end suture, is now generally utilized clinically, whereas the allograft is not generally applied in clinical cases, despite the fact that it has a higher potentiality for clinical application than the autograft. Since 1940s the nerve allograft has been actively studied. In the experiment in which a 3 in long allogeneic nerve segment was implanted in monkey peroneal nerve, Bentley and Hill (1940) described that motor responses appeared in the extensor muscles 250days postoperatively, and that histologically there were many nerve fibers in the graft 500 days after grafting. They further suggested the possibility that nerves obtained from cadavers could be used as allografts. However, follow-up studies by other investigators yielded no positive results; no appreciable regeneration was found to occur in the allograft. Spurling et al. (1945) found no nerve regeneration in

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eight cases of allografts, except for two cases in which only a few regenerating fibers were found. Davis et al. (1945) performed auto- and allografts of sciatic nerve in 470 cats, showing little, if any, nerve regeneration in the allograft. Barnes et al. (1946), in transplantation of 7-25 cm long nerves obtained from a cadaver 7-30 hr postmortem into 8 patients, failed to get promising results in any of the cases 202-732 days after surgery; they thought that it was not possible to use nerve allograft until the immunological reactions that occurred in the grafts and the histopathological changes following such reactions could be clarified. Verhoog and Bekkum (1971) pointed out the antigenicity of living Schwann cells. Seddon (1963) expressed his pessimistic opinion for nerve allograft in his lecture. On the other hand, fascicular allografts were recommended in the rat by Levinthal et al. (1978a, b). Different techniques of nerve grafting were also described (Millesi, 1969, 1981). The history of the nerve allograft is a series of endeavours to reduce or suppress the immune reactions to the graft. In the following sections, we will review various pretreatments of the graft and immunosuppression of the host that were performed in an attempt to cope with this problem. 4.2. ALCOttOL TREATMENT N ageotte (1917) first suggested the value of alcohol treatment for the nerve as the allograft. Huber (1919, 1920) grafted a rabbit sciatic nerve segment, which had been stored in 50% alcohol for about 4 weeks, into another rabbit. In examinations of the grafts for periods ranging from 2 days to 3 months, he stated that, while the cellular elements including Schwann cells in the graft were not viable, neurolemma sheaths were well preserved and regenerating axons could grow down through them to the distal stump. Although no micrograph was shown in this paper, and the histological techniques at that time were incomplete for the demonstration of regenerating nerve fibers in the graft, this statement is very suggestive in view of the present knowledge concerning the role of basal laminae in nerve regeneration. Later, Sweet (1929) examined the effectiveness of alcohol treatment in the dog by grafting 4 - 6 cm long sciatic nerve segments which were treated in 50-80% alcohol. In most cases a great amount of fibrous tissue was formed at both proximal and distal ends of the graft, while a fair nerve regeneration was seen only in a few grafts. He concluded that an alcoholized nerve graft was not justified as a choice of graft. Similarly, the alcohol treatment was regarded as useless by Sanders and Young (1942), and Gutmann and Sanders (1942) in the experiments using 2-5 cm long nerve grafts to the peroneal nerve of the rabbit. Contrary to these studies, klemme et al. (1943) claimed that nerve grafts from cadavers which had been treated by formaldehyde and alcohol were successful in 2 out of 3 cases. The failure of nerve regeneration through the alcohol-treated graft might be due partly to the denaturation of Schwann cells and myelin sheaths; denaturation rendered them difficult to remove by macrophages, thus they presumably became barriers to the growth of axons. Recently, alcohol-treated allografts were reexamined in the rabbit (Hirasawa et

al., 1989). By electron microscopy it was found that cellular elements of some nerve fibers had been eliminated prior to grafting, leaving behind Schwann cell basal laminae and associated connective tissues in the form of tubes. Some regenerating axons grew out through such empty "endoneurial tubes". However. the number of regenerating axons was not iarge enough, and in addition, it appears that by alcohol treatment the endoneurial connective tissue, including the Schwann cell basal laminae, loses its flexibility for the voluminous expansion.

4.3 S~fORAGE Bethe (1916) first made an attempt to use allografts stored in the refrigerator: he took 2-4 cm long tibial nerves of rabbit or dog, which had been stored at 2-6°C in a refrigerator for 3--12 days, implanted them into the defect of the tibial nerve, and examined the grafts from 4 days to 3 months after grafting, with some excellent results. Sanders and Young (1942) and Gutmann and Sanders (1942, 1943) grafted into the peroneal nerve of another rabbit a 2 cm long tibial nerve segment of the rabbit which had been stored in Ringer's solution at 2 C for 7-21 days. The histological examination showed that broken myelin sheaths were removed by a very large invasion of macrophages which were identified as "foam" cells loaded with phagocytosed myelin debris, and that the lymphocyte invasion was very much reduced in comparison with fresh allografts. They described good nerve regeneration through the graft; the graft had the appearance of the normal nerve 73 days after grafting. It is reasonable to assume that in these studies Schwann cells had been dead prior to grafting due to storage for a long period at 2~'C, while their basal laminae should have been well preserved. Therefore, the histology of their studies might have been the same as in our studies which showed the role of Schwann cell basal laminae in auto- and allograft (Ide, 1983; Ide et al., 1983; Osawa et al., 1986, 1989). The vigorous phagocytosis by macrophages and markedly reduced lymphocyte invasion are the same as seen in our studies. Tarlov and Epstein (1945) found good nerve regeneratkm in the allograft which had been stored in serum at 5'C for 1-2 days prior to implantation in the dog. The storage of the graft, however, was not effective in clinical use by Seddon and Holmes (1944): they reported that the graft was totally dead and under the process of removal by macrophages 426 days after grafting. Fresh allografts made at the same time became extensively collagenous 370--570 days after grafting, showing no sign of nerve regeneration. These discouraging outcomes in clinical applications of allografts halted the continuation of this kind of nerve allograft research. However, Sanders (1954) pointed out that the allogeneic nerve grafts which had been stored for a certain period in Ringer's solution, yielded good nerve regeneration, but freeze-dried and alcohol- or formalintreated grafts gave only poor results. These findings suggest that in Ringer-stored nerves, dead Schwann cells and myelin sheaths were eliminated, and Schwann cell basal laminae were probably left undamaged, serving as the pathways for regenerating

NERVE ALLOGRAFT

axons, whereas in gratis treated as above, cellular as well as acellular components were denatured, remaining unremoved thus hindering the growth of regenerating axons. Similarly, Therkelsen and Pool (1957) used a 22 mm sciatic nerve segment of dog stored for 7-9 days in a culture medium, and observed fair nerve regeneration in the "resutured" grafts 5-7 months after grafting. Most recently, the allograft of a sciatic nerve segment that had been stored in Lock's solution was performed using Lewis and DA rats, with only a little improvement (Chung and Chung, 1974). 4.4. FREEZING Schr6der and Seiffert (1970) compared nerve regeneration through 2-10 cm long fresh and frozen allografts in the dog sciatic nerve. By electron microscopic examinations 6-24 months after grafting, they found many regenerated nerves, and the formation of new perineurial sheaths around individual fibers or fiber bundles in the grafts as shown in our studies (Ide, 1983; Osawa et al., 1986, 1987). They proposed that Schwann cells do not always need to be alive in the graft. This is one of a few electron microscopic studies of allogeneic nerve graft done before the 1980s. Later, they used frozen allografts longer than 3 cm for clinical cases, but with no appreciable occurrence of nerve regeneration (Seiffert et al., 1972). Singh (1976) and Singh et al. (1977) performed experiments in which 4 - 7 c m long grafts kept frozen at - 7 0 ° C for 4-6 weeks were implanted in compatible (litter mates) and noncompatible dogs. Noncompatible frozen grafts exhibited little or no improvement in nerve regeneration. They concluded that for a long allograft (longer than 4cm) the histocompatibility was the most important factor. Systematic studies on the effect of freezing were carried out in genetically defined strains of rat (Fischer and Lewis) by Zalewski and Gulati (1982). The results showed that fresh isografts were successful, whereas frozen iso- and allografts had only a small number of axons in the proximal 1-2 cm. Thus they found no value in the freezing treatment of the graft. The same conclusion was drawn by Gulati (1988). They considered that Schwann cells and perineurial cells were required to be viable for successful nerve regeneration. The data from our studies are contrary to their conclusions. We will later describe our data in detail. 4.5. FREEZE-DRYING(LYOPHILIZATION) Freeze-drying, solely or in combination with irradiation, had been widely used in an attempt to reduce the immune reactions of the allograft in experimental and clinical studies. Weiss and Taylor (1943) reported good nerve regeneration through 1-2 crn long lyophilized nerve grafts in the rat. However, Jacoby et al. (1970), Seiffert et al. (1972), and Schr6der and Seiffert (1972) found no favorable results with 2-3 cm long grafts in the dog, or with 2-8 cm long grafts in the human. In the analysis of all the nerve transplantations using freeze-dried allografts since 1969, Kuhlendahl et al. (1972) found that

only poor or no nerve regeneration occurred in all the patients. Similarly, by follow-up studies it was concluded that there were no regeneration recoveries in any of the 74 clinical cases in which lyophilized homografts had been used (Penzholz, 1973). Despite these pessimistic results, Singh (1975) conducted transplantation of freeze-dried grafts (4-7 cm long) into the ulnar, median, and muscuiocutaneous nerves in 18 clinical cases. He claimed that 18-20 months after transplantation, there were signs of reinnervation in 7 cases as examined by EMG. Samii and Scheinpflug (1974) compared auto- and lyophilized homografts using rabbit nerves only 5 mm in length and found much fewer regenerating axons in the homograft than in the autograft. However, Mackinnon et al. (1984b), using 4.5 cm long freeze-dried sciatic nerve allografts in the rat, reported that a fair nerve regeneration was found 5 months after implantation through the graft as examined by light microscopy as well as electrophysiology. Regrettably, none of the studies employing freeze-dried allografts included the histological changes of the nerve after the freeze-drying treatment. It was not demonstrated whether the endoneurial connective tissues including Schwann cell basal laminae could be retained even after the freezedrying treatment. If these structures are damaged and disorganized by freeze-drying, there will be no possibility for axons to regenerate orderly and vigorously through the graft. Moreover it was not interred whether the dead (denatured) cellular components such as myelin sheaths could be removed by macrophages: if they persist for a long time, they would obstruct the pathways for regenerating axons to grow. 4.6. IRRADIATION Irradiation of the graft was introduced by Campbell et al. (1963). Since then, this treatment has been most frequently used to reduce the antigenicity of the graft (Marmor, 1963, 1964a, b). However, despite many experimental as well as clinical studies, the value of irradiation of the allograft has not been established. It should be noted that in most studies the allografts which were used for irradiation had been stored for various periods in a frozen state, or, in addition, treated by freeze-drying; therefore, the grafts underwent the combined effects of irradiation plus freezing, or irradiation plus freeze-drying. Campbell et al. (1963) reported a case showing recovery of sensory and motor functions in the ulnar nerve to which an 8.5 cm long allogeneic nerve had been grafted 1 year before. Marmor's experiment (1963) has become the standard for irradiation of the allograft. The graft was stored in a deep freezer at - 12°F, irradiated with 2,000,000 r.e.p, for l0 sec, and again kept in a frozen state until used. He implanted such a treated graft into the dog's peroneal nerve and noticed the recovery of function in the leg 6 months after implantation. As the next step, he applied irradiated nerves in clinical cases: defects of median and ulnar nerves were bridged with irradiated grafts of 3-5 inches in length in 2 patients. Motor and sensory functions recovered 4 months after grafting. Ashley et al. (1968), in the experiments

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in which irradiated short (1.5-3.0cm) and long (7-11.5 cm) allogeneic nerve segments were grafted into ulnar nerves of the monkey, noted a good nerve regeneration in the short grafts, but not in the long ones. Wilhelm and Ross (1972) performed in 17 cases transplantation of ulnar and median nerves which had been obtained from cadavers, lyophilized, irradiated, and then stored until used. They reported that good sensation was recovered in 6 cases after 5-12 months. Hiles (1972) also grafted nerves from cadavers, which had been treated in the same way as above in 2 patients. He considered, however, that the results were not yet conclusive nor convincing. Experimentally, irradiated nerves up to 4 5 cm in length were grafted in rabbit (Lewis and McLaurin, 1966, 1969), monkey (Ducker and Hayes, 1970), rat (Ikeda, 1966; Bucko and Steinmuller, 1974), goat (Robert, 1967), dog (Ikeda, 1966; Ducker and Hayes, 1968; Buch, 1970; Hirasawa et al., 1974b), guinea pig (Buch, 1970), and also clinically in some patients (Jacoby et al., 1970). Results of these studies were highly variable, and the conclusions drawn were not consistent nor confident. The histology of the graft was examined only by light microscopy, therefore the findings of the cellular changes of the graft were superficial and incomplete, or sometimes even lacking. Only Pollard and Fitzpatrick (1973b) carried out an electron microscopic study of the frozen-irradiated graft. They transplanted 4 cm long sciatic nerve segments from the DA to Wistar strain of rat, comparing the effect of irradiated plus freeze-dried grafts with nontreated ones. Like the nontreated grafts, the irradiated plus freeze-dried grafts showed no promotion of nerve regeneration; Schwann cells were denatured, being rendered difficult to remove by macrophages, and in addition there was an extensive proliferation of collagen fibrils which caused "neurolemmal tubes" to collapse, allowing only a few thin axons to grow through the graft. Although good nerve regeneration was not obtained, the role of "endoneurial tubes" (basal lamina tubes of Schwann cells) in nerve regeneration was suggested in this study. However, this important finding was apparently not taken into consideration in studies by other investigators that followed. Irradiated allografts continued to be studied without any detailed histological examination (Singh, 1975, 1976: Singh et al., 1977: Mackinnon et al., 1984b). 4.7, IMMUNOSUPPRESSIONOF THE Hos'r The suppression of the immune reactions in the host was carried out using immunosuppressive agents with or without the combination of irradiation and/or freezing of the grafts. Marmor et al. (1967) transplanted irradiated and nonirradiated sciatic nerve segments (1 cm long) into rats which had been administered Immuran (azathioprine, 6-(1-methyl-4nitro-5-imidazolyl)thiopurine) for 5 days, showing that the inflammatory response was markedly reduced even in nonirradiated grafts. The grafts that they employed were not allogeneic in a strict sense, because the nerves were implanted between Sprague-Dawley rats. Marmor (1970) proceeded to use irradiated grafts (3-5.5in long) for three patients who had been administered Immuran with

the result that there was no recovery of either sensory or motor functions. Gye et al. (1972) conducted the nerve allograft in human: allogeneic nerves from cadavers were stored at 0 - 1 0 C for 2 weeks, freezedried, and irradiated, and implanted into ulnar, median or tibial nerves of patients who were immunosuppressed with lmmuran. Grafts were 7 23 cm long. There were good sensory recoveries in 7 of 8 cases 7 14months after grafting, but reinnervation of muscles was poor. Pollard et aL (1971, 1973) reported good nerve regeneration as observed by light microscopy through irradiated graft implanted in hnmuran-immunosuppressed rat~ and monkeys. They used only the Wistar strata, again indicating that the graft was not altogeneic in the ral, but that it should be in the monkey. They claimed that a high density of regenerating axons was found in the graft. McLeod et aL (1975) performed nerve grafting in leprosy; they used ulnar, median and tibial nerves of cadavers, which had been freeze-dried, irradiated and stored at 4'C. Patients were administered immunosuppressive agents for a long time. Nine grafts out of 23 wine good or lair in the noxious sensory recovery. Pollard and Fitzpatrick (1973b~ made pioneering electron microscopic studies, in which 4 cm long sciatic nerve segments were transplanted from the DA to the Wistar strain of rats which had been administered Immuran plus the antilymphocyte serum. The immune reactions to the graft were markedly reduced by this treatment. The nerve regeneration was much more st,ccessful than in the simple allograft. They noted that the loss of donor Schwann cells occurred in both nontreated and immunosuppressed host despite the use of a high dosage of immunosuppressive agent, and that Schwann cell basal laminae were preserved as empty tubes. Although the growth of axons through basal lamina tubes was not clearly demonstrated, they considered that if it was possible to develop a nerve graft which consisted only of the "neurolemmal tube" and other supportive elements, immunosuppressive measurements might not be necessary. However. these important findings were given no attention by other investigators. Effects of immunosuppressive agents were investigated in a series by studies of Zalewski and his coworkers. Using well-defined rat strains with regard to histocompatibility complex, they showed that a 4cm long allograft was not rejected, but served as an effective graft in the rat to which the donor antigen had been injected at birth (Zalewski and Silvers, 1980). In a subsequent study, they found that the cellular elements of the allograft, including regenerating axons which had once been successfully retained in the immunologically tolerant rat were rejected within 4 weeks after the tolerance was abolished by injecting sensitized lymphocytes into the host (Zalewski et al., 1981). Similarly, the allograft which had been preserved in cyclosporin A-treated rats was rejected within 60 days after stopping the treatment (Zalewski and Gulati, 1981, 1984a). These studies showed that as long as the immunosuppressive treatment continued, allogeneic Schwann cells could be retained viable. This result was different from that of Pollard and Fitzpatrick (1973b), who showed that Schwann cells were lost even in Immuran-treated rats indicating that

N ~ w ALLOGRAFT

Immuran might not work for the preservation of Schwann cells. On the other hand, by experiments using rat strains well defined with MHC, such as Lewis (RT-11), Fischer (RT-11) and ACI (RT-la), Mackinnon et al. (1985) confirmed that there was only poor nerve regeneration in MHC-unmatched (Lewis/ACI) grafts (4 cm in length) 5 months after grafting. Next they demonstrated that by immunosuppression using azathioprine and hydrocortisone, relatively good nerve regeneration could be obtained, and that, contrary to results of Zalewski and Gulati (1981, 1984a), there was no rejection of donor Schwann cells 180 days after cessation of immunosuppression (Mackinnon et al., 1987). Furthermore, in contrast to the result of Zalewski and Gulati (1984b), they demonstrated that a low dose of cyclosporin A (5 mg/kg/day) could suppress the immune reaction of graft allogeneic nerve which had been aligned with the intact host nerve in the rat (Bain et al., 1988). 4.8. OTHER TREATMENTS

4.8.1. Cialit treatment Cialit (2-(acetylmercurimercapto)-benzoxazol-5carbonic acid sodium), an antiseptic agent, was used with the expectation that it might reduce the antigenicity of the graft. Since this drug is not used at present, the Cialit treatment is only historical. Afanassieff (1967) reported that 10 out of 20 clinical cases in which Cialit-treated nerves had been grafted showed an improvement in nerve regeneration. Similarly, Afanassief and Recht (1971) reported that 44 clinical cases grafted by Cialit-treated nerves showed some improvement 6 weeks to 15 months after grafting. Enya (1969) recommended the use of nerve grafts treated with Cialit in the allograft study in the rabbit. In their preliminary report, Seiffert et al. (1968) indicated the usefulness of Cialit treatment, comparing it to freezing or freeze-drying treatment. But later, when using 2-8 cm long allogeneic nerves treated with Cialit in dog and human, they did not obtain any favorable results (Seiffert et al., 1972; Schrrder and Seiffert, 1972). Although there were no histological data from electron microscopy, it can be presumed that Schwann cells which had been killed and denatured by Cialit treatment could not be phagocytosed by macrophages, nor did they serve as guides for growing axons. Therefore, even if the immune reactions were suppressed, the grafts were no longer very effective conduits of regenerating axons.

crating nerves grew out through them. Recently Gulati (1989) used fresh allogeneic carotid arteries (1 era long) to bridge the gap in sciatic nerve. The grafted aorta could be preserved by immunosuppression, and some regenerating nerves grew through this short tunnel of the aorta; however, it soon degenerated after ceasing the immunosuppression. Lyophilized human dura and fibrin membrane were tried for clinical use (Hirasawa et al., 1977), and the mesothelial or p~eudosynovial cell tube and the amnion membrane were designed for experimental studies (Lundborg and Hansson, 1981; Danielsen et al., 1983, 1988; Mackinnon et al., 1986), but no definitely positive results were reported in these studies. Millipore filters were also utilized experimentally. Novack et al. (1958) and Basset et al. (1959) used Millipore filter tubes (pores were 0.45/am in diameter) for 1-2.5 cm wide gaps in the sciatic nerve of the cat. Longer Millipore tubes (3.5 cm) were used by Campbell (1970) for ensheathing irradiated and lyophilized nerves. Although some nerve regeneration was reported to occur in these studies, the Millipore filter was fragile and caused severe adhesion with the surrounding connective tissue. Generally speaking, the use of nonbiological foreign materials such as Millipore filters should be avoided, because they persist unabsorbed for a long time, and are not flexible enough to change in accordance with the voluminous expansion of the regenerating nerves. In this sense, silicone tubes (Lundborg et al., 1982; Jenq and Coggeshail, 1986; Williams et al., 1988) or polyethylene tubes (Madison et al., 1987) are not suitable materials as guides for regenerating nerves. Molander et al. (1982) reported the use of polyglactin tubes which could be absorbed in due course after implantation. The use of other synthetic bioabsorbable nerve guides has been studied with some successful results (Reid et al., 1978; Seckel et al., 1984; Dellon and Mackinnon, 1988). As described below in detail, the basal lamina has properties of both flexibility and absorbability; by serving as a conduit for regenerating axons, the basal lamina tube can expand with the voluminous increase of regenerating nerves, and it gradually disintegrates with the maturation of the nerves. In addition, regenerating axons appear to preferably attach as scaffolds to the inner surface of the Schwann cell basal laminae. Therefore, the basal lamina is the ideal material as the scaffold for the growth of regenerating axons.

4.8.3. Predegeneration 4.8.2. Ensheathment Ensheathment of the graft with either artificial or biological material was performed for the purpose of providing regenerating axons with conduits to grow through, and at the same time with barriers against the invasion by immune cells while nutrients were accessible to the graft. Weiss (1943) utilized allogeneic frozen aortae or carotid arteries of rabbit, cat and monkey for the splicing of the graft to the host nerve stumps. He found that the grafted arteries remained unabsorbed for more than several months and regen-

Predegeneration (predenervation) of the graft was also employed in some studies, with the expectation that it reduces antigenicity and, in addition, enhances trophic effects of the graft. Cajal (1928) indicated the trophic influences of Schwann cells to the growth of regenerating axons in the distal stump of the severed nerve. However, Huber (1919) did not recognize any effect of predenervation in the auto- nor in the allograft using sciatic nerves of dog and rabbit. Similarly, Bentley and Hill (1936) did not obtain any favorable results in the autograft of the cat external popliteal nerve (nervus peroneus communis), and

(' h)E el al.

Bunnell and Boyes (1937) did not find any difference in the rate of growth of nerve fibers through fresh versus predenervated autografts 1-6 weeks after implantation. In our experiments on allograft (Osawa et al., 1989), myelin sheaths were more rapidly removed by macrophages in the predenervated than nonpredenervated graft. In the early period regenerating axons grew faster in the predenervated graft, but later the difference became indistinct 1 month after grafting. Das Gupta (1967) observed by electron microscopy cellular changes in predenervated and nonpredenervated nerves which were implanted in the intermuscular space in the Wistar rat: lymphoid cell infiltration was reduced and Schwann cell basal laminae were retained, apparently undergoing no immunological rejections in the predenervated graft. On the contrary, there was an extensive lymphoid cell infiltration in the nonpredenervated graft, resulting in the rejection of the graft 3 weeks after implantation. He used nerves predenervated 6 weeks prior to implantation: myelin sheath debris should have no longer been present in the graft on implantation. He thought that myelin sheaths, but not Schwann cells, had a strong antigenicity. It seems thal the results he presented were somewhat exaggerated, but this study is one of the few electron microscopic studies which describes the retention of Schwann cell basal laminae after cellular elements have been degenerated in the nerve graft. 4.9. BLO(O,D SUPPLY

The blood supply is critical for the viability of Schwann cells in the thick nerve graft. Tarlov and Epstein (1945) showed that new vascular supplies to the grafted nerve came through the surrounding sheath tissue as well as at the ends of the graft. Smith (1966) studied systematically the mode of blood supply to the graft, and noted that the blood supply to the nerve is via the "mesoneurium". Hassler (1969) demonstrated by microangiography that old blood vessels were rejected within l0 days postgrafting, which was followed by the formation of new blood vessels. Hirasawa et al. (1974a) found that many more new blood vessels were formed in the irradiated nerves than in the nonirradiated ones grafted heterogenicatly from human to the dog. In the autograft, in order to ensure the maintenance of blood supply, the nerves which are located nearby and measured as thick as the injured ones, can be transferred to bridge the gap of injured nerves, while keeping intact the main blood vessels (Koshima and Harii, 1985a, b; Takado et al., 1987). Such a "vascular nerve graft" is applicable only to the autograft. In the atlograft, there is no blood supply until new blood vessels are formed, during which time the graft should he supplied nutrients by diffusion. Therefore, nerves used as allografts should be as thin as possible and the area in which the graft is placed should be sufficiently vascularized. On the other hand, frozen nerve grafts, in which Schwann cells are all dead, are presumed not to require in the early period so rich a vascularity or nutritional supply as fresh grafts. In addition, it is conceivable that nutritients might be more readily diffusible in the frozen graft, because the epi- and perineurial sheaths are damaged.

5. ELECTRON MICROSCOPIC STUDIES OF NERVE REGENERATION IN THE AUTOAND ALLOGRAFT Among many electron microscopic studies on nerve degeneration and regeneration following ordinary nerve injuries by crushing or transection, only a few studies noted the significance of Schwann cell basal laminae for nerve regeneration. Nathaniel and Pease (1963b) observed regenerating axons in the basal lamina tubes from which Schwann cell processes had been detached probably owing in part to the cell retraction, From this observation they supposed the fundamental importance of basal laminae in nerve regeneration. Thomas (1964a) and Thomas and King (1974) also noted in denervation experiments the outgrowth of regenerating fibers through basal lamina tubes, which had been partly evacuated due to withdrawal of atrophied Schwann cells. With regard to allograft, electron microscopic studies are much fewer (Schr6der and Seiffert, 1970; Pollard and Fitzpatrick, 1973a, b). Pollard and Fitzpatrick (1973a) noted that some regenerating fibers grew through basal lamina tubes which were left intact after Schwann cells had been degenerated in the 4 cm long sciatic nerve grafted from the DA to the Wistar strain of the rat. In the autografts whici~ were conducted as controls to the allografts, regenerating axons were shown to elongate along the surface of living Schwann cells. In the companion paper (1973b), they observed that regenerating fibers also grew out through basal lamina tubes in the allogeneic nerves of the DA strain in the immunosuppressed host Wistar rats. Allogeneic Schwann cells appeared to degenerate even in the immunosuppressed host. while leaving the basal laminae intact. Das Gupta (1967) showed that basal laminae remained after Schwann cells had been eliminated in the grafts. though, in a strict sense, the grafts they used we~e not allogeneic but isogeneic. Thus, in early electron microscopic studies of nerve degeneration and regeneration, basal laminae of Schwann cells were noted to persist, and their important roles in nerve regeneration were suggested, but only vaguely, so that the role of basal laminae in guiding the regenerative axons has not been studied systematically. In clinical studies of nerve allograft, the histological examinations of the grafts were made only by light microscopy, therefore no attention was paid to the role of basal laminae. The systematic studies of nerve allograft have been made in our laboratory using sciatic nerves of mouse, rat and rabbit, and ulnar nerves of monkey. In this section we want to present the results of our studies which indicate the important roles of Schwann cell basal laminae in nerve regeneration in the allograft. Because these allograft studies were based on the results of the autograft which had been made with mouse sciatic nerve, we will first briefly describe the autograft study in the mouse 5.1 . AUTOGRAFT IN MICE

A small segment of the sciatic nerve (ddY) was autologously implanted after vation and freezing treatment. For the vation, the sciatic nerve was transected at

of mouse predenerpredenerthe upper

~

Nm,~w ALLOGIL~dq"

sections were cut and prepared for electron microscopic observations. Dead cellular elements including myelin sheaths Fa~section were phagocytosed by macrophagcs but basal lamiOF p r e d e n e r v a t t o n nae were not attacked and were left as empty tubes (Figs 4 and 5). It is notable that regenerating axons grew out through such empty basal lamina tubes (Fig. 6). In contrast, there were no regenerating axons growing out through the endoneurial connective tissue spaces outside basal lamina tubes. The growing 1 week tips of the axons were not accompanied by Schwann cells, but were always naked, being directly attached to the basal lamina. Immature Schwann cells migrated out from the proximal stumps of the host nerve, following behind the growing tips of the axons (Fig. 7). Although it has been demonstrated that Schwann cells can migrate without the guide of axons ~on in vitro (Abercrombie and Johnson, 1942; Crang and Blakemore, 1987) and in vivo (Thomas, 1966), it is clear that Schwann cells migrate following the growing axonal tip in the presence of axons. It has been shown that these immature Schwann cells were produced by mitosis of host axons at their proximal stump by stimulation of insults on the nerve (Hall and Gregson, 1975; Scaravilli et al., 1986). Basal lamina tubes often contained as many as 20 or more ~n the distal site axons per tube (Nageotte, 1932; Nomura, 1975). As the regenerating axons, as well as accompanying Schwann cells, became mature, axons were separated by Schwann cells into smaller fasicles or even individual axons (Fig. 8). However, regenerated axons which FIG. 3. This scheme depicts the experimental design for had grown in the common basal lamina tubes repredenervation and eryo-treatment of the nerve grafts used mained as nerve bundles, i.e. the smallest unit bundles in our autograft studies. The sciatic nerve was transected for of the nerve. Later, new perineurial sheath cells came predenervation. After 1 week, a certain length of the nerve to surround these unit nerve bundles, or even divide was excised from the distal portion, treated by freezing and thawing five times, and implanted into the original location. bundle fibers into individual ones (Fig. 9). Such Nerve segments available for grafting were less than 1 cm in separation of regenerating nerves by newly formed the mouse. For allograft experiments nerves were pretreated perineurial sheaths was described as neuromatous in almost the same way prior to transplantation in the neurotization by Schrfder and Seiffert (1970) and mouse, rat, rabbit and monkey (see the text). The grafts analyzed by Ahmed and Weller (1979). This experavailable in the experiments were ca. 3 cm for sciatic nerve iment clearly shows that Schwann cell basal laminae in the rat, 3.5 cm for the saphenous nerve in the rabbit, and serve as effective conduits for the growth of regener3-7 cm for the ulnar nerve in the monkey. ating axons. Similar results were reported by Hall (1986). This fact provides an important breakthrough in the study of nerve regeneration. Elimination of part of the thigh, keeping the distal stump apart from Schwann cells, retaining their basal laminae as guides the proximal stump to prevent their reunion. One for regenerating axons, would be a great benefit week later, a small segment (at most 1 cm long) was especially for the allograft, since basal laminae excised from the distal portion of the transected appear to elicit much less immune reaction than nerve, and treated by freezing five times at about cellular components. - 4 0 ° C to kill all the cellular elements including Schwann cells in the graft. Such freeze-treated nerve 5.2. ALLOGRAFTIN MICE segment was again implanted into its original location and sutured at the proximal and distal ends In order to examine the effectiveness of Schwann to the respective stumps of the host nerve (Fig. 3). cell basal laminae in allograft in mice, the same The suture of the distal end with the host nerve is experiments as above were made by using sciatic considered effective for nerve regeneration in the nerve transplants between C57BL and C3H, strains graft (Young et al., 1940; Sanders and Young, 1944; which differ from each other in the major histocomSanders, 1946). Mice at several intervals from 1 day patibility complex (H-2) (Iv~nyi, 1970). A nerve to 4 weeks or more after implantation were sacrificed segment 5-7 mm long was excised from the sciatic and fixed with the fixative containing 4% paraformal- nerve of the C57BL/6N strain, treated by freezing and dehyde and 1.25% glutaraldehyde in 0.1 i cacodylate thawing five times to kill the Schwann cells as in the buffer (pH 7.4). The grafts were resected, together case of autograft, and transplanted to the sciatic with the adjacent part of host nerve, postfixed in 1% nerve of the C3H/HeN strain at roughly the correosmium tetroxide solution, dehydrated in an ethanol sponding site where the nerve had been freshly transeries and embedded in Epon 812. Thin cross sectcd. The nerve sergment used in this experiment

after

/

~/mplantation

10

C:. IDE et ai.

had not been predenervated. The graft was sutured on both the proximal and distal stumps of the host sciatic nerve. Animals were fixed by perfusion through the heart with the fixative containing 2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M cacodylate buffer 4, 7, 14, 20, 30, 50, 70 and 100 days after grafting. Grafts and toe pads as well as lumbrical muscles of the operated side were processed for electron microscopic observations. The process of Schwann cell degeneration and subsequent nerve regeneration were the same as those described above. Schwann cells and myelin sheaths were fragmented and disintegrated into cell debris, while Schwann cell basal laminae remained apparently intact retaining the original form of tubes. Macrophages invaded these basal lamina tubes to phagocytose the cell debris, leaving basal laminae remaining almost undamaged (Fig. 10). It appeared that no immune reaction to the basal laminae took place. Regenerating axons and accompanying Schwann cells grew out through such basal lamina tubes vigorously (Figs 10 and 11). Separation and myelination of axons by Schwann cells were advanced in the same fashion as in the autograft (Fig. 12). Schwann cells containing an abundance of free ribosomes had no basal laminae of their own during migration. However, as the separation of the axons began, Schwann cells gradually produced their own basal laminae on the surface (Bunge and Bunge, 1983). From about 2 weeks postgrafting on, their myelin sheaths were found to form on thick axons. Fully developed myelin sheaths were attained by 5 weeks postgrafting. Along with the maturation as well as the voluminous increase of regenerating nerves, original basal lamina tubes were gradually fragmented and eventually disappeared 2-3 weeks after grafting as in the autograft (Fig. 12). The maturation of axons and Schwann cells proceeded in the same fashion as in the autograft. Axons further extended into the distal stumps of the host nerve, and arrived at end organs such as sensory corpuscles in the toe pad skin and motor endplates of lumbrical muscles 30 days after grafting (Fig. 13; Osawa et al., 1986). This study suggested that allogeneic Schwann cell basal laminae can act as effective pathways for the extension of regenerating axons as in autogeneic grafts. However, the grafts available in the mouse sciatic nerve were very short (less than 1 cm), compared to ordinary allograft experiments in other animals. 5.3. ALLOGRAFT IN RATS Using longer nerve grafts (ca. 3 cm) of the sciatic nerve, more systematic studies of allograft were performed between the Fischer 344 and the Brown Norway strains of rat (Osawa et al., 1989). These strains are different from each other in their major histocompatibility complex: the former has RT-P and the latter RT-1 n (Gill et al., 1987) or Ag-B1 and Ag-B3, respectively (Palm and Black, 1971). In this study, three types of grafts were performed: fresh (nonpretreated, NT), predenervated (PD), and predenervated plus frozen (PDC) grafts. For predenervation, the sciatic nerve was cut one week prior to use. Freezing of the graft was performed in the

same way as described in the autografl m mice. Approximately 3 cm long grafts were obtained from the Fischer 344 strain, and sutured with or without freezing treatment to the freshly transected sciatic nerve stumps of the host strain (Brown Norway) (Fig. 14). In the NT and PD grafts Schwann cells were alive, while they were dead in the PDC graft on implantation. As expected, various degrees of lymphoid cell infiltration were found in places in the NT and PD grafts, while there was only little lymphoid cell invasion in the PDC graft. The degradation of cellular components and their elimination by macrophages while retaining Schwann cell basal lamina undamaged in the form of tubes were the same in the PDC graft as in the cryotreated auto- and allograft in mice. The overall procedures of cell degradation and subsequent nerve regeneration were surveyed by light microscopy in Fig. 15. In NT and PD grafts, most Schwann cells appeared to be retained alive in the early periods, but were probably damaged within 7 10days after grafting. Some lymphoid cells were seen in direct contact with Schwann cells which exhibited apparently degenerative changes. However, it was not certain whether Schwann cells were always degraded b3 direct involvement of lymphocytes. The degradation of Schwann cells was advanced more rapidly in the proximal as well as in the distal portion than in the mid-portion of the graft. Except for the areas where severe cell infiltration occurred, basal laminae of Schwann cells were left almost undamaged, even in NT and PD grafts, after degradation of Schwann cells as in the case of PDC or freezing-treated autografl (Fig. 16). Within one week, regenerating axons entered the basal lamina tubes in all the three types of the grafts, and gradually increased in number as well as in volume. Some of the early regenerating axons appeared to be damaged: this finding indicates that once successfully regenerated axons could be degenerated in the allograft. This phenomenon might be due to the degeneration of host Schwann cells to which regenerating axons were associated. It was not easy to distinguish immature host Schwann cells which had moved along regenerating axons from the persisting donor Schwann cells. However, judging from the extensive degeneration of donor Schwann cells in the mid-portion of the graft (Fig. 16), it is reasonable to presume that almost all the donor Schwann cells disintegrate to be replaced by host Schwann cells. Basal lamina tubes were welt preserved in most places, serving as effective pathways for regenerating axons. Nerve regeneration in NT and PD grafts was retarded in the early period (Figs 17-19), but eventually became as good as in the PDC graft around 2 months postgrafting. As nerve regeneration advanced, basal laminae of donor Schwann cells were gradually disintegrated and had disappeared by 2 months after grafting. In the PDC graft, Schwann cell basal laminae were well preserved and vigorous axonal growth occurred through such basal lamina tubes. Regenerating axons were accompanied by immature Schwann cells from the proximal stump of the host nerve. Axons which had been surrounded by the common Schwann cells were separated into individual ones by proliferatkm

FIG. 1. Electron micrograph o f the normal peripheral nerve (mouse sciatic nerve). Schwann cells of both myelinated (A) and unmyelinated nerves (a) are invested by basal laminae (arrows) on the surface (23,000 x ). Scale bar = 1/zm. FIG. 2. Nerve regeneration after simple transection o f the sciatic nerve in mouse. This micrograph was taken from the toe pad skin 8 weeks after transection. Regenerating axons (a) are associated with Schwann cell processes (S), which reside within basal lamina tubes (arrows) (35,000 × ). Scale bar = 1/~m. 11

FIG. 4. Two days after autografting in mouse. Myelin sheaths (m) were extensively degraded, but the basal lamina of each myelinated fiber were preserved (arrows). A macrophage (M) having invaded the basal lamina tube phagocytoses myelin sheath debris (9500 × ). Scale bar = 1 tzm. FK;. 5. Seven days after autografting in mouse. The cell process (M) appears to be part of a macrophage that is leaving the basal lamina tube (arrows) after having phagocytosed myelin sheath debris. There are empty basal lamina tubes (B). A large macrophage (LM) containing myelin sheath debris is seen in the endoneurial space (9000 × ). Scale bar = 1 ~m.

12

FIG. 6. Seven days after autografting in mouse. Regenerating axons (A) are seen within basal lamina tubes (arrows). There are no axons growing out into the endoneurial connective tissue space (28,000 x). Scale bar = 1/~m. (C. Ide et al., Brain Res. 388 (1983), with the permission o f the publisher.) FIG. 7. Two weeks after autografting in mouse. Regenerating axons (A) are surrounded by new Sehwann cell processes (S) within a basal lamina tube (arrows). This old basal lamina appears to be undergoing degradation. Scale bar = I/~m.

13

FIG. 8. Two weeks after autografting in mouse. A group of regenerating axons which have grown through the same c o m m o n basal lamina tube is seen. Regenerating axons (A) are separated into individual fibers and are partly myelinated by Schwann cells (S). A new perineurial sheath (P) surrounds this unit nerve bundle (12,000 x ). Scale bar = ! ~m. (C. Ide, Archs Histol. ,lap. 46 (1983), with the permission of the publisher.) FLG. 9. Five weeks after autografting in mouse. Regenerating fibers (N) are separated into groups of several or even single fibers by extensively developed perineurial sheaths (P) (10,000 x ). Scale bar = 1 ~m. 14

FIG. 10. Seven days after allografting in mouse. Basal laminae of donor Sehwann cells are left undamaged, and regenerating axons (A) elongate through such a donor basal lamina tube (arrow). Empty basal lamina tubes can be seen (E) (31,000 x). Scale b a r = 1/~m. (T. Osawa et aL, Archs Histol. Jap. 49 (1986), with the permission of the publisher.) FIG. 11. Two weeks after allografting in mouse. Host Sehwann cell (S) accompanying axons has begun sorting out some axons (A). Arrows point to the donor Sehwann cell basal lamina. P, new perineurial cells. Collapsed basal lamina tubes (B) are seen (15,000 x ). Scale bar = 1 #m.

15

FIG. 12. Twenty days after allografting in mouse. Regenerating axons (A) in a unit bundle are sorted out and some of them are myelinated by Schwann cells (S). These nerves form a unit bundle, indicating their growth in a common basal lamina tube. The developing perineurial sheath (P) surrounds the bundle. There are some remnants of degenerated donor basal laminae (arrows) (7000 x ). Scale bar = 1/~m.

16

FIG. 13. Allografting in mouse. Nerve regeneration to the end organs such as Meissner corpuscle (a) and motor endplate of lumbrical muscle (b). a: Thirty days after grafting. A regenerating axon (A) has reached the denervated murine Meissner corpuscle (L) in the toe pad. E: epidermis, V: vessel (13,000 x ). b: One hundred days after grafting. This endplate has a regenerated axon terminal (A) which contains an abundance of mitochondria and vesicles. M: muscle fiber, S: Schwann cell (33,000 x ). Scale bar = 1/~m. (T. Osawa et al., Archs Histol. Jap. 49 (1986), with the permission of the publisher.)

17

FIG. 15. Allografting in the rat. These light micrographs show the histology of the grafts at the proximal portions. Vertical columns are from the left, NT (a-d), PD (e-h) and PDC (i-l), and horizontal columns from the top, 1 week (a, e, i), 2 weeks (b, f, j), 1 month (c, g, k) and 2 months (d, h, 1) after grafting. At 1 week, myelin sheaths were disintegrated and phagocytosed by macrophages (M) in PD and PDC, while they still remained apparently undegraded in NT. At 2 weeks, many profiles of regenerating nerves (arrows) are seen in PD and PDC, but not in NT. At I month, there are numerous regenerating nerves which are myelinated in PD and PDC, while regenerating nerves are smaller in number and still unmyelinated in NT. By 2 months, regenerating nerves are numerous and myelinated in all the three types of grafts (450 x ).

18

FIG. 16. Two weeks after allografting in the rat. These micrographs show the preservation of basal laminae of donor Schwann cells in NT, PD and PDC grafts at the middle portion of the graft, a: NT graft. Cellular elements (C) appear to have disintegrated by cytolysis, while basal laminae (arrows) remain unaffected (7000 x ). b: PD graft. Basal lamina tubes (arrows) of Schwann cells are well preserved; some are empty and others contain exogenous cell elements (C) (8500 x ). c: PDC graft. Basal lamina tubes (arrows) remain collapsed, and the profile of an exogenous cell process (C) is seen in one tube. No regenerating axons are seen in this area (9000 x ). Scale bar = 1/~m. (Osawa et al., J. Neurocytol., with the permission o f the editor and publisher.)

19

FIG. 17. Seven days after allografling in the rat. These micrographs show nerve regeneration at the proximal portion of the graft, a: N T graft. Regenerating axons (A) are surrounded by Schwann cells (S) in basal lamina tubes (arrows). A presumable macrophage (M) is present within a basal lamin tube (7000 x ). b: PD graft. Findings are the same as in the N T graft, but the number of axons appears to be larger. It has not been determined with confidence whether Schwann cells seen in the N T and PD grafts are those of donor or host animals. M, macrophage (7000 × ). c: PDC graft. It is obvious that basal laminae of donor Schwann cells are well preserved (arrows), and regenerating axons (A) grow out through such basal lamina tubes in the same way as they did in the autograft of the mouse (11,000 x ). Scale bar = 1 #m. (Osawa et al., J. NeurocTtol.. with the permission of the editor and publisher.)

20

FIG. 18. Two weeks after allografting in the rat. a: NT graft. The finding is the same as in the l-week stage, but regenerating axons (A) increased in number (6000 x ). b: PD graft. The number of axons per Schwann cell basal lamina tube (arrows) increased (9000 x ). c: PDC graft. Regenerating axons (A) are being separated into individual fibers and partly myelinated by Schwann cells (S). Each unit nerve bundle (UB) evidently shows that nerves grew through the common donor basal lamina tubes (arrows). A large unit bundle is surrounded by a new perineurial cell (P) (5000 x ). Scale bar = 1 #m.

21

FIG. 19. One m o n t h after allografting in the rat. a: N T graft. It is clear that regenerating axons (A) grow through basal lamina tubes (arrows). Schwann ceils (S) which have presumably migrated from the host proximal nerve stump accompany these axons. A part of an empty basal lamina tube (E) is seen. (8000 x ). b: PD graft. These small unit nerve bundles reflect the fact that each unit is a group of axons which have regenerated through the c o m m o n basal lamina tubes. Unlike the N T graft, there are many well-myelinated axons. Fragments of the basal lamina tube of donor Schwann cell are visible (arrows). There is no compartmentation by new perineurial cells in NT and PD grafts (3000 x). c: P D C graft. There are numerous myelinated (A) and unmyelinated fibers (a), and new perineurial sheaths (P) divide these fibers into smaller bundles (3000 x ). Scale bar = 1/zm.

FIG. 20. Two weeks after allografting in the rabbit. This section was obtained from the mid-portion of the graft. Many regenerating axons (A) are found within basal lamina tubes (large arrows). No distinct Schwann cell process is found. Some collapsed empty basal lamina tubes (small arrows) are seen in the endoneurial space (13,000 x ). Scale bar = 1 pm. FIG. 21. Two weeks after allografting in the rabbit, This section was also obtained from the mid-portion of the graft. A Schwann cell body (Sb) and cell processes (S) are seen associated with more than ten profiles of regenerating axons (A) within a common basal lamina tube. Basal laminae (arrows) o f presumably grafted Schwann cells are fragmented, partly surrounding new Schwann cells which are invested by their own basal laminae (13,000 x ). Scale bar = 1/~m.

23

FIG. 22. Fourteen weeks after allografting in the rabbit. This micrograph was taken at the mid-portion of the graft. There are many myelinated (A) and unmyelinated (a) axons in this unit bundle of regenerating nerves. The new perineurial sheath (P) consists of several layers of cell processes. Some empty basal lamina tubes are seen tarrows). A blood vessel (BV) is seen in the interspace between compartments. S: Schwann cells (5100×). Scale b a r = l # m FIG. 23. Fourteen weeks alter allografting in the rabbit. This micrograph was taken from a section or" the host saphenous nerve at the level 10 mm from the distal apposition site of the graft. Many myelinated (At and unmyelinated regenerating axons (arrows) are seen. There is no compartmentation in this level. S: Schwann cells (5100×). Scale bar : 1 ~tm,

24

FIG. 24. Five weeks after allografting in the monkey. Predenervated plus cryo-treated graft. This micrograph was taken from the graft at the level 10 mm distal to the proximal suture site. Basal laminae (arrows) of donor Schwann cells were well preserved after cellular elements had been degraded and removed (20,000 x ). Scale bar = 1 gin. FIG. 25. Five weeks after allografting in the monkey. Predenervated plus cryo-treated graft. This micrograph, which was taken at the level 5 mm distal to the proximal suture site, shows that many regenerating axons (A) grow out through donor basal lamina tubes (arrows). Schwann cell (S) is seen accompanying these axons. L: lymphocyte, V: vessel (5300 x ). Scale bar = 5 #m.

25

FIG. 26. Eight weeks after allografting in the monkey. Predenervated plus cryo-treated graft. This micrograph was obtained at the site 10 m m distal to the proximal suture site. N u m e r o u s regenerating axons (A) are in bundles, reflecting that they grew through c o m m o n basal lamina tubes. Remnants of disintegrated basal laminae of donor Schwann cells are identifiable (arrows). Myelination has begun on some axons. L: lymphocyte (4500 × ). Scale bar = 5 #m. FIG. 27. Five m o n t h s after allografting in the monkey. Predenervated plus cryo-treated graft. This micrograph was taken at 5 m m distal to the proximal suture site. There are many regenerating axons; some thick ones are well myelinated (A) in this unit bundle (UB1). New perineurial sheaths (P) are formed separating other unit bundles (UB) of regenerating axons (4500 × ). Scale bar = 5 p m .

26

FIG. 28. Five months after allografting in the monkey. Predenervated plus cryo-treated graft. This micrograph was taken from the host ulnar nerve at the wrist, i.e: about 13 c m distal to the proximal suture site. Many regenerating axons had grown into the host nerve; thick ones (A) are well myelinated, while thinner ones (a) are unmyelinated (4700 × ). Scale bar = 5/~m.

27

NERVEALLOGRAFT

29 transection

predenervation

/after

1 week\

"n-''°"'nol

NT

PD

" " " ' °:I

PDC

FXG. 14. This drawing shows the procedure of allo~'afting in the rat. The donor rat is Brown Norway (BN) and the host is Fischer 344 (F344). The predenervated nerve segments with (PDC) or without cryo-treatment (PD) were transplanted to the host rats, and the fresh nerve with no pretreatment was transplanted to other host rats (NT). See the text for the details of NT, PD and PDC grafts. (Osawa et aL, J. Neuroc2toL, with the permission of the editor and publisher.)

of Schwann cells. Bundles of axons which had grown through the same basal lamina tube remained as small units of nerve bundles. After the basal lamina tubes had disintegrated, new perineurial sheaths were formed separating nerve bundles into smaller ones or even individual fibers (Fig. 19c). Numerous regenerating axons were seen also beyond the graft through the distal portion of the host nerve. Since, even in the NT and PD grafts, basal laminae were preserved after the Schwann cells had been degenerated, the final achievement of nerve regeneration was the same as in the PDC graft. It can be concluded that basal laminae of Schwann cells can be as effective conduits for regenerating axons as those in the 3 cm long allograft in the rat.

5.4. ALLOGRAFTIN RABBITS Ten Japanese White (JW) rabbits and 12 New Zealand White (NW) rabbits were used as donors and recipients, respectively. One week prior to the transplantation, the saphenous nerve of the JW rabbits was transected for predenervation at the upper thigh level. For transplantation, a 35 mm long segment was excised from the distal portion of the transected saphenous nerve and treated by freezing and thawing five times as in the case of mouse and rat described above. These cryo-treated nerve segments were transplanted into the freshly prepared 30 mm gap of the saphenous nerve of N W rabbits. At the intervals of 1, 2, 4, 6, 8 and 14 weeks after transplantation, the grafts together with the adjacent host nerve segment were dissected out and fixed for the histological examination in a fixative containing

2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4). Two rabbits were used at each time point. The electron microscopic examinations were made mainly at the mid-portion of the graft. In one case (14weeks after transplantation), nerve regeneration into the host's distal stump was examined 1 cm from the distal apposition site (Tohyama et al., 1989). The process of nerve regeneration in these allogeneic cryo-treated nerve grafts in the rabbit was the same as in the cryo-treated auto- and allogeneic graft in the mouse and rat. Within 1 week after grafting, myelin sheath debris began to be phagocytosed by macrophages, while basal laminae of Schwann cells were left undamaged in the form of tubes. By 2 week after grafting, regenerating axons grew through such basal lamina tubes, which were usually attached to the inner surface of the basal laminae. They were at first naked, without Schwann cell investiment (Fig. 20), but later surrounded by accompanying Schwann cells at the mid-portion of the graft (Fig. 21). Regenerating axons increased in number forming a bundle in the common basal lamina tube, which was gradually separated into smaller bundles or even individual axons by Schwann cells. Separated thick axons were myelinated by 6 weeks after grafting. The number of myelinated fibers increased with time, and the characteristic compartmentation of regenerating nerves by new perineurial sheath cells became conspicuous 8 weeks after grafting (Fig. 22). Blood vessels were present not within, but in the space between the compartments. The ratio of myelinated nerves to the total number of regenerating nerves was roughly 10% at 8 weeks and 30% at 14 weeks after grafting. In the host distal nerve 10 mm

30

t . tDE et ai.

from the distal apposition site, there was an abuddance of regenerating axons .including myelinated ones 14 weeks after grafting (Fig. 23). These results indicate that the Schwann cell basal lamina tubes of cryo-treated allogeneic nerves can serve as conduits for nerve regeneration in a graft more than 30 mm long in the rabbit. 5,5, ALLOGRAFT IN MONKEYS

For the purpose of examining whether the Schwann cell basal lamina can be as effective a guide for regenerating axons in the primate as in the mouse and rat, allogeneic grafting was performed using the ulnar nerve in the crab-eating monkey (Macaea fascieularis) (Tajima et al., 1989). The ulnar nerve of the monkey consisted of 2--3 large fascicles of approximately 1-2 mm in diameter at the region of the elbow joint. One fascicle was cut at the point about 5 cm distal to the elbow joint for predenervation 7-10 days prior to grafting. A nerve segment 3-7 cm long was excised from the predenervated nerve, treated by freezing five times as described above, and transplanted into the ulnar nerve of another monkey at the level roughly corresponding to the site where the graft had been obtained. As the control, fresh, noncryo-treated nerve segments were grafted in the same way. For the histological examination, grafts were excised together with part of the host nerve 2, 5 and 8 weeks and 5 months after transplantation and processed for electron microscopic observation. Two grafts were used at each time point for both cryo-treated and noncryo-treated groups. In the control experiment, there were no regenerating nerves in the grafts 2-5 weeks after grafting. After 8 weeks, only a small number of regenerating nerves were found at the proximal portion (within 5 m m distal to the suture site) of the graft. The histological examination demonstrated that the graft was completely degenerated by necrosis and infiltrated by mainly macrophages 5 months after grafting. On the other hand, in the cryo-treated graft, donor Schwann cells and myelin sheaths were degenerated, and removed mainly by macrophages. Basal laminae of Schwann cells were left undamaged as empty tubes in the same fashion as in the auto- and allografts in mice and rats (Fig. 24). Many regenerating nerves extended through such basal lamina tubes of donor Schwann cells as far as 3 cm in the graft, 5weeks after grafting (Fig. 25). They were not myelinated. After 8 weeks, a large number of regenerating nerves were found throughout the graft and at the site approximately 1 cm distal to the proximal suture site. Separation of axons by Schwann cells had advanced and myelination had begun on some axons (Fig. 26). It is evident that regenerated nerves were separated into small bundles, reminiscent of their growth through the same basal lamina tubes. Five months later, thick regenerated axons were myelinated in various degrees in the graft (Fig. 27), and numerous thin unmyelinated and a small number of thick myelinated fibers were found in the host nerve at the wrist level, i.e. about 13 cm from the proximal suture site (Fig. 28). It was ascertained that the weak sensory response to the light touch by a fine needle was recovered in the hypothenar region of the hand.

These results show that Schwann cell basal laminae were left undamaged after the cellular elements were degraded in the cryotreated allografts, m d subsequently could serve as conduits for regenerating axons and accompanying host Schwann cells in the monkey.

6. CONSIDERATIONS ON THE MAIN C O M P O N E N T S OF THE GRAFT

6.1. SCHWANN CELLS The main problem of nerve allograft studies has been how to reduce the immune reactions of the graft, while hopefully preserving viable donor Schwann cells in the host. For this purpose, the immunosuppression of the host has been extensively studied. On the other hand, reducing the antigenicity of the graft itself has been attempted by various pretreatments such as freezing, freeze-drying, and/or irradiation, but no attention has been given to the fact that all the Schwann cells in the graft might be killed by such pretreatments. Moreover, it was not clear whether denatured Schwann cells and myelin sheaths could be eventually removed by macrophages or left for a long time serving as obstacles to the growth of regenerating axons. Rejection of Schwann cells in the allograft was first examined by Aguayo et al. (1977), who implanted a sural nerve segment of a patient with metachromatic leukodystrophy into a mouse which had been immunosuppressed by injection of antilymphocytic serum. The human Schwann celis in the graft were retained alive during immunosuppression, but were rapidly rejected by injection of sensitized lymphocytes following the discontinuation of antiserum treatment. Mononuclear cells which had invaded the graft, appeared to attack donor Schwann cells. By transplanting sciatic nerves of BALB/c or of C57/BL into trembler BALB/c mice, Pollard and McLeod (1981) showed that Schwann cells of C57/BL were rejected but those of BALB/c were kept alive in the trembler BALB/c mice. Because the graft used in this experiment contained no myelin sheaths, as the nerve used for the graft had been cut 12 weeks prior to the implantation, they concluded that Schwann cells but not myelin sheaths were antigenic m the nerve allograft. Regarding the antigenicity of nervous tissues, Seiffert and Schindler (1968) showed that the sciatic nerve and spinal cord are antigenic, causing lymphoid infiltration in the allogeneic transplantation. Comtet and Revillard (1979), by examining the histology of the allogeneic sciatic nerve which had been transplanted from the DBA/2 to the C57BL/6 strain of mice, observed more extensive cell infiltration and pyroninopbilic cell increase in the allograft than in the isograft. These immune reactions appeared very late in the nerve graft as compared to the skin graft which became necrotic within day 7-9 posttransplantation. The nervous tissues appeared to have less ability than the skin to sensitize the host animal. More detailed analysis of the antigenicity of the nerve allograft was performed by Mackinnon et at. (1982), who transplanted the sciatic nerve of Fischer 344

NERVEALIZ~RAFT (RT-11), Buffalo (RT-1 b) and ACI (RT-P) strains into Lewis (RT-1 l) strain, and examined the resulting cytotoxicity of the recipient lymphocytes using 5tCrrelease assay in which the 5~Cr-labelled splenocytes of the donor strain were lysed by cytotoxic lymphocytes of sensitized host animals, leading to the release of 51Cr. Sensitization occurred very late in the host rat grafted by the Fischer 344 nerve which was matched in major histocompatibility complex (MHC), as compared to the grafts of MHC-unmatched nerves of ACI and Buffalo strains. However, the rate of lymphocyte infiltration of the graft was almost the same among these three grafts. The authors supposed that, if the sensitization occurred sufficiently late, nerve regeneration might have taken place across the allograft prior to the appearance of immune reactions in the MHC-matched host, thus assuring the successful nerve regeneration without any immunosuppressive treatment. Recent studies have clarified the mechanisms of Schwann cell or glial cell rejections in the more strictly defined systems. Scaravilli and Jacobs (1981) showed that normal human Schwann cells could survive in Twicher mouse by use of a T-cell suppressive treatment. Sun and Wekerle (1986) showed that the astrocyte killing follows the rule of T-cell-mediated cytolysis in the in vitro experiment: astrocytes with myelin basic protein (MBP) antigen as well as Ia antigen on the surface were attacked by Ia-restricted MBP-specific T-lymphocytes. In the same way, they demonstrated that neonatal rat Schwann cells which were induced by IFN-7 (7-interferon) MBP and Ia antigens on the cell surface could be lysed by MBPspecific T-cells (Wekerle et al., 1986). IFN-y induces the expression of class II antigen in the human and rat Sehwann cells (Samuel et al., 1987a, b), whereas it induces class I antigen in the mouse Schwann cells (Steinhoff and Kaufmann, 1988). Schwann cells of neonatal mice express almost no class I nor class II antigen. By infecting mice with M. leprae and at the same time using IFN-y treatment, Schwann cells expressed M . leprae antigen as well as class I antigen on the surface. Such INF-y-stimulated M. lepraeprimed Schwann cells were lysed by CD8 + cytotoxic T-cells, but those with either M. leprae antigen or class I antigen were not. It has been thought that cytotoxic T-cells mediate the cellular mechanism of allograft rejection. However, recently an alternative mechanism has been postulated in which helper T-cells (Ly-I ÷ in mouse, and W3/25 + MRC OX8- in rat) play a main role in allograft rejection (Loveland and McKenzie, 1982; Lowry et al., 1983). The direct contact of T-lymphocyte with target cells is considered to be the feature of attack by lymphocytes which plays the major role in allograft rejection (Williams et al., 1964; Kalina and Berke, 1976; Lowry et al., 1983; Gregory and Alkinson, 1984; Gregory et al., 1984; Nocera et al., 1986; Forbes et al., 1988). Nocera et al. (1986), in the in vitro experiment using T-lymphocytes homing in a rejected human kidney allograft, showed that the cells responsible for the rejection of kidney allograft are helper-dependent cytotoxic T-lymphocytes, and that cytotoxic antibodies directed against the same specificity as that of the cytotoxic T-cells were found in the serum. It is highly probable that humoral antibodies

31

might be involved in the destruction of donor Schwann cells in the allograft (Mackinnon et al., 1982; Nocera et al., 1986): allograft rejection can be mediated by both humoral and cellular mechanisms. Macrophages were also shown to lyse donor cells in the heart transplantation (Christmas and MacPherson, 1982; Forbes et al., 1988). However, in the nerve allograft, there were no findings which suggested direct attack by macrophages on the living Schwann cells. Macrophages appeared to phagocytose only dead cellular elements, especially myelin sheath debris in the nerve allograft. As to the effectiveness of immunosuppression of the host animals, Pollard and Fitzpatrick (1973b) pointed out the fact that Schwann cells degenerated even in the immunosuppressed condition by the administration of Immuran, whereas Zalewski et al. (1981) reported that Schwann cells were rejected after abolishing immunological tolerance. However, recently Mackinnon et al. (1987) showed that a low dose azathioprine could suppress immune reactions and induce relatively good nerve regeneration and that there was no rejection reaction 180 days after cessation of the azathioprine administration. They also showed the effectiveness of a low dose (Smg/kg/day) of cyclosporin A in suppression of immune reaction in the rat (Bain et al., 1988). It is still uncertain whether donor Schwann cells can continue to survive for years after stopping the immunosuppression. If they are destined to be rejected eventually, such immune destructions of the donor Schwann cells might possibly result in deleterious effects on the regenerated axons. 6.2. BASALLAMINAE The basal lamina (or basement membrane) is an amorphous material formed on the external (adconnective tissue) surface of various cells including epithelial cells, vascular endothelial cells, striated and smooth muscle fibers, glial cells, and Schwann cells. The basement membrane which was first defined at the light microscopic level by Todd and Bowman (1857) and reviewed by Kefalides et al. (1979), consists of three sublayers as observed by electron microscopy: lamina lucida (or lamina rara), lamina densa, and reticular lamina. Basal lamina is a synonym of lamina densa, but is often used interchangeably with basement membrane. The lamina densa of the Schwann cell basement membrane measures approximately 30-40 nm in thickness, and the lamina lucida is as thick as the lamina densa. The reticular lamina is a thin ill-defined layer made of fine filamentous materials and occasional collagen fibrils. External to and partly continuous with the reticular lamina, is a thin layer of fine collagen fibrils, which is called the sheath of Plenk-Laidlaw (Plenk, 1927, 1934; Laidlaw, 1930). External to this layer, there is a thick layer of collagen fibrils, known as the sheath of Key-Retzius (Key and Retzius, 1873). The "endoneurial sheath or tube" used in the old literature presumably includes the basal lamina, sheath of Plenk-Laidlaw, and even part of sheath of KeyRetzius. The basal lamina described in this review means the lamina densa, but in most cases also includes other sublayers of the basement membrane.

32

C. IDE et al.

Basal laminae have been demonstrated to serve as the scaffolding for the orderly repair of tissue including muscle fibers, vascular endothelial cells, and alveolar epithelial cells (Vracko, 1972, 1978; Vracko and Benditt, 1972). With regard to nervous tissues, Nathaniel and Pease (1963a, b), Thomas (1964a, b), and Pollard and Fitzpatrick (1973a, b) inferred the possible involvement of Schwann cell basal laminae in nerve regeneration. Anderson et al. (1983) performed an electron microscopic study on nerve regeneration through culture-medium-stored or freeze-dried autogeneic nerve segments, but did not note the role of basal laminae in the growth of regenerating axons. Our electron microscopic study on nerve regeneration using the cryo-treated autograft was the first to clearly and systematically demonstrate the important role of Schwann cell basal laminae in guiding the direction of regenerating axons. This fact indicates that, instead of the ordinary graft containing living Schwann cells, the acellular nerve, i.e. the nerve segment consisting of only connective tissue components, can be used as a graft. In our subsequent aUo- and xeno- (hetero-) graft studies, basal laminae were shown to be retained, serving as very effective scaffolds for regenerating axons in the mouse (Osawa et aL, 1986~ 1987), rat (Osawa et al., 1989), rabbit (Tohyama et al., 1989) and monkey (Tajima et al., 1989). Judging from the fact that basal laminae remained undamaged for as long a time in the allograft as in the autograft after the cellular elements had been eliminated in the sciatic nerve of the mouse and rat, it appears that Schwann cell basal laminae disintegrate not by immunological rejections but by the ordinary metabolic turnover as suggested by other investigators (Walker, 1973; Price and Spiro, 1977). Basal laminae consist of the matrix of type IV collagen to which glycoproteins such as laminin (Timpl et al., 1979; Foidart et al., 1980), nidogen (Dziadek and Timpl, 1985), and entactin (Bender et al., 1981) are associated. Proteoglycans, such as heparan sulfate proteoglycan, are considered to be distributed throughout the sublayers, being most concentrated on the external zone of the reticular lamina (Kanwar and Farquhar, 1979a, b; Farquhar, 1981; Yokota et al., 1983). Histochemically, the basal laminae of Schwann cells are shown to be polarized regarding their constituents (Tohyama and Ide, 1984; Tohyama, 1985). Components of the basement membrane are known to be antigenic in the epidermo-dermal junction of the skin (Fine, 1988). It has been demonstrated that glomerular basement membranes contain various antigenic constituents (Kefalides, 1972; Bardos et al., 1976). In kidney transplantation, antibodies against tubular basement membrane were produced by lymphocytes isolated from the rejected allograft (Jordan et al., 1986). Mice immunized with dog insoluble glomerular and tubular basement membrane produced antibodies to each of these two basement membranes (Moulonguet-Doleris et al., 1981). Collagens obtained from the rat tail were species-specific and antigenic, although the titer of their antibody is low (Watson et al., 1954). All these studies show that the basement membrane is antigenic, possibly becoming the target of immune rejec-

tion in the allograft. However, contrary Io these results, Schwann cell basal laminae in the nerve aliograft remained almost intact as long as one month (Osawa et al., 1989), or more than 4 months (Pollard and Fitzpatrick, 1973a) after grafting; this may be due to the low antigenicity of the Schwann celt basement membrane. The growth of regenerating axons through basal lamina tubes is an interesting phenomenon. It has not been precisely determined as to whether Schwann cell basal laminae only provide mechanical pathways or have a neurotrophic influence on the growing axons. Regenerating axons are also able to grow through basal laminae of skeletal muscle fibers (Idc, 1984; Keynes et al., 1984; Glasby et al., 1986a, b, c; Fawcett and Keynes, 1986; Davies et al., 1987; Norris et at., 1988), or more generally in hollow structures such as denatured or fresh arteries (Weiss, 1943: Gulati, 1989) and artificial tubes (Reid et at., 1978: L'Jndborg et al., 1982; Molander et aL, 1982; Uzman and Villegas, 1983; Scaravilli, 1984; Seckel eta?., i984; Williams et al., 1988; Dellon and Mackinnot~, 1988). However, there is a difference in the way iJ~ which regenerating axons grow. In the Schwann cell basal lamina tubes, the regenerating axons are attached to the inner surface of the basal laminae alcq~g their entire length, whereas they are mostly detached from the inner wall of muscle basal laminae or :~rtificial tubes. This fact implies that Schwann cc!i basal laminae can serve as the most favorable footholds for the attachment of regenerating axons. The characteristic axonal growth on the basal lamina supports the concept of contact guidance for explaining the mechanism of growth of axons (Weiss, 1934: Nakai, 1956; Nakai and Kawasaki, 1959). Basal lamina components such as laminin and other associated components are probably responsible for the attachment of axons to the basal laminae (Foidart e: :~:.~ 1980: Edgar et al., 1984; Tohyama and Ide, !984i 7. CONCLUDING REMARKS Studies on nerve allograft have been reviewed according to various attempts that have been made for the purpose of reducing or suppressing the immune reactions to the graft. Despite various pretreatments of the grafts and/or use of immunosuppressive agents, which have been studied for almost a century, no definitely valuable improvement in nerve regeneration has yet been obtained. It was pointed out that detailed histological examinations by electron microscopy are largely neglected in almost all clinically oriented studies on nerve aUograft. From our recent electron microscopic studies on auto-, aIlo- and even heterografts of peripheral nerves which had been treated by predenervation and freezing prior to implantation, the basal laminae of Schwann ceils were shown to play important roles in nerve regeneration serving as conduits for growing axons in mouse, rat, rabbit, and monkey. The fact that basal laminae appear to undergo no obvious immune rejections in the allogeneic nerve is an interesting problem to be further studied in the context of nerve allograft. Acknowledgements--The authors wish to thank Professor P.

Langman for help with the manuscript, and E Yoshida for typing the manuscript.

NERVEALLOGRAFT

33

between Schwann cell function and extraceUular matrix production. Trends Neurosci. 6, 499-505. ABERCROMBIE,M. and JOHNSOn, M. L. (1942) The out- Bfh~6~sr,, O. V. (1891) Ober die Degenerations und Regenerations vor#inge am Nerven nach Verletzungen. Beitr. wandering of ceils in tissue culture of nerve undergoing path. Anat. 10, 321-387. Wallerian degeneration. J. exp. Biol. 19, 266-288. AFANmSlEFF,A. (1967) Premiers r6sultats de 20 homograffes Btm),mLL, S. and BovEs, J. H. (1937) Nerve grafts. A. J. Surg. 44, 64-75. de nerfs conserv6s par le Cilait (main et avan-bras). CAJAL, S. R. (1928) Degeneration and Regeneration of Presse m~d. 1409-1410. the Nervous System, pp. 236-238 and pp. 244-245. AEANASSlEFF,A. and RECnT, P. (1971) R6sultats de 44 (Facsimile of the 1928 edition.) Haler: London. homogreffes de nerfs conserves par le Cialit. Annls Chit. CALABRETTA,A. M., MUNOER, B. L. and GRAHAM,W. P. plast. 16, 284-287. (1973) The ultrastructure of degenerating rat sciatic AGUAYO,A. 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(1917) Die Oberbriickgrafts: Experimental studies in the dog and chimpanzee ung grol3er Nervenliicken. Beitrage zur Kenntnis der to define homograft limitations. J. Neurosurg. 32, Degeneration und Regeneration peripherischer Nerven. 236-243. J. Psychol. Neurol. 22, 267-318. DUEL, A. B. (1934) The operative treatment of facial palsy. BUCH, V. I. (1970) Experimental study of radiated vs fresh Br. reed. J. 2, 1027-1031. nerve homografts. Plastic reconstr. Surg. 45, 586-594. DYCK, P. J. and HOPKINS,A. P. (1972) Electron microscope BUCKO, C. D. and STEINMULLER,D. (1974) Reinnervation observations on degeneration and regeneration of unafter orthotopic grafting of peripheral nerve segments in myelinated fibers. Brain 95, 223-234. inbred rats. Plastic reconstr. Surg. 53, 326-331. DZIADEK, M. and TIMPL, R. (1985) Expression of nidogen BUNGE, R. P. and BUNGE, M. B. (1983) Interrelationship and laminin in basement membrane during mouse em-

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