Recollections of research in spinal cord regeneration

EXPERIMENTAL

NEUROLOGY

Recollections

71, l-5 (1981)

of Research WILLIAM

Brain

Research

in Spinal F.

Cord Regeneration1

WINDLE

Institute, Center for the Health Sciences, University Angeles 90024 and Denison University, Granville, Ohio Received

July

of California, 43023

Los

17, 1980

My interest in regeneration in the spinal cord arose 55 years ago when I was introduced to Theodore Koppanyi by our Professor of Physiology at Northwestern University Medical School. Koppanyi had done some research in Europe with Paul Weiss on central nervous regeneration in lower vertebrates and when he came to America, A. J. Carlson welcomed him into the Physiology Department of the University of Chicago. There he and Ralph Gerard planned to try to find out if regeneration could occur in the immature mammalian spinal cord as it did in the cord of animals lower in the phylogenic scale. They chose to sever the spinal cord of rat fetuses in utero and examine the offspring when they were born several days later. Ralph went to Europe soon after the experiments were started, and while he was away Koppanyi asked me to examine the first group of newborn rats and do the histopathology on those that had recovered function after their cords were severed in utero. He gave me several fruit jars of young rats in Formalin. Unfortunately, only two or three of the animals had knife cuts on the back and only one had a nick in its spinal cord. (It was naively believed that the knife wounds had healed with extraordinary rapidity in others.) I prepared a report and when Ralph returned from abroad he apologized for putting me to the trouble of working with such poor material, saying that the experiments had only started and had not been carefully controlled. He and Koppanyi went on with the work and in 1926 they reported (7) some 1 This paper was presented as the keynote address at the Fifth Biennial Conference on Regeneration in the Central Nervous System sponsored by the National Spinal Cord Injury Foundation and the Society for Neuroscience May 7-9, 1979. Supported by National Institutes of Health grant NS 15349-01 and a grant from the donor of the Wakeman Award.

0014-4886/81/010001-05$02.00/O Copyright 0 1981 by Academic Press. Inc. All tight of reproduction in any form reserved.

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WILLIAMF.

WINDLE

success on physiological grounds, but without histological verification of regeneration across the lesions of fetal and newborn rats. Davenport Hooker and John Nicholas at Yale promptly challenged (9) the findings of Gerard and Koppanyi. They interrupted continuity in the fetal rat spinal cord, mainly by coagulation through the uterus and fetal back with a cautery. After the rats were born the investigators saw hind limb movements that were coordinated with those of the forelimbs: these they believed were due to transmission by pressure stimuli rather than by conduction over spinal neurons. The technique produced large lesions. Most of the animals were killed soon after physiological testing, and the histological preparations, of course, revealed no regeneration in the damaged spinal cords. The negative results of Hooker and Nicholas were readily accepted as supporting the prevailing view that no regeneration takes place in the mammalian cord. I was inclined to share that view because of my uninspiring experience with the rats of theuniversity of Chicago investigators. Moreover, the findings of Oscar Sugar and Ralph Gerard (13) in 1940 did not spark my interest in regeneration. In the light of present knowledge, that study has assumed major importance, for it was the first to demonstrate that functional as well as structural regeneration can be made to occur in the spinal cord of young mammals. Would that I had had the foresight to appreciate the meaning of their findings at that time! But even Ralph Gerard gave the work scant consideration (only 11 lines) in his autobiography in The Neurosciences: Path of Discovery (6) shortly before he died. The experiments of Sugar and Gerard are remarkable not because they were performed on immature animals, but because they followed a suggestion of Ramon y Cajal30 years earlier (12) that implanting pieces of peripheral nerve into wounds of the brain and optic nerve might give the otherwise aborting sprouts of the central nerve fibers cordons of neurolemma cells that could produce a neurotropic substance and provide paths along which to continue growing. Where other investigators in Madrid, notably Tello (14), had failed to hold the nerve grafts effectively in place in the optic nerves, the Chicago scientists succeeded with their meticulous surgical technique. Some recent reports from the laboratories of Blakemore (2), Kao (lo), and Mei Liu (11) support the view that neurolemma-like cells can not only provide guidance, but secrete a growth factor that may enhance the process of regeneration. But I am way ahead of my story. Sugar and Gerard’s startling report received no enthusiastic acceptance by their contemporaries. Other groups published satisfying accounts of failure to obtain regeneration after transecting the mammalian spinal cord (1, 3, 4). The view of non-

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regeneratability of the severed cord had become dogma, accepted by neurologists, and reports of the occasional successful experiment were greeted by assumption that the operators had failed to transect the cord completely, or that they did not recognize spinal stepping. There the issue stood at midcentury. And that was the time I became actively involved in research on regeneration in the spinal cord. We had a government contract at the Institute of Neurology at Northwestern during the Second World War to study reactions of the spinal cord to injury. We investigated spinal cord concussion and effects of high-velocity missile wounds of the back, but we turned to other interests when the war ended and the contract was terminated. In 1947 we took with us to the University of Pennsylvania a project on central regulation of body temperature, which required transection of the neuraxis of cats and dogs at different levels including the spinal cord. One day William Chambers noticed that a dog whose cord had been severed weeks previously began to cry out each time its abdomen was compressed to evacuate the urinary bladder. Stimulation of the bladder wall after opening the abdomen produced the same pain response. The spinal cord was removed at necropsy and prepared by silver staining for histological examination. Regenerating nerve fibers, believed to be of central origin, were seen crossing the cut. Other spinal animals in the series that had been given the drug Piromen for several weeks also had regenerating fibers at the site of transection of the spinal cord. Chambers and I, joined by Carmine Clemente, Donald Scott, and Chan-nao Liu, began in earnest to investigate the phenomenon. I gave an informal report of our findings in Chicago in 1949 at Paul Weiss’s Conference on Genetic Neurology (15). The first formal presentation was at a joint meeting of Philadelphia and New York Neurological Societies (18). A representative of the Philadelphia Bulletin was present and his syndicated report created considerable public interest. Some neurologists, however, did not share our enthusiasm. I recall that at a later meeting in Atlantic City, Donald Munro of Boston went to the podium to discuss a paper of ours and requested that the authors add the words “in animals” to the title because, he said, we all know that it never occurs in man. Several years later, after I had joined the National Institute of Neurological Diseases and Blindness, the Director, Pearce Bailey, suggested that we organize a conference on Regeneration in the Central Nervous System. We did, and the proceedings of it appeared in 1955 (16). I prepared an invitational list of about three dozen people known to have had some- even remote- interest in the subject. Most of them accepted. Among the few who could not attend the Conference were LeGros Clark

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WILLIAM

F. WINDLE

and Francisco Tello, the latter because of declining health. Pearce Bailey, in welcoming the conferees, emphasized the importance of research on problems of damage in the central nervous system and called attention to the rising survival rate among victims of spinal cord injuries. The 1954 Conference on Regeneration in the Central Nervous System adjourned without having brought forth any new and striking discoveries, but it did publicize the importance of the field and the need to increase research efforts. Nevertheless, I am sure that few of those who attended left with an urge to use any of their precious time in research in this unpopular field. Within 5 years only four or five of the conferees were actively engaged in research in central nervous regeneration. Unknown to us, however, several Russian scientists found the proceedings of our conference stimulating. Academician L. A. Orbeli was impressed by the research with Piromen and arranged for manufacture of a similar pyrogenic substance in Moscow. He had our book translated and published in 1959, and thereafter, several articles describing research with the Russian drug, Pyrogenal, appeared. Pessimism prevailed on this side of the world. It seemed to me that more effort was spent trying to devise ways to prove that regeneration cannot occur in the damaged spinal cord than to seek basic information that could explain why it is that the process takes place so readily in lower vertebrates and not in mammals. The next conference was that organized by Jack French in 1960 on Basic Research in Paraplegia (5). In concluding my presentation, I declared belief that answers would not be found in hospital wards, but in laboratories of the basic sciences. Ralph Gerard, sensing my discouragment, admonished me not to lose hope and not to retreat. Again, but 9 years later, at the Seventeenth V.A. Conference on Spinal Cord Injury in New York, I spoke on the current status of the problem of regeneration of the spinal cord (17). Judging from the discussion of my paper, few of the listeners knew what I was talking about. William Geisler of Toronto was the only one who rose to the occasion to urge those with any influence to ask governments of their countries to increase their support of basic research in spinal cord regeneration. The outlook of the paraplegic was as grim as ever, and his physician cautioned against raising a false hope that it would improve. Great advances in rehabilitation medicine offered the alternative. Soon after the New York conference, I heard from Alan Reich, President of the National Paraplegia Foundation, who suggested that another conference on central nervous system regeneration be organized. I told him that I could not be very enthusiastic because the previous conferences had been so fruitless, but I finally agreed to help. The conference at The

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Breakers in Palm Beach in 1970 was a great success (8), as all of you know. And now, as we begin the fifth conference in the series sponsored by the National Paraplegia Foundation (now the National Spinal Cord Injury Foundation), my hopes are high. We have certainly learned ways to prevent total and permanent paraplegia from some injuries of the spinal cord. Whether we will ever be able to reconstruct the cord that has been severed remains to be seen, but, if we do, it will be because of knowledge acquired in the laboratories of medical scientists with animal subjects. REFERENCES 1. BARNARD, J. W., AND M. A. CARPENTER. 1950. Lack of regeneration in the spinal cord of the rat. J. Neurophysiol. 13: 223-228. 2. BLAKEMORE, W. F. 1977. Remyelination of CNS axons by Schwann cells transplanted from the sciatic nerve. Nature (London) 226: 68-69. 3. BROWN, J. O., AND G. P. MCCOUCH. 1947. Abortive regeneration in the transected spinal cord. J. Camp. Neural. 87: 131-137. 4. FEIGIN, I., E. H. GELLER, AND A. WOLF. 1951. Absence of regeneration in the spinal cord of the young rat. J. Neuropathol. Exp. Neural. 10: 420-42.5. 5. FRENCH, J. D., AND R. W. PORTER(Eds.). 1962. Basic Research on Paraplegia. Univ. of California Press, Los Angeles. 6. GERARD, R. W. 1975. The minute experiment in the large picture. Pages 457-474 in F. G. WORDEN et al., Eds., The Neurosciences: Path ofDiscovery. MIT Press, Cambridge. 7. GERARD, R. W., AND T. KOPPANYI. 1926. Studies on spinal cord regeneration in the rat. Am. J. Physiol. 76: 211-212. 8. GUTH, L., AND W. F. WINDLE. 1970. The enigma of central nervous regeneration. Exp. Neurol. 28(Suppl. 5): l-43. 9. HOOKER, D., AND J. S. NICHOLAS. 1930. Spinal cord section in rat fetuses. J. Comp. Neurol. 50: 413-467. 10. KAO, C. C., L. W. CHANG, AND J. M. B. BLOODWORTH, JR. 1977. Axonal regeneration across transected mammalian spinal cords: an electron microscopic study of delayed nerve grafting. Exp. Neurol. 54: 591-615. 11. LIU, H. M., E. S. BALKOVIC, M. F. SHEFF, AND S. I. ZACKS. 1979. Productionin vitro ofa neurotropic substance from proliferative neurolemma-like cells. Exp. Neural. 64: 271-283. 12. RAM~N y CAJAL, S. 1914. Degeneration y Regeneration de1 Sistema Nervioso. Nicolas Moya, Madrid. 13. SUGAR, O., AND R. W. GERARD. 1940. Spinal cord regeneration in the rat. J. Neurophysiol. 3: l-19. 14. TELLO, F. 1911. La influencia de1 neurotropismo en la regeneration de 10s centros nerviosos. Trab. Lab. Invest. Univ. Madrid. 9: 123-159. 15. WEISS. P. (Ed.). 1950. Genetic Neurology Univ. of Chicago Press, Chicago. 16. WINDLE, W. F. (Ed.). 1955. Regeneration in the Central Nervous System. Thomas, Springfield, Ill. 17. WINDLE, W. F. 1969. Regeneration in the spinal cord: current status of the problem. Proceedings

of the 17th Veterans

Administration

Spinal

Cord Injury

Conference,

New

York. 18. WINDLE, W. F., AND W. W. CHAMBERS. 1951. Regeneration in the spinal cord of the cat and dog. AMA Arch. Neural. Psychiat. 65: 261-262.