A Comparative Study of Response of Species to Peripheral-Nerve Injury

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1 A Comparative Study of esponse of Species to eripheral-nerve njury. Crush and Severance with rimary Suture CATAN DAVD G. ~LNE, Me, SA,* COLONEL GEOGE J. HAYES, MC, SA, t AND CATAN ATH S. MOSE, MC, SA Department of Experimental Surgery, Walter eed Army nstitute of esearch, Washington, D. C. C OMESSON or crush of an otherwise intact peripheral nerve has been used extensively to study peripheralnerve regeneration. -,6,7,9'~ Likewise, many experimental studies have been directed toward the effect of suture repair on regeneration.,5,7,n As discussed in the previous paper, s comparative studies of response of species to peripheral-nerve injuries have not been published. With this in mind, neural crush and severance with primary suture repair were carried out in a series of dogs, monkeys, baboons, and chimpanzees. The method and results of each type of injury will be described separately. The principles of laboratory animal care as promulgated by the National Society for Medical esearch were observed throughout these studies. Crush njury Method. eroneal and radial nerves were used for this portion of the study. The dogs, rhesus monkeys, and baboons were anesthetized by intravenous pentobarbital and the chimpanzees by intramuscular Sernylan and intravenous pentobarbital. Each nerve was then exposed over a length of - cm. The peroneal nerves were exposed in the popliteal fossa and the radial nerves at the level of the middle third of the upper arm. The functional threshold of stimulation was determined as described in art.s An Ochsner clamp was used to crush the nerve for a ~0-sec. period. The nerve was flattened over a 0.5 cm. distance by this method (Fig. ). The operative wound was then closed with silk. The crushed nerves were re-exposed at intervals of hours,,, 6,, and weeks after operation. Thresholds of stimulation were redetermined eceived for publication December, 96. * resent address: Department of Neurosurgery, niversity of Michigan Medical Center, Ann Arbor, Michigan. t Address: Walter eed General Hospital, Washington, D. C. 90 and the specimens of nerve were removed and pinned on paraffin blocks. After fixation in 0 per cent buffered formalin, each specimen was divided into proximal (A), central (B) and distal (C) blocks and embedded in paraffin. Cross sections from the proximal blocks were made approximately 5 ram. proximal to the center of the crushed area whereas cross sections of the distal blocks were made, 7.5, or 0.0 ram. distal from the crushed area. The central blocks were sectioned longitudinally. Hematoxylin and eosin, Masson, Bodian, and Morgan's myelin stains were used. At least two crushed nerves from each species were studied at each interval of time. A total of 60 nerves was thus compared. Method of Counting. Axonal counts were carried out on selected 5-micron cross sections stained by a modified Bodian technique. Axons proximal (A) and distal (C) to the area of crush were counted by means of an ocular square grid composed of 00 small squares. Ten squares in each field were selected at random and counted at a magnification of 970X. The average of these counts was then multiplied by the number of squares containing axons in each field. The calculated axons in each field were then added to give the total axons present in each cross section. The results are tabulated in Table. The ratio of proximal (A) to distal (C) counts was used as an index of regeneration. Two cross sections were counted completely and compared with the calculated counts: er Counted Calcu- Differ- Cent lated ence Difference Baboon O0-- roximal (A) 6,69 6,~ 6. Baboon 00- Distal (C) 5,75 6, Determination of numbers of axons by a method of calculation thus appeared reliable. ndoubtedly the use of a higher magnification would have yielded higher counts since the finer axons (less than /z) were difficult to resolve even at a magnification of 970

2 eripheral-nerve njury.. Crush and Severance 9 esults--crush After application of an Ochsner clamp the nerve was flattened to a thinness of ribbon (Fig. ). The normal contour of the nerve was almost completely restored 5 min. after crush (Fig. ). This type of crush injury resulted in wrist or foot drop with subsequent atrophy. These changes were obvious in the chimpanzees and baboons but more difficult to discern in the rhesus monkeys and dogs. The animals followed for intervals extending to or weeks appeared to have some return of function. However, no attempt was made to quantitate these clinical changes. The gross appearance of the older specimens was that of a small fusiform neuroma in continuity surrounded by fine connective-tissue adhesions. Histologic study of specimens removed hours after crush injury showed that most of the crushed axons and myelin were pushed to either side of the site of compression. ed blood cells and some polymorphonuclear cells were scattered throughout the edematous area of crush. The epineurium, perineurium, and most of the neurolemmal sheaths remained intact and retained their longitudinal orientation. Thus, FG. (left). eroneal nerve of a dog immediately after crush injury by an Oehsner clamp. FG. ~ (right). Same nerve shown in Fig., 5 minutes after crush injury. Contour of the nerve has been restored spontaneously. the continuity of the supporting structures of the crushed nerve was not interrupted by this injury. Study of the specimens removed and weeks after crush revealed degenerating myelin, axonal debris, and macrophages in the central area of crush and throughout the distal stump (Fig. ). Axonal regeneration occurred rapidly and by weeks axons penetrated the area of crush (Fig. ). The axons were of fine caliber (-0 t~) and often branched but maintained a predominantly longitudinal orientation. FG. 9. Chimp 99. Crushed area of radial nerve ~ weeks after injury. Note presence of macrophages and early appearance of fine axons. Bodian, X 79.

3 9~ D. G. Kline, G. J. Hayes and A. S. Morse By 6 weeks, axons were present distal to the area of crush (Fig. ). ndeed, where distal cross sections were taken close enough to the injury, axonal counts were greater than those in the proximal cross section (see Chimp 99 in Table ). By weeks axonal penetration of the distal segment was well advanced (Table ). Thresholds of conduction usually were restored to pre-injury levels by weeks although axons in the area of crush were still of fine caliber. The ~-week specimens had minimal evidence of injury. Axonal caliber and degree of myelination in the distal segments were almost normal although still reduced in the central crushed area (Fig. 5). Comparison of species revealed that remyelination in the higher primates, and in particular the chimpanzee, lagged behind that of the dog and the rhesus monkey. Thus a thin layer of myelin was seen in the distal segments of the canine and rhesus monkey at 6 weeks but was not evident until weeks in the baboons and chimpanzees. The apparent delay in remyeliuation could be correlated with increased connective tissue both in the crushed areas and in the distal segments of the baboon and especially in the specimens of the chimpanzee. Of particular interest, distal tubular collapse and endoneurial proliferation were most marked in the chimpanzee. These distal tubular changes were similar to those seen in the severed nerves of the chimpanzee. However, in spite of the comparative increase in connective tissue, axonal regeneration and carry through to the distal segment of the crushed nerves were as concentrated in the chimpanzee as in the other species studied (Table ) rimary Suture ]ethod. eroneal and ulnar nerves were used for this portion of the study. The dogs, rhesus monkeys, and baboons were anesthetized by intravenous pentobarbital and the chimpanzees by intramuscular Sernylan and intravenous pentobarbital. Sharp dissection was used to expose each nerve over a -era. distance. The peroneal nerves were exposed in the popliteal space and the ulnar nerves in the distal third of the upper arm. The threshold of stimulation was then determined. Two lateral epineurial sutures of 6-0 silk were placed as shown in Fig. 6 and the nerve was FO.. Baboon 00. Longitudinal section of radial nerve distal to crush 6 weeks after injury. Note longitudinal orientation of the axons. Bodian, X 79.

4 eripheral-nerve njury.. Crush and Severance 9 TABLE Axonalcoun~--crushse~es Nerve roximal (A) Distal (C) A/C Distance of C from Crush (mm.) Volts ~Weeks Dog 6T6 hesus 7~5 Baboon 05 Chimp 99 (br.) 0,659 5,50 6,0 6,00 Weeks Dog 9eT hesus 60 Baboon 00 Chimp 99 Chimp 99 6,65 9,00 6,~,90,90 5,50 7,7 5,500 ~,06, ~.9 $.5 ~Weeks Dog S hesus 70 Baboou 009 Chimp 97 (br.) 6,75,65 5,, 5,5 6,55,975 6,6~ Weeks Dog 7 hesus 6~ Baboon 00 Chimp 5 (br.) 7,7, 0, 7,55 6,~7 9,~6 7,~0 5,7.~0...s ~.0.0 = peroneal. = radial. (br.)= branch of radial. = no conduction. Fo. 5. Chimp 5. Crushed area of radial nerve weeks after injury. Bodian, X 79.

5 9 D. G. Kline, G. J. Hayes and A. S. Morse severed b e t w e e n t h e m. T h e lateral sutures were t h e n tied a n d several epineurial sutures were placed anteriorly and posteriorly. r e p l a c e m e n t of t h e lateral sutures minimized i n s t r u m e n t a l handling of t h e nerve, increased t h e chance of gaining proper fascicular alignment, a n d to a limited e x t e n t served to s t a n d a r d i z e t h e repair. W o u n d s were closed with -0 silk. T h e limbs were not immobilized. T h e animals were followed for intervals ranging from to weeks after operation and were t h e n anesthetized. T h e s u t u r e d nerves were exposed, restimulated, a n d removed. Histologic processing of each specimen was carried out as described u n d e r Severance s a n d Crush. E a c h specimen was graded for a n u m b e r of gross a n d histologic characteristics as shown in Table e. T h e m e t h o d for grading was as described in a r t. s Selected proximal and distal cross sections t a k e n a m e a s u r e d distance from t h e area of repair FG. 6. Suture model. eroneal nerve of chimpanzee with lateral epineurial sutures in place. Nerve was then severed between the lateral sutures. The sutures were tied and several anterior and posterior epineurial sutures were placed to complete the primary repair. TABLE Suture series Dog S S Weeks Gross neuroma Disorganization Carry through emyelination Connective tissue Distal collapse W59 7Y Weeks Gross neuroma Disorganization Carry through emyelination Connective tissue Distal collapse Weeks Gross neuroma Disorganization Carry through emyelination Connective tissue Distal collapse hesus 7S 7S ~ Baboon t S $ Graded on a 0 (least) to (most) basis. See. Severance for method of grading. = peroneal nerve. = radial nerve. = ulnar nerve. = no conduction S+ s.5 90 Chimp 0

6 eripheral-nerve njury.. Crush and Severance 95 FG. 7. Chimpanzee 97. Area of repair of peroneal nerve weeks after suture. Note proliferation of connective tissue and disorganization in the central (suture line) area. Masson, )< 7. were counted for axons. The method of counting was as described under Crush. esults--rimary Suture r i m a r y suture as executed in this study resulted in neuromas in continuity of variable size (Fig. 7 and Table ~). The classic picture of central (suture line) axonal disorganization interspersed with increased numbers of Schwann cells and fibroblasts and a proliferation of connective tissue predominated (Fig. ). As noted in the severed series of nerves, gross estimates of neuroma did not necessarily correspond with the amount of disorganization seen histologically. Study of specimens removed week after primary suture disclosed retrograde axonal fragmentation and myelin degeneration for FG.. Area of repair shown in Fig. 7. Graded for axonal disorganization. Bodian, X 96. (See Table.)

7 96 D. G. Kline, G. J. Hayes and A. S. Morse TABLE Axonal counts--suture series Distance of C Nerve roximal (A) Distal (C) A/C from Crush (ram.) Volts ~Weeks Dog 9Y hesus 597 Baboon 0 Chimp 9,50 7,7,5 0,7 Weeks Dog A59 hesus 60 Baboon 00 Chimp,9,679,50 6,59,65 7,50, 7, B Weeks Dog S~ hesus 57 Baboon 00 Chimp 97 5,0,70 5, 9,6 7,00 5,5 9,055, Weeks Dog 7Y hesus 66 Baboon 005 Chimp 9O 9,06,9 6,70 5,06 9,0,07,0 6, = peroneal. = ulnar. = no conduction. a short distance ( cm.) up the proximal stump with more extensive degenerative changes in the distal stump. Fibroblasts and Schwann cells were most concentrated in the central area of repair but were also present throughout the distal segment. Epineurial and perineurial thickening with connective-tissue proliferation at the site of repair was seen by ~ weeks. Axons of fine caliber began to penetrate the area of repair by ~ weeks in the dogs and rhesus monkeys and were well advanced into the distal stump of these species by 6 weeks (Table ). Axonal penetration of the area of repair at ~ weeks and axonal concentration in the distal stump at 6 weeks did not appear as concentrated in the sutured nerves of baboon and chimpanzee as in the specimens of canine and rhesus monkey. Axonal counts, made mm. distal to the area of repair and compared with proximal-stump counts, confirmed these impressions. A thin layer of myelin could be seen around most of the axons of the distal stump in the 6-week specimens of canine and rhesus monkey and many of the 6-week specimens of the baboon. Distal-stump remyelinafion, however, was not apparent in the 6-week specimens of the chimpanzee. Table summarizes the grades for multiple histologic characteristics evaluated in the -, -, and ~-week series of nerves. Although the basic pattern of repair was similar in all four species, connective-tissue proliferation and distal tubular collapse were most pronounced in the chimpanzee. Disorganization with branching, wandering axons in the area of repair was also a more distinctive feature in the higher primates than in the dogs and rhesus monkeys. emyelinafion of the distal segment of the sutured nerves of the chimpanzee lagged behind that of the other species as did restoration of a functional threshold of stimulation. Thresholds of stim-

8 eripheral-nerve njury. ulation, although present, were elevated at ~ weeks in the chimpanzee. n spite of the apparent differences of species in the pattern of regeneration axonal counts made mm. distal to the area of repair at weeks and 0.0 nm. distal to the area of repair at weeks did not differ significantly among the four species. Discussion Although the nerves of the chimpanzee responded to a crush injury with more connective tissue, less remyelination, and a slower restoration of functional conduction, axonal disorganization in the area of repair did not appear to be increased. ndeed, the ratio of proximal-stump to distal-stump axons (A/C) did not differ significantly among the species studied. These findings were not completely unexpected since the crush injury resulted in minimal distortion of the longitudinal connective-tissue framework. Thus, proliferation of the neurolemmal cells and axonal regeneration occurred in an organized fashion following the relatively intact connective-tissue framework. t is of interest to compare these findings with those seen in the severance and the severance and sutured series where the continuity of the supportive structures was interrupted and axonal regeneration occurred in a more disorganized fashion. Here, early (6 weeks) axonal penetration of the distal stump of the sutured nerve was slower in the chimpanzee and the baboon than in the dog and the rhesus monkey. Thus the ratio of proximal (A) to distal (C) counts was greater in the chimpanzee and baboon than in the dog and monkey. These observations are not original and support the concepts of Weiss and others, who have shown that a structured framework is not only necessary for axonal regeneration but is also a determinant of the direction regenerating axons take. Differences of species in the pattern of peripheral-nerve regeneration were present with all three types of injuries but were most apparent in the severance and the severance with suture series. These injuries interrupted. Crush and Severance 97 the continuity of the connective-tissue framework of the nerve and thus the path of regeneration was not predetermined. njuries which interrupt completely the continuity of a peripheral nerve arc common to man, and continue to present a challenge to those faced with their repair. Methods designed to repair this type of injury frequently have appeared promising when used on laboratory animals but have failed when tried on humannerve injuries. The findings described above indicate that there is a difference of species in the response of peripheral nerves to injury. Thus experimental methods of peripheralnerve repair which are successful in the lower order of mammals could fail when attempted on a higher order of mammals. The chimpanzee or baboon would thus seem to be a more reliable animal for testing various techniques of peripheral-nerve repair before attempting them on the human. ndoubtedly, further differences of species not elicited by the methods used in these studies exist and would seem to merit further investigation. Summary Multiple peripheral nerves in a series of dogs, rhesus monkeys, baboons, and chimpanzees were injured by crush or by severance with primary suture repair. Differences of species in response to a crush injury were minimal and consisted of a comparative increase in connective tissue and slower distal tubular remyelination in the chimpanzee than in the other species. Axonal counts distal to the crush injury, however, did not vary significantly among the species studied. Differences of species in response to severance and suture were more apparent than in the crush series. Connective-tissue proliferation and axonal disorganization in the area of repair were most marked in the chimpanzee. emyelination of the distal segment and restoration of a functional threshold of conduction in the sutured nerves of the chimpanzee also lagged behind that of the other species. Axonal counts of the proximal and distal stumps were compared. Axonal counts of the distal stumps of specimens removed 6

9 9 D. G. Kline, G. J. Hayes and A. S. Morse weeks after severance and suture were most concentrated in the dog and the rhesus monkey. Comparison of the axonal counts in the distal stump made at and weeks, however, showed no differences of species. Since differences of species in response to peripheral-nerve injury exist, it may be of value to test new techniques of peripheralnerve repair on higher primates before attempting them on the human. We wish to thank Dr. Walle J. Nauta and Dr. B. K. Chun for their valuable advice and Spec. Marvin Spangler and fc. Duane Munger for their technical assistance. eferences. BAcsc~,., and WYVN, G.M. The vascular pattern of peripheral nerve during repair after experimental crush injury. J. Anat., Lond., 95, 79: 9-.. BATON, A.A. An electron microscope study of degeneration and regeneration of nerve. Brain, 96, 5: CAGG, B. G., and THOMAS,. K. Changes in conduction velocity and fibre size proximal to peripheral nerve lesions. J. hysiol., 96, 57: DAVENOT, H. A., CHO, H., and DOLKAT,. E. The ratio of myelinated to unmyelinated fibers in regenerated sciatic nerves of macacus rhesus. J. comp. Neurol., 97, 67: GTH, L. egeneration in the mammalian peripheral nervous system. hysiol. ev., 956, 6: GTMANN, E., GTTMANN, L., MEDAWA,. B. and YONG, J. Z. The rate of regeneration of nerve. J. exp. Biol., 9, 9: GTMANN, E., and SANDES, F. K. ecovery of fibre numbers and diameters in the regeneration of peripheral nerves. J. hysiol., 9, 0: KLNE, D. G., HAYES, G. J., and MOSE, A.S. A comparative study of response of species to peripheral-nerve injury.. Severance. J. Neurosurg., 96, : AMON Y CAJAL, S. Degeneration and regeneration of the nervous system.. M. May, Transl. London: Oxford niv. ress, 9, : SANDES, F. K. The thickness of the myelin sheaths of normal and regenerating peripheral nerve fibers. roc. roy. Soc., 9, s.b. 5: WESS,. The technology of nerve regeneration: a review. Sutureless tubulation and related methods of nerve repair. J. Neurosurg., 9, : WESS,. Nervous system (neurogenesis). n: Analysis of development. B. H. Willicr,. A. Weiss, and V. Hamburger, Ed. hiladelphia: W. B. Saunders Co., 955, xii, 75 pp. (see pp. 6-0).. WEiss,., and HSCOE, H.B. Experiments on the mechanism of nerve growth. J. exp. Zool., 9,07: 5-95.