Leonard T. Yu, John England, Austin Sumner, Don Larossa, and William E Hickey
ELECTROPHYSIOLOGIC EVALUATION OF PERIPHERAL NERVE REGENERATION THROUGH Downloaded by: University of British Columbia. Copyrighted material.
ALLOGRAFTS IMMUNOSUPPRESSED WITH CYCLOSPORIN ABSTRACT A model was designed to evaluate the long-term in vivo electrophysiology of rat peripheral nerve transplants. The application of this model was demonstrated using cyclosporin (CSA) immunosuppression of recipient animals to facilitate peripheral nerve regeneration through nerve allografts. Isogenic Brown Norway (BN) rats [ RT1n ] were divided into three groups: two received Lewis (LE) rat [ RT1 • | allografts and one received BN isografts. One allograft recipient group received CSA immunosuppression for the duration of the investigation (150 days). Successful nerve regeneration in the isograft and the immunosuppressed allograft recipient groups was determined by immunohistochemical methods and serial in vivo electrophysiologic techniques to measure nerve conduction velocity and evoked compound muscle action potential amplitude. Statistical analysis of these results indicate that: (a) CSA immunosuppression of peripheral nerve allograft recipients facilitates peripheral nerve regeneration which is indistinguishable from isograft recipient controls at the functioning axon level; and (b) in vivo electrophysiologic monitoring in this model is particularly useful for long term peripheral nerve transplantation studies permitting serial assessment of regeneration with little morbidity.
Interest in mammalian peripheral nerve transplantation has been stimulated by recent advances in available immunosuppressiveagents. Traditionally, rat sciatic and peroneal nerves have been used for nerve allograft investigations. Although these nerves are extremely useful for studies of peripheral nerve repair, they have several disadvantages when adapted for nerve transplantation. In particular, there are few accurate methods to assess functional nerve regeneration quantitatively along these pathways. We present an alternative rat model designed specifically for peripheral nerve transplantation, which permits frequent and
reliable evaluation of nerve electrophysiology and demonstrates its application for peripheral nerve allografts, using long-term cyclosporin (CSA) immunosuppression.
MATERIALS AND METHODS EXPERIMENTAL GROUPS. Three groups of isogenic Brown Norway (BN) rats served as recipient animals. All animals (Charles River Laboratories, Kingston, NY)
Divisions of Plastic Surgery and Neurology, Hospital of the University of Pennsylvania, Philadelphia, PA, and Division of Neuropathology, Washington University School of Medicine, St. Louis, MO Materials in this paper were presented at the 33rd Annual Meeting of the Plastic Surgery Research Council, San Francisco, CA, May, 1988 Reprint requests: Dr. Larossa, Division of Plastic Surgery, Dept. of Surgery, Hospital of the University of Pennsylvania, 3400 Spruce St., Philadelphia, PA 19104 Accepted for publication April 17, 1990 Copyright © 1990 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
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were eight to 10 weeks old and weighed between 200 and 250 g at surgery. Group A (n = 12) received an isograft from BN donors. Group B (n = 8) received an allograft from inbred Lewis (LE) donors. Group C (n = 12) received an LE allograft and long term postoperative CSA immunosuppression. CSA (Sandoz Pharmaceuticals Corporation, E. Hanover, NJ) was administered subcutaneously at a daily dose of 15 mg/kg in olive oil for an induction period of 20 days and then biweekly for an additional 130 days. Animals were caged in pairs, fed a diet of standard laboratory chow and water, and were allowed to move freely after surgery. OPERATIVE PROCEDURE. The rat sciatic nerve is a Figure 1. Diagrammatic representation of graft recipmixed motor-sensory nerve. After emerging from the ient demonstrating site of proximal anastamosis in the thigh sciatic notch, it gives rise to a minor sensory branch in and distal anatamosis at the ankle. Sural nerve resection is the proximal thigh and then divides distally into three performed via the proximal incision (see text). major branches at the level of the knee (peroneal, sural, and tibial nerves). The entire length of the sciatic nerve, approximately 2.5 to 3 cm in a 250 g rat, can be and the proximal and distal sites of nerve coaptation. rapidly exposed through a dorsal thigh incision. The Nerve repair was performed using standard end-tosciatic nerve and the proximal portions of all three end epineural microsuture technique with 10-0 nylon. major branches are easily accessible through this inciBoth incisions were closed in single layers using a skin sion. The tibial nerve is the largest of these branches stapler. Elapsed time for recipient preparation and and travels deep to the gastrosoleus muscles of the both nerve repairs averaged approximately 20 min per calf. It gives rise to the plantar nerves at the ankle animal. which innervate the intrinsic muscles in the foot. A ELECTROPHYSIOLOGY. Nerve regeneration was evalsurgical approach to the distal tibial nerve and its uated by monthly in vivo electrophysiologic studies for proximal plantar branches requires a second incision five months using the method of Fullerton.1 Percutaneon the lateral aspect of the lower leg anterior to the ous platinum needle electrodes (Type E2, Grass InstruAchilles tendon. ment Co., Quincy, MA) were used for both stimulating Donor Harvest. A 4.50-cm. sciatic-tibial nerve graft and recording purposes. The proximal pair of stimulatwas harvested from a 250 g donor rat between the ing electrodes was positioned adjacent to the sciatic sciatic notch and the ankle. Peroneal and sural nerve nerve at the sciatic notch. The distal pair was posibranches were divided close to their takeoff from the tioned adjacent to the posterior tibial nerve at the main sciatic trunk, and the nerve was skeletonized ankle. from its adjacent investing tissue. Square wave stimulus pulses of 0.1 msec duration Recipient Preparation. Following adequate anesthewere delivered, and compound muscle action potensia (Nembutal 50 mg/kg i.p.) and using sterile techtials (CMAPs) were recorded, using an active electrode nique, a muscle-splitting incision was made through placed subcutaneously on the plantar surface of the the dorsal thigh to expose the sciatic nerve and its foot and an indifferent electrode inserted into the soft trifurcation. The tibial nerve was transected sharply tissue of the second digit. All CMAPs were obtained by with a Week blade and resected distally for approxsupramaximal nerve stimulation and recorded by a imately 1 to 1.5 cm. A segment of sural nerve (1.0 cm) TECA model TD-20 nerve stimulator (TECA Corporawas also resected at this level. Through the second tion, Pleasantville, NY). Permanent records were made incision at the ankle, the distal tibial/plantar nerve was of all evoked responses. The CMAP latency was measexposed anterior to the Achilles tendon and divided in ured from the stimulus artifact to the beginning of the similar fashion. A modified tendon retriever was passed first negative deflection and the amplitude from baseretrograde through this incision along the course of line to negative peak. The distance between the two the tibial nerve deep to the gastrosoleus muscles. In sites of stimulation was measured along the skin surthe correct plane, it would slide without resistance up face with the leg fully extended and a segmental nerve to the thigh. The proximal end of the nerve graft was conduction velocity (NCV) was calculated. This techplaced in the jaws of the tendon retriever and withnique permitted separate electrophysiologic measuredrawn into the newly created "tibial-tunnel" with the ments through the graft and distal native plantar knee in full extension. This permitted the graft to lie nerve. Limb temperature was carefully maintained bewithout tension or redundancy along the natural course tween 37 to 38°C by a thermistor probe inserted subcuof the native tibial nerve. taneously and a thermostatically controlled infrared Figure 1 illustrates the final position of the graft heat lamp.
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JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 6, NUMBER 4
ELECTROPHYSIOLOGIC EVALUATION OF ALLOGRAFTS/YU, ENGLAND, SUMNER, ET AL. HISTOLOGY. One animal from each group was sacrificed at eight 15, 30, 80, and 120 days postoperatively to allow harvest of the graft and histologic examination of longitudinal sections by light microscopy with hematoxylin and eosin stains. At the conclusion of the study, all remaining animals were sacrificed. Cryostat sections of the nerve grafts were examined by immunohistochemical methods to determine axon viability.2 Immunoperoxidase-positive axons were identified by light microscopic examination.
Postoperatively, no signs of wound infection, autocannibalism, or plantar ulceration were observed. Animals that received allografts without immunosuppression (Group B) demonstrated early histologic evidence of intense cellular graft rejection, followed by necrosis and scar formation within the nerve grafts (Fig. 2A). No animals in this group regained any electrophysiologic function during the study. Terminal immunohistochemical stains revealed an absence of viable axons in the body of these allografts and distal plantar nerves, indicating graft rejection and failure of functional axonal regeneration. Animals that received isografts (Group A) and immunosuppressed allografts (Group C) (Fig. 2B) demonstrated the classic histologic appearance of early postoperative Wallerian degeneration, followed by orderly regeneration of nerve fibers through the grafts and native distal nerves (Fig. 3). No signs of graft rejection could be detected by light microscopy in the immunosuppressed allograft recipients, and graft sections appeared histologically indistinguishable between these groups. Serial electrophysiologic studies demonstrated identical patterns of evoked CMAP organization in the
Figure 3. Immunosuppressed allograft segment 120 days post-transplantation. Note orderly regeneration of axons with minimal inflammation (H&E, xlOO). Downloaded by: University of British Columbia. Copyrighted material.
RESULTS
isograft and immunosuppressed allograft recipients. Small amplitude responses were detected as early as 60 days after transplantation. Uniform action potentials were evident by 120 days postgrafting. Resolution of differential temporal dispersion paralleled a rise in CMAP amplitude for each responding animal (Fig. 4). Mean values of CMAP amplitude increased in both groups until termination of the study (Fig. 5A). The rate of increase appeared more rapid in the cyclosporin-treated group, although a significant difference (p < 0.01 by t-test) was evident only at 90 days postgrafting. This disparity resolved by 120 days, and both groups produced statistically similar values for the remainder of the study. At 150 days after transplantation (Table 1), distal CMAP amplitude for Group C (allograft + CSA) averaged 4.8 mV (SEM 0.76), while Group A (isograft control) averaged 4.3 mV (SEM 0.61). Monthly, in vivo measurements of CMAPs were continued in the isograft group after 150 days to determine maximal amplitude return. A plateau level of 5.4 mV
•- •
. .B
Figure 2. A, Nonimmunosuppressed allograft segment 15 days post-transplantation. Note intense cellular inflammatory response during allograft rejection. B, Immunosuppressed allograft segment 15 days post-transplantation. Note typical Wallerian degeneration within allograft (H&E, xlOO).
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EVOKED MUSCLE ACTION POTENTIAL (Allograft + CSA)
DISTAL RESPONSE
k
PROXIMAL RESPONSE
• ISOGRAFT (N=7) A ALLOGRAFT + CSA (N=7) A ALLOGRAFT (N=5)
150
120
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DAYS POST-TRANSPLANTATION /u
1
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10
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.
1
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320
60
90
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120
150
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DAYS POST-TRANSPLANT
Figure 5. A, Postoperative mean compound muscle action potential (CMAP) amplitude from distal stimulus. B, Postoperative mean values for nerve conduction velocity (NCV). Reference values recorded from non-operated age matched controls. Error bars denote standard error of the mean |SEM|.
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A
Figure 4. Representative interval evoked compound muscle action potentials recorded from an immunosuppressed allograft recipient. Note progressive organization of response curve during nerve regeneration. Arrows indicate stimulus artifact.
ELECTROPHYSIOLOGIC EVALUATION OF ALLOGRAFTS/YU, ENGLAND, SUMNER, ET AL.
(A) Isograft (B) Allograft (C) Allograft + CSA
CMAP*
(SEM)t
NCV*
(SEM)
4.3 mV 0 4.8 mV
(0.61)
35.0 m/s 0 37.6 m/s
(2.7)
(0.76)
(2.7)
*Mean value of distal compound muscle action potential amplitudes. tStandard error of the means. *Mean value of nerve conduction velocities.
(SEM 0.65) was reached at 180 days and persisted up to 240 days after transplantation, when all animals in this group were sacrificed. Nerve conduction velocity in the conducting groups (Fig. 5B, Table 1) reached peak levels by 90 days and remained relatively constant for the remainder of the study (Group A 35.0 m/sec |SEM 2.7], Group C 37.6 m/sec |SEM 2.7]). Repeated measures tests were applied to the mean values of CMAP amplitude and NCV for both groups over the course of the study. No significant differences between the control isograft or immunosuppressed allograft recipient groups could be demonstrated. Each group displayed a significant difference from nonimmunosuppressed allograft recipients (p < 0.005).
At the conclusion of the study, motor and sensory nerve fibers were identified by neurofilament immunohistochemical stains in all grafts. However, significant numbers of axons in an organized fascicular pattern were evident in only the isograft and immunosuppressed allograft recipients. Although scattered axons were observed in the epineurium of all three groups, no evidence of fascicular axonal regeneration was seen in the nonimmunosuppressed allograft animals (Fig. 6).
DISCUSSION The disadvantages of tension at the primary repair of a peripheral nerve have demonstrated the need for nerve grafts to bridge large nerve gaps.3 However, the limited availability of donor autologous nerves has restricted the optimal reconstruction of these lesions by autografts. Although conduits of autologous 4 - 6 and synthetic7-9 material have been used to promote nerve regeneration, their clinical use has been confined to distal and relatively short nerve deficits.10 " Fora large proximal nerve gap, animal studies suggest that a conduit will not promote the same quality of peripheral nerve regeneration as a nerve autograft.12 Investigations of allogeneic peripheral nerve grafts,
I
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Table 1. In Vivo Electrophysiologic Values 150 Days Post-transplantation
•'•'o'«i .'it ' * . ' :
• * ,
'
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. ' *
*
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, '' '*
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Figure 6. Terminal stains for axonal viability 150 days post-transplantation. A, Isograft. B, Immunosuppressed allograft. C, Nonimmunosuppressed allograft. Note similarity of isograft and immunosuppressed allograft segments vs. absence of viable axons in rejected, nonimmunosuppressed allograft (neurofilament immunoperoxidase stain, X200).
'
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as an alternative to autografts, followed successful allotransplantation of other organs using recipient immunosuppression to prevent graft rejection. Hydrocortisone, 1314 azathioprine, 1314 and cyclosporin1516 have prevented peripheral nerve allograft rejection and promoted nerve regeneration. Zawleski and Gulati 1517 examined the survival of long (4 cm) peripheral nerve allografts transplanted across multiple minor histocompatability barriers in rats using CSA immunosuppression. Their model did not use a distal nerve repair to bridge a gap in continuity and although they were able to provide histologic evidence of nerve regeneration through the allografts, they did not evaluate functional return. Bain et a!.16 demonstrated nerve regeneration through 3-cm nerve allografts across an MHC barrier in rats immunosuppressed with CSA. Regeneration was documented by histologic examination and serial functional assessment using gait analysis. These values were comparable to control isograft recipients. At the termination of the study, an evaluation of nerve conduction was performed by surgical exposure of the graft and direct nerve stimulation. Walking track evaluation affords an accurate method to quantify integrated and functional end organ reinnervation serially following nerve regeneration.18"20 However, this method indirectly reflects local events that occur at the nerve fiber level. Nerve biopsy permits histologic examination of regenerating axons, but this invasive technique interferes with axonal continuity. In contrast, serial measurements of nerve electrophysiology provide a sensitive evaluation of axonal function and directly reflect local effects from inflammation and immunologic activity on regenerating nerve fibers.21 Although it is possible to perform accurate nerve electrophysiologic studies by surgical exposure and direct nerve stimulation, this method disturbs the bed of the graft and is impractical to perform repetitively in immunosuppressed animals over the long periods required for regeneration. The percutaneous in vivo technique described here permits frequent but relatively noninvasive evaluation of regenerating axonal electrophysiologic function, with little morbidity. Values of CMAP amplitude provide a measure of regenerating axon number, while differential temporal dispersion of the CMAP recording through the grafts indicates the degree of axonal maturity (i.e., remyelination) among the population of conducting fibers. The correlation of CMAP and serial NCV recordings throughout the postoperative period provides insight into the behavior of the entire body of regenerating axons during immunosuppression. This information may contribute to a broader understanding of local immune and inflammatory events that affect nerve function at the fiber level during regeneration. The present study supports the results presented by Bain et a!.16 in that regeneration through nerve
OCTOBER 1 9 9 0
allografts in our cyclosporin-treated animals did not differ significantly from isograft recipients. Maximum CMAP amplitudes reached plateau levels approximately five months after transplantation in both groups. This may indicate maximum reinnervation and may represent an endpoint for future studies using this model. The more rapid amplitude rise observed in the cyclosporin-treated group corroborates the improved functional (gait) testing in the early postgrafting period noted by Bain et al.16 This may represent local cyclosporin effects on inflammation, vascular ingrowth, and Schwann cell activity during regeneration. Peak nerve conduction velocity was evident three months after transplantation and was approximately 65 percent of normal (60 m/sec) values. This correlates with the decreased internodal distances and smaller diameter axons that are known to develop during regeneration. In contrast to the results of Bain et a!.,16 our nonimmunosuppressed allograft controls demonstrated graft rejection without any meaningful histologic or electrophysiologic evidence of regeneration. This discrepancy may be related to the size of the peripheral nerve gap that was bridged in each study. In our model, the distance between the ends of the proximal and distal native nerve spans a length of approximately 3 to 3.5 cm, extending from the thigh to the ankle. The model described by Bain et a!.16 involved the resection of a 1-cm segment of native nerve prior to repair by a 3-cm allo- or isograft confined to the thigh. Rat peripheral nerves display a strong capacity for regeneration. Even without accurate coaptation of proximal and distal nerve stumps, regenerating axons can occasionally bridge gaps several centimeters in length. Consequently, grafts longer than 4 cm have been recommended for use in nerve transplantation studies, as regeneration through shorter allograft controls has been observed after rejection.15.16,20.22,23 | n addition, immunohistochemical localization in this study revealed small numbers of axons that regenerated within the allograft epineurium. The presence of these scattered extrafascicular fibers demonstrates a potential route for aberrant regeneration around a rejected allograft and may partially explain the finding of nerve conduction in nonimmunosuppressed allografts that bridge a short nerve gap in other investigations. Transection of the sciatic nerve results in complete distal lower extremity paralysis and an insensate foot. In our experience, these rats are prone to plantar ulceration, infection, and self mutilation by autocannibalization. Such morbidities seriously interfere with methods used to evaluate end organ reinnervation. By preserving peroneal nerve sensation to the foot, such complications were avoided. The sural nerve is known to be a mixed nerve in the rat and may supply motor fibers to the proximal
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ELECTROPHYSIOLOGIC EVALUATION OF ALLOGRAFTSMJ, ENGLAND, SUMNER, ET AL. 7. 8. 9. 10.
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REFERENCES
18. 19.
Fullerton PM: Chronic peripheral neuropathy produced by lead poisoning in guinea pigs. ] Neuropath Exp Neurol 23:214, 1966 Hickey WF, Lee V, Trojanowski JQ, et al: Immunohistochemical application of monoclonal antibodies against myelin basic protein and neurofilament triple protein subunits. I Histochem Cytochem 31:1126, 1983 Millesi H: Nerve grafting. Clin Plast Surg 11:105, 1984 Chiu DTW, lanecka I, Krizek TJ, et al.: Autogenous vein graft as a conduit for nerve regeneration. Surg 91:226, 1982 Lundborg G, Hansson HA: Nerve regeneration through preformed pseudosynovial tubes. 1 Hand Surg 5:35, 1980 Gattuso |M, Davies AH, Glosby MA, et al. Peripheral nerve repair using muscle autografts. I Bone )oint Surg 708:524,
20.
21.
22. 23.
Molander H: Regeneration of a peripheral nerve through a polyglactic tube. Muscle Nerve 5:54, 1982 Seckel, BR, Chiu TH, Nyilas E, Sidman RL: Nerve regeneration through synthetic biodegradable nerve guides: Regulation by the target organ. Plast Reconstr Surg 74:173, 1984 Bora FW, Bednar JM, Osterman AL, etal: Prosthetic nerve grafts: A resorbable tube as an alternative to autogenous nerve grafting. I Hand Surg 12A:685, 1987 Norris RW, Glasby MA, Gattuso |M, Bowden REM: Peripheral nerve repair in humans using muscle autografts. J Bone Joint Surg 7OB:53O, 1988 Chiu DTW, Strauch B: A prospective clinical evaluation of autogenous vein graft as nerve conduit. Paper presented at the 68th Annual Meeting of the American Association of Plastic Surgeons, Scottsdale, AZ, 1989 Chiu DTW, Lovelace RE, Yu LT, et al: Comparative electrophysiologic evaluation of nerve grafts and autogenous vein grafts as nerve conduits: An experimental study. I Reconstr Microsurg 4:303, 1988 Pollard ID, Fitzpatrick L: A comparison of the effects of irradiation and immunosuppressive agents on regeneration through peripheral nerve allografts: An ultrastructural study. Acta Neuropath 23:166, 1973 Mackinnon SE, Hudson AR, Bain JR, et al: The peripheral nerve allograft: An assessment of regeneration in the immunosuppressed host. Plast Reconstr Surg 79:436, 1987 Zalewski AA, Gulati AK: Survival of nerve allografts in sensitized rats treated with cyclosporin A. ) Neurosurg 60:828, 1984 Bain |R, Mackinnon SE, Hudson AR, et al. The peripheral nerve allograft: An assessment of regeneration across nerve allografts in rats immunosuppressed with cyclosporin A. Plast Reconstr Surg 82:1052, 1988 Zalewski AA, Gulati AK: Failure of cyclosporin A to induce immunological unresponsiveness to nerve allografts. Exp Neurol 83:659, 1984 De Medinaceli L, Freed WJ, Wyatt RJ: An index of the functional condition of rat sciatic nerve based on measurements made from walking tracks Exp Neurol 77:634, 1982 Carlton IM, Goldberg NH: Quantitating integrated muscle function following reinnervation. Surg Forum 37:611, 1986 Bain [R, Mackinnon SE, Hunter DA: Functional evaluation of complete sciatic, peroneal, and posterior tibial nerve lesions in the rat. Plast Reconstr Surg 83:129, 1989 Yu LT, England J, Hickey WF, et al. Survival and function of peripheral nerve allografts after cessation of long-term cyclosporin immunosuppression in rats. Transpl Proc 21:3178, 1989 Zalewski AA, Silvers WK: An evaluation of nerve repair with nerve allografts in normal and immunologically tolerant rats. I Neurosurg 52:557, 1980 Zalewski AA, Silvers WK, Gulati AK: Failutre of host axons to regenerate through a once successful but later rejected long nerve allograft. I Comp Neurol 209:347, 1982
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intrinsic foot muscles. It variably conducted small amplitude, fast muscle action potentials following tibial nerve division. If the sural nerve were left undisturbed, these residual impulses would produce unexpectedly rapid measurements of NCV and would contribute to a mature pattern of CMAP during early regeneration. These could misinform the interpretation of a single direct conduction study performed at the termination of the investigation. Resection of a sural nerve segment at the time of transplantation routinely eliminated these ambiguous muscle action potentials for the duration of the study. These results demonstrate the value of the tibialtunnel graft model for extended postoperative evaluation of nerve regeneration through long peripheral nerve grafts. This model is particularly useful for peripheral nerve transplantation involving recipient immunosuppression. It permits direct serial assessment of electrophysiologic parameters of regeneration with minimal morbidity, and allows close monitoring of axonal function during all phases of peripheral nerve regeneration. The use of this technique with current methods of gait assessment may add valuable information to future investigations of peripheral nerve reconstruction.
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ELECTROPHYSIOLOGIC EVALUATION OF PERIPHERAL NERVE REGENERATION THROUGH Downloaded by: University of British Columbia. Copyrighted material.
ALLOGRAFTS IMMUNOSUPPRESSED WITH CYCLOSPORIN ABSTRACT A model was designed to evaluate the long-term in vivo electrophysiology of rat peripheral nerve transplants. The application of this model was demonstrated using cyclosporin (CSA) immunosuppression of recipient animals to facilitate peripheral nerve regeneration through nerve allografts. Isogenic Brown Norway (BN) rats [ RT1n ] were divided into three groups: two received Lewis (LE) rat [ RT1 • | allografts and one received BN isografts. One allograft recipient group received CSA immunosuppression for the duration of the investigation (150 days). Successful nerve regeneration in the isograft and the immunosuppressed allograft recipient groups was determined by immunohistochemical methods and serial in vivo electrophysiologic techniques to measure nerve conduction velocity and evoked compound muscle action potential amplitude. Statistical analysis of these results indicate that: (a) CSA immunosuppression of peripheral nerve allograft recipients facilitates peripheral nerve regeneration which is indistinguishable from isograft recipient controls at the functioning axon level; and (b) in vivo electrophysiologic monitoring in this model is particularly useful for long term peripheral nerve transplantation studies permitting serial assessment of regeneration with little morbidity.
Interest in mammalian peripheral nerve transplantation has been stimulated by recent advances in available immunosuppressiveagents. Traditionally, rat sciatic and peroneal nerves have been used for nerve allograft investigations. Although these nerves are extremely useful for studies of peripheral nerve repair, they have several disadvantages when adapted for nerve transplantation. In particular, there are few accurate methods to assess functional nerve regeneration quantitatively along these pathways. We present an alternative rat model designed specifically for peripheral nerve transplantation, which permits frequent and
reliable evaluation of nerve electrophysiology and demonstrates its application for peripheral nerve allografts, using long-term cyclosporin (CSA) immunosuppression.
MATERIALS AND METHODS EXPERIMENTAL GROUPS. Three groups of isogenic Brown Norway (BN) rats served as recipient animals. All animals (Charles River Laboratories, Kingston, NY)
Divisions of Plastic Surgery and Neurology, Hospital of the University of Pennsylvania, Philadelphia, PA, and Division of Neuropathology, Washington University School of Medicine, St. Louis, MO Materials in this paper were presented at the 33rd Annual Meeting of the Plastic Surgery Research Council, San Francisco, CA, May, 1988 Reprint requests: Dr. Larossa, Division of Plastic Surgery, Dept. of Surgery, Hospital of the University of Pennsylvania, 3400 Spruce St., Philadelphia, PA 19104 Accepted for publication April 17, 1990 Copyright © 1990 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
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were eight to 10 weeks old and weighed between 200 and 250 g at surgery. Group A (n = 12) received an isograft from BN donors. Group B (n = 8) received an allograft from inbred Lewis (LE) donors. Group C (n = 12) received an LE allograft and long term postoperative CSA immunosuppression. CSA (Sandoz Pharmaceuticals Corporation, E. Hanover, NJ) was administered subcutaneously at a daily dose of 15 mg/kg in olive oil for an induction period of 20 days and then biweekly for an additional 130 days. Animals were caged in pairs, fed a diet of standard laboratory chow and water, and were allowed to move freely after surgery. OPERATIVE PROCEDURE. The rat sciatic nerve is a Figure 1. Diagrammatic representation of graft recipmixed motor-sensory nerve. After emerging from the ient demonstrating site of proximal anastamosis in the thigh sciatic notch, it gives rise to a minor sensory branch in and distal anatamosis at the ankle. Sural nerve resection is the proximal thigh and then divides distally into three performed via the proximal incision (see text). major branches at the level of the knee (peroneal, sural, and tibial nerves). The entire length of the sciatic nerve, approximately 2.5 to 3 cm in a 250 g rat, can be and the proximal and distal sites of nerve coaptation. rapidly exposed through a dorsal thigh incision. The Nerve repair was performed using standard end-tosciatic nerve and the proximal portions of all three end epineural microsuture technique with 10-0 nylon. major branches are easily accessible through this inciBoth incisions were closed in single layers using a skin sion. The tibial nerve is the largest of these branches stapler. Elapsed time for recipient preparation and and travels deep to the gastrosoleus muscles of the both nerve repairs averaged approximately 20 min per calf. It gives rise to the plantar nerves at the ankle animal. which innervate the intrinsic muscles in the foot. A ELECTROPHYSIOLOGY. Nerve regeneration was evalsurgical approach to the distal tibial nerve and its uated by monthly in vivo electrophysiologic studies for proximal plantar branches requires a second incision five months using the method of Fullerton.1 Percutaneon the lateral aspect of the lower leg anterior to the ous platinum needle electrodes (Type E2, Grass InstruAchilles tendon. ment Co., Quincy, MA) were used for both stimulating Donor Harvest. A 4.50-cm. sciatic-tibial nerve graft and recording purposes. The proximal pair of stimulatwas harvested from a 250 g donor rat between the ing electrodes was positioned adjacent to the sciatic sciatic notch and the ankle. Peroneal and sural nerve nerve at the sciatic notch. The distal pair was posibranches were divided close to their takeoff from the tioned adjacent to the posterior tibial nerve at the main sciatic trunk, and the nerve was skeletonized ankle. from its adjacent investing tissue. Square wave stimulus pulses of 0.1 msec duration Recipient Preparation. Following adequate anesthewere delivered, and compound muscle action potensia (Nembutal 50 mg/kg i.p.) and using sterile techtials (CMAPs) were recorded, using an active electrode nique, a muscle-splitting incision was made through placed subcutaneously on the plantar surface of the the dorsal thigh to expose the sciatic nerve and its foot and an indifferent electrode inserted into the soft trifurcation. The tibial nerve was transected sharply tissue of the second digit. All CMAPs were obtained by with a Week blade and resected distally for approxsupramaximal nerve stimulation and recorded by a imately 1 to 1.5 cm. A segment of sural nerve (1.0 cm) TECA model TD-20 nerve stimulator (TECA Corporawas also resected at this level. Through the second tion, Pleasantville, NY). Permanent records were made incision at the ankle, the distal tibial/plantar nerve was of all evoked responses. The CMAP latency was measexposed anterior to the Achilles tendon and divided in ured from the stimulus artifact to the beginning of the similar fashion. A modified tendon retriever was passed first negative deflection and the amplitude from baseretrograde through this incision along the course of line to negative peak. The distance between the two the tibial nerve deep to the gastrosoleus muscles. In sites of stimulation was measured along the skin surthe correct plane, it would slide without resistance up face with the leg fully extended and a segmental nerve to the thigh. The proximal end of the nerve graft was conduction velocity (NCV) was calculated. This techplaced in the jaws of the tendon retriever and withnique permitted separate electrophysiologic measuredrawn into the newly created "tibial-tunnel" with the ments through the graft and distal native plantar knee in full extension. This permitted the graft to lie nerve. Limb temperature was carefully maintained bewithout tension or redundancy along the natural course tween 37 to 38°C by a thermistor probe inserted subcuof the native tibial nerve. taneously and a thermostatically controlled infrared Figure 1 illustrates the final position of the graft heat lamp.
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JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 6, NUMBER 4
ELECTROPHYSIOLOGIC EVALUATION OF ALLOGRAFTS/YU, ENGLAND, SUMNER, ET AL. HISTOLOGY. One animal from each group was sacrificed at eight 15, 30, 80, and 120 days postoperatively to allow harvest of the graft and histologic examination of longitudinal sections by light microscopy with hematoxylin and eosin stains. At the conclusion of the study, all remaining animals were sacrificed. Cryostat sections of the nerve grafts were examined by immunohistochemical methods to determine axon viability.2 Immunoperoxidase-positive axons were identified by light microscopic examination.
Postoperatively, no signs of wound infection, autocannibalism, or plantar ulceration were observed. Animals that received allografts without immunosuppression (Group B) demonstrated early histologic evidence of intense cellular graft rejection, followed by necrosis and scar formation within the nerve grafts (Fig. 2A). No animals in this group regained any electrophysiologic function during the study. Terminal immunohistochemical stains revealed an absence of viable axons in the body of these allografts and distal plantar nerves, indicating graft rejection and failure of functional axonal regeneration. Animals that received isografts (Group A) and immunosuppressed allografts (Group C) (Fig. 2B) demonstrated the classic histologic appearance of early postoperative Wallerian degeneration, followed by orderly regeneration of nerve fibers through the grafts and native distal nerves (Fig. 3). No signs of graft rejection could be detected by light microscopy in the immunosuppressed allograft recipients, and graft sections appeared histologically indistinguishable between these groups. Serial electrophysiologic studies demonstrated identical patterns of evoked CMAP organization in the
Figure 3. Immunosuppressed allograft segment 120 days post-transplantation. Note orderly regeneration of axons with minimal inflammation (H&E, xlOO). Downloaded by: University of British Columbia. Copyrighted material.
RESULTS
isograft and immunosuppressed allograft recipients. Small amplitude responses were detected as early as 60 days after transplantation. Uniform action potentials were evident by 120 days postgrafting. Resolution of differential temporal dispersion paralleled a rise in CMAP amplitude for each responding animal (Fig. 4). Mean values of CMAP amplitude increased in both groups until termination of the study (Fig. 5A). The rate of increase appeared more rapid in the cyclosporin-treated group, although a significant difference (p < 0.01 by t-test) was evident only at 90 days postgrafting. This disparity resolved by 120 days, and both groups produced statistically similar values for the remainder of the study. At 150 days after transplantation (Table 1), distal CMAP amplitude for Group C (allograft + CSA) averaged 4.8 mV (SEM 0.76), while Group A (isograft control) averaged 4.3 mV (SEM 0.61). Monthly, in vivo measurements of CMAPs were continued in the isograft group after 150 days to determine maximal amplitude return. A plateau level of 5.4 mV
•- •
. .B
Figure 2. A, Nonimmunosuppressed allograft segment 15 days post-transplantation. Note intense cellular inflammatory response during allograft rejection. B, Immunosuppressed allograft segment 15 days post-transplantation. Note typical Wallerian degeneration within allograft (H&E, xlOO).
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OCTOBER 1990
EVOKED MUSCLE ACTION POTENTIAL (Allograft + CSA)
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Figure 5. A, Postoperative mean compound muscle action potential (CMAP) amplitude from distal stimulus. B, Postoperative mean values for nerve conduction velocity (NCV). Reference values recorded from non-operated age matched controls. Error bars denote standard error of the mean |SEM|.
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A
Figure 4. Representative interval evoked compound muscle action potentials recorded from an immunosuppressed allograft recipient. Note progressive organization of response curve during nerve regeneration. Arrows indicate stimulus artifact.
ELECTROPHYSIOLOGIC EVALUATION OF ALLOGRAFTS/YU, ENGLAND, SUMNER, ET AL.
(A) Isograft (B) Allograft (C) Allograft + CSA
CMAP*
(SEM)t
NCV*
(SEM)
4.3 mV 0 4.8 mV
(0.61)
35.0 m/s 0 37.6 m/s
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*Mean value of distal compound muscle action potential amplitudes. tStandard error of the means. *Mean value of nerve conduction velocities.
(SEM 0.65) was reached at 180 days and persisted up to 240 days after transplantation, when all animals in this group were sacrificed. Nerve conduction velocity in the conducting groups (Fig. 5B, Table 1) reached peak levels by 90 days and remained relatively constant for the remainder of the study (Group A 35.0 m/sec |SEM 2.7], Group C 37.6 m/sec |SEM 2.7]). Repeated measures tests were applied to the mean values of CMAP amplitude and NCV for both groups over the course of the study. No significant differences between the control isograft or immunosuppressed allograft recipient groups could be demonstrated. Each group displayed a significant difference from nonimmunosuppressed allograft recipients (p < 0.005).
At the conclusion of the study, motor and sensory nerve fibers were identified by neurofilament immunohistochemical stains in all grafts. However, significant numbers of axons in an organized fascicular pattern were evident in only the isograft and immunosuppressed allograft recipients. Although scattered axons were observed in the epineurium of all three groups, no evidence of fascicular axonal regeneration was seen in the nonimmunosuppressed allograft animals (Fig. 6).
DISCUSSION The disadvantages of tension at the primary repair of a peripheral nerve have demonstrated the need for nerve grafts to bridge large nerve gaps.3 However, the limited availability of donor autologous nerves has restricted the optimal reconstruction of these lesions by autografts. Although conduits of autologous 4 - 6 and synthetic7-9 material have been used to promote nerve regeneration, their clinical use has been confined to distal and relatively short nerve deficits.10 " Fora large proximal nerve gap, animal studies suggest that a conduit will not promote the same quality of peripheral nerve regeneration as a nerve autograft.12 Investigations of allogeneic peripheral nerve grafts,
I
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Table 1. In Vivo Electrophysiologic Values 150 Days Post-transplantation
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Figure 6. Terminal stains for axonal viability 150 days post-transplantation. A, Isograft. B, Immunosuppressed allograft. C, Nonimmunosuppressed allograft. Note similarity of isograft and immunosuppressed allograft segments vs. absence of viable axons in rejected, nonimmunosuppressed allograft (neurofilament immunoperoxidase stain, X200).
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as an alternative to autografts, followed successful allotransplantation of other organs using recipient immunosuppression to prevent graft rejection. Hydrocortisone, 1314 azathioprine, 1314 and cyclosporin1516 have prevented peripheral nerve allograft rejection and promoted nerve regeneration. Zawleski and Gulati 1517 examined the survival of long (4 cm) peripheral nerve allografts transplanted across multiple minor histocompatability barriers in rats using CSA immunosuppression. Their model did not use a distal nerve repair to bridge a gap in continuity and although they were able to provide histologic evidence of nerve regeneration through the allografts, they did not evaluate functional return. Bain et a!.16 demonstrated nerve regeneration through 3-cm nerve allografts across an MHC barrier in rats immunosuppressed with CSA. Regeneration was documented by histologic examination and serial functional assessment using gait analysis. These values were comparable to control isograft recipients. At the termination of the study, an evaluation of nerve conduction was performed by surgical exposure of the graft and direct nerve stimulation. Walking track evaluation affords an accurate method to quantify integrated and functional end organ reinnervation serially following nerve regeneration.18"20 However, this method indirectly reflects local events that occur at the nerve fiber level. Nerve biopsy permits histologic examination of regenerating axons, but this invasive technique interferes with axonal continuity. In contrast, serial measurements of nerve electrophysiology provide a sensitive evaluation of axonal function and directly reflect local effects from inflammation and immunologic activity on regenerating nerve fibers.21 Although it is possible to perform accurate nerve electrophysiologic studies by surgical exposure and direct nerve stimulation, this method disturbs the bed of the graft and is impractical to perform repetitively in immunosuppressed animals over the long periods required for regeneration. The percutaneous in vivo technique described here permits frequent but relatively noninvasive evaluation of regenerating axonal electrophysiologic function, with little morbidity. Values of CMAP amplitude provide a measure of regenerating axon number, while differential temporal dispersion of the CMAP recording through the grafts indicates the degree of axonal maturity (i.e., remyelination) among the population of conducting fibers. The correlation of CMAP and serial NCV recordings throughout the postoperative period provides insight into the behavior of the entire body of regenerating axons during immunosuppression. This information may contribute to a broader understanding of local immune and inflammatory events that affect nerve function at the fiber level during regeneration. The present study supports the results presented by Bain et a!.16 in that regeneration through nerve
OCTOBER 1 9 9 0
allografts in our cyclosporin-treated animals did not differ significantly from isograft recipients. Maximum CMAP amplitudes reached plateau levels approximately five months after transplantation in both groups. This may indicate maximum reinnervation and may represent an endpoint for future studies using this model. The more rapid amplitude rise observed in the cyclosporin-treated group corroborates the improved functional (gait) testing in the early postgrafting period noted by Bain et al.16 This may represent local cyclosporin effects on inflammation, vascular ingrowth, and Schwann cell activity during regeneration. Peak nerve conduction velocity was evident three months after transplantation and was approximately 65 percent of normal (60 m/sec) values. This correlates with the decreased internodal distances and smaller diameter axons that are known to develop during regeneration. In contrast to the results of Bain et a!.,16 our nonimmunosuppressed allograft controls demonstrated graft rejection without any meaningful histologic or electrophysiologic evidence of regeneration. This discrepancy may be related to the size of the peripheral nerve gap that was bridged in each study. In our model, the distance between the ends of the proximal and distal native nerve spans a length of approximately 3 to 3.5 cm, extending from the thigh to the ankle. The model described by Bain et a!.16 involved the resection of a 1-cm segment of native nerve prior to repair by a 3-cm allo- or isograft confined to the thigh. Rat peripheral nerves display a strong capacity for regeneration. Even without accurate coaptation of proximal and distal nerve stumps, regenerating axons can occasionally bridge gaps several centimeters in length. Consequently, grafts longer than 4 cm have been recommended for use in nerve transplantation studies, as regeneration through shorter allograft controls has been observed after rejection.15.16,20.22,23 | n addition, immunohistochemical localization in this study revealed small numbers of axons that regenerated within the allograft epineurium. The presence of these scattered extrafascicular fibers demonstrates a potential route for aberrant regeneration around a rejected allograft and may partially explain the finding of nerve conduction in nonimmunosuppressed allografts that bridge a short nerve gap in other investigations. Transection of the sciatic nerve results in complete distal lower extremity paralysis and an insensate foot. In our experience, these rats are prone to plantar ulceration, infection, and self mutilation by autocannibalization. Such morbidities seriously interfere with methods used to evaluate end organ reinnervation. By preserving peroneal nerve sensation to the foot, such complications were avoided. The sural nerve is known to be a mixed nerve in the rat and may supply motor fibers to the proximal
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REFERENCES
18. 19.
Fullerton PM: Chronic peripheral neuropathy produced by lead poisoning in guinea pigs. ] Neuropath Exp Neurol 23:214, 1966 Hickey WF, Lee V, Trojanowski JQ, et al: Immunohistochemical application of monoclonal antibodies against myelin basic protein and neurofilament triple protein subunits. I Histochem Cytochem 31:1126, 1983 Millesi H: Nerve grafting. Clin Plast Surg 11:105, 1984 Chiu DTW, lanecka I, Krizek TJ, et al.: Autogenous vein graft as a conduit for nerve regeneration. Surg 91:226, 1982 Lundborg G, Hansson HA: Nerve regeneration through preformed pseudosynovial tubes. 1 Hand Surg 5:35, 1980 Gattuso |M, Davies AH, Glosby MA, et al. Peripheral nerve repair using muscle autografts. I Bone )oint Surg 708:524,
20.
21.
22. 23.
Molander H: Regeneration of a peripheral nerve through a polyglactic tube. Muscle Nerve 5:54, 1982 Seckel, BR, Chiu TH, Nyilas E, Sidman RL: Nerve regeneration through synthetic biodegradable nerve guides: Regulation by the target organ. Plast Reconstr Surg 74:173, 1984 Bora FW, Bednar JM, Osterman AL, etal: Prosthetic nerve grafts: A resorbable tube as an alternative to autogenous nerve grafting. I Hand Surg 12A:685, 1987 Norris RW, Glasby MA, Gattuso |M, Bowden REM: Peripheral nerve repair in humans using muscle autografts. J Bone Joint Surg 7OB:53O, 1988 Chiu DTW, Strauch B: A prospective clinical evaluation of autogenous vein graft as nerve conduit. Paper presented at the 68th Annual Meeting of the American Association of Plastic Surgeons, Scottsdale, AZ, 1989 Chiu DTW, Lovelace RE, Yu LT, et al: Comparative electrophysiologic evaluation of nerve grafts and autogenous vein grafts as nerve conduits: An experimental study. I Reconstr Microsurg 4:303, 1988 Pollard ID, Fitzpatrick L: A comparison of the effects of irradiation and immunosuppressive agents on regeneration through peripheral nerve allografts: An ultrastructural study. Acta Neuropath 23:166, 1973 Mackinnon SE, Hudson AR, Bain JR, et al: The peripheral nerve allograft: An assessment of regeneration in the immunosuppressed host. Plast Reconstr Surg 79:436, 1987 Zalewski AA, Gulati AK: Survival of nerve allografts in sensitized rats treated with cyclosporin A. ) Neurosurg 60:828, 1984 Bain |R, Mackinnon SE, Hudson AR, et al. The peripheral nerve allograft: An assessment of regeneration across nerve allografts in rats immunosuppressed with cyclosporin A. Plast Reconstr Surg 82:1052, 1988 Zalewski AA, Gulati AK: Failure of cyclosporin A to induce immunological unresponsiveness to nerve allografts. Exp Neurol 83:659, 1984 De Medinaceli L, Freed WJ, Wyatt RJ: An index of the functional condition of rat sciatic nerve based on measurements made from walking tracks Exp Neurol 77:634, 1982 Carlton IM, Goldberg NH: Quantitating integrated muscle function following reinnervation. Surg Forum 37:611, 1986 Bain [R, Mackinnon SE, Hunter DA: Functional evaluation of complete sciatic, peroneal, and posterior tibial nerve lesions in the rat. Plast Reconstr Surg 83:129, 1989 Yu LT, England J, Hickey WF, et al. Survival and function of peripheral nerve allografts after cessation of long-term cyclosporin immunosuppression in rats. Transpl Proc 21:3178, 1989 Zalewski AA, Silvers WK: An evaluation of nerve repair with nerve allografts in normal and immunologically tolerant rats. I Neurosurg 52:557, 1980 Zalewski AA, Silvers WK, Gulati AK: Failutre of host axons to regenerate through a once successful but later rejected long nerve allograft. I Comp Neurol 209:347, 1982
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intrinsic foot muscles. It variably conducted small amplitude, fast muscle action potentials following tibial nerve division. If the sural nerve were left undisturbed, these residual impulses would produce unexpectedly rapid measurements of NCV and would contribute to a mature pattern of CMAP during early regeneration. These could misinform the interpretation of a single direct conduction study performed at the termination of the investigation. Resection of a sural nerve segment at the time of transplantation routinely eliminated these ambiguous muscle action potentials for the duration of the study. These results demonstrate the value of the tibialtunnel graft model for extended postoperative evaluation of nerve regeneration through long peripheral nerve grafts. This model is particularly useful for peripheral nerve transplantation involving recipient immunosuppression. It permits direct serial assessment of electrophysiologic parameters of regeneration with minimal morbidity, and allows close monitoring of axonal function during all phases of peripheral nerve regeneration. The use of this technique with current methods of gait assessment may add valuable information to future investigations of peripheral nerve reconstruction.
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