Noboru Maeda, Naoki Ishiguro, Goro Inoue, Takayuki Miura, and Kimiya Sugimura
NERVE REGENERATION IN RAT COMPOSITE-TISSUE ALLOGRAFTS ABSTRACT
Cyclosporin A (CyA), an effective immunosuppressive agent, has been used in human organ transplants and has improved results, compared with conventional immunosuppression. Recently, the agent has been used successfully in the experimental study of composite-tissue allografts in animal models. Previous studies of composite-tissue allografts, using dog or rat models treated with various immunosuppressive regimens, resulted in limited success because of infection or drug toxicity.12 Hewitt, Black, and Fraser3 first reported success in the control of rejection with CyA in composite-tissue allografts using a rat hind-limb model. These findings have been confirmed by other authors. Fritz, Swartz, and Rose4 for example, reported success with CyA in suppressing rejection in composite allografts across defined major histocompatibility barriers. Kim, Aziz, and Oyer5 also reported CyA to be superior to conventional agents such as azathioprine and predonisolone, in preventing rejection of transplanted limbs in rats. The goal of transplantation surgery in compositetissue allografts is functional recovery. Daniel, Egerszegi, and Samulack6 reported studies of transplanta-
Downloaded by: Universite de Sherbrooke. Copyrighted material.
Reinnervation of composite-tissue allografts was examined in a rat hind-limb transplant model under Cyclosporin A (CyA) immunosuppression. Two groups of animals were studied. Group 1 consisted of 10 composite-tissue allografts using inbred Fisher rats (F344) as recipients and inbred Lewis rats (LEW) as donors. Recipient animals received 15 mg/kg/day CyA subcutaneously for three weeks and then twice weekly for three months. Group 2 consisted of 10 composite-tissue isografts undergoing no immunosuppression, and using F344 as both donors and recipients. Eight months after operation, electromyographic, histologic, and morphometric assessments of nerve regeneration were made. There were no significant electromyographic differences between these two experimental groups in amplitude and conduction velocity, but the levels were lower than in controls. The histologic patterns of nerve regeneration within the composite-tissue transfers were similar in the two groups. However, the fiber population in the regenerated nerves in the two groups was different from that in the normal sciatic nerve. This study confirmed the reinnervation of composite-tissue allografts under CyA immunosuppression. The quality of regeneration in allografts was similar to that in isografts, but the histologic and electromyographic properties of regenerated nerves were different from those of normal sciatic nerves.
tion of the upper extremity in primates, and recognized the sensory recovery and reinnervation of donor muscles. Samulack and colleagues8 demonstrated neuroanatomic evidence of reinnervation. Guzman-Stein and Shons9 also confirmed reinnervation of muscle and skin in a rat-limb transplant model. Nerve regeneration is essential for functional recovery in composite tissue allografts. Samulack, Dykes, and Munger10 have demonstrated axon-receptor continuity and quantified nerve regeneration in primateextremity allotransplanted tissue immunosuppressed with CyA neurophysiologically. The present investigation confirmed nerve regeneration under immunosuppression with CyA in rat composite-tissue allografts electrophysiologically and histologically, and assessed it morphometrically.
MATERIALS AND METHODS ANIMAL MODEL. Transplantation of compositetissue allografts in the rat hind limb was done using
Departments of Orthopaedic Surgery and Neurology, Nagoya University School of Medicine Reprint requests-. Dr. Maeda, Dept. of Orthopaedic Surgery, Nagoya Daini Red Cross Hospital, 2-9 Myokencho, Showaku, Nagoya 466, Japan Accepted for publication April 15,1991 Copyright © 1991 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
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JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 7, NUMBER 4
two strains of inbred rats, F344/Hok and LEW/Hkm adult males, weighing 200 to 250 g. This combination represents a minor histocompatibility difference. Two groups of animals were studied. Group 1 consisted of 10 cases of allotransplanted composite tissue: F344 received LEW limb allografts. Recipient animals received 15 mg/kg/day CyA subcutaneously daily for three weeks and then twice weekly for three months. Group 2 consisted of 10 cases of isotransplanted composite tissue, undergoing no immunosuppression and using F344 rats as both donor and recipient. All animals were maintained in clean cages and no antibiotics were administered. Intraperitoneal pentobar-
bital (50 mg/kg) was used for general anesthesia. An aseptic technique skin incision was made over the anteromedial surface of the recipient hind limb. The femoral vessels were carefully dissected and divided at midfemoral level and occluded with microvascular clamps. The femoral and sciatic nerves were also dissected and divided at the same level. The hind limb was amputated at the midfemoral level, except that skin and muscular structure were divided as far distally as possible. The donor limb was similarly dissected and amputated, and was transplanted at the recipient limb stump. Donor and recipient femurs were fixed with an 18-gauge needle intramedullarly, and the muscle layers were repaired. The femoral vessels were anastomosed with 10-0 nylon. The sciatic nerve was repaired with 10-0 nylon epineural sutures. To monitor circulation and skin rejection in the grafted limb, the donor skin was attached only in the foot. The remainder of the skin covering the donor limb was removed so that only the donor limb proximal to the ankle was covered with recipient skin.
298
Morphometric evaluation. Morphometric evaluation of the nerve fibers regenerated into the distal transplanted sciatic nerve was carried out by photography at 900 x magnification in regular fashion, using an image analyzer (NACL TVIP-2000) linked to a personal computer using morphometry software. The density and fiber-size frequency distribution of myelinated fibers were calculated based on a minimum number of 300 fibers. The results were compared using the Student T-test, and a p value of less than 0.05 was considered statistically significant.
RESULTS A total of 20 vascularized rat hind-limb transplantations was performed. All transplants were successfully vascularized and survived without evidence of rejection. All animals were followed for eight months, except for one animal with an allogeneic transplant which died at seven months after transplantation for unspecified reasons, with no evidence of rejection of hind limb and graft versus host disease. In the feet, normal characteristics of hair and nail growth were observed in all allogeneic and isogeneic groups. ELECTROMYOGRAPHIC ASSESSMENT.
The results of
the electromyographic studies are shown in Figure 1. There were no significant differences in electromyographic amplitude and conduction velocity in controls between allogeneic and isogeneic groups. In the transplanted composite tissue, the amplitude was lower than in the controls (p < 0.05) in both groups. However, no significant difference was detected in the electromyographic amplitude between allogeneic and isoMETHODS OF ASSESSMENT. Electromyographic assess- geneic groups. Similarly, the conduction velocity was ment. Eight months after transplantation, electro- slower than in controls (p < 0.05) in both groups. myographic recordings were made using Medelec type However, no significant difference was detected in conMS6. Both sciatic nerves on the experimental and duction velocity between allogeneic and isogeneic control sides were exposed. A double-hooked stimu- groups. lating electrode was placed around the sciatic nerve HISTOLOGIC STUDIES. On light microscopic evalproximal and distal to the suture site. Recordings were uation, the histologic findings in allogeneic transmade with a recording needle electrode in the calf plants were identical to those of isogeneic transplants. muscles. Similar recordings were made on the control The normal architecture in regeneration of the sciatic side. Distal motor latencies and amplitudes of the first nerve was maintained and well vascularized. No active two positive and negative peaks were measured. Nerve inflammatory cell infiltration was present. A high denconduction velocities across nerve suture sites were sity of well-myelinated smaller fibers was noted, comcalculated. pared with controls. Histologic studies. After electromyographic recordOn electron microscopic evaluation (Fig. 2), there ings, the donor sciatic nerve, 2-cm distal to the suture were well-myelinated smaller fibers in an organized site, was transected and fixed in 2 percent glutaralde- extracellular collagen matrix in both allografts and hyde in 0.025 M cacodylate buffer, pH 7.4. The speci- isografts. men was postfixed in osmium tetroxide and embedMORPHOMETRIC EVALUATION. The density of myelided in epoxy resin. Toluidine blue was used to stain nated fibers in the allografts was not significantly difl-|xm-thick transverse sections for light microscopy. ferent from that of isografts. However, it increased Lead citrate and uranyl acetate were used to stain for significantly, compared with the controls (Fig. 3). electron microscopy. Figure 4 shows the fiber-size frequency distribu-
Downloaded by: Universite de Sherbrooke. Copyrighted material.
SURGICAL PROCEDURE.
OCTOBER 1991
COMPOSITE-TISSUE ALLOGRAFTS/MAEDA, ISHIGURO, INOUE, ET AL.
(mV)
p
NERVE REGENERATION IN RAT COMPOSITE-TISSUE ALLOGRAFTS ABSTRACT
Cyclosporin A (CyA), an effective immunosuppressive agent, has been used in human organ transplants and has improved results, compared with conventional immunosuppression. Recently, the agent has been used successfully in the experimental study of composite-tissue allografts in animal models. Previous studies of composite-tissue allografts, using dog or rat models treated with various immunosuppressive regimens, resulted in limited success because of infection or drug toxicity.12 Hewitt, Black, and Fraser3 first reported success in the control of rejection with CyA in composite-tissue allografts using a rat hind-limb model. These findings have been confirmed by other authors. Fritz, Swartz, and Rose4 for example, reported success with CyA in suppressing rejection in composite allografts across defined major histocompatibility barriers. Kim, Aziz, and Oyer5 also reported CyA to be superior to conventional agents such as azathioprine and predonisolone, in preventing rejection of transplanted limbs in rats. The goal of transplantation surgery in compositetissue allografts is functional recovery. Daniel, Egerszegi, and Samulack6 reported studies of transplanta-
Downloaded by: Universite de Sherbrooke. Copyrighted material.
Reinnervation of composite-tissue allografts was examined in a rat hind-limb transplant model under Cyclosporin A (CyA) immunosuppression. Two groups of animals were studied. Group 1 consisted of 10 composite-tissue allografts using inbred Fisher rats (F344) as recipients and inbred Lewis rats (LEW) as donors. Recipient animals received 15 mg/kg/day CyA subcutaneously for three weeks and then twice weekly for three months. Group 2 consisted of 10 composite-tissue isografts undergoing no immunosuppression, and using F344 as both donors and recipients. Eight months after operation, electromyographic, histologic, and morphometric assessments of nerve regeneration were made. There were no significant electromyographic differences between these two experimental groups in amplitude and conduction velocity, but the levels were lower than in controls. The histologic patterns of nerve regeneration within the composite-tissue transfers were similar in the two groups. However, the fiber population in the regenerated nerves in the two groups was different from that in the normal sciatic nerve. This study confirmed the reinnervation of composite-tissue allografts under CyA immunosuppression. The quality of regeneration in allografts was similar to that in isografts, but the histologic and electromyographic properties of regenerated nerves were different from those of normal sciatic nerves.
tion of the upper extremity in primates, and recognized the sensory recovery and reinnervation of donor muscles. Samulack and colleagues8 demonstrated neuroanatomic evidence of reinnervation. Guzman-Stein and Shons9 also confirmed reinnervation of muscle and skin in a rat-limb transplant model. Nerve regeneration is essential for functional recovery in composite tissue allografts. Samulack, Dykes, and Munger10 have demonstrated axon-receptor continuity and quantified nerve regeneration in primateextremity allotransplanted tissue immunosuppressed with CyA neurophysiologically. The present investigation confirmed nerve regeneration under immunosuppression with CyA in rat composite-tissue allografts electrophysiologically and histologically, and assessed it morphometrically.
MATERIALS AND METHODS ANIMAL MODEL. Transplantation of compositetissue allografts in the rat hind limb was done using
Departments of Orthopaedic Surgery and Neurology, Nagoya University School of Medicine Reprint requests-. Dr. Maeda, Dept. of Orthopaedic Surgery, Nagoya Daini Red Cross Hospital, 2-9 Myokencho, Showaku, Nagoya 466, Japan Accepted for publication April 15,1991 Copyright © 1991 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
297
JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 7, NUMBER 4
two strains of inbred rats, F344/Hok and LEW/Hkm adult males, weighing 200 to 250 g. This combination represents a minor histocompatibility difference. Two groups of animals were studied. Group 1 consisted of 10 cases of allotransplanted composite tissue: F344 received LEW limb allografts. Recipient animals received 15 mg/kg/day CyA subcutaneously daily for three weeks and then twice weekly for three months. Group 2 consisted of 10 cases of isotransplanted composite tissue, undergoing no immunosuppression and using F344 rats as both donor and recipient. All animals were maintained in clean cages and no antibiotics were administered. Intraperitoneal pentobar-
bital (50 mg/kg) was used for general anesthesia. An aseptic technique skin incision was made over the anteromedial surface of the recipient hind limb. The femoral vessels were carefully dissected and divided at midfemoral level and occluded with microvascular clamps. The femoral and sciatic nerves were also dissected and divided at the same level. The hind limb was amputated at the midfemoral level, except that skin and muscular structure were divided as far distally as possible. The donor limb was similarly dissected and amputated, and was transplanted at the recipient limb stump. Donor and recipient femurs were fixed with an 18-gauge needle intramedullarly, and the muscle layers were repaired. The femoral vessels were anastomosed with 10-0 nylon. The sciatic nerve was repaired with 10-0 nylon epineural sutures. To monitor circulation and skin rejection in the grafted limb, the donor skin was attached only in the foot. The remainder of the skin covering the donor limb was removed so that only the donor limb proximal to the ankle was covered with recipient skin.
298
Morphometric evaluation. Morphometric evaluation of the nerve fibers regenerated into the distal transplanted sciatic nerve was carried out by photography at 900 x magnification in regular fashion, using an image analyzer (NACL TVIP-2000) linked to a personal computer using morphometry software. The density and fiber-size frequency distribution of myelinated fibers were calculated based on a minimum number of 300 fibers. The results were compared using the Student T-test, and a p value of less than 0.05 was considered statistically significant.
RESULTS A total of 20 vascularized rat hind-limb transplantations was performed. All transplants were successfully vascularized and survived without evidence of rejection. All animals were followed for eight months, except for one animal with an allogeneic transplant which died at seven months after transplantation for unspecified reasons, with no evidence of rejection of hind limb and graft versus host disease. In the feet, normal characteristics of hair and nail growth were observed in all allogeneic and isogeneic groups. ELECTROMYOGRAPHIC ASSESSMENT.
The results of
the electromyographic studies are shown in Figure 1. There were no significant differences in electromyographic amplitude and conduction velocity in controls between allogeneic and isogeneic groups. In the transplanted composite tissue, the amplitude was lower than in the controls (p < 0.05) in both groups. However, no significant difference was detected in the electromyographic amplitude between allogeneic and isoMETHODS OF ASSESSMENT. Electromyographic assess- geneic groups. Similarly, the conduction velocity was ment. Eight months after transplantation, electro- slower than in controls (p < 0.05) in both groups. myographic recordings were made using Medelec type However, no significant difference was detected in conMS6. Both sciatic nerves on the experimental and duction velocity between allogeneic and isogeneic control sides were exposed. A double-hooked stimu- groups. lating electrode was placed around the sciatic nerve HISTOLOGIC STUDIES. On light microscopic evalproximal and distal to the suture site. Recordings were uation, the histologic findings in allogeneic transmade with a recording needle electrode in the calf plants were identical to those of isogeneic transplants. muscles. Similar recordings were made on the control The normal architecture in regeneration of the sciatic side. Distal motor latencies and amplitudes of the first nerve was maintained and well vascularized. No active two positive and negative peaks were measured. Nerve inflammatory cell infiltration was present. A high denconduction velocities across nerve suture sites were sity of well-myelinated smaller fibers was noted, comcalculated. pared with controls. Histologic studies. After electromyographic recordOn electron microscopic evaluation (Fig. 2), there ings, the donor sciatic nerve, 2-cm distal to the suture were well-myelinated smaller fibers in an organized site, was transected and fixed in 2 percent glutaralde- extracellular collagen matrix in both allografts and hyde in 0.025 M cacodylate buffer, pH 7.4. The speci- isografts. men was postfixed in osmium tetroxide and embedMORPHOMETRIC EVALUATION. The density of myelided in epoxy resin. Toluidine blue was used to stain nated fibers in the allografts was not significantly difl-|xm-thick transverse sections for light microscopy. ferent from that of isografts. However, it increased Lead citrate and uranyl acetate were used to stain for significantly, compared with the controls (Fig. 3). electron microscopy. Figure 4 shows the fiber-size frequency distribu-
Downloaded by: Universite de Sherbrooke. Copyrighted material.
SURGICAL PROCEDURE.
OCTOBER 1991
COMPOSITE-TISSUE ALLOGRAFTS/MAEDA, ISHIGURO, INOUE, ET AL.
(mV)
p
