Akira Yanai, Kiyonori Harii, and Katsuyuki Okabe
PREVENTING DENERVATION ATROPHY OF A GRAFTED MUSCLE ABSTRACT
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Electrodes were implanted in the grafted muscles of rabbits to generate continuous stimulation for the purpose of preventing denervation atrophy. Denervation atrophy could be prevented, to some extent, in dissected muscles with vascular pedicles not occluded during the experiment. Denervation atrophy was found to be promoted, rather than prevented, in grafted muscles with occluded vascular pedicles. The crucial conditions are varying stimulation and the long duration of occlusion (90 min). Overly strong stimulation of muscle contraction appears to result in "fatigue" of grafted muscles in which blood circulation has been blocked for more than the 90 min used in the reported experiment. Further investigations are necessary of the conditions of stimulation and the point in time at which stimulation should commence after grafting. Free muscle transfer has come into wide clinical use in recent years, parallel with general advances in microsurgical tissue transfer.12 However, a serious problem has remained in that grafted muscles may be affected by denervation atrophy during the period between grafting and the regeneration of nerves. It would therefore be highly beneficial to find a means of preventing muscle atrophy during such a period. In an attempt to investigate the possibility of preventing denervation atrophy of grafted muscle, we conducted an experimental study using rabbits whose muscles were denervated and implanted with electrodes for continuous exogenous stimulation. We examined the resulting degree of atrophy over time.
The rabbits were anesthetized with an intravenous Nembutal injection through the auricular vein and then subjected to continuous GOF anesthesia by means of orotracheal intubation. 89 The bilateral inguinal region of each rabbit was shaved, and the skin extending from the femur region to the inguinal region was incised. A stimulating electrode consisted of two stainless steel pipes and woven Teflon-coated stainless wire was employed. Each pipe was 7 mm long and had an outer diameter of 0.6 mm. The pipes were lined up about 3 mm apart and then placed parallel to sandwich muscle at the time of implantation (Fig. 1). The Teflon coating covering those sections of the wire passing through the pipes was removed. Two electrodes, one implanted close to the muscle origin and the other implanted close to the muscle insertion, were used to MATERIALS AND METHODS stimulate the muscle (Fig. 2). The electrode nearest the neuromuscular junction served as the cathode. A pair Experiment I (Continuous Stimulation of Dissected Mus- of stainless steel wires running from the electrodes to cles). Forty-six rabbits weighing 2.5 to 3.5 kg were an electrode unit on the rabbit's cranium were laid out used. The test material was the caput breve of the subcutaneously. The electrode unit was enclosed in rectus femoris muscle which originates from the spina resin to form a "boat," with a concave bottom to fit the iliaca anterior inferior and inserts at the patella. The rabbit cranium (Fig. 3A). Ten or more holes were drilled muscle is spindle-shaped, easily dissected, and pos- in the cranium with an airtome for positioning of an sesses large nutrient vessels and a large motor nerve. electrode unit that was set in place with resin (Fig. 3B). It therefore is considered a good model for experimen- Stimulation routes were protected with spring rings to offset movement of the rabbit in its cage (Fig. 4). The tal free muscle transfers.3-7
Department of Plastic Surgery, School of Medicine, luntendo University, Tokyo, Japan and Department of Plastic and Reconstructive Surgery, University of Tokyo Hospital Reprint requests-. Dr Yanai, Dept. of Plastic Surgery, School of Medicine, luntendo University, 2-2-1, Hongo, Bunkyo-ku, Tokyo 113, Japan Accepted for publication October 15, 1990 Copyright © 1991 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
85
APRIL 1991
Figure I. Implantable electrode. A, Stainless steel wire is passed through two lined up stainless steel pipes. B, Status of electrode after implantation.
exogenous stimulating equipment consisted of an MEC stimulator and an isolator. Rabbits were divided into the following two groups: Group 1 (Experiment 1-1). The right rectus femoris muscle of the rabbit was denervated by severing a 1-cm length of the femoral nerve close to the inguinal ligament. The wound was closed and the muscle biopsied one week (subgroup I-lw), two weeks (subgroup I-2w), three weeks (subgroup I-3w), one month (subgroup I-lm), two months (subgroup I-2m), and three months (subgroup I-3w) postoperatively. Changes in the weight of muscles were measured, and a histologic examination performed. The left rectus femoris muscle was left intact to act as a control. Each subgroup consisted of six rabbits, for a total of 36 rabbits. Group 2 (Experiment 1-2). In this group of 10 rabbits, the bilateral rectus femoris muscles were denervated. A pair of electrodes was implanted in the right muscle and bipolar stimulation consisting of 30 cycle, 1 msec pulses for 2 sec at 300 sec intervals was continually applied for two weeks. The left muscle was left untouched. A hyperbolic square wave was used. Visible muscle contractions were first obtained with stimulation of 3 mA, but this had to be increased with time to 8 to 10 mA by the final day to maintain visible contrac-
tions. Five of the 10 rabbits in this group survived the stimulation for two weeks. Experiment II (Continuous Stimulation of Grafted Mus-
cles). Test materials were similar to those used in Experiment I. Twenty rabbits divided into two equal groups of 10 each were used. The left rectus femoris muscle was exposed and an electrode implanted in each side of the muscle. The motor nerve of the muscle was severed close to the inguinal ligament. A skin incision was made in the cheek and neck to expose the facial nerve, common carotid artery, and external jugular vein (Fig. 5A). A free muscle with a neurovascular pedicle was then prepared (Fig. 5B). Muscle grafting was implemented by fixing one end at the posterior margin of the mandible and the other at the dermis of the upper lip. The donor site selected was on the side contralateral to the recipient site, in view of the relationship between the recipient site and the location of the muscle neurovascular pedicle. The grafted muscle was maintained at the same length it had been in the donor site. The common carotid artery and the muscle external iliac artery were joined by end-to-side anastomosis, the external jugular vein and the external iliac vein by end-to-end anastomosis, and the facial nerve and
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JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 7, NUMBER 2
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PREVENTING DENERVATION ATROPHY/YANAI, HARII, OKABE
B Figure 2. Rectus femoris muscle of rabbit. A, Dissection of muscle. B, Implantation of the electrodes in both ends of muscle.
Figure 4. State in which continuous stimulation is performed.
the femoral nerve by suturing (Fig. 5C). The surgical microscope was a Zeiss OPMi, and the nylon suture with needle S&T 10V-43. Group 1 (Experiment II-l). The left rectus femoris muscle was harvested and grafted to the right face. The right rectus femoris muscle was denervated, as in Experiment 1, by removing a 1-cm length of the femoral nerve, and was used as a control. Group 2 (Experiment 11-2). As in Group I, the left rectus femoris muscle was harvested and grafted to
the right face. It was then subjected to continuous stimulation for three weeks (Fig. 5D). Again, as in Experiment II-l, the right rectus femoris muscle was denervated and used as a control. In both Groups 1 and 2, vessel occlusion was limited to 90 min. The strength of isometric muscle contraction and the weight of the muscles were measured at three weeks postoperatively in both groups. Histologic examinations were conducted simultaneously and a histogram of muscle fiber diameters was compiled. Iso-
B Figure 3. Electrode unit. A, Electrode unit imbedded in resin. B, Unit affixed to cranial bone with resin.
87
JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 7, NUMBER 2
APRIL 1991
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Figure 5. Free muscle grafting and installation of stimulation system. A, Recipient site. Common carotid artery, external jugular vein, and facial nerve are exposed. B, Free rectus femoris muscle with two electrodes prior to grafting. Clips are affixed to external iliac vessels. C, Grafting of rectus femoris muscle to face. Anastomoses of arteries and veins, and suturing of nerves has been completed. D, Electrode system has been positioned and skin closure completed.
metric contraction was measured at a preload tension of 50 g and stimulation of 100 Hz/50V. Five of the 10 rabbits in Group 1 and three of the 10 in Group 2 successfully survived all procedures in Experiments II-1 and II-2.
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Experiment I. Changes in the weight of the denervated muscles in Group 1 are shown in Figure 6. One 3M 2M IW 2W 3W IM week after the operation, the denervated muscle weighed about 11 percent less than the normal control muscle, Time and in the second or later weeks, displayed significant Figure 6. Experiment 1-1: Changes in weight of disatrophy (2w-. 32 percent, 3w: 39 percent, lm: 47 per- sected rectus femoris muscle after denervation. cent, 2m: 49 percent, 3m: 51 percent). On the basis of the Group 1 results, we considered that it would be permissible to investigate muscle atrophy for less than two weeks, to judge whether or not electric stimulation was effective for prevention of denervation atrophy. This is why we decided on a twoweek period of continuous electric stimulation for
Downloaded by: NYU. Copyrighted material.
B
PREVENTING DENERVATION ATROPHY/YANAI, HARM, OKABE
Table 1. Experimental Results, Experiment 1-2. Changes in Weight of Denervated Muscle with Stimulation Rabbit 1-2-G-l G-2 G-3 G-4 G-5
Weight (stimulated)
Weight (control)
59 g
50 g
8.0
6.5 5.5 5.4 6.1
6.5 5.9
6.5
Differences + 18% + 23% + 18% + 9% + 6.6% + 14.9% ± 6.9%
than the denervated control muscle, while the continuously stimulated grafted muscle (Group 2) had a weight practically equal to that of the denervated control muscle. Isometric contraction strength of the nonstimulated grafted muscles (Group 1) was about 40 g and that of stimulated grafted muscles (Group 2) about 20 g (Fig. 8). Isometric contraction strength of denervated control muscles in both Groups 1 and 2 was about 300 g. Histologic examination indicated an increase in endomysium connective tissue and the presence of adipose tissue in the group of non-stimulated grafted muscles. Contrary to this, significant atrophy and even degenerative necrosis of some fibers were seen in continuously stimulated grafted muscles, even when samples were obtained from the central parts of muscle (Fig. 9). Figure 10 is a comparative histogram of grafted muscle fiber diameters. Fibers of stimulated grafted muscles showed the smallest diameters, and fibers of control denervated muscles the largest diameters. Histochemically and histologically, there were no significant differences between Groups 1 and 2. The results indicated that atrophy would be promoted, rather than prevented, by continuous stimulation of grafted muscles under the prescribed conditions.
DISCUSSION Control count ! 758 M=38.6 S =15.3
Figure 7. Experiment 1-2: Comparison of diameters of dissected muscle fibers with post-denervation stimulation and without stimulation.
In 1970, Tamai et al.10 carried out experiments which validated that free muscle transfer by suturing small vessels and nerves was feasible. Clinical application of this concept was first achieved by Harii and colleagues in 1973" when they carried out a gracilis muscle transfer in a case of facial paralysis, resulting in successful dynamic reconstruction. Following this, free muscle transfer has come into wide use for regaining of motility in such cases as functional deficits of limb or face, once thought impossible to achieve.1213
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Group 2. The results, shown in Table 1, indicate that Table 2. Experimental Results, Experiment II. Changes there was a greater decrease in weight in the control in Weight of Grafted Muscle With and Without Stimulation (Three Weeks after Denervation) group than in the stimulated muscle subgroup, while Weight in Group 1, non-stimulated muscle weighed about 32 Rabbit Weight (control) Differences percent less than the normal muscle control (see II-1 No. 1 8.0 g 60 g + 33.3% Fig. 6). (without No. 2 8.5 6.0 + 41.7% Figure 7 is a histogram of muscle fiber diameters stimulation) 7.7 No. 3 + 24.2% 6.2 in Group 2. Significant differences between stimulated 8.4 No. 4 6.2 + 23.5% denervated muscle and control non-stimulated denerNo. 5 7.8 5.8 + 34.5% vated muscle suggest that denervation atrophy could, 11-2 No. 6 6.5 g 6.2 g + 4.8% (with No. 7 to some extent, be prevented through continuous elec6.4 + 12.3% 5.7 stimulation) No. 8 6.8 5.8 + 17.2% tric stimulation of the denervated muscle. Experiment II. Changes in weight of the grafted and denervated control muscles are shown in Table 2. The non-stimulated grafted muscle gained more weight
JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 7, NUMBER 2
APRIL 1991
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Figure 8. Experiment II: Measurement of isometric contraction (50 g in stationary state). A, Normal rectus femoris muscle, about 500 g. B, Control muscle (three weeks after denervation), about 300 g. C, Grafted muscle (with stimulation, three weeks after denervation), 40 g. D, Grafted muscle (with stimulation, three weeks after denervation), about 20 g.
\
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Figure 9. Histology of denervated muscle. A, Control muscle (three weeks after denervation). B, Grafted muscle (no stimulation, three weeks after denervation). C, Grafted muscle (with stimulation, three weeks after denervation).
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—
C i
. - . . •
i
30-
i t h stimulation (II—No. 7 ) 20" count '. 533 M=28.5 10-
0
10"
S =14.6
20
40
60
80
100
120
Diameter (M)
•ithout stimulation ( I —No 2 ) count : 391 M=46. 1
0-
20-
S =20.02
30-
Figure 10. Experiment II-2. Comparison of diameters of grafted muscle fibers with post-denervation stimulation and without stimulation.
Currently, fundamental studies are continually being conducted on such subjects as the reinnervation process of grafted muscles, the strength of reinnervated muscles, and the safe time span for vessel occlusion. 3514 - 16 Regarding the strength of a grafted muscle, many factors are involved, such as the surgical technique, the condition of the recipient site (selection of the recipient nerve in particular), and the period of vessel occlusion. It is necessary that a grafted muscle be able to restore its power sufficiently to function well. In this study, we probed the possibility of preventing denervation atrophy by means of electric stimulation of muscles (dissected and grafted muscle). We were of the opinion that it was necessary for stimulation to be applied during the time when reinnervation was in progress. Nerve stimulation and muscle contraction through the use of implanted electrodes have seen wide application in the fields of orthopedic surgery, neurosurgery, and cardiosurgery.17-22 Many of these efforts, however, have been intended for treatment of such upper motor neuron disorders as phrenic paralysis and hemiplegia due to encephalomyelopathy. In almost all such cases, the nerves stimulated are regenerated nerves. The final goal of our study was to prevent atrophy of grafted muscles. We therefore conducted experiments on preventing acute-phase atrophy of denervated muscles due to peripheral nerve disorder. Since stimulation was applied immediately after denervation and prior to completion of reinnervation, we selected muscle stimulation rather than nerve stimulation. In Experiment I, we found that atrophy of dissected muscle could be prevented to some extent but in Experiment
II, we failed to prevent denervation atrophy through stimulation of grafted muscles. We used "sandwich" type electrodes in our experiments in order to obtain stable and effective stimulation. We would not have been able to apply stable stimulation, had we used needle electrodes or plate electrodes, which are used clinically in EMG examinations. Our "sandwich" electrode could be fixed in the muscle, to provide relatively stable and effective stimulation. However, even using this innovation of ours did not help us complete all procedures of continuous stimulation in some rabbits. In particular, we lost many rabbits in Experiment II-2 in which we had to overcome two peaks, free muscle grafting and continuous stimulation. Infection was the main cause of animal loss during continuous stimulation, and even small shocks, as from anesthetization, resulted in rabbit death. We lost some rabbits for these reasons in Experiments I and II-l, but these were quickly replaced. The main difference in results between dissected muscles and grafted muscles could be explained by vessel occlusion and the duration of such occlusion. Duration of occlusion may be the more important element. It is likely that "fatigue" may be the more significant problem in grafted muscles in which blood circulation has been blocked for some interval. A future solution to the problem of fatigue may be found through clarification and selection of the propertiming for beginning stimulation. In our experiments, it was difficult to distinguish histologically and histochemically between type 1 fibers and type 2 fibers, because the manipulation of muscles within three weeks after denervation was involved. Problems for future study include the following. 1. At what time after grafting should stimulation of grafted muscle begin? 2. What patterns (shape of wave, intervals, frequency, duration, length, strength, etc.) should we select for effective stimulation? 3. At what point and how should we evaluate the degree of muscle atrophy?
REFERENCES Harii K: Microvascular Tissue Transfer. Tokyo, New York: Igaku-
shoin, 1983 O'Brien BM, Morrison WA: Reconstructive Microsurgery. New York: Churchill Livingstone, 1987 Yamada A: Experimental study on free muscle transplantation with neurovascular anastomosis in rabbits. I Jpn Soc Plast Reconstr Surg 2:147, 1982 Terzis IK, Sweet RC, Dyke RW: Recovery of function in free muscle transplants using microneurovascular anastomoses. I Hand Surg 3:37, 1978 Yasunaga H: An experimental study on free muscle transplantation. | Jap Soc Orthoped Surg 58:1267, 1984 91
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JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 7, NUMBER 2
7 8. 9. 10. 11. 12. 13 14.
Frey M, Gruber H, Havel M, et a\.\ Experimental free muscle transplantation with microneurovascular anastomoses. Plast Reconstr Surg 71:689, 1983 GuelinckxPJ, Dom R, Bex M, etal.-. Rectus femoris muscle grafts performed with and without vascular anastomosis: An experimental study in the rabbit. Br 1 Plast Surg 37:584, 1984 Darr NL: Rabbit intubation and halothane anesthesia. Lab An Sci 24:617, 1974 Yanai A, Harii K, Okabe K, et a\.\ An original method for endotracheal intubation of rabbits. Res Exp Med 186:117, 1986 Tamai S, Komatsu S, Sakamoto H, et al.-. Free muscle transplantation in dogs, with microsurgical neurovascular anastomoses. Plast Reconstr Surg 46:219, 1970 Harii K, Ohmori K, Torii S: Free gracilis muscle transplantation, with microneurovascular anastomosis for the treatment of facial paralysis. Plast Reconstr Surg 57:133, 1976 Manktelow RT, McKee N: Free muscle transplantation to provide active finger flexion. I Hand Surg 3:416, 1978 Serafin D, Smith P|: Vascularized transplantation of skeletal muscle. I Microsurg 1:529, 1980 Yoshioka K: An experimental study of free muscle graft. ) lap Soc Orthoped Surg 55:1384, 1981
15.
Kubo T, Ikuta Y, Tsuge K: Free muscle transplantation in dogs by microneurovascular anastomosis. Plast Reconstr Surg 55: 495, 1976 16. Frey M, Gruber H, Freilinger G: The importance of the correct resting tension in muscle transplantation: Experimental and clinical aspects. Plast Reconstr Surg 71:510, 1983 17. Lieberman IS: Serial phrenic nerve conduction studies in candidates for diaphragm pacing. Arch Phys Med Rehabil 61: 528, 1980 18. Sigrist GVM: An implantable myography electrode for recording muscle activity in freely moving small animals. IEEE Trans Biomed Eng BME-29:730, 1982 19. Glenn WL: Long-term ventilatory support by diaphragm pacing in quadriplegia. Ann Surg 183:566, 1976 20. Hoffer JA: Implantable electrical and mechanical interfaces with nerve and muscle. Ann Biomed Eng 8:351, 1980 21. Meier RH: Evaluation of cervical spinal cord injured patients for electrophrenic respiration. Arch Phys Med Rehabil 60:186, 1979 22 Nemoto K, Williamas HB, Nemoto K, et al: The effects of electrical stimulation on denervated muscle using implantable electrodes I Reconstr Microsurg 4:251, 1988
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APRIL 1991
PREVENTING DENERVATION ATROPHY OF A GRAFTED MUSCLE ABSTRACT
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Electrodes were implanted in the grafted muscles of rabbits to generate continuous stimulation for the purpose of preventing denervation atrophy. Denervation atrophy could be prevented, to some extent, in dissected muscles with vascular pedicles not occluded during the experiment. Denervation atrophy was found to be promoted, rather than prevented, in grafted muscles with occluded vascular pedicles. The crucial conditions are varying stimulation and the long duration of occlusion (90 min). Overly strong stimulation of muscle contraction appears to result in "fatigue" of grafted muscles in which blood circulation has been blocked for more than the 90 min used in the reported experiment. Further investigations are necessary of the conditions of stimulation and the point in time at which stimulation should commence after grafting. Free muscle transfer has come into wide clinical use in recent years, parallel with general advances in microsurgical tissue transfer.12 However, a serious problem has remained in that grafted muscles may be affected by denervation atrophy during the period between grafting and the regeneration of nerves. It would therefore be highly beneficial to find a means of preventing muscle atrophy during such a period. In an attempt to investigate the possibility of preventing denervation atrophy of grafted muscle, we conducted an experimental study using rabbits whose muscles were denervated and implanted with electrodes for continuous exogenous stimulation. We examined the resulting degree of atrophy over time.
The rabbits were anesthetized with an intravenous Nembutal injection through the auricular vein and then subjected to continuous GOF anesthesia by means of orotracheal intubation. 89 The bilateral inguinal region of each rabbit was shaved, and the skin extending from the femur region to the inguinal region was incised. A stimulating electrode consisted of two stainless steel pipes and woven Teflon-coated stainless wire was employed. Each pipe was 7 mm long and had an outer diameter of 0.6 mm. The pipes were lined up about 3 mm apart and then placed parallel to sandwich muscle at the time of implantation (Fig. 1). The Teflon coating covering those sections of the wire passing through the pipes was removed. Two electrodes, one implanted close to the muscle origin and the other implanted close to the muscle insertion, were used to MATERIALS AND METHODS stimulate the muscle (Fig. 2). The electrode nearest the neuromuscular junction served as the cathode. A pair Experiment I (Continuous Stimulation of Dissected Mus- of stainless steel wires running from the electrodes to cles). Forty-six rabbits weighing 2.5 to 3.5 kg were an electrode unit on the rabbit's cranium were laid out used. The test material was the caput breve of the subcutaneously. The electrode unit was enclosed in rectus femoris muscle which originates from the spina resin to form a "boat," with a concave bottom to fit the iliaca anterior inferior and inserts at the patella. The rabbit cranium (Fig. 3A). Ten or more holes were drilled muscle is spindle-shaped, easily dissected, and pos- in the cranium with an airtome for positioning of an sesses large nutrient vessels and a large motor nerve. electrode unit that was set in place with resin (Fig. 3B). It therefore is considered a good model for experimen- Stimulation routes were protected with spring rings to offset movement of the rabbit in its cage (Fig. 4). The tal free muscle transfers.3-7
Department of Plastic Surgery, School of Medicine, luntendo University, Tokyo, Japan and Department of Plastic and Reconstructive Surgery, University of Tokyo Hospital Reprint requests-. Dr Yanai, Dept. of Plastic Surgery, School of Medicine, luntendo University, 2-2-1, Hongo, Bunkyo-ku, Tokyo 113, Japan Accepted for publication October 15, 1990 Copyright © 1991 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
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APRIL 1991
Figure I. Implantable electrode. A, Stainless steel wire is passed through two lined up stainless steel pipes. B, Status of electrode after implantation.
exogenous stimulating equipment consisted of an MEC stimulator and an isolator. Rabbits were divided into the following two groups: Group 1 (Experiment 1-1). The right rectus femoris muscle of the rabbit was denervated by severing a 1-cm length of the femoral nerve close to the inguinal ligament. The wound was closed and the muscle biopsied one week (subgroup I-lw), two weeks (subgroup I-2w), three weeks (subgroup I-3w), one month (subgroup I-lm), two months (subgroup I-2m), and three months (subgroup I-3w) postoperatively. Changes in the weight of muscles were measured, and a histologic examination performed. The left rectus femoris muscle was left intact to act as a control. Each subgroup consisted of six rabbits, for a total of 36 rabbits. Group 2 (Experiment 1-2). In this group of 10 rabbits, the bilateral rectus femoris muscles were denervated. A pair of electrodes was implanted in the right muscle and bipolar stimulation consisting of 30 cycle, 1 msec pulses for 2 sec at 300 sec intervals was continually applied for two weeks. The left muscle was left untouched. A hyperbolic square wave was used. Visible muscle contractions were first obtained with stimulation of 3 mA, but this had to be increased with time to 8 to 10 mA by the final day to maintain visible contrac-
tions. Five of the 10 rabbits in this group survived the stimulation for two weeks. Experiment II (Continuous Stimulation of Grafted Mus-
cles). Test materials were similar to those used in Experiment I. Twenty rabbits divided into two equal groups of 10 each were used. The left rectus femoris muscle was exposed and an electrode implanted in each side of the muscle. The motor nerve of the muscle was severed close to the inguinal ligament. A skin incision was made in the cheek and neck to expose the facial nerve, common carotid artery, and external jugular vein (Fig. 5A). A free muscle with a neurovascular pedicle was then prepared (Fig. 5B). Muscle grafting was implemented by fixing one end at the posterior margin of the mandible and the other at the dermis of the upper lip. The donor site selected was on the side contralateral to the recipient site, in view of the relationship between the recipient site and the location of the muscle neurovascular pedicle. The grafted muscle was maintained at the same length it had been in the donor site. The common carotid artery and the muscle external iliac artery were joined by end-to-side anastomosis, the external jugular vein and the external iliac vein by end-to-end anastomosis, and the facial nerve and
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JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 7, NUMBER 2
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PREVENTING DENERVATION ATROPHY/YANAI, HARII, OKABE
B Figure 2. Rectus femoris muscle of rabbit. A, Dissection of muscle. B, Implantation of the electrodes in both ends of muscle.
Figure 4. State in which continuous stimulation is performed.
the femoral nerve by suturing (Fig. 5C). The surgical microscope was a Zeiss OPMi, and the nylon suture with needle S&T 10V-43. Group 1 (Experiment II-l). The left rectus femoris muscle was harvested and grafted to the right face. The right rectus femoris muscle was denervated, as in Experiment 1, by removing a 1-cm length of the femoral nerve, and was used as a control. Group 2 (Experiment 11-2). As in Group I, the left rectus femoris muscle was harvested and grafted to
the right face. It was then subjected to continuous stimulation for three weeks (Fig. 5D). Again, as in Experiment II-l, the right rectus femoris muscle was denervated and used as a control. In both Groups 1 and 2, vessel occlusion was limited to 90 min. The strength of isometric muscle contraction and the weight of the muscles were measured at three weeks postoperatively in both groups. Histologic examinations were conducted simultaneously and a histogram of muscle fiber diameters was compiled. Iso-
B Figure 3. Electrode unit. A, Electrode unit imbedded in resin. B, Unit affixed to cranial bone with resin.
87
JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 7, NUMBER 2
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Figure 5. Free muscle grafting and installation of stimulation system. A, Recipient site. Common carotid artery, external jugular vein, and facial nerve are exposed. B, Free rectus femoris muscle with two electrodes prior to grafting. Clips are affixed to external iliac vessels. C, Grafting of rectus femoris muscle to face. Anastomoses of arteries and veins, and suturing of nerves has been completed. D, Electrode system has been positioned and skin closure completed.
metric contraction was measured at a preload tension of 50 g and stimulation of 100 Hz/50V. Five of the 10 rabbits in Group 1 and three of the 10 in Group 2 successfully survived all procedures in Experiments II-1 and II-2.
100. \Oo
c CD
o
m
n
RESULTS
V^§o
o
f \ So""---CL 0 Oo 0 0
0
oo D°
0 0
oo_
Experiment I. Changes in the weight of the denervated muscles in Group 1 are shown in Figure 6. One 3M 2M IW 2W 3W IM week after the operation, the denervated muscle weighed about 11 percent less than the normal control muscle, Time and in the second or later weeks, displayed significant Figure 6. Experiment 1-1: Changes in weight of disatrophy (2w-. 32 percent, 3w: 39 percent, lm: 47 per- sected rectus femoris muscle after denervation. cent, 2m: 49 percent, 3m: 51 percent). On the basis of the Group 1 results, we considered that it would be permissible to investigate muscle atrophy for less than two weeks, to judge whether or not electric stimulation was effective for prevention of denervation atrophy. This is why we decided on a twoweek period of continuous electric stimulation for
Downloaded by: NYU. Copyrighted material.
B
PREVENTING DENERVATION ATROPHY/YANAI, HARM, OKABE
Table 1. Experimental Results, Experiment 1-2. Changes in Weight of Denervated Muscle with Stimulation Rabbit 1-2-G-l G-2 G-3 G-4 G-5
Weight (stimulated)
Weight (control)
59 g
50 g
8.0
6.5 5.5 5.4 6.1
6.5 5.9
6.5
Differences + 18% + 23% + 18% + 9% + 6.6% + 14.9% ± 6.9%
than the denervated control muscle, while the continuously stimulated grafted muscle (Group 2) had a weight practically equal to that of the denervated control muscle. Isometric contraction strength of the nonstimulated grafted muscles (Group 1) was about 40 g and that of stimulated grafted muscles (Group 2) about 20 g (Fig. 8). Isometric contraction strength of denervated control muscles in both Groups 1 and 2 was about 300 g. Histologic examination indicated an increase in endomysium connective tissue and the presence of adipose tissue in the group of non-stimulated grafted muscles. Contrary to this, significant atrophy and even degenerative necrosis of some fibers were seen in continuously stimulated grafted muscles, even when samples were obtained from the central parts of muscle (Fig. 9). Figure 10 is a comparative histogram of grafted muscle fiber diameters. Fibers of stimulated grafted muscles showed the smallest diameters, and fibers of control denervated muscles the largest diameters. Histochemically and histologically, there were no significant differences between Groups 1 and 2. The results indicated that atrophy would be promoted, rather than prevented, by continuous stimulation of grafted muscles under the prescribed conditions.
DISCUSSION Control count ! 758 M=38.6 S =15.3
Figure 7. Experiment 1-2: Comparison of diameters of dissected muscle fibers with post-denervation stimulation and without stimulation.
In 1970, Tamai et al.10 carried out experiments which validated that free muscle transfer by suturing small vessels and nerves was feasible. Clinical application of this concept was first achieved by Harii and colleagues in 1973" when they carried out a gracilis muscle transfer in a case of facial paralysis, resulting in successful dynamic reconstruction. Following this, free muscle transfer has come into wide use for regaining of motility in such cases as functional deficits of limb or face, once thought impossible to achieve.1213
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Group 2. The results, shown in Table 1, indicate that Table 2. Experimental Results, Experiment II. Changes there was a greater decrease in weight in the control in Weight of Grafted Muscle With and Without Stimulation (Three Weeks after Denervation) group than in the stimulated muscle subgroup, while Weight in Group 1, non-stimulated muscle weighed about 32 Rabbit Weight (control) Differences percent less than the normal muscle control (see II-1 No. 1 8.0 g 60 g + 33.3% Fig. 6). (without No. 2 8.5 6.0 + 41.7% Figure 7 is a histogram of muscle fiber diameters stimulation) 7.7 No. 3 + 24.2% 6.2 in Group 2. Significant differences between stimulated 8.4 No. 4 6.2 + 23.5% denervated muscle and control non-stimulated denerNo. 5 7.8 5.8 + 34.5% vated muscle suggest that denervation atrophy could, 11-2 No. 6 6.5 g 6.2 g + 4.8% (with No. 7 to some extent, be prevented through continuous elec6.4 + 12.3% 5.7 stimulation) No. 8 6.8 5.8 + 17.2% tric stimulation of the denervated muscle. Experiment II. Changes in weight of the grafted and denervated control muscles are shown in Table 2. The non-stimulated grafted muscle gained more weight
JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 7, NUMBER 2
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Figure 8. Experiment II: Measurement of isometric contraction (50 g in stationary state). A, Normal rectus femoris muscle, about 500 g. B, Control muscle (three weeks after denervation), about 300 g. C, Grafted muscle (with stimulation, three weeks after denervation), 40 g. D, Grafted muscle (with stimulation, three weeks after denervation), about 20 g.
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Figure 9. Histology of denervated muscle. A, Control muscle (three weeks after denervation). B, Grafted muscle (no stimulation, three weeks after denervation). C, Grafted muscle (with stimulation, three weeks after denervation).
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Figure 10. Experiment II-2. Comparison of diameters of grafted muscle fibers with post-denervation stimulation and without stimulation.
Currently, fundamental studies are continually being conducted on such subjects as the reinnervation process of grafted muscles, the strength of reinnervated muscles, and the safe time span for vessel occlusion. 3514 - 16 Regarding the strength of a grafted muscle, many factors are involved, such as the surgical technique, the condition of the recipient site (selection of the recipient nerve in particular), and the period of vessel occlusion. It is necessary that a grafted muscle be able to restore its power sufficiently to function well. In this study, we probed the possibility of preventing denervation atrophy by means of electric stimulation of muscles (dissected and grafted muscle). We were of the opinion that it was necessary for stimulation to be applied during the time when reinnervation was in progress. Nerve stimulation and muscle contraction through the use of implanted electrodes have seen wide application in the fields of orthopedic surgery, neurosurgery, and cardiosurgery.17-22 Many of these efforts, however, have been intended for treatment of such upper motor neuron disorders as phrenic paralysis and hemiplegia due to encephalomyelopathy. In almost all such cases, the nerves stimulated are regenerated nerves. The final goal of our study was to prevent atrophy of grafted muscles. We therefore conducted experiments on preventing acute-phase atrophy of denervated muscles due to peripheral nerve disorder. Since stimulation was applied immediately after denervation and prior to completion of reinnervation, we selected muscle stimulation rather than nerve stimulation. In Experiment I, we found that atrophy of dissected muscle could be prevented to some extent but in Experiment
II, we failed to prevent denervation atrophy through stimulation of grafted muscles. We used "sandwich" type electrodes in our experiments in order to obtain stable and effective stimulation. We would not have been able to apply stable stimulation, had we used needle electrodes or plate electrodes, which are used clinically in EMG examinations. Our "sandwich" electrode could be fixed in the muscle, to provide relatively stable and effective stimulation. However, even using this innovation of ours did not help us complete all procedures of continuous stimulation in some rabbits. In particular, we lost many rabbits in Experiment II-2 in which we had to overcome two peaks, free muscle grafting and continuous stimulation. Infection was the main cause of animal loss during continuous stimulation, and even small shocks, as from anesthetization, resulted in rabbit death. We lost some rabbits for these reasons in Experiments I and II-l, but these were quickly replaced. The main difference in results between dissected muscles and grafted muscles could be explained by vessel occlusion and the duration of such occlusion. Duration of occlusion may be the more important element. It is likely that "fatigue" may be the more significant problem in grafted muscles in which blood circulation has been blocked for some interval. A future solution to the problem of fatigue may be found through clarification and selection of the propertiming for beginning stimulation. In our experiments, it was difficult to distinguish histologically and histochemically between type 1 fibers and type 2 fibers, because the manipulation of muscles within three weeks after denervation was involved. Problems for future study include the following. 1. At what time after grafting should stimulation of grafted muscle begin? 2. What patterns (shape of wave, intervals, frequency, duration, length, strength, etc.) should we select for effective stimulation? 3. At what point and how should we evaluate the degree of muscle atrophy?
REFERENCES Harii K: Microvascular Tissue Transfer. Tokyo, New York: Igaku-
shoin, 1983 O'Brien BM, Morrison WA: Reconstructive Microsurgery. New York: Churchill Livingstone, 1987 Yamada A: Experimental study on free muscle transplantation with neurovascular anastomosis in rabbits. I Jpn Soc Plast Reconstr Surg 2:147, 1982 Terzis IK, Sweet RC, Dyke RW: Recovery of function in free muscle transplants using microneurovascular anastomoses. I Hand Surg 3:37, 1978 Yasunaga H: An experimental study on free muscle transplantation. | Jap Soc Orthoped Surg 58:1267, 1984 91
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PREVENTING DENERVATION ATROPHY/YANAI, HARM, OKABE
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7 8. 9. 10. 11. 12. 13 14.
Frey M, Gruber H, Havel M, et a\.\ Experimental free muscle transplantation with microneurovascular anastomoses. Plast Reconstr Surg 71:689, 1983 GuelinckxPJ, Dom R, Bex M, etal.-. Rectus femoris muscle grafts performed with and without vascular anastomosis: An experimental study in the rabbit. Br 1 Plast Surg 37:584, 1984 Darr NL: Rabbit intubation and halothane anesthesia. Lab An Sci 24:617, 1974 Yanai A, Harii K, Okabe K, et a\.\ An original method for endotracheal intubation of rabbits. Res Exp Med 186:117, 1986 Tamai S, Komatsu S, Sakamoto H, et al.-. Free muscle transplantation in dogs, with microsurgical neurovascular anastomoses. Plast Reconstr Surg 46:219, 1970 Harii K, Ohmori K, Torii S: Free gracilis muscle transplantation, with microneurovascular anastomosis for the treatment of facial paralysis. Plast Reconstr Surg 57:133, 1976 Manktelow RT, McKee N: Free muscle transplantation to provide active finger flexion. I Hand Surg 3:416, 1978 Serafin D, Smith P|: Vascularized transplantation of skeletal muscle. I Microsurg 1:529, 1980 Yoshioka K: An experimental study of free muscle graft. ) lap Soc Orthoped Surg 55:1384, 1981
15.
Kubo T, Ikuta Y, Tsuge K: Free muscle transplantation in dogs by microneurovascular anastomosis. Plast Reconstr Surg 55: 495, 1976 16. Frey M, Gruber H, Freilinger G: The importance of the correct resting tension in muscle transplantation: Experimental and clinical aspects. Plast Reconstr Surg 71:510, 1983 17. Lieberman IS: Serial phrenic nerve conduction studies in candidates for diaphragm pacing. Arch Phys Med Rehabil 61: 528, 1980 18. Sigrist GVM: An implantable myography electrode for recording muscle activity in freely moving small animals. IEEE Trans Biomed Eng BME-29:730, 1982 19. Glenn WL: Long-term ventilatory support by diaphragm pacing in quadriplegia. Ann Surg 183:566, 1976 20. Hoffer JA: Implantable electrical and mechanical interfaces with nerve and muscle. Ann Biomed Eng 8:351, 1980 21. Meier RH: Evaluation of cervical spinal cord injured patients for electrophrenic respiration. Arch Phys Med Rehabil 60:186, 1979 22 Nemoto K, Williamas HB, Nemoto K, et al: The effects of electrical stimulation on denervated muscle using implantable electrodes I Reconstr Microsurg 4:251, 1988
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