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Peripheral nerve reconnection: Immediate histologic consequences of distributed mechanical support
EXPERIMENTAL
NEUROLOGY
(1983)
81,459-468
Peripheral Nerve Reconnection: Consequences of Distributed LUIS DE MEDINACELI
Immediate Mechanical
AND WILLIAM
Histologic Support
J. FREED’
Adult Psychiatry Branch, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032 Received February 16, I983 We tested the possibility of improving the quality of axonal repair after peripheral nerve transection by suppression of all mechanical stressesfrom the zone of repair. A method was developed involving mechanical support of the sciatic nerve from below in a grooved chamber with additional support provided by suturing the nerve to a rubber sleeve. Histological studies of silver-impregnated specimens from rabbit sciatic nerve using this technique revealed that the axons’ tips were closely reconnected with minimal disruption in their direction or in the alignment of fibers and without extraneous material between the severed stumps. Comparison of these results with those of current microsurgical techniques showed the unequivocal superiority of this method in terms of immediate histologic aspects.The course of Walletian degeneration was not modified.
INTRODUCTION It is generally considered axiomatic that the technical quality of the repair of severed peripheral nerves has some influence on the eventual outcome. There is considerable evidence that a wide gap (3, 5, 9, 10, 16, 20), the presence of blood or foreign material (16), fibrous scarring (6, 12, 16, 18), erroneous alignment of the stumps (1, 7, 11, 17), or any other mechanical interference with axonal regrowth can result in less than optimal regeneration. Thus, the ideal peripheral nerve reconnection is generally considered to be that which places each severed axon in close contact with its distai end without distortion and without intervening foreign material. Current microsurgical techniques stress the superiority of perineurial over epineurial repair, ’ We thank Richard Jed Wyatt, Barry J. Hoffer, Anthony Seaber, Miguel de Medinaceli, Alan Fine, and the Division of Special Mental Health Research staff for their invaluable assistance and encouragement during many phases of this project. We also thank Eleanor Krauthamer for preparing the histologic materials and Denise Ondrish for preparing the manuscript. 459 0014-4886/83 $3.00 Copyright 0 1983 by Academic Press. Inc All rights of reproduction in any form reserved.
460
DE
MEDINACELI
AND
FREED
essentially due to the better fascicular alignment (2). Even a well done perineurial repair, however, leaves a massive disorganization at the axonal level as shown by the histologic appearance of the site of any nerve microsuture (8, 14, 19). The purpose of our work was to study the possibility that axonal disorganization could be decreased through the use of a new technique of reconnection of the stumps involving distribution of the mechanical stress on the nerve to regions removed from the transection site (Fig. 1). Cooling was routinely used for slowing the metabolic phenomena with the idea that this might enhance the quality of the repair. Mild transient cooling is known to be harmless to nerve trunks (4). MATERIALS
AND
METHODS
Forty albino rabbits weighing 2 to 3 kg were anesthetized with phenobarbital (Nembutal), i.p., and xylazine (Rompun), i.m. The sciatic trunk was exposed and dissected from hip to knee. The nerve was laid in the groove of a small copper cooling chamber coated with rubber (the finger of a rubber glove). The nerve was sutured to the chamber with a stitch of 6-O silk through the epineural sheath 3 mm from the extremity of the groove and then through the rubber coating at the edge of the groove. Three other identical stitches were made as shown in Fig. 2a. When they were tied the nerve was firmly sutured to the chamber so that there was no longitudinal tension on the nerve in the center of the preparation. Local temperature was recorded from
FIG. 1. Theoretical basis of distributed mechanical support. a-Current techniques of repair concentrate stresses in the zone of repair. We considered this an adverse situation. b-Our method aims at diverting all mechanical stresses laterally, a few millimeters from the zone of transection.
NERVE RECONNECTION
461
b
FIG. 2. Illustration of the preparative procedure. a-The nerve was placed in the groove of a brass cooling chamber coated with a rubber sheet. Stitches were passed through the rubber and nerve to fix the nerve in place and to prevent retraction. b-After transection and reconnection the rubber was cut out around the nerve: a small piece of rubber remained attached to the nerve and prevented retraction and separation of the stumps.
the superior wall of the chamber directly from the metal under the rubber coating. In four animals no cooling was used and the temperature remained about 30°C throughout the experiment. In 36 animals the temperature of the chamber was lowered to a stable 15°C by circulation of cold water. The temperature of the nerve was estimated to be between 17 and 20°C depending on the size of the nerve and the duration of the cooling. The nerve was then lifted slightly from the bottom of the groove and transected with a pair of scissors. Because the nerve was fixed to the chamber no retraction of the stumps occurred. The bleeding of the stumps varied from none to quite heavy. In the first 16 animals no attention was paid to the bleeding and the reconnection was made at once by letting the stumps fall back together. In the 24 other casesno reconnection was made before bleeding had ceased. Blood was removed with a small piece of blotting paper or a few threads of cotton, avoiding contact with the surfaces of transection.
462
DE MEDINACELI
AND FREED
Reconnection was always easy because the stumps fell back in the groove to the correct position. With the use of characteristic features such as minute superficial blood vessels, the alignment could be made very precisely. The temperature was then returned to normal by interrupting the water circulation. The average duration of cooling was 10 min. From 30 min to 1 h after the transection, the rubber of the chamber was cut out around the nerve and the chamber removed. A small rectangular piece of rubber remained attached to the nerve and prevented retraction and separation of the stumps (Fig. 2b). The wound was momentarily closed to avoid drying. Twenty-five nerves were removed from the body from 1 to 6 h after injury. They included seven nerves for which the technical quality of the repair had been considered especially good, those where cooling had not been used (four nerves) and 14 others chosen at random. They were transported with the small rubber support into Bouin’s fluid. After 24 h the rubber was removed, the specimens were coded, embedded in paraffin, and sectioned longitudinally at 7 Km. Selected sections were stained with silver by the method of Holmes (15) and the remaining sections were stained with solachrome cyanin by the method of Page (15). The 15 remaining animals were used to test the soundness of the reconnection. Twenty hours after the injury they were anesthetized again. The four small stitches were cut and the rubber support removed; the nerve was then free of sutures and other material. No special precautions were taken in the further handling and care of the animals. The nerve was checked 6 to 8 days later. Nine additional nerves were used for control experiments: (a) Four nerves were repaired by conventional microscopic perineurial suturing with 9-O silk, (b) in two animals the rubber support was removed 10 h after reconnection, and (c) in three animals the nerve was not transected but was only cooled at 15°C for 1 h. These nine controls were not included among the 40 experimental animals. FIG. 3. Histologic appearance of unsatisfactory repairs (Holmes’ stain). a-A conventional microscopic suturing 2 h after repair. The external appearance of this fascicle was good under the operating microscope and this portion of the fascicle was selected as having the best appearance. Note the distortion of the subfascicles and large pools of blood and foreign material. The gap, indicated by the double arrowheads, is at its smallest here. The average width of the gap was about 400 Frn (bar = 200 pm). b-An unsatisfactory repair with distributed mechanical support, 6 h after reconnection. The axons are only slightly distorted at their extremities and there is little blood between the stumps. A thick, dense scar membrane separating the stumps is evident (bar = 200 pm). c-Another repair with distributed mechanical support 2 h after reconnection. The general appearance is very good with little distortion of the axons, no blood between the stumps, and a fairly close approximation of the axons. A scar membrane is evident (bar = 100 pm). d-A higher magnification of the same specimen. The gap between the stumps is evident although not very wide (10 to 20 pm). There is a thin, but nearly continuous, scar membrane that separates the two stumps (bar = 20 pm).
NERVE
RECONNECT
ION
463
464
DE MEDINACELI
AND FREED
RESULTS Macroscopic Observations
Within 3 to 6 min after reconnection, the gap became less apparent which was attributed to coagulation occurring between the stumps. The gap was usually reduced to a small pink line that was difficult to discern with the naked eye. That appearance remained unchanged during the first few hours. General Microscopic Findings
Attention was focused on six points: (i) the general appearance of the reconnection-did the fascicles face each other with the axons undistorted or were the axons irregular and distorted; (ii) was there an interposition of foreign material (epineurium, fibrous tissue) between the stumps; (iii) were large amounts of blood present in the region of the gap; (iv) what was the distance between the tips of the axons of the two stumps; (v) was a visible line or membrane-like formation present between the stumps; and (vi) in the late specimens (over 4 h), were there early signs of traumatic or Wallerian degeneration? Sutures. One hour after conventional repair, the sutured nerves presented a gap ranging from 100 to 400 pm. That gap was filled with blood, various cell debris, and fragments of epineurial or perineurial sheaths. Disruption of axonal direction was conspicuous in all cases (Fig. 3a). Repairs through Distributed Mechanical Support. (i) Unsatisfactory reconnections [ 18 nerves (Fig. 3b-d)]. In 12 cases the histologic appearance was poor. The fascicles were brushed against each other, and epineural sheath or fibrous tissue was interposed. Large pools of blood or plasma were often seen between the stumps. Nevertheless, these reconnections looked much more precise than those obtained by conventional suturing. In six cases the general appearance was better in that there was little or no distortion of the axons, no interposition of foreign material, and little or no blood. In all 18 cases there was rarely close approximation of the tips of the axons and the extremities of the stump were lined by a continuous formation of scarlike membrane which we considered to be collagen or fibrin formations. Between FIG.4. Histologic appearance of satisfactoryrepairs with distributed mechanical support (Holmes’ stain). a-General appearance of a satisfactory repair 2 h after injury. Compare with Fig. 3a and c. Here the area of injury (arrowheads) is only faintly visible although the axons are evidently distorted (bar = 100 pm). b-Another satisfactory repair. The tips of some axons come very close together. The point of transection is recognizable only as an indistinct bluish zone indicated by the arrowheads. There is no interposed foreign material and no continuous scar membrane (bar = 20 pm). c-e-Three higher magnifications of areas where there is very close apposition between axon tips from opposite sides of the gap. In each case, close examination revealed that the axons did not actually make contact. There was always a small gap, sometimes less than I pm in width, which is not always apparent in the photographs. The points of transection are indicated by arrowheads and the small arrows indicate areas of close apposition between axons. The bars are 8. 20, and 20 pm, respectively.
NERVE
RECONNECTION
465
466
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MEDINACELI
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FREED
the two scar membranes there usually was an evident gap, ranging from 10 to 20 pm. (ii) Satisfactory reconnections (seven nerves) (Fig. 4). These nerves were the last to be repaired in the series. Satisfactory results were attributed to a better technique bearing essentially on the position of the stitches. This controlled the elimination of longitudinal stresses from the repair zone. Better removal of blood from the surface of transection was also considered beneficial. The general appearance of these reconnections was fair, with slight distortion or brushing of the axons in two cases. No foreign material was seen between the stumps except some blood which was found between the stumps in two cases. In three nerves the general aspect was very good with no blood visible. It was usually possible to find at least one large zone where the tips of the transected axons of both stumps came very close to each other. Also, there was no scar membrane or formation visible between the stumps, at least in the zones of good connection. Neither fusion nor true contact between the axons was seen. There were signs of degeneration in the two specimens removed after 6 h (swelling and breaking down of the axons). Soundness of Repair In all 15 animals from which the rubber support had been removed after 20 h, the check at 1 week showed a satisfactory appearance of the nerve. No secondary separation of the stumps was observed. The zone of repair was difficult to locate precisely as it was not marked by any change in caliber but only by a slight change in color. In the two animals from which the rubber support had been removed after 10 h, complete separation of the stumps was observed at 1 week. Influence of Cooling The absence of permanent damage to the nerves caused by transient cooling was confirmed in the three control nerves that were cooled at 15°C (temperature of the chamber walls) for 1 h. In all three animals a transient clinical deficit was observed for 4 to 6 days (loss of the stretching reflex of the toes, diminution of muscular force). After 6 days, normal function was recovered. We did not attempt to determine whether the transient deficits were due to the manipulations of the nerve or to the cooling itself. Histologic examination of those nerves at the 15th day showed that the fibers were intact. The influence of cooling on the quality of the repair was unclear; we noticed only that in the four cases where the nerves were not cooled, the microscopic aspect was not good. DISCUSSION These observations suggest that a significant improvement in peripheral nerve repair quality can be brought about solely by improvements in the
NERVE
RECONNECTION
467
mechanical aspects of the repair technique. In this study we used a reconnection technique that distributes the mechanical tension on the nerve away from the transection site. In particular, lateral stresses are relieved by a surrounding chamber and the longitudinal stresses are displaced to at least a few millimeters away from the transection site. When reconnection was achieved under those conditions, there was a dramatic improvement in the anatomic manifestations of the repair. Minimal whorling or disruption of the axons’ alignment, minimal interposition of foreign material, and close apposition of the tips of the severed axons characterized these reconnections. Cooling the nerve did not bring any evident modifications in the conditions of the experiment. No fusion of axons was observed and signs of degeneration were seen in the late specimens. After 20 h the reunion was solid enough to hold without further external support; however, tensile strength is known to require 3 to 4 weeks to return to normal (13). It was thus decided that in further experiments the delay before removal of the support should be longer. We suggest that this method of distributed mechanical support should have a favorable influence on the final outcome of peripheral nerve injuries by decreasing the size and the irregularity of the scar. REFERENCES 1. BRUSHART, T. M., AND M. M. MESULAM. 1980. Alteration in connection between muscle and anterior horn motoneurons after peripheral nerve repair. Science 208: 603-605. 2. BUCK-GRAMCKO, D. 1979. Microsurgery of peripheral nerves. Page 80 in T. L. LIE, Ed., Microsurgery. Excerpta Medica, Amsterdam/Oxford. 3. DEINEKA, D. 1908. L’influence de la temperature ambiante sur la regeneration des fibres nerveuses. Folia Neural. Biol. 2: 13-24. 4. DENNY-BROWN, D., R. D. ADAMS, C. BRENNER,AND M. M. DOHERTY. 1945. The pathology of injury to nerve induced by cold. J. Neuropathol. Exp. Neural. 4: 305-323. 5. DUCKER, T., L. KEMPE, AND G. HAYES. 1969. The metabolic background for peripheral nerve surgery. J. Neurosurg. 30: 270-280. 6. EDSHAGE, S., AND J. J. NIEBAUER. 1966. Evaluation of freezing as a method to improve cut surfaces in peripheral nerves preparatory to suturing. Plast. Reconstr. Surg. 37: 196-202. 7. GAZE, R. M. 1970. The Formation of Nerve Connections. Academic Press, New York, pp. 70-75. 8.
GOTO, Y. 1967. Experimental study of nerve auto-grafting by funicular suture. Arch. Jap. Chir.
36: 478-484.
9. GUTHRIE, D. M. 1962. Regenerative growth in insect nerve axons. J. Insect. Physiol.
8:
79-92.
10. GUTMANN, E. 1942. Factors affecting recovery of motor function after nerve lesions. J. Neural. Psychiatry 5: 8 l-95. 11. JACOBSON,M. A. 1978. Developmental Neurobiology. Plenum Press, New York, pp. 357363.
12. LANE, J. M., F. W. Bow, AND D. PLEASURE. 1978. Neuroma scar following peripheral nerve transection. J. Bone Joint Surg. (Boston) 60: 197-203.
DE MEDINACELI
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AND FREED
13. MUKHERJEE, S. 1953. Tensile strength of nerves during healing. Br. J. Surg. 41: 192-195. 14. ORGEL, M., AND J. TERZIS. 1977. Epineurial versus perineurial repair: an ultrastructural and electrophysiological study of nerve regeneration. Pk. Reconstr. Surg. 60: 80-9 1. 15. RALIS, H. M., R. A. BEESLEY, AND Z. A. RALIS. 1973. Techniques in Neurohistoiogy. Bunerworths, London. 16. RAM~N Y CAJAL, S. 1928. Pages 188 and 361 in R. MAY (Trans. and Ed.) Degeneration and Regeneration ofthe Nervous System. Oxford Univ. Press, London and New York. 17. SPERRY,R. W. 1941. The effect of crossing nerves to antagonistic muscles in the hind limb of the rat. J. Comp. Neural. 75: l-19. 18. SUNDERLAND, S. 1952. Factors influencing the course of regeneration and the quality of the recovery after nerve suture. Brain 75: 19-54. 19. WISE, A., C. TOPUZLLJ, P. DAVIS, AND L. KAYE. 1969. A comparative analysis of macroand microsurgical neurorrhaphy technics. Am. J. Surg. 117: 566-572. 20. YOUNG, J. 1948. The growth and differentiation of nerve fibres. Symp. Sot. Exp. Biol. 2: 57-73.
NEUROLOGY
(1983)
81,459-468
Peripheral Nerve Reconnection: Consequences of Distributed LUIS DE MEDINACELI
Immediate Mechanical
AND WILLIAM
Histologic Support
J. FREED’
Adult Psychiatry Branch, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032 Received February 16, I983 We tested the possibility of improving the quality of axonal repair after peripheral nerve transection by suppression of all mechanical stressesfrom the zone of repair. A method was developed involving mechanical support of the sciatic nerve from below in a grooved chamber with additional support provided by suturing the nerve to a rubber sleeve. Histological studies of silver-impregnated specimens from rabbit sciatic nerve using this technique revealed that the axons’ tips were closely reconnected with minimal disruption in their direction or in the alignment of fibers and without extraneous material between the severed stumps. Comparison of these results with those of current microsurgical techniques showed the unequivocal superiority of this method in terms of immediate histologic aspects.The course of Walletian degeneration was not modified.
INTRODUCTION It is generally considered axiomatic that the technical quality of the repair of severed peripheral nerves has some influence on the eventual outcome. There is considerable evidence that a wide gap (3, 5, 9, 10, 16, 20), the presence of blood or foreign material (16), fibrous scarring (6, 12, 16, 18), erroneous alignment of the stumps (1, 7, 11, 17), or any other mechanical interference with axonal regrowth can result in less than optimal regeneration. Thus, the ideal peripheral nerve reconnection is generally considered to be that which places each severed axon in close contact with its distai end without distortion and without intervening foreign material. Current microsurgical techniques stress the superiority of perineurial over epineurial repair, ’ We thank Richard Jed Wyatt, Barry J. Hoffer, Anthony Seaber, Miguel de Medinaceli, Alan Fine, and the Division of Special Mental Health Research staff for their invaluable assistance and encouragement during many phases of this project. We also thank Eleanor Krauthamer for preparing the histologic materials and Denise Ondrish for preparing the manuscript. 459 0014-4886/83 $3.00 Copyright 0 1983 by Academic Press. Inc All rights of reproduction in any form reserved.
460
DE
MEDINACELI
AND
FREED
essentially due to the better fascicular alignment (2). Even a well done perineurial repair, however, leaves a massive disorganization at the axonal level as shown by the histologic appearance of the site of any nerve microsuture (8, 14, 19). The purpose of our work was to study the possibility that axonal disorganization could be decreased through the use of a new technique of reconnection of the stumps involving distribution of the mechanical stress on the nerve to regions removed from the transection site (Fig. 1). Cooling was routinely used for slowing the metabolic phenomena with the idea that this might enhance the quality of the repair. Mild transient cooling is known to be harmless to nerve trunks (4). MATERIALS
AND
METHODS
Forty albino rabbits weighing 2 to 3 kg were anesthetized with phenobarbital (Nembutal), i.p., and xylazine (Rompun), i.m. The sciatic trunk was exposed and dissected from hip to knee. The nerve was laid in the groove of a small copper cooling chamber coated with rubber (the finger of a rubber glove). The nerve was sutured to the chamber with a stitch of 6-O silk through the epineural sheath 3 mm from the extremity of the groove and then through the rubber coating at the edge of the groove. Three other identical stitches were made as shown in Fig. 2a. When they were tied the nerve was firmly sutured to the chamber so that there was no longitudinal tension on the nerve in the center of the preparation. Local temperature was recorded from
FIG. 1. Theoretical basis of distributed mechanical support. a-Current techniques of repair concentrate stresses in the zone of repair. We considered this an adverse situation. b-Our method aims at diverting all mechanical stresses laterally, a few millimeters from the zone of transection.
NERVE RECONNECTION
461
b
FIG. 2. Illustration of the preparative procedure. a-The nerve was placed in the groove of a brass cooling chamber coated with a rubber sheet. Stitches were passed through the rubber and nerve to fix the nerve in place and to prevent retraction. b-After transection and reconnection the rubber was cut out around the nerve: a small piece of rubber remained attached to the nerve and prevented retraction and separation of the stumps.
the superior wall of the chamber directly from the metal under the rubber coating. In four animals no cooling was used and the temperature remained about 30°C throughout the experiment. In 36 animals the temperature of the chamber was lowered to a stable 15°C by circulation of cold water. The temperature of the nerve was estimated to be between 17 and 20°C depending on the size of the nerve and the duration of the cooling. The nerve was then lifted slightly from the bottom of the groove and transected with a pair of scissors. Because the nerve was fixed to the chamber no retraction of the stumps occurred. The bleeding of the stumps varied from none to quite heavy. In the first 16 animals no attention was paid to the bleeding and the reconnection was made at once by letting the stumps fall back together. In the 24 other casesno reconnection was made before bleeding had ceased. Blood was removed with a small piece of blotting paper or a few threads of cotton, avoiding contact with the surfaces of transection.
462
DE MEDINACELI
AND FREED
Reconnection was always easy because the stumps fell back in the groove to the correct position. With the use of characteristic features such as minute superficial blood vessels, the alignment could be made very precisely. The temperature was then returned to normal by interrupting the water circulation. The average duration of cooling was 10 min. From 30 min to 1 h after the transection, the rubber of the chamber was cut out around the nerve and the chamber removed. A small rectangular piece of rubber remained attached to the nerve and prevented retraction and separation of the stumps (Fig. 2b). The wound was momentarily closed to avoid drying. Twenty-five nerves were removed from the body from 1 to 6 h after injury. They included seven nerves for which the technical quality of the repair had been considered especially good, those where cooling had not been used (four nerves) and 14 others chosen at random. They were transported with the small rubber support into Bouin’s fluid. After 24 h the rubber was removed, the specimens were coded, embedded in paraffin, and sectioned longitudinally at 7 Km. Selected sections were stained with silver by the method of Holmes (15) and the remaining sections were stained with solachrome cyanin by the method of Page (15). The 15 remaining animals were used to test the soundness of the reconnection. Twenty hours after the injury they were anesthetized again. The four small stitches were cut and the rubber support removed; the nerve was then free of sutures and other material. No special precautions were taken in the further handling and care of the animals. The nerve was checked 6 to 8 days later. Nine additional nerves were used for control experiments: (a) Four nerves were repaired by conventional microscopic perineurial suturing with 9-O silk, (b) in two animals the rubber support was removed 10 h after reconnection, and (c) in three animals the nerve was not transected but was only cooled at 15°C for 1 h. These nine controls were not included among the 40 experimental animals. FIG. 3. Histologic appearance of unsatisfactory repairs (Holmes’ stain). a-A conventional microscopic suturing 2 h after repair. The external appearance of this fascicle was good under the operating microscope and this portion of the fascicle was selected as having the best appearance. Note the distortion of the subfascicles and large pools of blood and foreign material. The gap, indicated by the double arrowheads, is at its smallest here. The average width of the gap was about 400 Frn (bar = 200 pm). b-An unsatisfactory repair with distributed mechanical support, 6 h after reconnection. The axons are only slightly distorted at their extremities and there is little blood between the stumps. A thick, dense scar membrane separating the stumps is evident (bar = 200 pm). c-Another repair with distributed mechanical support 2 h after reconnection. The general appearance is very good with little distortion of the axons, no blood between the stumps, and a fairly close approximation of the axons. A scar membrane is evident (bar = 100 pm). d-A higher magnification of the same specimen. The gap between the stumps is evident although not very wide (10 to 20 pm). There is a thin, but nearly continuous, scar membrane that separates the two stumps (bar = 20 pm).
NERVE
RECONNECT
ION
463
464
DE MEDINACELI
AND FREED
RESULTS Macroscopic Observations
Within 3 to 6 min after reconnection, the gap became less apparent which was attributed to coagulation occurring between the stumps. The gap was usually reduced to a small pink line that was difficult to discern with the naked eye. That appearance remained unchanged during the first few hours. General Microscopic Findings
Attention was focused on six points: (i) the general appearance of the reconnection-did the fascicles face each other with the axons undistorted or were the axons irregular and distorted; (ii) was there an interposition of foreign material (epineurium, fibrous tissue) between the stumps; (iii) were large amounts of blood present in the region of the gap; (iv) what was the distance between the tips of the axons of the two stumps; (v) was a visible line or membrane-like formation present between the stumps; and (vi) in the late specimens (over 4 h), were there early signs of traumatic or Wallerian degeneration? Sutures. One hour after conventional repair, the sutured nerves presented a gap ranging from 100 to 400 pm. That gap was filled with blood, various cell debris, and fragments of epineurial or perineurial sheaths. Disruption of axonal direction was conspicuous in all cases (Fig. 3a). Repairs through Distributed Mechanical Support. (i) Unsatisfactory reconnections [ 18 nerves (Fig. 3b-d)]. In 12 cases the histologic appearance was poor. The fascicles were brushed against each other, and epineural sheath or fibrous tissue was interposed. Large pools of blood or plasma were often seen between the stumps. Nevertheless, these reconnections looked much more precise than those obtained by conventional suturing. In six cases the general appearance was better in that there was little or no distortion of the axons, no interposition of foreign material, and little or no blood. In all 18 cases there was rarely close approximation of the tips of the axons and the extremities of the stump were lined by a continuous formation of scarlike membrane which we considered to be collagen or fibrin formations. Between FIG.4. Histologic appearance of satisfactoryrepairs with distributed mechanical support (Holmes’ stain). a-General appearance of a satisfactory repair 2 h after injury. Compare with Fig. 3a and c. Here the area of injury (arrowheads) is only faintly visible although the axons are evidently distorted (bar = 100 pm). b-Another satisfactory repair. The tips of some axons come very close together. The point of transection is recognizable only as an indistinct bluish zone indicated by the arrowheads. There is no interposed foreign material and no continuous scar membrane (bar = 20 pm). c-e-Three higher magnifications of areas where there is very close apposition between axon tips from opposite sides of the gap. In each case, close examination revealed that the axons did not actually make contact. There was always a small gap, sometimes less than I pm in width, which is not always apparent in the photographs. The points of transection are indicated by arrowheads and the small arrows indicate areas of close apposition between axons. The bars are 8. 20, and 20 pm, respectively.
NERVE
RECONNECTION
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466
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the two scar membranes there usually was an evident gap, ranging from 10 to 20 pm. (ii) Satisfactory reconnections (seven nerves) (Fig. 4). These nerves were the last to be repaired in the series. Satisfactory results were attributed to a better technique bearing essentially on the position of the stitches. This controlled the elimination of longitudinal stresses from the repair zone. Better removal of blood from the surface of transection was also considered beneficial. The general appearance of these reconnections was fair, with slight distortion or brushing of the axons in two cases. No foreign material was seen between the stumps except some blood which was found between the stumps in two cases. In three nerves the general aspect was very good with no blood visible. It was usually possible to find at least one large zone where the tips of the transected axons of both stumps came very close to each other. Also, there was no scar membrane or formation visible between the stumps, at least in the zones of good connection. Neither fusion nor true contact between the axons was seen. There were signs of degeneration in the two specimens removed after 6 h (swelling and breaking down of the axons). Soundness of Repair In all 15 animals from which the rubber support had been removed after 20 h, the check at 1 week showed a satisfactory appearance of the nerve. No secondary separation of the stumps was observed. The zone of repair was difficult to locate precisely as it was not marked by any change in caliber but only by a slight change in color. In the two animals from which the rubber support had been removed after 10 h, complete separation of the stumps was observed at 1 week. Influence of Cooling The absence of permanent damage to the nerves caused by transient cooling was confirmed in the three control nerves that were cooled at 15°C (temperature of the chamber walls) for 1 h. In all three animals a transient clinical deficit was observed for 4 to 6 days (loss of the stretching reflex of the toes, diminution of muscular force). After 6 days, normal function was recovered. We did not attempt to determine whether the transient deficits were due to the manipulations of the nerve or to the cooling itself. Histologic examination of those nerves at the 15th day showed that the fibers were intact. The influence of cooling on the quality of the repair was unclear; we noticed only that in the four cases where the nerves were not cooled, the microscopic aspect was not good. DISCUSSION These observations suggest that a significant improvement in peripheral nerve repair quality can be brought about solely by improvements in the
NERVE
RECONNECTION
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mechanical aspects of the repair technique. In this study we used a reconnection technique that distributes the mechanical tension on the nerve away from the transection site. In particular, lateral stresses are relieved by a surrounding chamber and the longitudinal stresses are displaced to at least a few millimeters away from the transection site. When reconnection was achieved under those conditions, there was a dramatic improvement in the anatomic manifestations of the repair. Minimal whorling or disruption of the axons’ alignment, minimal interposition of foreign material, and close apposition of the tips of the severed axons characterized these reconnections. Cooling the nerve did not bring any evident modifications in the conditions of the experiment. No fusion of axons was observed and signs of degeneration were seen in the late specimens. After 20 h the reunion was solid enough to hold without further external support; however, tensile strength is known to require 3 to 4 weeks to return to normal (13). It was thus decided that in further experiments the delay before removal of the support should be longer. We suggest that this method of distributed mechanical support should have a favorable influence on the final outcome of peripheral nerve injuries by decreasing the size and the irregularity of the scar. REFERENCES 1. BRUSHART, T. M., AND M. M. MESULAM. 1980. Alteration in connection between muscle and anterior horn motoneurons after peripheral nerve repair. Science 208: 603-605. 2. BUCK-GRAMCKO, D. 1979. Microsurgery of peripheral nerves. Page 80 in T. L. LIE, Ed., Microsurgery. Excerpta Medica, Amsterdam/Oxford. 3. DEINEKA, D. 1908. L’influence de la temperature ambiante sur la regeneration des fibres nerveuses. Folia Neural. Biol. 2: 13-24. 4. DENNY-BROWN, D., R. D. ADAMS, C. BRENNER,AND M. M. DOHERTY. 1945. The pathology of injury to nerve induced by cold. J. Neuropathol. Exp. Neural. 4: 305-323. 5. DUCKER, T., L. KEMPE, AND G. HAYES. 1969. The metabolic background for peripheral nerve surgery. J. Neurosurg. 30: 270-280. 6. EDSHAGE, S., AND J. J. NIEBAUER. 1966. Evaluation of freezing as a method to improve cut surfaces in peripheral nerves preparatory to suturing. Plast. Reconstr. Surg. 37: 196-202. 7. GAZE, R. M. 1970. The Formation of Nerve Connections. Academic Press, New York, pp. 70-75. 8.
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