Peripheral nerve reconnection: Mechanical, thermal, and ionic conditions that promote the return of function

EXPERIMENTAL

NEUROLOGY

81,469-487

(1983)

Peripheral Nerve Reconnection: Mechanical, Thermal, and Ionic Conditions That Promote the Return of Function LUIS DE MEDINACELI, RICHARD JED WYATT, Adult

AND WILLIAM

Psychiatry Branch, National Institute Saint Elizabeths Hospital, Washington, Received

February

J. FREED’

of Mental Health, D.C. 20032

16. I983

Current microsurgical techniques for repairing peripheral nerves do not take into account a number of factors that are of importance. A technique was developed that considered 17 factors, the major ones of which are proper alignment of the stumps, sharpness of cut, and prevention of physical and chemical damage. The clinical results after transection of rats’ sciatic nerve were assessedin terms of functional recovery. When all 17 conditions were met, our technique was significantly superior to microsutures, suggesting the possible use of this method to improve the return of function after peripheral nerve injury. INTRODUCTION

In previous studies of sciatic nerve transection in rabbits and rats, we described a sutureless method of repair that eliminates both transverse and longitudinal stresses,thereby producing a precise reunion of the nerve stumps. Histological studies showed longitudinal misalignment (whorling) to be minimal, and the gap between axon tips was sometimes reduced to a few micrometers without interposition of foreign material (11). In the best cases, some electrical activity in the distal stump, possibly ephaptic in nature, could be evoked by electrical stimulation of the proximal stump ( 12). These repairs were actually conceived as one facet of a more complete technical approach to peripheral nerve repair, termed reconnection. Numerous studies of nerve lesions suggest several physical and chemical factors that may influence the results, such as type of trauma (57) precision of repair (28) interposition of Abbreviations: SF&sciatic functional index, CPI-constant pattern of injury. ’ We thank Darlene de Medinaceti and Miguel de Medinaceli for their participation in conceiving this project, Alan Fine for his suggestions and comments, Denise Ondrish, Paul Byrne, and Phillip Johnson for their valuable technical help. This work was done in the Preclinical Neurosciences Section. 469 0014-4886/83 $3.00 Copyright Q 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.

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extraneous material (48), mechanical stresses at the suture line (29, 42, 45), ion shifts (1, 63), penetration of free calcium into the axoplasm (52, 53), and outflow of axoplasm from severed fibers and similar movements of myelin (14, 15, 34). We hypothesized that an approach taking into consideration all those factors would yield clinical results different from those obtained with nerve repair carried out with microsutures. A number of experiments determined the relative importance of some of those factors. The results were assessed in terms of functional recovery because function is the ultimate criterion of success and is possible to quantitate (13). MATERIALS

AND

METHODS

Subjects and Surgery Male albino rats initially weighing 270 to 650 g were used in groups of seven animals, except in groups IX (N = 5) and X (N = 4). Table 1 summarizes the procedures used for groups I through VIII. In all animals with transected nerves, the nerve was totally severed. Group Z (Crush). The left thigh was opened under general anesthesia and the sciatic nerve trunk crushed 45 s at midthigh with maximum strength in the bare jaws of a serrated forceps. Group ZZ (Suture). The left sciatic nerve trunk was transected at midthigh with a pair of scissors and immediately sutured with two to five perineurial stitches of 9-O nylon monofilament (Prolene). TABLE

1

Comparison of Experimental and Control Treatments with Functional Results Experimental treatments Group

Control treatments

VIII

Change of medium without transection Crush Suture

I II III VII IV VI V

Freezing

Sharp cut

Change of medium

Final results (96 of deficit) 0

0 0 + + +

0 0 + 0 +

0 + 0 + +

-5 -78 -61’ -73’ -55* -47+ -24

* Groups III, VII, IV, and VI were not significantly different from group II (suture).

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Sutureless Reconnection-General Technique. The left thigh was opened from hip to knee and the sciatic trunk gently dissected. The superficial blood vessels coursing longitudinally in the interfascicular spaces were removed, together with the mesoneurium between the main nerve branches, for a length of 1 cm. The nerve was laid on a fragment of surgical glove approximately 5 mm wide and sutured to it by four epineurial stitches of 6-O silk. These two pairs of stitches were made in such a way that when they were tied all mechanical tension of the nerve between the sutures was eliminated (Fig. 1). The nerve was entirely relaxed, so that at first it could not be straightened (Fig. 2a). An elastic contraction soon took place and after a few minutes the nerve was straightened (Fig. 2b). A small chamber or thermic coil was placed under the preparation. The temperature of the chamber, controlled by circulation of fluid, was initially maintained at 35°C. The nerve was transected in the middle of the preparation. No retraction of the stumps occurred because of the fixation of the nerve to the rubber. The stumps fell back into contact almost by themselves (Fig. 2~). After 8 to 12 min, coagulation between and around the stumps consolidated their reunion enough to allow removal of the preparation from the chamber. The rubber flaps were folded over the nerve and held by one stitch (Fig. 2d). The cuff was left in place for an average of 20 days. On the day of operation and on removal of the cuff, 100,000 U penicillin were administered. Several variations were included in this general technique, bearing on temperature, method of cutting the nerve, and fluids used to soak the preparation. Group ZZZ(Ringer’s, No Freezing, Rough Section). The temperature of the chamber was lowered in 2 to 3 min from 35 to 10°C. The nerve was then cut with a pair of scissors. One minute after reconnection the temperature was increased (during 5 to 10 min) to 35°C. Lactated Ringer’s solution at the appropriate temperature was used throughout the experiment to prevent drying.

FIG. 1. Application of the antiretraction device. The nerve is laid on a small piece of thin rubber and tied to it on one side by two stitches. Two other stitches are passed through the other extremity. When tied, they will eliminate all mechanical tension in the center of the preparation.

FIG. 2. Steps in sciatic nerve reconnection. a-The nerve trunk has been gently dissected and the main longitudinal vesselsrunning between the fascicles removed. The nerve was then sutured to a piece of rubber in such a way that all mechanical tension was displaced longitudinally. bElastic contraction occurred rapidly and the nerve straightened, slightly increasing in diameter. c-The nerve was then transected and the stumps realigned. In this case there was, however, a slight imperfection at the peroneal nerve reunion site. d-A rubber flap was folded around the nerve and fixed by one stitch. If edema were to occur there would be no compression because the nerve had room to expand; however, the cuff effectively prevents retraction.

Group IV (Ringer’s, Freezing, Sharp Cut). Lactated Ringer’s was used throughout the experiment but the temperature was lowered at the rate of 1“C/min from 35°C to complete crystallization of the nerve. The nerve was then cut with a vibrating blade (a fragment of a razor blade set at the tip of a metal engraving tool). The speed of vibration was 120 Hz. Pilot experiments had shown that a neat cut could be obtained with this device only when the nerve was hard. This occurred for temperatures from -0.5 to -3S”C measured by a microprobe inserted in the nerve and could be easily judged by very slight pressure on the nerve trunk. Immediately after the cut, the preparation was thawed by warming the chamber to 3 to 5°C and simultaneously soaking the nerve with lactated Ringer’s at 4°C. The groove left by the blade was about 0.5 mm wide and was clearly visible at first, but in about 1 min the

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stumps creeped back into contact. After 1 min at 3 to 5°C the temperature was increased to 35°C usually in 60 to 90 s. The chamber was then maintained at 35°C for 10 to 12 min until full coagulation. Group V (Change of Medium, Freezing, Sharp Cut). The procedure was identical to that of group IV but the fluids used to soak the preparation were different. From 35 to 15°C i.e., 20 min, the nerve was soaked with Ringer’s without calcium [NaCl, 150 IIIM; KCl, 4 mM; pH 6.00 (fluid A)]. From 15°C to crystallization the nerve was soaked in fluid B: NaCl, 20 mM; KH2P04 120 mM; ethylene-glycol-bis-P-amino-ethylether-Nit acid, 5 mrq and imidazole, 30 ITIM; pH adjusted to 6.35 with KOH. After transection the nerve was immediately thawed with a few drops of the same solution at 3°C to which had been added lysophosphatidylcholine (0.5 mg/ml) and epinephrine tartrate (0.4 mg/ml) (fluid C). No other fluids were added during the remainder of the procedure. Group VI (Change of Medium, Freezing, Rough Cut). The procedure was the same as in group V and the same fluids were used but the nerve was transected with scissors (two animals), with several strokes of a surgical knife held by hand (two animals), or with the vibrating blade applied to an insufficiently hard nerve trunk (three animals). Group VZZ (Change of Medium, No Freezing, Rough Cut). The same fluids as in group V were used but the temperature was lowered to 10°C only, and the nerve was transected with scissors. Group VZZZ (Change of Medium). A 27-gauge needle was inserted in two places into the sciatic nerve at midthigh and an average total of 0.3 ml fluid C (see group V) was rapidly injected. This distended a portion of the nerve trunk at least 1 cm long. The wound was then closed. Group IX (CUB. In a group of five animals the longitudinal blood vessels were removed by dissection. The nerve was sutured on a rectangle of rubber but was left uncut. The antiretraction cuff was folded around the nerve and the wound closed. Group X (Cooling). In a last group of four animals the nerve was laid on the chamber and the temperature lowered to 10°C during 25 min, then the chamber was removed and the wound closed. Testing

Nerve function was evaluated by the sciatic functional index (WI), which is based on measurements of rats’ walking tracks and is expressed in percentage of functional deficit ( 13). Animals in groups I to VII were tested preoperatively and on postoperative days 1, 4, 7, 11, 15, 18, 25, 32, 39, 46, 53, 60, 67, and 74. Group VIII was not tested after the 39th day. Groups IX and X were tested preoperatively and on days 1, 2, and 4 postoperatively. Ninety-eight

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tracks obtained on the 32nd and 74th days for all animals in groups I to VII were coded and measured by a “blind” observer who was unaware of the object of the experiment. The same tracks were again measured by the principal observer who also measured in a nonblind manner all other tracks. Normality was defined as the preoperative performance of 36 healthy rats chosen at random. Twelve months after repair, animals taken at random in groups II (suture), V (change of medium, freezing, sharp cut), and VI (change of medium, freezing, rough cut) were killed. The nerves were fixed in Bouin’s fluid, embedded in paraffin, and cut longitudinally at 6 pm. Selected sections were stained with silver by the method of Holmes (49). RESULTS After injury to the sciatic nerve trunk at midthigh and to day 25, all animals except those in groups I (crush) and VII (change of medium) displayed a similar impairment of function. This constant pattern of injury (CPI) is shown graphically as a solid black line and represents the mean f SE of the SFI of groups II to VII (Figs. 3 to 5). The mean SFI for group I (crush) is shown as a function of time in Fig. 3. This group rapidly improved from day 18. By day 25, no abnormality could be detected by clinical examination, but the SFI was less than before the operation until day 53. Plotting of individual performances indicated a small variability in this group (Fig. 4a). Group II (suture) showed only a slow and incomplete recovery by 2.5 months (Fig. 3). Individual differences in this group were large (Fig. 4b). One animal in group II presented a very rapid recovery. In group V (change of medium, freezing, sharp cut), all animals showed an unequivocal recovery on day 32 (Figs. 3, 4~). This recovery differed from that of group I (crush) in that it began 10 to 12 days later. The results at 2.5 months also showed an average difference of 24% with group I (crush) and/ or normality. Compared with group II (suture), group V (change of medium, freezing, sharp cut) showed differences from the day 32 onward. The average difference in results at 2.5 months was 54%. As already mentioned, however, one individual response in group II (suture) was as good as the average response in group V (change of medium, freezing, sharp cut); another one was almost as good (Fig. 4b). None of groups III, IV, VI, and VII had substantial recovery (Fig. 5), and there was no sharp recovery of function such as that observed in group I (crush) or V (change of medium, freezing, sharp cut). The animals in group VIII (change of medium without transection) had a total impairment of function for 7 days but starting at about day 10 a very rapid recovery occurred

475

NERVE RECONNECTION

-Group

VIII

-Group -Group

I (Crush) V (Ch.of sharp

(Change med. cut)

of medium) freezing.

FIG. 3. Results of the principal groups. Functional capacity (SFI) is expressed as the percentage deficit (means) as a function of time. The initial impairment of function was similar for all animals; thus the “constant pattern of injury” (CPI), shown in solid black, represents the mean (SE) deficit of all rats with a lesion. Rapidly, however, two groups branched away from the CPI: group VIII (change of medium) recovered first, and group I (crush) recovered soon after. Recuperation in group V (change of medium, freezing, sharp cut) was parallel but occurred later and was not as complete as after crush. Group II (suture) displayed only a slight recuperation.

in all animals (Fig. 3). Neither cuff and modification of blood supply (group IX) nor simple cooling to 10°C (group X) caused an impairment of the nerve function (Fig. 5). The overall results and procedures for each treatment group are summarized in Table 1. Statistical Analysis The mean (+-SE) SFI of the normal animals was -0.19 + 2.48. For measurements of the 98 tracks by different observers the intraclass correlation coefficient (70) was 0.77 (F(97,98) = 7.86, P < 0.001). A two-way analysis of variance showed significant main effects of surgical treatments (F(6,42) = 15.99, P < 0.001) and measures (F(7, 294) = 31.10, P < 0.001). Interaction effects were also significant (F(42,294) = 2.99, P < 0.001). Individual comparisons by the Schefl% test showed that group I (crush) was

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significantly better than group II (transection and suture) from day 18 (t = 3.37, P = 0.001) through day 74 (t = 2.69, P = 0.008). Groups III, IV, VI, and VII were never different from group II (transection and suture). Group V (change of medium, freezing, sharp cut) was significantly different from group I (crush) on day 25 (t = 2.26, P = 0.023) but not on day 32 (t = 0.98, P = 0.67) and thereafter (P > 0.5). The difference between group V (change of medium, freezing, sharp cut) and group II (transection and suture) did not quite attain a statistically significant difference on day 39 (t = 1.87, P = 0.058), but group V was significantly greater from day 46 (t = 2.05, P = 0.039) through day 74 (t = 1.98, P = 0.045). Aspect of Repair The repair at the time of the operation in groups II to VII was classified in one of three categories (Fig. 6a-c). The long-term postoperative appearance

FIG. 4. Individual performance of animals in groups I, II, and V. a-The SFI for group I (crush) as a function of time. Individual performances were very homogeneous. b-The SFI for group II (suture) as a function of time. Compared with a, individual performances after suture were highly variable. The mean WI for group V is shown by the dotted line for comparison. c-The SFI for group V (change of medium freezing, sharp cut) as a function of time. Individual performances were homogeneous but delayed compared with group I (crush). This group could be described as an optimized suture group, in which each functional result was as good as the best of the sutured animals. The mean SFI for group I is shown by the dotted line for comparison.

NERVE

RECONNECTION

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, d

.

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111 (Ringers,

-Group

IV

(Ringers. nge

Pre-op

I

4

7

II

I3

18

23

32

39

46

no

freezing.

freezing. of medium, of medium,

33

rough sharp

cut)

cur)

freezing. rough CM) no freezing. rough

60

67

cut)

74

Days

FIG. 5. Functional recovery for groups in which the reconnection procedure was only partial. Statistically these groups were not different from group II (suture). Thus an ensemble of conditions was necessary to get consistently good results; if some factors were omitted, the results were not better than those obtained by suturing.

of the nerve in group II (suture) was not investigated, but in groups III to VII the appearance of the nerve was also evaluated at cuff removal (Fig. 6d-f). Comparison of these evaluations with the functional result at 2.5 months showed that there was no relationship between the appearance of the nerve under the operating microscope or at cuff removal and the final outcome. Histology showed extensive fiber regrowth in all cases (Fig. 7). There was very little evidence of scar formation or gross physical disturbance of the nerve trunk. The region of the injury could not be distinguished as there was no visible disruption in the axons’ alignment. It was not possible to find characteristic differences between specimens of different groups. DISCUSSION There are many factors that have been demonstrated to be important in nerve repair. We hypothesized that devising a technique using these factors could lead to improved results. The technique of reconnection was designed

FIG. 6. Appearance of nerves at the time of oper; ition (a-c) and at cuff removal (d-f). Appearance of the nerve at operation was rated as good (gap minimal or hardly visible, little or no whorling, little or no defect in alignment of fascicles, a); p oar (gap visible or defect in fascicles alignment or whorling, b); or mediocre (intermediary aspe:ct on whole or most of the repair, c). At cuff removal, appearance of the nerve was rated as 6;ood (transection site impossible to determine, d); poor (transection site marked by changes in caliber and tissue aspect, pellucid zone, e); or mediocre (intermediary aspect on whole or part of the transection site, f).

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, ;;r’, ,*;;

a

.$ 1

b

I’. l -a

C

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to take into consideration physical factors (imprecision of the repair, interposition of extraneous material and tension at the point of repair, irregularity of the cut, additional crush damage, outflow of axoplasm), and chemical factors (ion shifts and regulatory mechanisms). An antiretraction device was used to deal with physical factors. Whorling of axons and misalignments favor misdirection (28, 48) thus generating improper connections between the central nervous system and the periphery (6). Errors of connections are shown by the absence of proportionality between number of fibers growing back to the periphery and return of function (24, 43) and may be responsible for poor results (36, 56). Consequently, current microsurgical techniques try to achieve precise reconstitution at the fascicular level (39,60). Clearly, however, these techniques are not technically satisfactory at the axonal level (11) and the outcome is only partially improved (7, 21, 74). Blood, fat, collagen, perineurium, pus, or stitches between the stumps may hamper regrowth, whether directly or by favoring formation of a scar (30,48). Such interpositions may favor fiber wandering rather than constitute a real barrier (67). Tension at the suture line and surgical trauma of the stump also have an adverse influence (29, 41, 42, 45). The antiretraction device was used to fulfill the three above requirements, by diverting mechanical stresses away from the zone of repair. Cuffs have often been used [for review see (66)], and sutureless reunions have proved feasible in animal (73) and man (54). However, we found that countering retraction was indispensable to avoid important interpositions of extraneous substances in the gap, and to obtain precise axonal reunion (11). The device was removed about day 20 because return of tensile strength occurs within 3 to 4 weeks (46). Cooling was used because of four physical factors. There is a substantial longitudinal extension of fiber damage proximal and probably distal to traumatic injury (55, 57, 69). Several ways of removing these damaged portions by sharply trimming the stumps have been proposed (9,16), but it is impossible to obtain even or smooth stump surfaces through the use of the sharpest blade held by hand ( 16). The irregularity of the stumps diminishes the precision of repair. Moreover, additional crush damage is inevitably produced by pressure when scissors or razor blades are used to trim nerves. These iatrogenic crush damages could be partially responsible for the traumatic degeneration described histologically after apparently sharp cuts (33, 48). Because this FIG. 7. Long-term histologic aspects of the zone of repair after different procedures: a-suture; b-animal with satisfactory functional result (group V, change of medium, freezing, sharp cut); c-incomplete protocol (group VI, change of medium freezing, rough cut). No characteristic difference could be found between the specimens. The functional result was, however, quite different as indicated by the respective tracks of the same animals, just before killing, one year after repair.

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damage extends several tenths of a millimeter on each side of the cut, even the most precise reunion in fact leaves a large interposition of dead or dying tissue. Outflow of axoplasm at the site of injury is well known (34, 40, 7 l), although it is not clear if this phenomenon has any effect on the long-term results. We hypothesized that cold would temporarily harden the axoplasm, diminish its movements, and thereby improve the long-term result. Freezing had been considered by others (17) as a possible means of improving the quality of the cut, but was rejected because it damages the nerve, We thought that precise monitoring of the temperature would minimize this damage. Very smooth surfaces were obtained with a vibrating blade, but only when the nerve was hardened by freezing. The device could not cut a normal nerve due to the contrast between the hard elastic sheath and its fragile contents. Last, cold was used to diminish active regulatory mechanisms. Moderate cold does not suppress movement of ions and nerve activity at low temperature has been demonstrated (38). Cold, however, greatly slows metabolism by decreasing enzymatic activity. Because we thought that the use of special fluids would be beneficial (see below) we had to apply a temperature below 15°C to avoid triggering reactions of the axons’ regulatory systems. The rate of cooling was selected because the speed of 1“C/min is often used in tissue freezing (3 1, 68), and also because the corresponding delay was considered sufficient to insure good diffusion into the nerve stumps of the low-molecularweight substances of fluid B (see below). Cold duration in this study was considered too short to produce the slowing down of Wallerian degeneration that has been long known in hibernating (10,37,6 1) or heterothermic animals (20, 35). Although seldom studied, ions shifts are known to occur after a nerve injury. They are immediately signaled by disorderly firing of the fibers, irreversible depolarization of the membrane, and the presence of a lesion current (1, 4, 19, 63, 65). Calcium ion shifts have been shown to harm the cytoplasm (8, 26, 27, 52) and could trigger Wallerian degeneration (53). We hypothesized that diminishing the ion shifts would decrease the extent of the damage to the fibers. Special chemical fluid media were used to minimize chemical damage. Penetration of calcium into the axoplasm can be effectively prevented by the use of the chelating agent EGTA (ethylene-glycol-bis-P-amino-ethylether N,N’amino-tetracetic acid) (53). We tried to lessen other ion shifts by soaking the preparation in a medium similar to that axoplasmic composition (2) and blocking the regulatory systems by the use of cold. The composition of this medium (fluids B and C) was also designed to prevent the damaging decrease of pH which otherwise accompanies freezing of physiological solutions (62). Interposition of myelin has been clearly described at the site of an injury

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( 14, 15, 34). When examining slides from a previous study on reconnections (1 l), we noticed that axon tips often seemed covered by some capping substance. Pilot histological studies showed that myelin was always diffusely present at the site of reunion. The administration of lysophosphatidylcholine was designed to disrupt, and possibly wash away this myelin (25). Discussion of Results The reliability of the SFl in evaluating nerve injury was shown in a previous study (13). The functional recovery observed in group I (crush) was consistent with the values given by other authors after a similar lesion (22, 72). The almost sudden return of function followed by a period of slow improvement corresponded to what has been described clinically (22, 72) histologically (44), or with the use of tracers (18). We considered this sharp functional recovery to be the ideal result, since secondary improvement by further arrival of fibers at the periphery or by adjustment of the centers is minimal (36, 59, 72). It is generally agreed that after transection the return of function is highly variable both in time and in quality (23,24). Therefore, experimental severance and suture is considered an unreliable technique to study nerve regrowth (32, 47). The widely spread values of group II (suture) coincided with this view. Four other groups of animals, III, IV, VI, and VII, yielded the same pattern of evolution, with globally poor results and one or two occasional satisfactory returns of function, thus indirectly confirming the results of group II (suture). These values are consistent with those of other investigators (3, 22, 43). In particular the absence of a sharp increase in functional return after transection and suture has been described (22, 72). Group V (change of medium, freezing, sharp cut), i.e., the group with all previously cited conditions combined, presented a sharp increase in function followed by a slow further improvement, parallel with that of group I (crush), although the recovery occurred later and was not as complete. Comparison of the evolution of these two groups with that of the other animals led us to conclude that if no unequivocal return of function was observed in the rat by the 32nd day after sciatic injury at midthigh, the subsequent evolution would be unsatisfactory and the results at 2.5 months would be poor or mediocre. We attributed this to misdirection of the sprouts. We attributed the loss of function that followed injection of fluid C in the nerve [group VIII (change of medium)] to disruption of myelin without axonal degeneration, because the mechanical strain of the injection itself is harmless (13) and recovery occurred before new fibers could have regrown to their peripheral targets. Such transient loss of function without degeneration was described by Waller on himself (64) and has been shown to occur after compression or injection of drugs in the nerve (5, 50, 5 1).

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The results of groups II to VII indicated that satisfactory results are occasionally observed with any method of repair, suture (one good result out of seven), precise reunion alone (2/7), precise reunion and sharp cut (l/7), precise reunion and modification of medium (3/l 4). The combination of several factors, however, seemed necessary to obtain consistently good results (7/7). The lack of correspondence between the observed aspect of the repair and the functional results was interpreted as an indication that current microsurgical techniques, although satisfactory at the fascicular level and apparently precise under the operating microscope, still belong to the field of tissue surgery. What seems to be needed in peripheral nerve repair is precision at the axonal level, together with other requirements specific to cellular surgery. The decisive factors for good results could not be analyzed in detail in this study. Nevertheless, we think that our observations emphasize the crucial importance of correct initial path finding by the new fibers. In the peripheral nervous system it is not possible to arrest or even lessen the vigorous regrowth of axons (58), unless the cell body itself is injured. Fibers, however, are very easily misrouted by any obstacle and seem to branch when their progression is hampered (48), thus increasing the probability of misdirection. We hypothesize that wandering and branching is directly proportional to the extent of the trauma. Any injury, whether physical or chemical, which extends on both sides of the region of trauma will favor wandering of new fibers just by increasing the length of this damaged region. Thus, factors that limit the extension of the damaged region should at the same time decrease the probability of fiber wandering and axon branching. We believe that our technique was successful primarily for that reason, i.e., because the extent and severity of damage to the nerve was minimized. REFERENCES 1. ADRIAN, (Biol.)

E. D. 1930. The effects of injury on mammalian nerve fibers. Proc. R. Sot. Lond. 106: 596-618.

2. BAKER, P., A. HODGKIN, AND T. SHAW. 1962. Replacement of axoplnsm of giant nerve libres with artificial solutions. J. Physiol. (London) 164: 330-354. 3. BALLANTYNE, J., AND M. CAMPBELL. 1973. Electrophysiological study after surgical repair of sectioned human peripheral nerves. J. Neural. Neurosurg. Psychiatry 36: 797-805. 4. BORGENS,R., L. JAFFE, AND M. COHEN. 1980. Large and persistent electrical currents enter the transected lamprey spinal cord. Proc. Natl. Acad. Sci. U.S.A. 17: 1209-1213. 5. BRISCOE,C. 1925. The diagnosis of unilateral phrenic nerve paralysis. Luncet 208: 376381. 6. BRUSHART, T., AND M. MESULAM. 1980. Alteration in connections between muscle and anterior horn motoneurons after peripheral nerve repair. Science 208: 603-605. 7. CABAUD, E., W. RODKEY, H. MCCAROLL, S. MUTZ, AND J. NIEBAUER. 1976. Epineurial and perineurial fascicular nerve repairs: a critical comparison. J. Hand Surg. 1: 13 1- 137.

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8. CHAMBERS, R., AND E. CHAMBERS. 196 1. Explorations into the Nature of the Living Cell. Harvard Univ. Press, Cambridge, Mass., pp. 131-140. 9. CLARK, G. 1964. A method of preparation of nerve ends for suturing. Plast. Reconst. Surg. 34: 233-235. 10. COURRIER,R. 1926. La degenkescence Wallerienne chez les chauves-souris hibemantes. C. R. Sot. Biol. (Paris) 94: 1385-1388. 11. DEMEDINACELI, L., AND W. FREED. 1983. Peripheral nerve reconnection: immediate histologic consequences of distributed mechanical support. Exp. Neural. 81: 459-468. 12. DE MEDINACELI, L., AND W. FREED. 1982. Peripheral nerve repair with distributed mechanical support. Sot. Neurosci. Absfr. 8. 26. 13. DE MEDINACELI, L., W, FREED, AND R. WYAI-~. 1982. An index of the functional condition of rat sciatic nerve based on measurements made from walking tracks. Exp. Neural. 77: 634-643. 14. DE RENYI, G. 1929. The structure of cells in tissue as revealed by microdissection. II. The physical properties of the living axis cylinder in the myelinated nerve fiber of the frog. J. Camp.

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15. DE RENYI, G. 1929. The structure of cells in tissue as revealed by microdissection. III. Observations on the sheaths of myelinated nerve fibers of the frog. J. Camp. Neural. 48: 293-3 10. 16. EDSHAGE, S. 1964. Peripheral nerve suture. Acta Chirurg. Stand. Suppl. 331: 2-104. 17. EDSHAGE, S. 1966. Evaluation of freezing as a method to improve cut surfaces in peripheral nerves preparatory to suturing. Plast. Reconstr. Surg. 37: 196-202. 18. FORMAN, D., AND R. BERENBERG. 1978. Regeneration of motor axons in the rat sciatic nerve studied by labeling with axonally transported radioactive proteins. Brain Res. 156: 213-225. 19. GALVANI, L. 1792. Pages 34-37 in MUTINAE, Ed., De Viribus Electricitatis in Motu Musculari Commentarius. Bologna. 20. GAMBLE, H., F. GOLDBY, AND G. SMITH. 1957. Effect of temperature on the degeneration of nerve fibres. Nature (London) 179: 527. 2 1. GRABB, W., S. BEMENT, G. KOEPKE, AND R. GREEN. 1970. Comparison of methods of peripheral nerve suturing in monkeys. Plast. Reconstr. Surg. 46: 3 l-4 1. 22. GUTMANN, E. 1942. Factors affecting recovery of motor function after nerve lesions. J. Neural.

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