quantitative analysis of regenerated crossed sciatic nerves in parabiotic rats

QUANTITATIVE ANALYSIS O F REGENERATED CROSSED SCIATIC NERVES IN

PARABIOTIC RATS

JEAN FOSTER AND HOWARD A. MATZKE Department of Anatomy, University of Eansm, Lawrence

THREE FIGURES

INTRODUCTION

The present investigation of sciatic nerve regeneration in parabiotic rats was prompted by the observations of Matzke and Kamrin ( ' 5 3 ) . These workers found that the number of fibers in the distal stumps of crossed, regenerating sciatic nerves of parabionts was slightly higher than that in the regenerating sciatic nerves of single animals. Available evidence concerning nerve regeneration suggests that while central factors are more important in the extension of the axons out to the periphery, peripheral factors are also important in the establishment of proper terminal connections, in the maturation of nerve fibers and in the functional restoration of the nerve. Matzke and Kamrin ( ' 53 ) suggested that it is unlikely that a nerve would regenerate as successfully into foreign tissue as into its own if a chemotactic influence was exerted by peripheral end-organs. They suggested that the phenomenon of crossed nerve regeneration adds further sup- port to the growing evidence that the control of regeneration is central rather than peripheral.

'This investigation was supported in part by Research Grant B609 of the National Institute of Neurological Diseases and Blindness, U. S. Public Health Service.

A portion of the paper was submitted by the senior author to the Faculty of the Graduate School of the University of Kansas in partial fulfillment of the require- ments for the degree of Master of Arts.

213

214 J. FOSTER AND H. A. MATZKE

Prior to the investigation of Matzke and Kamrin ('53) and Matzke and Fedinec ('55), only one study of sciatic nerve crosses in parabiotic ra ts has been found. Morpurgo ('23) joined 20 pairs of rats in lateral parabiosis and established that extensive regeneration and functional recovery occurs in sciatic nerves crossed between the animals. I n spite of the loss of some of the parabionts, presumably due to intoxication, 9 pairs survived long enough to give useful results. Morpurgo reported perfect reneurotization of the peripheral stump and the return of both sensory and motor functions in 1% to two months. More recent histological and electrical studies (Weiss and Campbell, '44; Berry and Hinsey, '46; and Gut- mann and Sanders, '43) indicate that morphological and functional restitution of cut and sutured nerves is never entirely complete. Complete restoration implies return of fiber number, restoration of fiber diameter, remyelination and the es tabIishment of adequate peripheral connections hy all axons. Theoretically, the preceding conditions must be met before the nerve is restored to a perfect condition of function.

One objective of the present study was to determine the numbers of axons in the proximal and distal stumps of the sciatic nerves in a group of single rats whose left sciatic nerve was cut and sutured and in a group of ra ts composed of parabionts. I n the latter group, one rat supplied the proximal portion of the shared sciatic nerve while its partner supplied the distal portion. A second objective was to compare the processes of regeneration in the nerves of the two groups aiid to determine whether or not they are as generally successful when the axons terminate in the distal stump of an- other animal possessing a genetic background similar to that of i ts partner.

MATERIALS AND METHODS

Twenty-one day old Sprague Dawley litter-mate Albino rats were used in these experiments. The animals were placed in parabiosis essentially after the method of Bunster and Meyer ('33). The sciatic nerves of the adjacent extremities

NERVE REGENERATION 215

were sectioned. The proximal portion of the nerve from the right animal was sutured to the distal nerve segment of the left animal. The sutured nerve was protected with a polyethylene tube. The left sciatic nerves of 17 single control animals were similarly sectioned and sutured.

Of 56 pairs united, 25 survived. Eleven pairs were discarded due to faulty nerve unions o r poor histologic sections. Six single animals and 5 parabionts each were sacrificed at 21 and 42 days. Five singles and 4 parabionts were sacrificed at 100 days. During the survival period the response to nociccptive

TABLE 1

Fiber counts in single animals and parabionts

BINQLE ANIMALS PARABIONTS

Proximal Distal Proximal Distal

21 days Mean 6367 3792 5259 2465 Range 5677 - 7033 3014 - 5621 3088 - 6688 1576 - 3327

42 days Mean 5874 4460 6407 3459 Rangr 4881 - 7062 3020 - 6053 5610-7155 3127-4266

100 days Mean 6568 4969 6028 4279 Range 5502 - 7800 4326 - 5428 4200 - 71 59 2782 - 5832

stimuli applied to the involved area was periodically tested. At the time of sacrifice the regenerated nerve was exposed and stimulated electrically. The nerves were then removed, placed in Bodian’s No. 2 fixative and the sections stained after Romanes’ silver method. A sampling method was used to estimate the total number of axons in each of the sections taken proximal and distal to the suture. Approximately one eighth of the total area of each section was counted. The sampling error mas determined to be no greater than rt 77%.

RESULTS

The fiber counts of the proximal and distal segments of the sciatic nerves of single animals and parabionts are shown in


table 1. Figure 1 is a comparison of distal fiber counts of single and parabiont rats.

The average proximal counts ranged from 5874 to 6568 in single animals and from 5259 to 6407 in parabionts. The range of average distal counts was from 3792 to 4969 in single animals and from 2465 to 4279 in parabionts. Proximal axon counts varied only slightly from the normal fiber count of 6227 which was calculated by taking the mean of the counts by Matzke and Kamrin ('53) and Birren and Wall ('56), 6497 and 5958, respectively. Increases in proximal fiber counts in

0 0 6000

0 8g m S 0 5000 0 5

% 4000 - -

,o- _ _ _ _ _ _ _ - - - - - - - - - -

0 =Singles =zoo0 - . r7)srsbiont.s --- =Single mean - =Pw&biorittnern

21 42 100

a 1000 ~ I I

Days a.F Regeneration

Fig. 1 Numbers of axons in distal segments of nerves.

both groups between 21 and 100 days are indicative of proliferation of proximal fibers (table 1). Although profuse branching has been reported by some investigators, Weiss and Campbell ('44) have cited evidence to show that restriction in the form of arterial tubulation prevents proliferation of proximal axons. The polyethylene tubing used in these experiments may also have prevented extensive proliferation.

Reinnervation of the distal segment occurred more rapidly in regenerating nerves in single animals than in those of parabionts (see fig. 2). At 21 days the distal nerve segments of single animals contained 60.8% of the number of fibers pre-


sent in the normal sciatic nerve while only 39.6% were seen in the distal segment of parabionts. At 42 days these per- centages had increased to 71.6% for single animals and 55.6% for parabionts. Fiber counts at 100 days showed further increases in total axon numbers (see table 1). Distal nerve segments of single animals possessed 79.9% of the number of fibers in the normal nerve, while those of parabionts had 68.7% of the normal fiber count.

Days o f ‘Reg cnerat i o n

Fig. 2 Percentage of normal number of nxons in the distal segments of the nerves.

It is probable that the degeneration of severed fibers in the distal stump is overlapped by the outflow of new axons from the proximal stump. Numerous macrophages were found in the distal stumps of all 21 and 42-day animals (see fig. 3). At all time intervals, however, macrophages were more prevalent in nerves shared by parabionts. It is interesting to note that at 42 days macrophage activity had almost ceased in single animals, while the macrophages present in parabionts equaled the number found in 21 day single animals. Furthermore, macrophage activity persisted in three 100-day parabionts. In single animals the macrophage count perhaps can be taken


as a rough index l o the persistence of the inflammatory process through which the debris from degenerating axons is cleared away. I n parabionts, however, the height and persistence of the inflammatory process may indicate that the nerves have been traumatized during the period of regeneration.

Functional reinnervation was demonstrated in all 100-day animals. Electrical stimulation of the nerve proximal to the suture line resulted either in plantar o r dorsi-flexion of the foot. I n addition, a crossed response was demonstrated in three of the 100-day parabionts, i.e., pinching the leg contain-

Fig. 3 Number of niacrophages in distal nerve segments.

iiig the distal nerve segment with a sharp forceps caused the animal's partner to squeal. I n the animals in which crossed response was detected there were fiber counts of 3498, 5330 and 5832 in the distal segment of the nerve. Also, these were cases in which the macrophage activity was low or absent after 100 days.

At 42 days all single animals responded to electrical stimulation of the nerve with moderate flexion of the foot. I n the parabiont group, however, one pair did not respond to stimulation, three pairs showed only slight foot flexion, and one pair responded moderately. No crossed response was elicited from 42-day parabionts.


In 21-day animals, only one of the singles responded to electrical stimulation. Very slight plantar flexion was observed. None of the parabionts responded. Although such tests are difficult to quantitate, it seems evident that functional reinnervation has begun to return in single animals, and in some cases in parabionts, at 42 days. Considerable function was recovered by both single and parabiontic rats at 100 days.

No evidence of foreign tissue reaction was found in parabiont nerves. No giant cells, areas of necrosis or extensive fibrosis were seen within the fascicles of the nerves. Exten- sive proliferation and fibrosis of epineurial connective tissue was seen in parabiont regenerated nerves. The epineurial connective tissue sheaths of these nerves were composed almost entirely of dense collagenous tissue. I n contrast the epineu- rium of the nerves of single animals was made up of a thin sheath of dense collagenous tissue immediately surrounding the fascicles bordered by loose collagenous tissue and adipose tissue.

DISCUSSION

In the study of nerve regeneration in parabionts the follow- ing facts are of special interest. The process of regeneration on the whole is essentially the same in parabionts and in single animals. There is evidence in the present experiments that the total numbers of axons in the distal segment of parabiont nerves may equal the number found in single animals (see table 1). Likewise, functional recovery in parabionts is similar to that seen in single animals.

In the two groups of 100-day animals differences in the regeneration rate and in the total numbers of fibers present in the distal stump may be influenced by mechanical factors that alter the course of regeneration in the parabiont group. The regenerating sciatic nerve between members of a parabiotic pair is subjected to greater mechanical stress than a nerve regenerating in a single animal. Presumably in the paired animals frequent changes in the position of the limbs result in movement of the polyethylene tubing containing the sutured nerve causing a widening of the gap bptween the ends of the

220 J. FOSTER AND H. A. MATZIiE

stumps. As would be expected in such cases (Young, '421, delay and imperfection of recovery occurred in t,hree of the 100- day parabionts as evidenced by fiber counts of 3953, 2782 and 3498. It was observed that 800,448 and 251 macrophages persisted in these cases, while parabiont nerves containing 5330 and 5832 fibers had 85 and 0 macrophages. Supporting the view that tension widened the gap between the ends of the nerve stumps is the observation that macrophage activity was consistently higher on the whole in parabionts than in single animals (see fig. 3). Prolongation of the inflammatory process due to frequent changes in position, tension and trauma may have resulted in increased macrophage activity and the persistence of histogenous and hematogenous macrophages in some parabionts.

Moreover, it was noted that in three of the 100-day parabionts there was marked thickening of the epineurial connective tissue. This condition was not observed in any of the single animals. The proliferation of connective tissue can be visualized as resulting from trauma to the outer surface of the nerve caused by movement, tension, perhaps even buckling, of the nerve against the edge of the tube.

Further evidence of gaps between the stumps was the presence of numerous disoriented axons in the epineurial connective tissue in the distal nerve segments of some of the 21, 42 and 100-day parabionts. According to Young ('42) criss-crossing of fibers occurs when a gap is present. Inva- sion of epineurial connective tissue could easily result from such disorientation. In addition, twice as many small blood vessels were counted within nerve fascicles of parabiont distal segments as in those of single animals. Seemingly these blood vessels would have more opportunity to enter the distal stump of the nerve when the gap between the proximal and distal stumps had increased.

The technical problems of nerve union between parabiont s have not been completely solved. Tantalum foil coiled to form a close fitting tube (Weiss, '44) may prove to be a better ma- terial for protection of the suture area. With this technique


Matzke and Kamrin ('53) were able to achieve excellent reinnervation of the distal stumps. Weiss, however, described cases where buckling of tantalum cuffs resulted in complete failure of regeneration due to scar tissue penetrating through holes created by wrinkling of the cuffs. In parabionts the pos- sibility of buckling is greater than in single animals. Young and Medawar (cited by Young, '42) allowed concentrated fibrinogen to clot over the apposed ends of stumps of small nerves. Such a procedure might eliminate some of the technical difficulties encountered when protective cuffs are em- ployed.

It is obvious from the results in table 1 that some blocking of axons occurred, probably due to the extent of the scar area within the gap. It is interesting to note, however, that a steady outflow of axons continued in spite of the height and persistence of the inflammatory process in the distal stump. This seems to strengthen the view that strong central influences control the outflow of axoplasm in its movement toward the periphery. Furthermore, peripheral factors important to the establishment of adequate terminal connections and the maturation of nerve fibers (Evans, '53) must have been operative in parabionts as well as in single animals. Otherwise functional recovery would have been impossible.

It is probable that successful regeneration of axons can be expected in all litter-mate animals in which parabiotic union has been satisfactorily performed. Such experiments are somewhat analogous to those of Gutmann and Sanders ( '43). These investigators studied the regeneration of nerve auto- grafts and nerve homografts in the peroneal nerves of rab- bits. They were able to demonstrate that in many instances regeneration in homografts was as satisfactory as in auto- grafts. Apparently discordant factors which prevent homo- grafting in human tissue are either present in lesser amount or absent in the tissues of other animal species. Litter-mate rats were used in the present experiments. For this reason it might be expected that the homograft tissue in the parabiotic


animals would be more closely related genetically to the tissue of the host animal than would that of an ordinary homograft.

The results of these experiments cannot be compared di- rectly with the findings of Morpurgo ('23) since no reference was found to the performance of axon counts. Morpurgo observed considerable recovery of function, and similar observations were made in the present experiments. Functional recovery in the present investigations, however, was accom- panied by lack of coordination and by stiffness of ankle movement in both single and parabiotic rats (similar to that described by Sperry in '45). Although, in a number of individual cases, extensive regeneration of axons was achieved, the axon counts in the present experiments were lower on the whole than those of Matzke and Kamrin ('53). It is suggested that the difference in the results may be due to variations in the technique of joining the nerves and the presence of gaps in the nerve unions of some of the parabiotic rats. Also, the pre- liminary investigations of Matzke and Kamrin included only a small number of cases.

SUMMARY A N D CONCLUSIONS

It has bcen established that regeneration proceeds essentially in the same manner in the sciatic nerves of single animals and parabionts.

Axon counts after 21, 42 and 100-day regeneration periods show many cases in which fiber regeneration in surgically united animals has been equal to or superior to that in single animals. Functional recovery similar to that in single animals also has been observed in parabionts.

Under the conditions of the present experiments, regeneration was more rapid in single animals than in parabionts. Like- wise, functional recovery in single animals after 42 days ex- ceeded that in parabionts. There is evidence to show that mechanical factors may be responsible f o r this difference.

It has been demonstrated that in 21-day parabionts 39.6 per cent of the normal number of axons have reached the distal seapent of the regenerating nerves. Single animal distal


nerve segments contained 60.8 per cent of the normal fiber count at 21 days. Similar variations at 42 and 100 days indicate that “scar delay” was probably longer in the nerves of parabionts.

Regeneration proceeded less rapidly in parabionts than in single animals. After 100 days, however, the total number of axons in parabiont distal nerve segments was 86 per cent of the number found in the distal nerve segments of single animals.

Considering the influence of mechanical factors involved in parabiont nerve regeneration, the number of axons restored in paired animals compared very favorably with the number regenerated in single animals.

LITERATURE CITED

BIRREN, J. E., AND D. WALL 1956 Age changes in conduction velocity, re- fractory period, number of fibcrs, connective tissue space and blood vessels in sciatic nerves of rats. J. Comp. Neurol., 104: 1-16.

The recovery of diameter and impulse conduction in regenerating nerve fibers. Ann. N. Y. Acad. Sci., 4 7 : 559-574.

BUXSTER, E., A N D R. K . MEYER 1933 An improved method of parabiosis. Anat. Rec., 57: 339-343.

EVANS, D. H. L. Investigations on the nature of peripheral factors in- fluencing the process of motor nerve regeneration. J. Comp. Neurol.,

GUTNANN, E., AXD F. K. SANDERS Recovery of fiber numbers and diameters in the regeneration of peripheral nerves. J. Physiol., 2/32 : 489-518.

MATZKE, H., AND B. B. KAMRIN 1953 Regeneration of resected and crossed sciatic nerves in parabiotic rats. Science, 128: 623-624.

MATZKE, I€., AND A. FEDINEC 1955 The spread of tetanus toxin along the sciatic nerve of the rat. Anat. Rec., 221: 335-336.

MORPURGO, B. 1923 Nerve uuion in parabiotic rats. ( I n German) Klinische Wochschrift, 2 * : 129.

SPERRY, R. W. The problem of central nervous reorganization a f te r nerve regeneration and muscle transposition. Quart. Rev. Biol., 20 : 311-369.

WEISS, P. Sutureless reunion of servered nerves with elastic cuffs of tantalum. J. Neurosurg., 1: 219-225.

WEISS, P., AND C. J. CAMPBELL 1944 Nerve fiber counts and muscle tension a f te r nerve regeneration in the rat. Am. J. Physiol., 140: 616-626.

YOUNG, J. Z. 1942 The functional repair of nervous tissue. Physiol. Rev., 22:

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quantitative analysis of regenerated crossed sciatic nerves in parabiotic rats

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