Axons regenerated through silicone tube splices

EXPERIMENTAL

NEUROLOGY

92,6 I-74 ( 1986)

Axons Regenerated

through

Silicone Tube Splices

II. Functional Morphology

R.DOUGLASFIELDSANDMARKH.ELLISMAN Department of Neurosciences, School of Medicine, University of California, San Diego, La Jolla, California 92093 Received May 28, 1985: revision received November 5, 1985 The recovery of axons regenerated through silicone tube splices was studied with electron microscopic and morphometric methods. Regenerated nerves contained both myelinated and unmyelinated axons of near normal morphology. The number and diameter of axons increased with postoperative time, and size-frequency histograms demonstrated that regeneration occurred in all major fiber groups. Remyelination occurred between about 4 and 6 weeks. Some of the smallest regenerated axons had unusually thick myelin sheaths, but overall regenerated axons had a slightly thinner sheath compared with similar-size normal fibers, although the ratio of sheath thickness to axon size was within the normal limits of g = 0.65 to 0.8 by 6 weeks. Axons did not, however, regain their normal size within IO months of surgery. This deficit was apparently the primary factor limiting conduction velocity in these regenerated axons. Q 1986 Academic

Press, Inc.

INTRODUCTION In the previous paper we reported the conduction properties of rat sciatic axons which had regenerated across a l-cm gap through a silicone tube. In this paper we report complementary studies of the morphology of the regenerated axons from the same nerves in which conduction properties were measured. Measurements of conduction properties of these samples prior to fixation for electron microscopy will provide a better understanding of the struc’ We thank J. LeBeau and F. Longo for instruction in the silicone tube splice technique. This work was supported in part by grants from the National Multiple Sclerosis Society, Muscular Dystrophy Association, the National Institutes of Health (NINCDS N 147 18). and the National Science Foundation to M. H. E. Dr. Fields’ current address is Dept. of Neurology, Stanford Univ. Medical Center, Stanford, CA. 94305. Please address reprint requests to Dr. Ellisman. 61 0014-4886/86 $3.00 Copyright 0 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.

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tural and functional relationships of axons undergoing regeneration with this tubation procedure. METHODS Nerves were removed from the electrophysiologic recording chamber and fixed by immersion in cold fixative containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.15 A4 cacodylate buffer at pH 7.3. Samples were postfixed 1 to 2 h in 1% osmium tetroxide, washed in buffer, dehydrated in an ethanol and acetone series, and embedded in epoxy resin. Sections were cut and stained with lead citrate and uranyl acetate, and examined with a JEOL 1OOB or 1OOCX transmission electron microscope. Transverse sections were taken of several nerves in which the tubation procedure was successful at points proximal and distal to the tube and within the tube at 2-, 5-, and &mm points along the gap. For the purposes of quantitative analysis, some standard sampling point was needed that could be (i) located precisely, (ii) was free from any possible influences of transition zones at the ends of the tube, and (iii) would best correlate with the physiologic measurements of conduction properties of axons in the tube. Because of the limited spatial resolution of the physiologic techniques, the midpoint seemed most suitable. Transverse sections of the regenerated nerves were cut at the midpoint of the grafted region (i.e., 5 mm from the cut end of the nerve). These were examined with TEM and micrographs were taken at 5,000 times magnification. Montages of the nerves were constructed from 8 X IO-in. prints for morphometric analysis and quantification of the numbers and types of regenerated axons. Measurements were made of the diameter of axons in a subsample taken along a transect across the width of the regenerated nerve. For greater accuracy, the axon in cross section was considered an ellipse, and measurements of the major and minor axes were made. The cross-sectional area of the axonal core (i.e., not including the myelin sheath) was calculated, and this result was reported as the diameter of a circle having an equivalent area. Any axon with a major axis greater or equal to three times the minor axis was excluded from the analysis to avoid axons cut at oblique angles. To evaluate the extent of remyelination, axon caliber and myelin thickness were measured and the total fiber diameter was calculated from those measurements to express the myelin thickness as the ratio of fiber diameter to axon core diameter (g ratio). To statistically evaluate differences in g ratios among different fibers, large and small fibers in each nerve were measured independently, so as to separate the bimodal population of fibers in the nerve into two discrete, normally distributed populations as required with a parametric two-way analysis of variance (ANOVA).

MORPHOLOGY OF REGENERATING AXONS

63

RESULTS Nerves regenerated for more than 47 days contained both myelinated and unmyelinated axons of normal morphology (see Fig. 1). At earlier time points, axons were observed undergoing myelination by Schwann cells (Fig. 3A, B), or were unmyelinated. The development of these axons was preceded by the deposition of extracellular matrix material, and the migration of Schwann cells into the silicone cuff in the 1st few weeks following surgery. Gradients in development were visible along the tube at the earliest time points, but such gradients in fiber size and myelin thickness were not obvious at later stages. The number and diameter of axons (sampled at the 5-mm point) increased with postoperative time. In the earlier stages of regeneration, the nerves contained Schwann cells in a loosely packed and unorganized arrangement. Unmyelinated and myelinated axons became segregated as regeneration proceeded, and groups of fibers were bundled into fascicles in later stages (Fig. l A-E). Regenerated axons at all ages followed more tortuous courses than normal nerves, as seen by the Iarge number of axons transected at oblique angles in these nerves. Size frequency histograms (Fig. 2) show that many axons larger than 1 pm in diameter were unmyelinated at 47 days of regeneration. After 2 months of regeneration, very few unmyelinated axons larger than 1 /*rn in diameter were seen. The median size of unmyelinated axons at all stages was about 0.75 pm. The median size of myelinated axons was seen to increase as regeneration proceeded, but fibers did not attain axon diameters greater than 7pm, even after 10 months of recovery. The form of size-frequency histograms for nerves regenerated longer than 3 to 4 months was similar to that of normal nerves. This indicated that all major fiber types had undergone regeneration by that time, and had attained nearly normal relative sizes. but the absolute size of axons in all fiber groups was less than in control nerves. These morphological results were in agreement with the size and shape of the compound action potential which develops a normal bimodal waveform after 3 to 4 months. The thickness of the myelin sheath was nearly normal for the diameter of axons after about 6 weeks of regeneration (Figs. 4, 5). However, there was a small but statistically significant difference between the g ratio for small and large fibers of regenerated nerves (mean g = 0.67 and 0.76; SD 0.13 and 0.062, respectively, P -=c0.001). Similarly the g ratio increased from 0.66 to 0.70 rfr0.068 and 0.033) for the small and large normal fibers. Secondly, regenerated fibers had a statistically significant larger g ratio compared with controls of comparable size (0.7 1 and 0.67; 2 0.114 and 0.054, respectively, P < 0.003). This slight difference between regenerated and control axons of comparable

FIG. 1. A-E-cross sections of the sciatic nerve during regeneration through the silicone tube tube splices at different days after implantation (A, 47; B, 67; C, 98; D, 138; E, 302 days). F is a control preparation for comparison.

66

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FIG. 2. A-F-size/frequency histograms for axon core diameters. Note the increase in number and size of fibers with regeneration time, and the remyelination of axons larger than about 1 pm. The size-frequency histograms become bimodal after 3 months (98 days). By IO months (302 days) the histograms were similar to controls, indicating that the major fiber groups of normal nerve had been restored (A, N = 76; N = 99; C, N = 65; D, N = 122; E, N = 9.5).

size was also seen from Fig. 5. Thus, the myelin sheath was thinner on small or regenerated axons. Some regenerated axons had extreme g ratios of less than 0.4. These were always very small axons of less than 2 pm in diameter. DISCUSSION The results of this study, together with the electrophysiological studies on these nerves prior to fixation for electron microscopy (cf. previous paper), show that axons will regenerate across a l-cm gap inside a silicone tube splice, with the restoration of conduction properties and the formation of functional neuromuscular junctions. A lasting conduction deficit was measured in electrophysiologic experiments, together with higher electrical resistance and decreased excitability.

FIG. 3. A-the normal morphology of myelinated and unmyelinated axons in the sciatic nerve of a control rat. A = axons, S = Schwann cells. B-an axon regenerated after 47 days undergoing remyelination.

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100

300

no0 DAYS REGENERATION

FIG. 4. Myelin thickness (measured as the ratio of total fiber diameter to axon core diameter), showing that regenerating axons had a myelin sheath thickness within normal limits even in the earliest stages of recovery (g = 0.5 to 0.9). The difference between small and large regenerated and control nerves was statistically significant by a two-way ANOVA (P < 0.001).

Size-frequency histograms generated from electron micrographs show that axons do not regain normal size. For example, the alpha motor axons are 6 to 7 pm in diameter in regenerated nerves after 10 months, compared with 7 to 9 pm in control nerves. The small size of regenerated axons would be associated with a higher axial resistance, and therefore a lower conduction velocity (6, 13, 30, 40, 43). Other studies show that axons regenerating across a gap, or through a grafted or sutured lesion, never achieve their normal size (1, 3, 12, 15, 48). Regen-

0-o Axon

Diameter

(rm)

FIG. 5. The relation between myelin thickness (g ratio) and fiber diameter for normal (0) and regenerated (0) nerves. There were fewer small thickly myelinated axons in normal nerves. Regenerated axons had thinner sheaths (higher g ratio) than normal nerves of comparable diameter.

MORPHOLOGY

OF

REGENERATING

AXONS

69

eration that follows crushing lesions is more complete (26, 38) primarily because the pathways toward appropriate target organs are less disrupted (45. 62). In addition to this factor, the growth of axons regenerating through silicone tubes has been shown to be stunted by the constricting and ischemic effects of the tube itself; the problem is accentuated as the length of the gap increases and vascularization is further restricted ( 14). In Ducker and Hayes’s studies of this problem, the optimum diameter for maximum fiber growth was achieved with tubes having a cross-sectional area 2.5 times that of the nerve, which is approximately twice as large as that used in these studies. Significant improvements in conduction velocity would follow if techniques could be devised that would increase the diameter of axons regenerated through this device. Clinical studies show that a decreased conduction velocity may produce only slight functional impairments. More profound deficits will result from failure to conduct the temporal pattern of spikes faithfully (55). Discontinuities in fiber size can slow, block, or reflect impulses, depending on the changes in cable properties and impedance (25,52). In the case in which axons are sharply constricted, such as in the regenerated region inside the Silastic tube, efferent conduction would be especially susceptible to conduction block, and longterm sensory impairments might be expected unless other factors act to compensate for impedance mismatching at the lesion. The observations of Sanders that the fibers proximal to a lesion develop thicker than normal myelin (43, 44) and the adjustments in internodal spacing and nodal morphology (9, 11, 13, 18, 24, 27, 29, 31, 35, 37, 39, 41, 53, 56, 57, 58, 61) to alter membrane impedance of axons in other situations are possible examples of compensatory processes that could promote conduction through such a region. Similar problems occur at branch points along axons. Although branching was not measured directly, other research indicates extensive branching in the processes of axonal outgrowth toward target sites (36, 46). These data show that the g ratio is not constant for different size fibers of either regenerated or control nerves, as it often reported, but smaller fibers have proportionately thicker myelin sheaths than larger fibers. The earliest studies of this question also showed a parabolic-like relationship between myelin thickness and fiber diameter (43) with the smallest fibers having g ratios well below 0.4. Although more recent literature has in some instances concurred with this nonlinear relation between g ratio and axon diamater (4, 10, 19-2 1, 28) other research has shown that the relation is linear ( 16, 17, 22, 23); or that there is no simple relation between myelin thickness and diameter, but that g quotients show a wide variance within a range of 0.5 to 0.9 in normal axons (2, 47, 49, 55, 59, 60). The question has been a matter of some controversy because Rushton (42) used Sanders’s (43) parabolic relation for g and fiber diameter to derive formulae relating conduction velocity

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ELLISMAN

(29) to axon diameter. The validity of these relations has therefore been questioned in cases in which the g ratio does not appear to follow a parabolic function of axon diamater, such as in some small myelinated fibers in the central nervous system (55, 60). The reasons for this discrepancy in g ratios may include differences in sampling technique (lo), methods (light or electron microscopic) (56, 60) or biological differences among nerves of different species and ages (8), or failure to take into consideration other interrelated parameters (19). Friede and Beuche (5, 19) recently showed that the g ratio of normal nerves has features of both parabolic and linear distributions, with wide scatter for the small axons, which those authors describe as a “cornucopiashaped curve.” Their studies show that a better relation can be obtained when internodal distances are taken into consideration (19, 50). This is because regenerated nerves typically have shorter internodal distances (5, 12, 24, 27, 3 1, 35, 37) and therefore require thinner myelination to maintain an adequate safety factor. The increased g ratio observed in these studies would therefore predict that internodal distances are also reduced in these fibers regenerated through a Silastic cuff. The exponential nature of the g ratio is more apparent in studies of regenerated nerves, whether the lesion is through crushing freezing or sectioning (5, 43, 44, 48). Recent studies (5, 48) attribute this parabolic shape to the presence of very small regenerated axons that are more abundant in early stages of regeneration with abnormally thick myelin sheaths. Axons of this type (with g ratios ~0.4 and diameters ~2 pm) were also revealed in these studies. Those authors suggest that such axons represent atrophied fibers that have become myelinated, but have failed to reach their appropriate end organs, and are undergoing degeneration which reduces the diameter of the axon. An alternate explanation might be that this is a functional adaptation to compensate for the lower space constant due to the increased axial resistance of these very small axons. All other factors being equal, the space constant is proportional to the square root of the ratio of the resistance of the axolemma and the axial resistance of the fiber. The fewer numbers of fibers having g ratios less than 0.4 in later stages of regeneration and in normal nerves of adults may therefore reflect the growth of very small myelinated axons, rather than the loss of atrophic fibers. As such axons acquire a larger diameter and concomitant reduction of axial resistance, the myelin need not be as thick to maintain the same space constant. Also, the sheath made by a Schwann cell will have an absolute minimum thickness, but the diameter of an axon may vary continuously. Therefore, a small g ratio woclld result if very small axons (~0.25 pm) are myelinated by only two layers of membrane, which has an incremental thickness of approximately 13 nm per layer. Either of these processeswould explain the presence of thickly myelinated small fibers in normal nerves, which may be seen, but are infrequent (10, 19, 23, 43, 49). Also

MORPHOLOGY

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morphologic indications of myelin resorption and autolysis of axons are not characteristic of these axons. These explanations predict that nerves would have a greater proportion of small, thickly myelinated fibers in juvenile animals in which more axons would be encountered in the left tail region of the curve. It should be observed that sampling methods that fail to take into account the bimodal distribution of fiber diameters in many peripheral nerves, will easily fail to reveal the difference in g ratio for small and large fibers. If the larger fibers are preferentially sampled in the plateauing portion of the parabola, a linear relation will be observed, whereas if only a portion of the smaller mode is sampled, and mixed with the measurements of the larger mode, no relation between g and fiber diameter will be observed. Despite these differences in myelin thickness between normal and regenerated nerves, the thickness of the myelin sheath was nearly normal for regenerated axons after 6 weeks. The g ratio varies from 0.5 to 0.9 in axons from normal nerves (7, 19,23,47,49,5 1,54), and differences of this magnitude will affect conduction velocity by about 5% (34, 5 1,54, 58,60). This is another indication that the conduction velocity is primarily limited by the small size of these regenerated axons. These morphologic measurements are in accordance with electrophysiologic measurements showing that refractory periods and the membrane time constants were nearly normal in regenerated axons after about 2 months. The higher axial resistance of small growing axons in the earliest stages of regeneration seem to be the main reason for the abnormally long time constant in the earliest stages of regeneration, but higher membrane capacitance due to somewhat incomplete remyelination and shorter internodes and perhaps nodal morphology could also contribute to this increased time constant. The very small, heavily myelinated fibers do not apparently contribute significantly to the compound action potential so the physiologic characteristics of this interesting class of axons are unknown. The long time constant and associated structural abnormalities are temporary and representative of only the first stages of regeneration and functional recovery. It is interesting that in later stages of regeneration, the time constant of excitation is not significantly different between normal and regenerated nerves, yet regenerated axons are smaller, have a slightly thinner myelin sheath, and presumably shorter inter-nodal distances. Apparently these and other factors combine so as to compensate for the functional effects of these morphologic changes. More detailed research on these parameters and on possible remodeling of nodal and internodal membrane using freeze-fracture and electrophysiologic methods (4, 32, 33) could provide a better understanding of these processes. These studies show that recovery after this tubation treatment is similar to that obtained when nerve stumps are joined directly by epineural suture. These results should be useful to continuing studies using this tubation pro-

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cedure as an in situ experimental remyelination.

AND

ELLISMAN

chamber for studies of regeneration and

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