Regeneration of the perineurium after microsurgical resection examined with immunolabeling for tenascin-C and alpha smooth muscle actin

Abstract

The regenerative process of the perineurium and nerve function were examined using an in vivo model of perineurium resection in the rat sciatic nerve. Our hypothesis is that the regenerative process of the perineurium can be demonstrated by immunolabeling for tenascin-C and alpha smooth muscle actin after microsurgical resection of the perineurium in vivo. A total of 38 Lewis rats were used. Eight-week-old animals were assigned to one of two groups: the epi-perineurium removal group or the sham group. Under operative microscopy, the sciatic nerve was dissected from surrounding tissues at the thigh level from the ischial tuberosity to the fossa poplitea. The epi-perineurium was carefully removed by cutting circumferentially and stripping distally for 15 mm. For CatWalk® dynamic gait analysis, only right sciatic nerves underwent surgery; the left sciatic nerves were left intact. For pathological and electrophysiological tests, both the right and left sciatic nerves underwent surgery. Analysis of data was performed at each time interval with a two-group t-test. P<0.05 was considered statistically significant. After resection of a 15-mm section of the epi-perineurium, immediate endoneurial swelling occurred in the outer portion and spread into the central portion. Although demyelination and axonal degeneration were found in the swollen area, remyelination and recovery of electrophysiological function were seen after regeneration of the perineurium. An immunohistological and electron microscopic study revealed that the perineurium regenerated via fusion of the residual interfascicular perineurium and endoneurial fibroblast-like cells of mesenchymal origin. CatWalk gait analysis showed not only motor paresis but also neuropathic pain during the early phases of this model.

Fig. 1

The epi-perineurium was stripped distally for 15 mm (white arrow) and removed microsurgically, taking care to minimize damage to the axons.

Fig. 2

Mean intensity of paw placement. At days 2 and 7, the group that underwent epi-perineurium removal showed a significantly lower mean intensity of paw placement than the sham group (*P < 0.05).

Fig. 3

Duration of stance phase (A) and swing phase (B). Two days after surgery, the time that the lesioned paw was in contact with the floor (stance phase) was reduced to 0.43 ± 0.39 and the swing phase increased to 2.69 ± 1.25 of the preoperative value, with no significant differences between groups.

Fig. 4

Individual paw parameters: print area (A), max area (B). Two days after surgery, there were significant (P < 0.05) differences between groups in terms of print area (A) and max area (B). In the following weeks, these parameters gradually increased in the group that underwent epi-perineurium removal, with no significant differences between groups (*P < 0.05).

Fig. 5

Time course of latency of CMAPs. The CMAP latency of the anterior tibialis muscle was significantly longer for the epi-perineurium removal group than for the sham group 20 days after surgery. At day 42, the nerve tended to recover in the epi-perineurium removal group (*P < 0.05).

Fig. 6

In the sham group, normal sciatic nerve was observed in sections stained for toluidine blue (bar = 50 μm) (A), electron microscopy (bar = 5 μm) (B).

Fig. 11

Immunohistochemical study of TN-C (4F10TT) expression in the sham group and the epi-perineurium removal group. In the sham group, TN-C (4F10TT) was expressed in the perineurium (curved arrows) (A). At 2 days after surgery, TN-C (4F10TT) was overexpressed in the nerve fascicle (black arrows), especially in the outer portion (B). At 7 days, TN-C (4F10TT) was overexpressed in the outer portion of the endoneurium (black arrows), particularly at the edge of the nerve fascicles (C). At 20 days, TN-C (4F10TT) was expressed in the periphery of the nerve fascicles with adhesion (black arrows), and was not expressed in the nerve fascicles (D). At 42 days, TN-C (4F10TT) surrounded the nerve fascicle (black arrows) and appeared to be an original perineurium (E) (bar = 200 μm).

Fig. 12

Immunohistochemical study of TN-C (4C8MS) expression in the sham group and the epi-perineurium removal group. In the sham group, TN-C (4C8MS) was expressed only at vessel walls (arrows). At 2 days after surgery, TN-C (4C8MS) was weakly expressed in the outer portion of the nerve (arrows) (B). At 7 days, TN-C (4C8MS) was weakly expressed at the edge of the nerve fascicles (arrow) (C). After 20 days, there were no appreciable differences in the expression pattern of TN-C (4C8MS) in terms of spatial distribution or intensity between the treated and sham groups (D,E).

Fig. 13

Immunohistochemical study of α-SMA expression in the sham group and the epi-perineurium removal group. In the sham group, α-SMA was expressed only at vessel walls (black arrows). At 2 days after surgery, α-SMA was expressed in the endoneurium, especially around vessels (arrows) (B). At 7 days, α-SMA was expressed at the edge of the nerve fascicles (arrows) (C). At 20 days, α-SMA was expressed in the endoneurium but not in the perineurium (D). There was a significant difference in the percentage area expressing α-SMA in the endoneurium removal group at days 2, 7 and 20 compared with the sham group. At 42 days, α-SMA was expressed only at the vascular wall, and the expression patterns matched those observed for the sham group (E). (bar = 200 μm).

Fig. 7

At 2 days after removal of the epi-perineurium, marked endoneurial swelling in the outer portion was observed in sections stained for toluidine blue (bar = 500 μm) (A). In the central portion of the nerve fascicle, most of the myelinated nerve fibers remained intact (bar = 50 μm) (B). However, in the swollen outer portion, degeneration of the nerve fibers was noted (arrow) (bar = 50 μm) (C). An englobed myelinated nerve (arrow) outside the endoneurium was observed by electron microscopic examination (bar = 2 μm) (D).

Fig. 8

At 7 days after removal of the epi-perineurium, the area of endoneurial swelling became larger (bar = 500 μm) (A). The central portion of the endoneurium remained intact (bar = 50 μm) (B), but demyelinated and degenerated nerve fibers were noted in the outer portion of the endoneurium (arrows) (bar = 50 μm) (C). Electron microscopy revealed an englobed myelinated nerve and loss of collagen fibers in the extracellular matrix (bar = 2 μm) (D).

Fig. 9

At 20 days after removal of the epi-perineurium, the area of endoneurial swelling spread further (bar = 500 μm) (A), and degenerated nerve fibers (arrow) were noted in toluidine blue-stained sections in the outer portion (bar = 50 μm) (B) and in the central portion (arrow) (bar = 50 μm) (C) of the endoneurium. However, regeneration of the perineurium by the laminar structure of endoneurial fibroblast-like cells (arrows) were observed by electron microscopic examination (bar = 2 μm) (D).

Fig. 10

At 42 days after removal of the epi-perineurium, differences in the outer and central portions diminished (bar = 500 μm) (A), and the whole nerve was slightly swollen. In the subperineurial area, re-myelinated nerve fibers were noted in sections stained for toluidine blue (arrows) (bar = 50 μm) (B) and in electron microscopy (arrows) (bar = 2 μm) (D). However, nerve fibers that spread to the outside were also observed (arrows) (bar = 50 μm) (C).

Fig. 14

The number of myelinated nerve fibers in the center and periphery of both groups. The number of myelinated nerve fibers decreased after resection of the epi-perineurium. There was a significant difference between the center and periphery in the epi-perineurium removal group at day 7 (*P < 0.05). Recovery of the number of myelinated nerve fibers after epi-perineurium removal was observed after day 20.

Fig. 15

Time course of wet weight muscle changes in both groups. Percentages of wet muscle weights of the sum of tibialis anterior and gastrocnemius muscles to total body weights in each group are shown (*P < 0.05).