Correlation Between the Clinical and Histopathological Results in Experimental Sciatic Nerve Defect Surgery

Abstract

Background and Objectives: Peripheral nerve defect regeneration is subject to ongoing research regarding the use of conduits associated with various cells or molecules. This article aims to correlate histopathological and clinical outcomes at the end of a 12-week experiment performed on a rat sciatic nerve model and show which repair method has the best results. Materials and Methods: Forty male Wistar rats were divided into four groups to compare the results of four different methods of reconstruction for sciatic nerve defect: (1) nerve graft-control group, (2) empty aortic conduit, (3) aortic conduit filled with platelet-rich plasma (PRP) and (4) aortic conduit filled with mesenchymal stem cells. There were three clinical examinations: a sensitivity test, a mobility test and a footprint test. After 12 weeks, the nerves were excised and assessed microscopically using conventional Hematoxylin and Eosin staining (HE), special stains and immunohistochemistry (IHC). Results: Nerve regeneration was observed in all batches, both from the clinical and histopathological assessment; the two types of examinations correlated for each batch. Immunohistochemistry and special staining offered a more complete image of the nerve regeneration results. Conclusions: Superior nerve regeneration was achieved using an aortic conduit in combination with either PRP or stem cells, while the empty aortic conduit recorded lesser results.

Keywords: PRP; experimental model; histopathology; nerve regeneration; stem cells.

Figure 1

Macroscopic aspects of nerves after regeneration. (A) Narrow sciatic nerve-auto-graft site. (B) See-through sciatic nerve in the distal end (marked *). (C) Robust, opaque sciatic nerve at the site of the nerve repair.

Figure 2

Batch 1, proximal level: (A) Normal nerve bundles—longitudinal and transverse sections (HE, 4×); (B) Masson’s trichrome stain: There are striated muscle peripherally (star), the connective tissue of the perineurium (arrow) and nerve fibers with attenuated, pale purple–red myelin sheaths. The suture is seen on the right side of the image (10×); (C) Gömori stain: fine reticulin fibers in a longitudinal plane of section (10×); (D) PGP 9.5 immunostain is strongly positive in a cytoplasmic pattern in most of the nerve fibers (10×); (E) S100 stain: strong nuclear and cytoplasmic positivity in the majority of the nerve fibers; moderate intercellular edema (40×).

Figure 3

Batch 1, distal level: (A) HE-moderate nervous regeneration; intra- and intercellular edema are readily apparent (10×); the circle highlights the suture site (B) PGP 9.5-overall cytoplasmic positivity, with varying intensity among the nerve fibers, in a mosaic pattern (10×); (C,D) S100-moderate nuclear and cytoplasmic positivity, in a heterogeneous pattern; lack of staining in the damaged nerve fibers (20×–40×); (E) Masson’s trichrome stain highlighting the connective tissue meshwork. The myelin sheaths display weak discontinuous staining (20×); (F) Gömori stain: prominent intercellular edema; nerve fibers show architectural distortion and attenuated reticulin meshwork (20×).

Figure 4

Batch 2, point of junction between proximal and distal levels: (A) Atrophic striated muscle (arrow), fibrotic aortic conduit and thin nerve fibers showing weak-to-moderate regeneration (star). The circle highlights the suture (HE, 10×); (B) Masson’s trichrome stain: The nerve fibers are disorganized and interrupted, displaying faint myelin sheath staining (40×). (C) PGP 9.5 shows moderate cytoplasmic staining. Many fibers are immunonegative and display vacuolizations (arrow). Viable striated muscle is focally visible in the periphery (arrowhead). The suture can be seen in the upper right corner (20×); (D) S100 shows moderate nuclear and cytoplasmic positivity (20×).

Figure 5

Batch 3, proximal and distal levels: (A) HE-proximal level (lower section) and distal level (upper section): Similar nerve calibers and optimal nerve regeneration. Distally there are gaps through which nerve bundles penetrate the surrounding tissue (arrow, 4×); (B) proximal and distal levels showing strong PGP9.5 staining and similar caliber (4×); (C) S100-intense staining in the majority of the distal nerve fibers and their growth into adjacent tissue (10×); (D) CD34-numerous capillaries among the distal nerve fibers (arrowheads, 40×); (E) Masson’s trichrome stain highlighting the connective tissue meshwork of the distal region (10×); (F) Gömori silver stain highlighting in black the fine reticulin fibers surrounding the capillaries in the distal portion of the nerve (arrowheads, 40×).

Figure 6

Batch 4, distal level: (A) Delicate mesenchymal tissue with regenerating nerve fibers (HE, 4×). Inset shows loose mesenchymal tissue with numerous capillaries and scattered stellate cells displaying a stem phenotype (HE, 10×); (B) S100-prominent nerve fiber regeneration; some expansion throughout the aortic conduit and extension into adjacent tissue. There is positive internal control in the peripheral adipose tissue (4×); (C) PGP9.5-same growth pattern as seen with S100 immunostain (4×); (D) PGP9.5-strong cytoplasmic positivity in the nerve fibers extending into the endoluminal mesenchymal tissue (40×); (E) CD34-focal, moderate-to-intense membranous positivity in the stellate cells, highly suggestive of their stem phenotype (20×). Inset displays positive mesenchymal star-shaped cells (40×); (F) Ku80 stain is negative in the regenerating portion of the nerve. This finding suggests that DNA repair might be impaired in the long run (20×); (G) Masson’s trichrome stain highlights the connective tissue; faint myelin sheath staining in most of the nerve fibers (10×); (H) Gömori stain underlines the fine reticulin meshwork extending into the mesenchymal tissue, paralleling the growth of the nerve fibers and capillaries (20×).

Figure 7

Nervous regeneration in the distal portions of the 4 batches (B1–B4), comparative aspects: Batches 1, 3 and 4 show superior nervous growth as compared to batch 2; periarterial expansion is a common feature seen in (B2–B4) (S100, 10×).