Plasticity of peptidergic innervation in healing rabbit medial collateral ligament

Abstract

Background: Denervation substantially impairs healing of the medial collateral ligament (MCL). Because normal ligaments are sparsely innervated, we hypothesized that neuropeptide-containing neurons would sprout or proliferate after ligament transection, followed by later regression with healing, in a manner analogous to blood vessels.

Methods: We transected the right MCL in 9 mature female New Zealand white rabbits and killed 3 rabbits at 2, 6 or 14 weeks. Alternate sets of 12-mm serial sections of healing MCL scars were examined by fluorescent immunohistochemistry for substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and pan-neuronal marker PGP9.5.

Results: Normal MCLs had few peptidergic fibres located in the epiligament in a perivascular pattern. At 2 weeks, PGP9.5-, SP-and CGRP-positive fibres had increased in the epiligament adjacent to the injury. By 6 weeks, there were increases in CGRP-and PGP9.5-positive fibres in epiligament and scar, with similar but less marked increases in SP-positive fibres. At 14 weeks, there was notable regression of immunostained peptidergic nerve fibres in the scar.

Conclusion: This experiment shows evidence for a remarkable plasticity of ligament innervation after injury, supporting the idea that neuronal factors play a fundamental role in wound healing.

FIG. 1. Low-power view of a longitudinal section of rabbit medial collateral ligament at 6 weeks postinjury, showing the transition zone between normal ligament matrix and scar. Note the organized parallel bundles of crimped collagen fibres in the normal matrix at left (arrow) and the disorganized noncrimped collagen fibres in the scar at right (arrowhead) (hematoxylin–eosin stain, scale bar = 100 μm).

FIG. 2. Photomontage of typical high-power images of immunohistochemically stained nerve fibres (arrows) at different time points after injury. (A) At 2 weeks, CGRP fibres appear very fine in the early scar. (B) At 6 weeks, CGRP-immunoreactive growth cone–like structures (large arrow head) and sprouting appear, as well as a predominantly perivascular distribution of fibres. (C) At 14 weeks postinjury, there are fewer CGRP-immunoreactive fibres in the scar, with most found again in the epiligament. (D) All SP-positive profiles were found to be perivascular at 2 weeks after injury. (E, F) Similar to the appearance at 2 weeks, SP-positive profiles were only found associated with proliferating vessels within the scar at later time points. (G) Typical image of NPY-containing fibres associated with small arterial vessels in a 6-week postinjury scar. (H) At 6 weeks postinjury, staining for the marker PGP9.5, which stains all nerve fibres, revealed a pattern that combined aspects of the other markers, with most fibres in a perivascular location, and some free in the scar matrix (scale bar = 20 microns). CGRP = calcitonin gene-related peptide; NPY = neuropeptide Y; PGP9.5 = protein gene product 9.5; SP = substance P; v = vessel lumen.