Neuronal Mechanisms in Tendon Healing

The incidence of tendon injuries is increasing. Tendon healing and modelling are frequently protracted, often leading to pain and impaired function so called overuse syndromes. Presumably, the sensory nervous system may contribute both to promotion of repair as well as to inhibition of healing and tissue modeling. Knowledge of the obstructive influence of systemic neuropathy, chronic inflammation and immobilization on repair and modeling as well as of how and when mobilization should be applied is vital in order to improve the healing process. This study in the rat was designed in order to further explore the local regulatory role of the sensory neuropeptides, SP and CGRP, in tendon repair and modeling. Histological and immunohistochemical analysis over 16 weeks in a tendon rupture model detected a peak occurrence of neuronal SP/CGRP in the healing area at weeks 2-4 coinciding with a maximum increase in organized collagen. Quantitative assessments (RT-PCR) of gene expression at 1 and 2 weeks disclosed a conspicuous increase in the mRNA levels of SP(NK1) and CGRP-receptors (CRLR and RAMP 1) in the healing tendon at 2 weeks. The increased occurrence of sensory neuropeptides and up-regulation of their receptor expression during the maximum deposition of organized collagen suggest a regulatory role in tendon tissue proliferation. At 2 weeks a concurrent elevation in the expression of extracellular matrix (ECM) proteins (collagen type I and III, versican, decorin and biglycan), growth factors (BDNF, bFGF, IGF-1) and inflammatory mediators (COX 1, COX 2, HIF-1α) was seen. The effects of increased physical activity on tendon healing at week 4 were assessed according to the diameter of organized collagen and the occurrence of SP/CGRP. Wheel running seemed to stimulate the rate of nerve retraction from the healing area and a subsequent decrease in the SP/CGRP occurrence. Concomitantly, an increased diameter of longitudinally organized collagen as well as an increased maturity of the repair tissue was observed. Further investigation on the neuronal involvement during plaster-immobilized tendon healing established a decreased diameter of longitudinally organized collagen and a less mature healing area at 4 weeks postrupture. This was paralleled with a marked decrease at week 2, in the expression of SP/CGRP-receptors to levels similar to intact controls. The findings indicate that immobilization reduces the tissue susceptibility to neuropeptide stimulation. Two weeks of immobilization also decreased the expression of ECM molecules, growth factors and inflammatory mediators, whereas these effects were not observed at week 1 implying that a short period of immobilization does not seem detrimental to the healing process. To investigate whether impaired healing seen in different neuropathic conditions may be due to reduced occurrence of sensory neuropeptides, capsaicin-denervated animals were assessed with respect to the development of biomechanical tissue properties during tendon healing. Lower levels of SP in the dorsal root ganglia (DRG) correlated with a decrease in transverse area, ultimate tensile strength and stress at failure in the healing Achilles tendon. Nociception, sensitivity to mechanical and thermal stimuli, after denervation correlated with the levels of SP in peripheral nerves, and was subsequently used as a surrogate for local SP occurrence. A high sensitivity to noxious stimuli at early time-points, possibly as a reflection of local SP concentrations, correlated with later improved biomechanical tissue properties, indicating that early SP occurrence is critical for the subsequent healing process. High sensitivity to noxious stimuli preoperatively was correlated with high levels of SP and CGRP centrally as well as peripherally later during the healing process. Such findings imply that postoperative pain and also subsequent healing capacity, reflected by release of sensory neuropeptides, could be partially foreseen by preoperative assessments of pain sensitivity. In a model of systemic chronic inflammation involving the Achilles tendon, the occurrence of sensory neuropeptides and inflammatory cells was restricted to the paratenon and bone tendinous junction, suggesting a role in paratenonitis and enthesitis. Neither nerves containing neuropeptides nor inflammatory cells were seen in the tendon proper. These findings imply that regulation of inflammation occurs in the tendon envelope and that there might be a connection to the development of tendinosis. The results of this thesis demonstrate that the regulation of tendon repair and promotive effects of early mobilization seem closely related to a functionally intact and quickly responding peripheral sensory nervous system. The findings suggest that physical activity leading to mechanical loading of the healing tendon tissue is a prerequisite for the up-regulation of sensory neuropeptide receptors, increasing the tissue susceptibility to neuronal stimuli. Prolonged immobilization seems detrimental to the healing process. Dysregulation of the nervous system by systemic diseases such as neuropathy or chronic inflammation seems to impair tendon healing and modelling. In the future it might be possible to employ pharmacological and/or physical means to promote neuronal pathways and thereby stimulate tendon healing during normal as well as pathological conditions. LIST OF PUBLICATIONS I. Bring DK-I, Heidgren ML, Kreicbergs A, Ackermann PW. Increase in sensory neuropeptides surrounding the Achilles tendon in rats with adjuvant arthritis. J Orthop Res. 2005 Mar;23(2):294-301. II. Bring DK-I, Kreicbergs A, Renstrom PA, Ackermann PW. Physical activity modulates nerve plasticity and stimulates repair after Achilles tendon rupture. J Orthop Res. 2007 Feb;25(2):164-72. III. Bring DK-I, Reno C, Renstrom P, Salo P, Hart DA, Ackermann PW. Joint immobilization reduces the expression of sensory neuropeptide receptors and impairs healing after tendon rupture in a rat model. J Orthop Res. 2009 Feb;27(2):274-280. IV. Bring DK-I, Reno C, Renstrom P, Salo P, Hart DA, Ackermann PW. Prolonged immobilization compromises up-regulation of repair genes after tendon rupture in a rat model. Scand J Med Sci Sports. Accepted for publication. V. Bring DK-I, Paulson Kent, Renstrom P, Salo P, Hart DA, Ackermann PW. Decreased substance P levels after denervation correlate with impaired tendon repair. Submitted to Wound Repair And Regeneration.