Peptidergic innervation of periarticular tissue : a study in the rat

Painful musculo-skeletal disorders pose a tremendous burden on the healthcare system, the main reason being the limited knowledge of the underlying pathomechanisms and lack of specific treatment. Hypothetically, the peripheral nervous system in the musculo-skeletal tissues plays an important role in the regulation of pain, inflammation, vasoactivity and also tissue repair. The present study in the rat was designed to explore the expression of neuropeptides in periarticular tissues wider normal conditions and after injury.

A combined approach based on radioimmunoassay (RIA), immunohistochemistry (IHC) and high performance liquid chromatography (HPLC), disclosed the presence of autonomic (NPY, VIP, NA); sensory (SP, NKA, CGRP, GAL, SOM); and opioid (LE, ME, MEAP, MEAGL, N/OFQC) mediators in tendon, ligament and joint capsule. In tendon, deltaopioid receptors were also detected. The peptidergic nerve fibres occurred in abundance in the paratenon, epiligament and beneath the lining layer of the synovium, whereas the proper structures were almost devoid of nerve fibres. This suggests that the neuronal regulation of periarticular tissues highly depends on the innervation of the surrounding structures. RIA showed that the ratio of NPY (sympathetic)/ VIP (parasympathetic) was 8-times higher in ligaments than in tendons and in capsules. The ratio of SP and CGRP (nociceptive)/ GAL, NEAP, N/OFQ (anti-nociceptive) was 10-times higher in tendons and twice as high in capsules compared to ligaments. The distribution and levels in the different tissues may represent the homeostatic state of vasoactivity and nociception, and also indicate the susceptibility to physico-chemical stress.

In healing of experimental Achilles tendon rupture, nerve regeneration and neuropeptide expression at different time points (1-16 weeks) were analysed by IHC including semi-quantification. Analysis of markers for new (GAP) and mature fibres (PGP) disclosed extensive nerve fibre ingrowth into the rupture site. A peak nerve fibre expression during weeks 2-6 (regenerative phase) was followed by nerve fibre withdrawal during weeks 8-16 (remodeling phase). New nerve ingrowth is presumably a prerequisite for delivery of neuronal mediators required for tissue repair. Analysis of specific neuropeptides showed an increased number of SP and CGRP fibres one week post-rupture (inflammatory phase) around blood vessels surrounded by inflammatory cells. This complies with a nociceptive and pro-inflammatory role. During the regenerative phase, the expression of SP and CGRP peaked in the rupture site of proper tendon, seen as free sprouting nerve endings among fibroblasts and newly formed vessels, which may prove to reflect a role in cell proliferation. An initial sparse occurrence of GAL, NPY and VIP was followed by a peak expression at the early remodeling phase, i.e., around week six, which may represent modulatory effects on SP and CGRP required to end the nociceptive, inflammatory and regenerative processes.

Nociception according to hind paw withdrawal at thermal and mechanical stimuli was related to the expression of sensory neuropeptides during healing as assessed by semi-quantitative IHC. Increased expression of SP/CGRP during weeks 1-2 coincided with increased thermal sensitivity on the injured side. Conversely, increased GAL expression during weeks 4-6 coincided with decreased thermal sensitivity, and notably, also with increased mechanical sensitivity. A strong relationship between neuropeptide expression and thermal sensitivity was found by considering the combined rates of change of the three neuropeptides (SP + CGRP - GAL) during healing. The finding indicates that nociception is regulated by the speed by which the expression of potentiating and inhibitory neuropeptides is altered, rather than the absolute tissue levels.

From the present study, it seems obvious that periarticular tissues are supplied with a complex peptidergic network, which presumably takes part in maintaining tissue homeostasis, but also by its plasticity, i.e. nerve ingrowth and temporal alteration m neuropeptide expression, is capable of responding to injury. Interestingly, ingrowth of new nerves seems to be a fundamental feature of tissue repair. In a future perspective, neuronal mediators may prove to be useful in targeted pharmacotherapy and tissue engineering of painful and degenerative rnusculo-skeletal disorders.

List of scientific papers

I. Ackermann PW, Finn A, Ahmed M (1999). "Sensory neuropeptidergic pattern in tendon, ligament and joint capsule. A study in the rat" Neuroreport 10(10): 2055-60
https://pubmed.ncbi.nlm.nih.gov/10424674

II. Ackermann PW, Li J, Finn A, Ahmed M, Kreicbergs A (2001). "Autonomic innervation of tendons, ligaments and joint capsules. A morphologic and quantitative study in the rat. " J Orthop Res 19(3): 372-8
https://pubmed.ncbi.nlm.nih.gov/11398848

III. Ackermann PW, Spetea M, Nylander I, Ploj K, Ahmed M, Kreicbergs (2001). "An Opioid System in Connective Tissue. A study of achilles tendon in the rat. " J Histochem Cytochem 49(11): 1387-96 (In Print)
https://pubmed.ncbi.nlm.nih.gov/11668192

IV. Ackerman WP, Ahmed M, Kreicbergs A (2001). "Early nerve regeneration after Achilles tendon rupture- a prerequisite for healing?" Journal of Orthopaedic Research (In Print)

V. Ackermann WP, Ahmed M, Lundeberg T, Kreicbergs A (2001). "Sensory neuropeptide expression and nociception during Achilles tendon healing." (Submitted)