Summary

Intramuscular administration of Platelet Rich Plasma into a denervated muscle stimulates the muscle's satellite cells and triggers changes at the neuromuscular junction that may promote a faster and more effective reinnervation.

Abstract

Peripheral nerve injuries (PNI) are one of the most prevalent lesions of the musculoskeletal system worldwide. Even with the advances in these treatments aimed at accelerating nerve growth, regeneration of this tissue is still slow and limited, favoring prolonged denervation in the target muscle. This denervation leads to different processes such as muscle atrophy and affection of the neuromuscular junction (NMJ). This work aims to analyze the effect of intramuscular Platelet-Rich Plasma (PRP) application in a denervated muscle due to peripheral nerve injury. The hypothesis of this study is based on taking advantage of the biological action and stimulation of PRP to attenuate the effects generated in the muscle by the denervation induced after PNI.

Eighty Wistar rats (2-3 months of age) were divided into four groups: non-treatment group (N = 18), saline group (N = 18) and PRP group (N = 18). A fourth group of animals was used as blood donors to prepare PRP (N = 26). The intervention was performed on one of the hind limbs of the animal, using the contralateral limb as a healthy control for different analyses. The common peroneal nerve was dissected and resected, and the stumps were sutured to prevent reinnervation. PRP pools from donors were prepared each day of the intervention. The number of erythrocytes, leukocytes, and platelets from blood and PRP, were assessed using a hematology analyzer. Intramuscular infiltrations of saline or PRP (200 µL) were performed into the tibialis anterior muscle. PRP activation with CaCl2 (10%). Infiltrations were applied every week, from surgery to sacrifice. The NT group underwent surgery but did not receive any treatment. Sacrifices were performed 2, 4 and 6 weeks after surgery. Denervated and non-denervated anterior tibialis anterior muscles were collected, weighted, frozen and stored at -80°C until histological processing. This processing included immunohistochemical and western blot analysis to assess the effects on muscle satellite cells (SCs) and the NMJ. Comparisons were performed by ANOVA or Kruskal-Wallis one-way ANOVA for nonparametric data. Data were considered statistically significant when P values were less than 0.05.

The mean platelet concentration of PRP was 1202.7×103 ± 335.8 platelets/µL, achieving a platelet enrichment of 1.5 times the blood platelet concentration, without leukocytes and erythrocytes. The PRP group showed changes in the nAChR clusters of the NMJ, with a significant decrease in the e subunit (adult type) and a significant increase in the ? subunit (fetal type), compared to the other groups (p < 0.05). In addition, the PRP group also showed significantly higher levels of the protein Pax7 (p < 0.05), indicating SCs stimulation. Finally, the PRP group suffered less and slower weight loss than the control groups (saline and no-treatment), suggesting less muscle atrophy (p < 0.05).

The findings suggest the importance of treating the denervated muscle as a consequence of injury to the nerve that innervates it, as well as the nerve itself. It could promote a faster and more effective reinnervation, enhancing and accelerating the process that would improve the patient's clinical outcomes.