Spatiotemporally-guided defunctionalization of nociceptive nerves with capsaicin enhances structural repair in a murine Achilles tendon rupture model

Chengyu Zhuang, Rong Zhou, Yifan Qu, Renhao Yang, Y / Xu, W W Yan, Kai Yang, Lianfu Deng

Chronic Achilles tendinopathy, a prevalent and debilitating condition, impairs daily function and quality of life. While healthy tendons lack internal nerves, pathological ingrowth of sensory nerve fibers into the tendon is now recognized as a key source of chronic pain. These sprouting nerves act as nociceptors and release pain-associated neuropeptides like Substance P (SP) and Calcitonin Gene-Related Peptide (CGRP), perpetuating pain and neurogenic inflammation. Paradoxically, sensory nerves are also essential for early healing by mediating acute inflammatory responses. This highlights a time-dependent dual role: early, timely nerve ingrowth aids necessary inflammatory repair, whereas their late-phase failure to retract maintains a state of chronic pain. Consequently, modulating this spatiotemporal dynamic to facilitate late-phase retraction while not disrupting early-phase ingrowth emerges as a key therapeutic goal. Capsaicin, a TRPV1 agonist, can selectively defunctionalize sensory nerve endings, offering a potential tool to achieve this goal. Transgenic Advilin-Cre; mTmG mice were used to trace sensory nerves, visualized in 3D via iDISCO tissue clearing and light-sheet microscopy. An Achilles tendon rupture-and-repair model was established. Healing was assessed at post-operative days 3, 7, and 21 using H&E staining, polarized light microscopy, and immunofluorescence for nerves (Tuj1, PGP9.5), blood vessels (CD31, α-SMA), macrophages (F4/80), and neuropeptides (SP, CGRP). To intervene, high-dose capsaicin was injected locally from day 3 to day 6 post operation. Its effects on tissue histology, neural/neuropeptide distribution, vascularity, and inflammation were evaluated on days 7 and 21. Imaging confirmed nerves reside in the paratenon, not the healthy tendon body. After injury, nerve ingrowth peaked in the proliferative phase (day 7) and retracted during remodeling (day 21). Vascular patterns mirrored this growth-regression cycle. Capsaicin treatment accelerated healing, resulting in denser, better-organized tissue with less macrophage infiltration by days 7 and 21. It effectively suppressed the distribution and intensity of SP and CGRP within the healing tendon. Furthermore, capsaicin promoted the retraction of both nerves and blood vessels in the later phase and reduced macrophage presence at day 21, indicating a late anti-inflammatory effect. This study delineates the temporal progression and histological distribution of sensory nerves during tendon healing, linking their persistent ingrowth and neuropeptide release to chronic pain. Capsaicin, by selectively targeting TRPV1-positive fibers, successfully reduced pain mediators, expedited the regression of nerves and vessels during remodeling, and improved tissue maturation. This supports the therapeutic concept of guiding timely nerve retraction to resolve pain while promoting quality repair. Unlike transient anesthetics like lidocaine, capsaicin induces a longer-lasting defunctionalization, offering a novel mechanistic approach. These findings provide preclinical evidence for targeting nerve-tendon crosstalk, paving the way for innovative treatments for chronic Achilles tendinopathy that address both pain and healing.