Neuroimmune braking mechanisms in exercise-related musculoskeletal injury: from nociceptors to tissue regeneration

Abstract

Background

Musculoskeletal injuries are a frequent occurrence in sports medicine and rehabilitation, and their healing process needs an accurate response from inflammation. Recent studies show that sensory neurons play a critical role in tissue repair through neuropeptides, which is quite different from their usual function as simple pain detectors. The current review summarizes studies conducted during the last ten years concerning neuroimmune crosstalk mechanisms in musculoskeletal injury healing, in which the role of sensory neurons in macrophage polarization is emphasized.

Methods

We reviewed preclinical studies on the neuro-immune interaction after musculoskeletal injury, emphasizing the CGRP-RAMP1-thrombospondin-1 axis and parallel pathways including TGF-β-mediated macrophage specification and TAFA4-IL-10 signaling. Based on rodent data regarding skin wounds, muscle injury, peripheral nerve injury, and other pertinent models, a theoretical model relating mechanical loading and neuroimmune inhibition was established.

Results

CGRP-RAMP1-Thrombospondin 1 signaling pathway has been identified in preclinical studies as an candidate pathway for shifting macrophages from a pro-inflammatory (M1) to a pro-repair (M2) phenotype, hence balancing the process of pathogen clearance against tissue repair process. Parallel systems, such as TGF-β-mediated macrophage specification and TAFA4-IL-10 signaling, provide redundancy and robustness. Controlled loading, as seen with active recovery, causes stimulation of sensory neuron terminals, leads to the release of neuropeptides, triggers the neuroimmune braking process, and promotes proper M1-to-M2 conversion. The understanding of these mechanisms explains why patients have always been advised to undertake active recovery rather than rest following tendon/ligament damage.

Conclusion

This review proposes a new conceptual framework in which the neuroimmune axis may serve as a key controller of musculoskeletal repair. These biological mechanisms offer a potential explanation for the medical choice of active recovery over complete rest following specific injuries; nevertheless, there is no direct evidence in humans, and this theory needs to be tested in human studies.

Introduction

Musculoskeletal injuries are the most frequently seen presentation in the practice of sports medicine and rehabilitation. Skeletal muscle, comprising about 40–45% of total body weight, is highly vulnerable to both acute injury, such as a strain or contusion, to the complete rupture of a muscle, as well as chronic damage due to overuse of tendons, ligaments, and bones [1]. These injuries have a considerable impact on the world population, with millions of people who are athletes and physically active experiencing impaired functionality, prolonged time of recovery, and danger of re-injury [2].

In its essence, the successful restoration of tissues after the musculoskeletal injury means that the inflammatory responses need to be perfectly coordinated to provide a cure. In accordance with the classical concept of exercise immunology expressed by the open window hypothesis and J-shaped model, the predominant concern was systemic changes in circulating immune cells during exercise [3]. Even though these models have contributed greatly towards understanding the risk of infection and immunological surveillance, their main drawback is ignoring the intricate local micro environment which is really where healing takes place. One severe lacuna in our knowledge of the function of the nervous system is its regulation of local inflammation and tissue repair processes [4].

Historically, the dominant conception has been that nociceptive sensory neurons are specific types of pain receptors - sentinels with the main role of perceiving tissue injury and warning the central nervous system of danger [5]. Nevertheless, in the past ten years, the viewpoint has drastically changed. Peripheral neurons no longer have been viewed as passive bystanders of the inflammation because they actively direct the actions of immune cells by releasing neuropeptides [6]. The dialogue between them functions in a local manner, not dependent on reflexes originating in the brain, and constitutes an instant response mechanism that regulates the inflammatory environment [7].

This paradigm shift is especially clinically relevant in sports medicine. Through decades of empirical observation, it has been proposed that active recovery, a controlled and progressive loading during the healing process, is more effective than immobilizing a tendon or ligament after an injury [8]. However, the mechanism behind this has been mysterious. The new insight into the neuroimmune interactions currently offers a convincing explanation, i.e., controlled mechanical stimulation of sensory neurons endings in the damaged tissue causes the activation of neuropeptides secretion that directly orders local macrophages to switch between pro-inflammatory and pro-repair phenotypes [9].

As is evident, most of the experimental findings mentioned above have been obtained from a few preclinical trials using rodent models, especially with respect to wounds, muscle injury, and peripheral nerve injury. Despite the strong support for these studies in terms of proof of concept, their application to musculoskeletal injuries in humans (especially tendons, ligaments, and bones) is still highly speculative. We will therefore make a clear distinction between direct experimental findings, plausible mechanistic pathways, and translational hypotheses requiring further validation.

The purpose of this review is to generalize the developments of the last ten years in the field of our knowledge of neuroimmune braking mechanisms in exercise-induced musculoskeletal injury. First of all, we will set the basis of immunity underlying tissue repair, particularly, the importance of macrophages polarization dynamics. Thereafter, we follow the history of the development of understanding of how nociceptors functions, starting with pain sensation and moving towards immune modulation, including the key finding of the CGRP-RAMP1-thrombospondin-1 pathway as the main signal pathway [9]. The parallel mechanisms of TGF-β-mediated macrophage specification [10, 11], TAFA4-IL-10 signaling are explored as alternate pathways. Lastly, we bring all these basic processes together with the clinical application of sports rehabilitation suggesting a conceptual model whereby active recovery is viewed as an intervention on endogenous neuroimmune braking systems [11].

Immunological foundations of musculoskeletal repair

The inflammatory time course following muscle injury

The muscle injury, whether it is caused by the direct blow or eccentrics or even repetitive strain injury, triggers a very organized inflammatory cascade, which takes place over days or weeks. Conceptually, this process may be divided into three overlapping stages: the damage stage, the inflammatory stage, and the repair/remodeling stage [12].

Right after injury, mechanical destruction of muscle fibers and vascular structures releases intracellular material into the extracellular space. There are damage-associated molecular patterns such as high-mobility group box 1 (HMGB1), ATP, uric acid, mitochondrial DNA (DAMPs) [13]. These molecules are identified by pattern recognition receptors present on resident immune cells and endothelial cells, and cause a fast onset of an inflammatory response.

The first circulating immune cells at the injury site are neutrophils, which usually infiltrate the area 1–2 h after the injury and reach the peak at 24–48 h after the injury [14]. These cells play a variety of roles, i.e., they engulf cellular debris, release proteolytic enzymes to digest damaged extracellular matrix, and generate reactive oxygen species that clean the injury site. Neutrophils also release chemotactic agents that attract monocytes and prolong the inflammatory cascade. Nonetheless, their existence is temporary, as chronic presence of neutrophils can be linked to the delay in healing and overexposure to tissue injury [3, 15].

The infiltration of injured muscles by monocytes starts within 24–48 h when they differentiate into macrophages after entering the tissue. The following days see a massive increase in the macrophage population, such that by 3–5 days it is the most prevalent type of immune cell found in the healing muscle [16]. The expansion corresponds to ongoing recruitment of circulating monocytes as well as proliferation of tissue-resident macrophages locally.

Macrophage polarization: The M1-to-M2 switch

Macrophages have remarkable functional plasticity whereby they can adopt different activation states when responding to micro-environmental cues [17]. Traditionally, the dichotomy was used to differentiate between pro-inflammatory M1 macrophages and anti-inflammatory/pro-repair M2 macrophages, but it is currently understood that these are not discrete categories, but instead form a continuum of functional states.

Th1 cytokines (interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α)) and products of microbes (lipopolysaccharide (LPS)) via Toll-like receptors can induce M1 macrophages [18]. Such cells have increased levels of inducible nitric oxide synthase (iNOS), pro-inflammatory cytokine (TNF-α, IL-1β, IL-6) secretion, and strong antimicrobial action. When it comes to muscle damage, M1 macrophages are prevalent during the early inflammatory stage where they remove cellular debris and produce chemical signals that initiate the activation of muscle satellite cells [19].

M2 macrophages are found in a continuum of activation stages related to repairs of tissues, regulation of the immune system and termination of inflammation. Arginase-1 expression is characteristic of and extracellular matrix component secretion characterizes the so-called alternate activation (M2a) [20]. IL-10 and glucocorticoids encourage a regulatory phenotype (M2c) that is geared towards immunosuppression and clearing of debris. On days 3 through 7 after muscle injury, the change from M1 to M2 dominance has been noted, reflecting a transition from active inflammation to repair mechanisms [21].

The significance of the temporal switch the significance of these studies was shown by manipulating the population of macrophages. Removal of macrophages in the early inflammatory phase would significantly delay the restoration of muscles as it implies that the M1 functions should be performed to achieve successful healing [4, 22]. In contrast, lack of switching to M2 dominance leads to chronic inflammation, excessive fibrosis and dysfunction repair [23]. Therefore, timing and efficiency of the switch between M1 and M2 with high precision can be considered as the factor determining the outcome of healing.

The evolving concept of neuroimmune dialogue

Nociceptors: From pain sensors to immune regulators

Nociceptors are specialized sensory neurons with peripheral endings that can innervate almost any tissue such as skin, muscle, joints, and viscera [24]. Cell bodies exist in the dorsal root ganglia (DRG) and trigeminal ganglia and axons go to peripheral target and spinal cord dorsal horn. The traditional role given to these neurons has been nociception i.e. the recognition of noxious stimuli such as extreme temperatures, mechanical stimuli, and chemical irritants, and the transmission of this information to the central nervous system to produce pain sensation [9, 25].

Nevertheless, increasing evidence within the last ten years has established the fact that nociceptors have a likewise critical effector role. Apart from transmitting afferent signals to the spinal cord, their peripheral terminals also secrete a number of neuropeptides with local actions in neighboring cells, such as immune cells, vascular endothelial cells and tissue-resident stromal cells [26]. These afferents place nociceptors into the position of major players in regulating tissue homeostasis and inflammation.

Other neuropeptides of nociceptors are calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal peptide (VIP) and galanin, amongst others [27]. The peptides reside in dense-core vesicles at nerve terminal and they are released when neurons are activated in a calcium-dependent fashion. It is important to note that neuropeptide release may not necessarily be induced by traditional nociceptive stimuli but also by inflammatory factors contained within the injured tissue microenvironment such as prostaglandins, bradykinin, and cytokines [28].

The molecular language of neuroimmune communication

Neuropeptide-induced activities of immune cells are controlled by particular G-protein coupled receptors (GPCRs) present on target cells. CGRP uses a heterodimeric receptor consisting of calcitonin receptor-like receptor (CLR), and receptor activity-modifying protein 1 (RAMP1) to signal [29]. Without RAMP1, the receptor does not work; it defines ligand specificity and allows the expression of the receptor complex on the cell surface.

RAMP1 expression has been reported on various types of immune cells and is especially high on myeloid cells (neutrophils, monocytes, macrophages). The pro-healing effects of CGRP in musculoskeletal injury models are primarily mediated via RAMP1 on myeloid cells, as demonstrated by conditional knockout experiments. Later research has demonstrated that CGRP activation of myeloid cell behavior and function requires the presence of RAMP1 and affects a variety of cellular mechanisms such as chemotaxis, generation of cytokines, and phagocytosis [30].

Although substance P has classically been associated with neurogenic inflammation, which enhances vascular permeability and attracts leukocytes, it appears that its role in wound healing is much more complex.

The CGRP-RAMP1 axis: a core neuroimmune braking pathway

Landmark discovery: CGRP sensory neurons promote tissue healing

In the year 2024, Lu et al. published a pioneering article which essentially contributed to the knowledge of how sensory neurons control the process of tissue repair [9]. These findings provide direct genetic evidence from mouse skin and acute muscle injury models. Direct evidence in human tendons, ligaments, or bone is currently lacking. By combining genetic mouse models with high-tech imaging and mechanistic in vitro investigations, they were able to show that NaV1.8⁺ nociceptive sensory nerves are necessary to heal skin wounds and restore muscles after an acute injury to their regular state.

The absence of NaV1.8⁺ sensory neurons (Nav1.8^cre/Rosa26^tdT ) in mice caused significant defects in tissue repair. Six days after injury, skin wound areas demonstrated delayed migration of the epithelium, lower numbers of proliferating keratinocytes were mostly open. The process of muscle regeneration after volumetric muscle loss injury was also adversely affected, as more fibrotic tissues were deposited and fewer new muscle fibers were formed [9, 10].

It was clear what caused this defect when the researchers studied the functioning of sensory neurons during healing. In order to see whether sensory neuron endings grew into the granulation tissue of healing wounds and injured muscle using the Nav1.8^cre/Rosa26^tdT mice, wherein NaV1.8⁺ neurons express tdTomato fluorescent protein, they found that sensory neuron endings actively invaded the granulation tissue of healing wounds and wounded muscle, forming the innervation of the injured tissues along with the repair process (Fig. 1A). Such in vasive neurons also exhibited high expression of the neuropeptide CGRP, which is proposed as a mediator of neuroimmune interactions in healing [9].

Fig. 1.

Overview of CGRP-mediated neuroimmune regulation in musculoskeletal injury repair. A Sensory neuron sprouting into injured tissue. Direct evidence from mouse skin and muscle injury models [9]. NaV1.8⁺ sensory neuron endings (red) invade the granulation tissue of healing wounds and injured muscle. B CGRP-RAMP1-TSP-1 signaling pathway. Solid arrows indicate steps experimentally validated in at least one preclinical model (mouse skin or muscle injury): CGRP release from sensory neuron terminals, binding to RAMP1 on myeloid cells, upregulation of thrombospondin-1 (TSP-1), promotion of neutrophil apoptosis and efferocytosis, and induction of M2 macrophage polarization (CD206, Arg1). Dashed arrows indicate plausible steps that have not yet been directly demonstrated in the indicated context or represent extrapolations to human tissues (e.g., tendon, ligament, bone). C Three-phase working model of neuroimmune regulation after musculoskeletal injury. The time windows (0–24 h, 24–72 h, ≥ 72 h) are based on rodent data and are presented as a hypothesized timeline (shaded box). Validation in human musculoskeletal injuries, particularly in hypocellular tissues such as tendons and ligaments, is required

CGRP acts on myeloid cells via RAMP1

In order to find out if CGRP mediates the healing properties of sensory neurons, the researchers produced mice lacking the ability of immune cells to respond to CGRP by eliminating the RAMP1 gene selectively in the myeloid cell population (LysMᶜʳᵉ⁺/⁻/Ramp1ᶠˡ/ᶠˡ). The mice reproduced the healing deficits of Nav1.8ᶜʳᵉ/Rosa26ᴰᵀᴬ mice, such as the lack of closure of the skin wounds and impaired recovery after injury to the muscles. It was shown that CGRP helps with the healing process by acting exclusively on myeloid cells [3, 9].

This conclusion was verified by bone marrow transplantation experiments. Wild type mice that were reconstituted with Ramp1 -/- bone marrow cells had decreased healing as compared to those that received wild type bone marrow cells, but the transplant of wild type cells into ramp1 -/- mice restored the healing impairment [9]. Taken together, these findings proved that CGRP signaling via RAMP1 on hematopoietic cells, mainly neutrophils and monocytes/macrophages is required to support normal tissue repair.

The thrombospondin-1 mechanism

The mechanism via which CGRP regulates myeloid cell activity was explained by transcriptomic profiling of macrophages challenged with CGRP. It was found that this led to the upregulation of thrombospondin-1 (TSP-1) which is a multicellular protein with various roles in cell adhesion, motility, and inflammation [9].

The function of TSP-1 occurs in an autocrine and paracrine fashion to help mediate the effect of CGRP on myeloid cells. CGRP-inducible TSP-1 induces the process of apoptosis of neutrophils faster and facilitates their removal by macrophages (efferocytosis), which is one of the important stages of alleviating inflammation. In macrophages, TSP-1 has been shown to cause polarization towards an M2-like, pro-repair phenotype with the expression of CD206 and release of anti-inflammatory cytokines [9]. These mechanistic insights were obtained from mouse skin and muscle injury models; whether TSP-1 plays a similar role in human tendon or ligament healing remains unknown.

The importance of the role played by TSP-1 was established by showing that recombinant TSP-1 was able to restore the healing defects of Ramp1-deficient myeloid cells but that TSP-1-neutralizing antibodies prevented the pro-healing actions of CGRP [9]. It thus defined TSP-1 as the key downstream effector of neuroimmune signaling mediated by CGRP (Fig. 1B).

Current evidence base and its limitations

The results mentioned above have been experimentally validated in a number of preclinical models; including those focusing on skin wound repair and acute muscle damage in mice. It is demonstrated that NaV1.8⁺ sensory neuron ablation and myeloid-specific RAMP1 deletion impair the healing process, while CGRP-induced upregulation of TSP-1 expression promotes neutrophil apoptosis and M2 polarization. Nonetheless, there are no current scientific studies which have provided direct evidence that the above pathway may operate in human cases of musculoskeletal injuries, especially tendon, ligament and bone related injuries. While there are some plausible assumptions regarding the application of the said pathway as well as its timeline and the effects of exercise on it, they are not yet thoroughly verified. Further studies are required to confirm the involvement of the CGRP-RAMP1-TSP-1 pathway in the healing process in humans and during active rehabilitation programs.

Parallel neuroimmune regulatory pathways

TGF-β-mediated macrophage specification

Whereas CGRP gives fast and short term control of macrophage phenotype it has been shown that sensory neurons play a role in more gradual changes to macrophage specification, mediated by TGF-β signaling. In 2025, Kolter et al., in a study published in Immunity, showed that sensory neurons determine the characteristics of tissue-resident macrophages by direct contact and the on-site synthesis of TGF-β [5, 10].

With sophisticated imaging and single-cell transcriptomics, they demonstrated the fact that macrophages in near proximity to sensory nerve ends gain a particular transcriptional pattern, defined by the presence of tissue repair-related genes as well as immune-regulatory genes. The macrophage identity of the peri-neural zone requires TGF-β signaling; elimination of TGF-β signaling in macrophages prevents them from acquiring their peri-neural state and disrupts tissue homeostasis [10].

It expands the neuroimmune talk beyond acute regulation to the level of tissue residency on a long-term basis. In repeated exercise or chronic musculoskeletal conditions, TGF-β-mediated specifications of macrophages might be able to clarify the reason why regularly active people have different muscle immune environments compared to those who are less active. This mechanism of an “immune memory”, may impact responses to future injuries.

TAFA4-IL-10 pathway

A different parallel pathway has been described in literature by Hoeffel et al. in 2021 reporting that a small part of non-peptidergic sensory neurons generate a neuropeptide TAFA4 [10]. In contrast to the CGRP, which is kept in dense core vesicles and excreted during the neuronal activity, TAFA4 is constantly released and exerts an influence on macrophages to induce the production of IL-10.

TAFA4 signaling via an unknown receptor on macrophages triggers the transcriptional program that promotes IL-10 expression and anti-inflammatory phenotype. The absence of TAFA4 in mice demonstrates poor inflammatory resolution and slow tissue repair after injury, which suggests that this pathway acts alongside the CGRP signaling to provide strong neuroimmune control [10].

The TAFA4-IL-10 axis could prove especially useful in helping sustain the anti-inflammatory milieu at the latter stages of repair, alongside the more immediate effects of CGRP. These parallel pathways are together redundant and guarantee that neuroimmune regulation remains strong in case of perturbations of any one signaling system.

TRPV1 as an upstream regulator

Assuming that neuropeptides represent the so-called instructions delivered by sensory neurons, which factors decide when such instructions will be provided? Recent studies suggest that the transient receptor potential vanilloid 1 (TRPV1) channel acts as a key upstream activator of activity-triggered neuropeptide secretion [31].

TRPV1 is a non-selective cation channel found in a subpopulation of nociceptive sensory neurons, which is sensitive to various types of stimuli such as noxious heat (> 43 deg), low pH, irritants like capsaicin, and endogenous inflammatory mediators like prostaglandins and bradykinin [32]. When tissue damage occurs, the inflammatory environment offers numerous TRPV1-activating cues, inducing calcium influx in sensory neuron terminals and causing the release of neuropeptides.

The latest finding of Jiang et al. has proven that TRPV1-mediated CGRP release is an important step in the bone repair after dental implantation of the bone [33]. It was found that the activation of TRPV1 increased CGRP release and stimulated the polarization of M2 macrophages through the PI3K/AKT pathway to enhance osseointegration. On the other hand, TRPV1 inhibition increased inflammation and compromised bone healing process. This evidence is limited to a rat dental implant model. Extrapolation to other bone injuries (e.g., fracture) or to soft tissue healing should be made with caution.

The dose dependent manner of TRPV1 activation holds significant consequences on prescribing exercise. Light to moderate controlled stimulation such as gentle warmth or mild mechanical stimulation would be useful to induce the release of potentially useful neuropeptides. Extreme (or long-term) stimulation can produce neurogenic inflammation and pain sensitization [34]. It offers a molecular explanation of how active recovery interventions have a therapeutic window.

Exercise as a neuroimmune modulator

How exercise activates the neuroimmune axis

The physical exercise is capable of generating several signals able to elicit sensory neuron activity and involve the neuroimmune regulatory process. In the process of muscle contraction and joint motion mechanical pressures are passed across the tissues, stimulating mechanosensitive ion channels of sensory nerves such as Piezo2 and TRPV4 [35]. The concomitant rise in metabolic demand results in local buildup of metabolites, including lactate, adenosine, and protons, to activate other receptor families, including acid-sensing ion channels (ASICs), and G protein-coupled receptors such as GPR81 [36]. These mechanisms are inferred from basic neurophysiology studies; direct evidence that exercise-induced activation of this axis occurs in injured human tendons or ligaments is not yet available.

Exercise-induced temperature variations local muscle warming-offer another activation stimulus to thermosensitive channels such as TRPV1. Mechanical, metabolic and thermal signals combined together form a multi-modal activation profile which is intrinsically different to the isolated noxious stimuli considered within classic pain models [37].

Equally important is the fact that the intensity and pattern of exercise dictate what kind of neuronal activation will be elicited. Gentle and rhythmical movements, such as walking or cycling, lead to short-term and irregular excitation of sensory neurons with a propensity on neuropeptide release, but do not cause continuous pain signals. Conversely, very intense eccentric contractions or exercises executed using an inflamed tissue might induce extended and high rate firing, which encourages pain and neurogenic inflammation [38].

Active recovery versus immobilization: Divergent macrophage outcomes

Neuroimmune framework mechanistically explains the clinically better outcomes of active recovery as compared to complete immobilization after musculoskeletal injury, which has been clinically documented over a long period.

Due to the absence of mechanical loading and joint movement in immobilized limbs, sensory neurons are deprived of their typical activating stimuli. There is a decrease in sensory neuron activity and reduction of CGRP release at the terminals in damaged tissue and the loss of neuroimmune braking signal. The resulting effect is the continued predominance of M1 macrophages, the slow transition into an M2 phenotype, the inability to engulf dead neutrophils (apoptosis), and chronic inflammation [39]. The animal study supports the findings that immobilization after muscle damage slows down the process of macrophage polarization and hinders the functional recovery [40].

On the other hand, controlled active recovery, or the systematic loading within a therapeutic window, provides mechanical cues, which activate residual sensor neurons located and surrounding the injured tissue. It leads to the release of CGRP, activation of RAMP1 signaling on infiltrated myeloid cells and effective conversion between M1 and M2 populations. As Kobayashi and others have shown, treadmill running after injury to the sciatic nerve raised M2 macrophages in the injured nerve and reduced the neuropathic pain behaviors [41]. Like this, Detloff and others found that early physical activity after spinal cord injury prevented emergence of neuropathic pain by minimizing macrophage accumulation in the dorsal root ganglia [42].

This relationship between CGRP signaling and human active recovery responses must be emphasized to be hypothetical. The model outlined above is purely a hypothesis which should be tested for humans. On the other hand, one should accept that at the moment, there is not enough direct data based on experiments with humans which would confirm the connection between the CGRP-RAMP1-TSP-1 signaling system and successful rehabilitation. The theory that the neuroimmune braking mechanism stimulates bone repair through controlled loading is very plausible; however, it cannot be confirmed by any experiments carried out with humans up until now.

The dose response relationship is essential. Loading too much during insufficient tissue repair may cause injury to the tissue as well as increase inflammation. Lack of loading does not activate the neuroimmune system of braking. It highlights the relevance of an accurate prescription of rehabilitation that is defining the best timing, intensity and duration of loading based on each injury type and the individual patient.

Evidence from systematic reviews

However, the systematic reviews referred to above (by Sleijser Koehorst et al., concerning peripheral nerve injury and by Lutke Schipholt et al., concerning joint and nerve release) are mainly concerned with nerve injury models. Even though these studies give useful information about the interaction between the nervous and immune systems, one must be careful in directly linking these data to muscle, tendon, or ligament healing. Thus, we believe that these studies serve more as general proof of the effect of mechanical stimuli on neuroimmune interaction than direct evidence of their effect in specific tissues.

The growing body of preclinical data regarding the effects of exercise on the neuroimmune system has undergone a systematic review. In a systematic review and meta-analysis Sleijser-Koehorst et al. searched through the effects of aerobic exercises on the neuroimmunology of animals with a traumatic injury to the peripheral nerves and found 40 studies involving 139 comparisons [43] .

Their meta-analyses showed that exercise had a significant effect on reducing pro-inflammatory mediators, as well as on promoting regenerative factors. In wounded nerves, exercise was shown to reduce TNF-α and increase insulin like growth factor-1 (IGF-1) and growth associated protein 43 (GAP43) which are indicators of regeneration. Exercise lowered the level of microglial and astrocyte activation markers in the spinal cord. Notably, these changes were correlated with better functional outcomes [43].

Another systematic review of this kind by Lutke Schipholt et al. discussed joint and nerve mobilization technique, and found that such technique has evidence on decreased spinal cord glial fibrillary acidic protein (GFAP), lower DRG IL-1beta, and higher spinal cord IL-10 after those manual interventions [44]. Although the study heterogeneity and risk of bias were problematic to the evidence quality, such results support the idea that mechanical maneuvers can alter the neural-immune response. To clarify the tissue-specific and model-specific nature of the available evidence, we summarise the main preclinical studies according to tissue/injury model, species, signaling pathway, key outcomes, and translational relevance to humans (Table 1). The phrase “direct evidence” implies that the discovery has been proven experimentally using the indicated model. The term “Extrapolation” refers to the fact that the mechanism has not been tested in this particular tissue, although there is a hypothesis regarding its involvement in the process. Unless otherwise stated, the data on humans is unavailable(Table 2).

Table 1.

Summary of key preclinical evidence for neuroimmune pathways across different tissues and injury models

Tissue/injury model	Species	Signaling pathway / intervention	Main outcomes	Evidence type for humans
Skin wound healing	Mouse	CGRP-RAMP1-TSP-1 (Nav1.8⁺ neuron ablation, myeloid RAMP1 deletion)	Delayed epithelial migration, reduced keratinocyte proliferation, increased fibrosis; CGRP induces TSP-1 → promotes neutrophil apoptosis and M2 polarization	Direct evidence in rodents only. Whether same pathway operates in human skin wounds unknown.
Acute muscle injury (VML)	Mouse	CGRP-RAMP1-TSP-1 (as above)	Impaired muscle regeneration, increased fibrosis; CGRP signaling via RAMP1 promotes healing	Direct evidence in rodents only. Relevance to human muscle recovery unknown.
Peripheral nerve injury (sciatic nerve)	Rat, mouse	Aerobic exercise (treadmill, swimming)	Decreased TNF-α, increased IGF-1 and GAP43; reduced microglial and astrocyte activation in spinal cord	Direct evidence in rodents only. Hypothesised to alleviate neuropathic pain; human mechanistic data lacking.
Peripheral nerve injury (sciatic nerve)	Mouse	Voluntary wheel running	Increased M2 macrophages in injured nerve, reduced neuropathic pain behaviours	Direct evidence in rodents only. Whether active recovery in humans engages similar mechanisms unclear.
Spinal cord injury	Rat, mouse	Early physical activity	Reduced macrophage accumulation in DRG, prevention of neuropathic pain	Direct evidence in rodents only. Extrapolation to human spinal cord rehabilitation requires caution.
Sensory neuron-macrophage specification	Mouse	TGF-β signaling (neuron-derived)	Maintains specific transcriptional profile of perineural tissue-resident macrophages; loss of TGF-β disrupts this state	Direct evidence in rodents only. Whether repeated exercise creates “immune memory” via this mechanism remains speculative.
Skin / muscle (TAFA4)	Mouse	TAFA4-IL-10	Constitutive TAFA4 release → IL-10 production by macrophages → promotes resolution of inflammation and tissue repair	Direct evidence in rodents only. Role in human musculoskeletal injury unknown.
Bone healing (dental implant)	Rat	TRPV1 → CGRP → PI3K/AKT → M2 polarization	TRPV1 activation increases CGRP release, promotes M2 polarization, enhances osseointegration	Direct evidence in rat dental model only. Extrapolation to other bone injuries (e.g., fracture) is hypothetical.
Tendon / ligament injury	No direct evidence	Hypothesised: mechanical load → sensory neuron → CGRP → M2 polarization	No direct experimental evidence; clinical experience supports active recovery but mechanisms remain hypothetical	No direct evidence; extrapolation from muscle/skin models. Human studies required.
Bone injury (non-dental)	No direct evidence	Hypothesised as above	No direct experimental evidence (except dental implant model above)	No direct evidence; extrapolation from muscle/skin models. Needs validation in fracture models.

Table 2.

Summary of key characteristics of the three stages of neuroimmune regulation after musculoskeletal damage – evidence derived from rodent models (skin, muscle, nerve)

Phase	Time window	Sensory neuron activity	Dominant neuropeptides	Macrophage phenotype	Key cellular events
Injury perception	0–24 h	Activation by DAMPs; TRPV1-mediated Ca²⁺ influx	Substance P (initial)	M1 predominance iNOS, TNF-α, IL-1β, IL-6	Neutrophil infiltration; DAMP release; initial debris clearance
Neuroimmune instruction	24–72 h	Active sprouting into injured tissue; sustained CGRP release	CGRP (peak)	M1→M2 transition early iNOS, followed by Arg1, CD206	CGRP-RAMP1 signaling; TSP-1 upregulation; neutrophil apoptosis and efferocytosis
Remodeling stabilization	72 h onward	Gradual normalization of terminal density	TAFA4, TGF-β	M2 predominance Arg1, CD206, IL-10, TGF-β	ECM remodeling; angiogenesis; satellite cell activation; long-term macrophage specification

Integrating framework: the neuroimmune axis in musculoskeletal rehabilitation

Three-phase model of neuroimmune regulation

Using the evidence presented in the synthesis above, we suggest a three- stage model of neuroimmune regulation after musculoskeletal injury (Fig. 1C). The timing (0–24 h, 24–72 h, etc.) is based on rodent muscle and nerve injury models and may differ in human tissues, particularly in hypocellular tissues such as tendons and ligaments(Table 3).

Table 3.

Summary of key characteristics of the three stages of neuroimmune regulation after musculoskeletal damage

Phase	Time window	Sensory neuron activity	Dominant neuropeptides	Macrophage phenotype	Key cellular events
Injury perception	0–24 h	Activation by DAMPs; TRPV1-mediated Ca²⁺ influx	Substance P (initial)	M1 predominance iNOS, TNF-α, IL-1β, IL-6	Neutrophil infiltration; DAMP release; initial debris clearance
Neuroimmune instruction	24–72 h	Active sprouting into injured tissue; sustained CGRP release	CGRP (peak)	M1→M2 transition early iNOS, followed by Arg1, CD206	CGRP-RAMP1 signaling; TSP-1 upregulation; neutrophil apoptosis and efferocytosis
Remodeling stabilization	72 h onward	Gradual normalization of terminal density	TAFA4, TGF-β	M2 predominance Arg1, CD206, IL-10, TGF-β	ECM remodeling; angiogenesis; satellite cell activation; long-term macrophage specification

The initial stage of injury perception (0–24 h)

Local sensory endings of neurons are activated by DAMPs released due to tissue damage through TRPV1 and other channels. Early release of neuropeptides (especially substance P) causes leukocytes recruitment and vasodilatation. Neutrophils invade, and macrophages start to collect, taking on their first M1 phenotype. During this stage, active inflammation needed to remove debris is present.

Neuroimmune instruction (24–72 h) is the second stage

The sensory neuron endings grow into the injured tissue to form direct neuroinnervation of the healing area. The persistence of CGRP activates RAMP1 receptors residing on myeloid cells leading to TSP-1 upregulation. It is what starts the braking program i.e. inhibition of high recruitment of neutrophils, speeding up neutrophil cell death and efferocytosis, and starting macrophage M1-to-M2 polarization. This is another crucial time point when the neuroimmune signaling decides the course of healing.

Remodeling stabilization phase (72 h onwards)

Predominating during this phase are M2 macrophages, which secrete IL-10, TGF-β, and IGF-1 to promote angiogenesis, fibroblast, and satellite cells proliferation. The sensory neuron TGF-signaling defines the long term identity of tissue-resident macrophages and forms an immune memory that could be used to respond to any subsequent injury. Signal transduction through TAFA4 keeps the anti-inflammatory state.

Future directions and unanswered questions

Fundamental questions

Nevertheless, regardless of such an impressive advancement, there are still basic questions that need answering. Sensory neuron diversity and its distinct role in neuroimmune regulation is not yet well-understood. Single-cell transcriptomic investigations have just started to uncover the diversity of DRG neurons, but it has been difficult to identify how these subtypes relate to distinct roles in tissue repair [45].

The aftermath of neuroimmune instructions of macrophages remains to be determined. Will CGRP-polarized M2 macrophages revert back into quiescence, die via apoptosis, or remain as tissue-resident cells that are able to remember the injury that originally polarized them? The observations of TGF-β imply that it is likely to last for a long time, however, the length, and constancy of such specification have not been defined.

The sex difference in neuroimmune regulation is an important area of knowledge that remains open. The biology of pain has documented significant sex differences, with females being more susceptible to most forms of chronic pain. Whether such differences can be extended to cover the neuroimmune regulation of tissue repair and whether this explains the reported sex differences in the healing of injuries requires systematic research [46].

Although a study in peripheral nerve injury examined RAMP1 knockdown in Schwann cells, the relevance of this finding to the proposed neuroimmune braking mechanism in musculoskeletal repair is unclear, and the present review focuses on the well-established role of RAMP1 in myeloid cells.

Translation to humans

The application of these preclinical findings to humans presents both possibilities and challenges. The determination of neuropeptides levels in a healing human body is invasive and difficult to achieve. Measurement through surrogate markers in the circulatory system or easily obtained fluids such as synovial fluid and wound exudate could possibly be another source of information, but it should first be validated. There is no human research which has measured the effect of CGRP-mediated neuroimmune braking in an injured tendon, ligaments, or muscles during recovery.

Imaging techniques that are being developed now can help assess neuroimmune processes without resorting to invasive measures. PET radiotracers specific to TSPO enable quantification of neuroinflammation [50]. Novel radiotracers for CGRP receptors or other molecules involved in the neuroimmune pathway can be used as a window into pathway dynamics.

Genetics in humans is yet another solution. Variability in the genetic makeup of genes coding for proteins of the neuroimmune system, like RAMP1, TAC1 (Substance P), and CALCA (CGRP), may be linked to differing outcomes of injury repair processes. If this is the case, it will be used to identify individuals who would respond well to certain treatment protocols [37].

Therapeutic opportunities and challenges

This new knowledge of neuroimmune regulation offers many therapeutic opportunities. Perhaps the most directly, the engineered CGRP (eCGRP) created by Lu et al. can be viewed as a possible biologic treatment in those situations where the neuroimmune signaling is dysfunctional (e.g., in diabetic neuropathy, aging, or severe denervation injury). Such strategies might be used to address the deficit of neuroimmune signals in injured tissues by maximizing delivery allowing them to remain locally and continue their action.

Also promising are small molecule modulators of the pathway. The drugs acting as TRPV1 agonist/antagonist, RAMP1 modulator, and those targeting downstream signaling nodes may be used to fine-tune neuroimmune reactions. Nevertheless, the two-fold nature of these molecules in the signaling of pain and other physiological activities should be taken into account when it comes to on-target toxicity.

One of the first opportunities might be in improving the current rehabilitation methods. If we understand how movement and load triggers neuroimmune braking processes, we may create more exact, evidence-based protocols that achieve the highest possible gains with the lowest possible risks. It is a change over to the empirical prescription to the rehabilitation based on mechanism.

Limitations

Several important limitations must be acknowledged when interpreting the proposed neuroimmune braking framework.

First and foremost, direct human evidence for the CGRP-RAMP1-TSP-1 pathway in musculoskeletal injuries is currently absent. All key mechanistic insights – including sensory neuron sprouting, RAMP1 signaling in myeloid cells, and TSP-1-mediated macrophage polarization – are derived exclusively from rodent models of skin wound healing and acute muscle injury. Whether the same pathway operates in human tendons, ligaments, or bone remains entirely unknown, and extrapolation should be made with great caution.

Second, the translational validity of rodent models to human musculoskeletal repair is inherently limited. Human tendons and ligaments are hypocellular, have slower metabolic turnover, and experience different mechanical loading patterns compared to rodent muscle or skin. Findings from mouse muscle injury models may not directly apply to load-bearing connective tissues.

Third, most of the preclinical studies cited in this review did not perform sex-disaggregated analyses. Given well-documented sex differences in pain perception, immune responses, and tissue healing, the absence of sex-specific data limits our ability to assess whether the proposed neuroimmune braking mechanism functions equally in males and females.

Fourth, the hypothesized link between active recovery (controlled mechanical loading) and the engagement of the neuroimmune braking system remains speculative. While the model provides a plausible mechanistic explanation for clinical observations, no study has directly measured CGRP release, RAMP1 activation, or macrophage polarization in human musculoskeletal tissues during active rehabilitation after injury.

Fifth, the proposed three-phase timeline (0–24 h, 24–72 h, ≥ 72 h) is based on rodent data and may differ substantially in humans, particularly in tissues with poor vascularity or slow healing responses.

In summary, the neuroimmune braking model presented here is a hypothesis-driven framework intended to guide future research, not a validated clinical paradigm. Rigorous human studies – including tissue biopsy, imaging, or genetic association studies – are urgently needed to test its relevance to human musculoskeletal injury and repair.

Conclusions

A new paradigm shift has occurred within the last ten years in terms of our knowledge of how the nervous system together with the immune system controls the repair of tissues after an injury to the musculoskeletal system. Active teachers rather than merely passive detectors of pain, nociceptive sensory neurons have been identified as active players in the immune response. We hypothesize that these neurons may act as braking mechanisms by releasing CGRP, acting on RAMP1 receptors on myeloid cells, which leads to macrophage polarization into M2 phenotypes, converting inflammation into regeneration. One downstream mediator, thrombospondin-1, coordinates various components of this response to include inhibition of excessive neutrophils aggregation, enhancement of efferocytosis, and direction toward pro-repair activities of macrophage polarization.

The TGF-β-mediated differentiation of macrophages pathway and the TAFA4-IL-10 signaling pathway are parallel pathways that act complementary to the CGRP axis with redundancy and strong regulation. Channels of TRPV1 play a vital role as an upstream regulator and connect the inflammatory microenvironment with the release of neuropeptides and direct linkage between the state of the tissue and neuroimmune output.

This model provides a plausible mechanistic explanation for the long-standing clinical observation that active recovery often leads to better outcomes than immobilization after certain musculoskeletal injuries. Based on preclinical models, controlled movement may trigger sensory neurons, engages the neuroimmune braking mechanism and facilitates smooth M1-to-M2 transition. This clarifies the importance of the so-called dose of loading that is either not sufficient or can lead to re-injuries and increased inflammation.

Looking towards the future, the application of neuroimmune principles to the practice of sports medicine and rehabilitation has the potential to enhance results after a musculoskeletal injury. Through the best prescription of exercise, additional physical modes including, but not limited to, development of specific biologic therapies and the use of the neuroimmune system remains a breakthrough of regenerative medicine. The next challenge is to translate these preclinical findings into evidence-based clinical practice–a goal that will require rigorous human studies, avoid long-term disability, and get back to work and active lives the injured population.

Abbreviations

Arg1

Arginase-1

ATP

Adenosine triphosphate

BDNF

Brain-derived neurotrophic factor

CGRP

Calcitonin gene-related peptide

CLR

Calcitonin receptor-like receptor

DAMPs

Damage-associated molecular patterns

DRG

Dorsal root ganglia

eCGRP

Engineered calcitonin gene-related peptide

ECM

Extracellular matrix

GAP43

Growth associated protein 43

GDNF

Glial cell line-derived neurotrophic factor

GFAP

Glial fibrillary acidic protein

HMGB1

High-mobility group box 1

IFN-γ

Interferon-γ

IGF-1

Insulin-like growth factor-1

IL

Interleukin

iNOS

Inducible nitric oxide synthase

LPS

Lipopolysaccharide

NGF

Nerve growth factor

NK1R

Neurokinin-1 receptor

PET

Positron emission tomography

RAMP1

Receptor activity-modifying protein 1

SP

Substance P

TAFA4

TAFA chemokine-like family member 4

TGF-β

Transforming growth factor-β

TNF-α

Tumor necrosis factor-α

TRPV1

Transient receptor potential vanilloid 1

TSP-1

Thrombospondin-1

TSPO

Translocator protein

VIP

Vasoactive intestinal peptide

VML

Volumetric muscle loss

Authors’ contributions

EX: Conceptualization, Writing – original draft, Visualization, Funding acquisition. EX: Visualization, Writing – original draft. YH: Writing – review & editing. TC: Writing – review & editing, funding acquisition. CL and ZW: Conceptualization, Writing – review & editing, Funding acquisition, Resources.

Funding

This study was supported by Ministry of Education Humanities and Social Sciences Program in China (21YJA890030).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors have approved the final version of the manuscript.

Competing interests

The authors declare no competing interests.