Role of neurogenic inflammation in intervertebral disc degeneration

Abstract

In healthy intervertebral discs (IVDs), nerves and blood vessels are present only in the outer annulus fibrosus, while in degenerative IVDs, a large amount of nerve and blood vessel tissue grows inward. Evidence supports that neurogenic inflammation produced by neuropeptides such as substance P and calcitonin gene related peptide released by the nociceptive nerve fibers innervating the IVDs plays a crucial role in the process of IVD degeneration. Recently, non-neuronal cells, including IVD cells and infiltrating immune cells, have emerged as important players in neurogenic inflammation. IVD cells and infiltrating immune cells express functional receptors for neuropeptides through which they receive signals from the nervous system. In return, IVD cells and immune cells produce neuropeptides and nerve growth factor, which stimulate nerve fibers. This communication generates a positive bidirectional feedback loop that can enhance the inflammatory response of the IVD. Recently emerging transient receptor potential channels have been recognized as contributors to neurogenic inflammation in the degenerative IVDs. These findings suggest that neurogenic inflammation involves complex pathophysiological interactions between sensory nerves and multiple cell types in the degenerative IVDs. Clarifying the mechanism of neurogenic inflammation in IVD degeneration may provide in-depth understanding of the pathology of discogenic low back pain.

Key Words: Intervertebral disc degeneration; Discogenic low back pain; Neurogenic inflammation; Neuropeptides; Neurotrophins; Transient receptor potential channels

Core Tip: The outer layer of the annulus fibrosus and the vertebral endplate are innervated by nociceptive nerves fibers. Injury to the annulus fibrosus or endplate excites nociceptive stimuli, releasing neuropeptides such as substance P and calcitonin gene related peptide, which results in neurogenic inflammation within the intervertebral disc. Neurogenic inflammation further leads to progressive disc degeneration and chronic discogenic pain through complex interactions between the nervous and immune systems.

INTRODUCTION

The research history of neurogenic inflammation dates back to 1874 when Goltz realized that the sciatic nerve in dogs contains vasodilator fibers in addition to normal constrictor nerves. The term neurogenic inflammation is now widely used to describe the mechanism by which sensory nerve contributes to inflammation[1,2]. Neurogenic inflammation exists not only in the skin and joints but also in the viscera, trachea, heart, bladder, and reproductive organs[3]. Although the term neurogenic inflammation has hardly been mentioned in published literature on the intervertebral disc (IVD) pathology, a large number of studies have implicated the role of neurogenic inflammation in IVD degeneration (IVDD). Neurogenic inflammation of the IVD is a complex process in which neuropeptides like substance P (SP) and calcitonin gene related peptide (CGRP) are released from afferent neurons innervating the IVD in response to IVD injury or degeneration[4-6]. This process is characterized by complex interactions between blood vessels, the immune system, and the nervous system, leading to changes in the structure and function of the IVD and ultimately to discogenic low back pain (DLBP)[7-12].

Low back pain (LBP) is one of the most important medical and social issues in the world today[13]. It is estimated that up to 84% of the population suffers from LBP at some point in their lives, while 10% are chronically disabled[14]. The components that make up the lumbar spine, such as muscles, ligaments, vertebrae, facet and sacroiliac joints, IVDs, and neurovascular structures, are susceptible to different stressors, and each of these factors, individually or in combination, can contribute to LBP[15]. Multiple studies have shown that LBP caused by IVDD, also known as DLBP, is the most common type of LBP. It is one of the main causes of disability and has significant socioeconomic impacts[16].

The development of IVDD is a complex and multifaceted situation. Numerous studies have suggested that multiple factors such as aging, abnormal mechanical stress, trauma, infection, nutrition, genetics, obesity, and gender are associated with IVDD[17-21]. Due to the complex and multifactorial nature of IVDD, little is known about its pathogenesis and risk factors, which seriously hinders the proper stratification of patients with LBP and limits the development of personalized treatments. This article reviewed the current knowledge on lumbar IVD innervation, the role of neuropeptides released by sensory nerve fibers innervating IVD in causing neurogenic inflammation and subsequent IVDD and DLBP, and the role of transient receptor potential (TRP) channels in IVD neurogenic inflammation.

ANATOMY OF IVD

The IVD is a functional unit that connects the vertebrae of the spine. Each IVD consists of three structures. A soft gelatinous nucleus pulposus (NP) in the center is surrounded by a tough peripheral lamellar annulus fibrosus (AF) and sandwiched between two cartilage endplates (CEP). The components of the disc work together to promote the movement of the spine and act as a shock absorber between the two vertebral bodies[22]. The cell density of the human IVD is very low and its renewal is very slow. As a result, they heal slowly and limited after injury and exhibit progressive and age-related changes[12].

The AF consists of 15-25 concentric angle-ply layers that cross each other obliquely in space. The AF is divided into inner and outer zones with different biochemical and cellular compositions as well as biomechanical properties. The outer layer of the AF is composed of fibroblasts that produce type I collagen, along with small amounts of collagen types III, V, and VI. The cells in the inner layer of AF are more like chondrocytes, producing mainly type II collagen and proteoglycans such as aggrecan[23].

The NP is a jelly-like structure rich in aggrecans restricted within the endplate and AF. It consists of chondrocyte-like cells that produce polysaccharide/mucoprotein molecules such as chondroitin sulfate, collagen, and elastin fibers[24]. The main function of the NP is to redistribute the applied load to the rest of the surrounding IVD.

The CEP is a thin, incomplete layer of cartilage that covers the area of the vertebral body surrounded by a ring apophysis[25]. Its thickness is between 0.1 and 1.6 mm, and it is the largest near the NP and inner AF and absent in the outer AF[26]. The CEP adjacent to the IVD consists mainly of fibrocartilage and is closely bound to the nuclear and annulus regions. However, the portion adjacent to the vertebral body consists mainly of hyaline cartilage, which is weakly attached to the subchondral bone[25].

Because the blood supply within the IVD is limited to the outer AF, and the nearest blood supply to most disc tissue in adults is located in the subchondral bone of the vertebral body, metabolite exchange mainly comes from the diffusion of the vertebral body through CEP[25,27]. In this way, the IVD microenvironment is often described as harsh because of its limited nutrients (glucose and oxygen), low pH, and large changes in osmolarity. The dense extracellular matrix of IVD mechanically inhibits the ingrowth of blood vessels through high physical pressure and chemically through high aggrecan content[12]. The combination of high aggrecan content and secretory inhibitors can prevent the ingrowth of nerves and blood vessels in non-degenerative IVDs[28,29]. AF, CEP, and immunosuppressive molecular factors are defined as the blood-NP barrier, which separates NP from the host immune system[30].

INNERVATION OF IVD

The prerequisite for DLBP is that IVD has innervation, but it is generally believed that normal NP and inner annular zones do not have innervation[7]. However, early opinions were divided as to whether the outer annulus was innervated or not[31]. Malinsky[32] found a variety of free nerve endings and some button-like terminals in the outer layers of the lumbar AF and noted that partially and fully encapsulated mechanoreceptors were confined to the AF surface. Since then, researchers have generally agreed with Malinsky’s classic observation that the superficial layers of the normal AF have sensory nerve endings[33], that the nerve penetration of the human AF is about 3 mm deep[34], and that it involves three outer lamellar layers[35].

Numerous animal studies have shown that the nerve fibers that innervate the IVD come from multiple dorsal root ganglion (DRG) neurons. There are two pathways between the AF and the DRGs: One through the sinuvertebral nerve (SVN) and the other through the sympathetic trunk[36-40]. It was agreed that SVN consists of a fine sympathetic branch from the grey ramus communicans and a fine sensory spinal branch from the ventral ramus[41]. The SVN re-enters the vertebral canal through each intervertebral foramen and is located in front of the nerve root with segmental blood vessels[42]. It is commonly believed that up to three segments of the lumbar SVN overlap, which may explain the poor localization of LBP[33]. In addition, a double origin of the disc nerve fibers, the somatic nerve, and the sympathetic nervous system, reflects a similar pattern with some enteric organs, leading to the belief that low back pain is a visceral pain[33].

In degenerative IVDs in humans and animals, especially in painful IVDs in humans, a significant increase in innervation has been observed, with nociceptive nerve fibers growing into inner AF and even to the NP, often accompanied by the ingrowth of blood vessels (Figure 1)[7,11,33,43,44]. The immunohistochemical patterns of nerve fibers and neurons innervating pathological IVDs are the same as those reported under normal conditions. Therefore, the difference in disc innervation patterns in the normal state compared to the degenerative state is quantitative rather than qualitative[7,33].

Figure 1 Schematic of a healthy disc and a degenerative disc. A healthy disc is composed of the annulus fibrosus, nucleus pulposus, and cartilaginous endplates (left side). Nerve fibers and blood vessels are distributed only in the outer layer of the annulus fibrosus and in the vertebral endplate. A degenerative disc shows loss of border between the annulus fibrosus and nucleus pulposus, reduction of nucleus pulposus cells, and ingrowth of blood vessels and nerve fibers into the inner layer of degenerative disc (right side). There are significantly increased nerve fibers and blood vessels in the vertebral endplate.

In addition to the peripheral portion of the AF, the vertebral endplate is innervated by nociceptive nerves[45]. The basivertebral nerve (BVN) plays an important role in the transmission of pain sensation in the endplate[46,47]. BVN is a branch of SVN that enters the vertebral body through the central vascular foramen along with the basivertebral vessels and branches around the vertebral endplate[48]. With the degeneration of the IVD, IVD cells and infiltrating immune cells release inflammatory cytokines such as interleukin (IL)-1, IL-6, IL-8, tumor necrosis factor (TNF)-α, and chemokines such as CC motif chemokine ligand (CCL) 2, CCL3, and C-X-C motif chemokine 10[4-10]. Through convection, crosstalk occurs between the IVD and subchondral bone marrow, and the vertebral endplate gradually undergoes degenerative changes, displaying Modic changes on magnetic resonance imaging (Figure 2)[49,50]. Inflammatory cytokines, especially IL-1, stimulate the expression of angiogenic factors and neurotrophic factors, which trigger the ingrowth of nociceptive nerves into the vertebral endplate (Figure 1)[7].

Figure 2 The relationship between severe disc degeneration and Modic changes in the endplates. A: Lateral radiograph of the lumbar spine showed significant narrowing of the L4/5 intervertebral space, osteophyte formation at the edges of the vertebral bodies, and sclerosis of the upper and lower endplates; B and C: T2-weighted and T1-weighted magnetic resonance imaging revealed bone marrow hypersignal in the upper and lower endplates of the L4/5 disc, respectively, indicating Modic changes type 2 in the vertebral endplates (bone marrow fatty degeneration).

Studies have shown that the degree of endplate lesions is related to the severity of back pain and is closely related to the degree of IVDD[51]. The density of vertebral endplate innervation is directly proportional to the degree of vertebral endplate injury, further providing evidence for its role in chronic LBP[52]. A histopathological study found that those pathologically altered vertebral endplates, such as structural defects or endplate lesions, had a higher innervation density than unaltered endplate nerves[53]. The reports on the improvement of LBP after radiofrequency ablation of the BVNs and lumbar fusion surgery in patients with endplate-related LBP reinforce this important viewpoint[54-57].

NEUROGENIC INFLAMMATION IN IVDD

Nociceptive nerve fibers in lumbar IVD

It is widely recognized that normal IVDs are innervated by sensory (mainly nociceptive) and postganglionic sympathetic (vasomotor efferent) nerve fibers[5,7]. The sensory nerve fibers innervating the lumbar disc consist mainly of small myelinated A-delta fibers and unmyelinated C-fibers[33]. These tiny fibers express SP and CGRP, which are nociceptors associated with inflammatory pain[7]. They are nerve growth factor (NGF)-dependent and express high-affinity tyrosine receptor kinase (Trk) A[5]. After experimental IVD puncture and inflammation induction, the number of CGRP-positive activated DRG neurons innervating lumbar IVD increases[36]. Studies have indicated that almost all nociceptive nerve fibers in human and rat IVDs are peptide-containing nerve fibers, suggesting that nerve fibers associated with inflammation may transmit pain originating from degenerative IVDs[58,59].

An early study showed that nerve fibers observed in human painful discs express growth associated protein 43 (GAP-43)[60]. GAP-43 is associated with axonal growth and synaptic plasticity and plays an important role in neuronal development and regeneration[61]. During progressive degeneration of IVD, GAP-43-positive nerve fibers from DRG neurons penetrate to different depths of IVD. The inflammation of the IVD in rats induced by complete Freund’s adjuvant[62,63] or exposure of the NP to the outside of the AF[62] can increase the expression of GAP-43 in DRG neurons innervating the IVD, suggesting that IVD inflammation may promote axonal growth of DRG neurons. In addition, the studies also found in rats[64] and humans[65] that mRNA and protein levels of GAP-43 were significantly higher in the degenerative IVDs than in the control group. These results suggest that GAP-43 may be an effective marker for identifying neoinnervation in degenerative IVD[64,65].

Neuropeptides

Neurogenic inflammation is mediated by the release of neuropeptides such as SP and CGRP from sensory nerve fibers[1-3]. In rodents, A-delta and C-fiber afferents cannot be distinguished according to the mode of stimulation. Both types of afferents can be distinguished based on the electrophysiological and chemical properties of DRG neurons. The C-fiber population is characterized by a sensitivity to capsaicin, associated with expression of the TRPV1 receptor, a high activation threshold, and a long-sustained action potential insensitive to tetrodotoxin (an Na+ channel blocker). The A-delta fiber population is insensitive to capsaicin but sensitive to tetrodotoxin[66].

SP and CGRP are classical neuropeptides that act directly on vascular endothelial cells and smooth muscle cells, thereby mediating vascular events. SP increases vascular permeability, followed by plasma extravasation and edema[1]. The release of SP induces mast cells to release vascular endothelial growth factor (VEGF), which promotes angiogenesis and inflammatory cell infiltration[67]. CGRP is an extremely potent microvascular vasodilator, which contributes to most neurogenic vasodilation and participates in the recruitment of inflammatory cells[67]. SP and CGRP both act through their respective G-protein-coupled receptors: Neurokinin-1 receptor (NK1R) for SP and CGRP receptor complex for CGRP[4].

Nerve fibers expressing CGRP and SP are distributed in the outer AF of healthy IVDs, and in degenerative or painful IVDs, these nerve fibers reach the inner AF and NP[33,60]. In the IVD, sensory nerve fibers are mixed with autonomic nerve fibers that contain additional neuropeptides such as neuropeptide Y, noradrenaline and vasoactive intestinal peptide, which may be involved in vascular regulatory function or IVD cell biology[6,68].

Clinically, neurogenic inflammation of degenerative lumbar IVDs may be present in patients with DLBP. Histological studies of painful IVDs have shown many features of neurogenic inflammation, such as infiltration of mast cells and macrophages, nerve fiber ingrowth, and new angiogenesis[69,70]. Immunostaining studies have shown that the expression of CGRP and SP is upregulated in the sensory nerves innervating painful or degenerative discs[33,60,69] and degenerative vertebral endplate[45,53]. It has been reported that the expression of SP and CGRP is significantly increased in painful degenerative IVDs[9,71-74]. In addition, neurogenic inflammation of the lumbar IVD in rats has been identified. Capsaicin was applied to the ventral portion of the L5-L6 disc after intravenous injection of Evans blue in rats, and dye extravasation was observed in the groin skin (L2 dermatome). Because the ventral portion of the L5-L6 IVD in rats is mainly innervated by the neurons of L2 DRG, this phenomenon is caused by the axonal reflex of the dichotomizing fibers from L2 spinal nerve[36].

SP

SP is an 11-amino acid protein that belongs to the tachykinin protein family. SP is typically described as a neurotransmitter that preferentially binds to the NK1R, which is expressed in two splicing variants: The full-length isoform and the truncated isoform[75,76]. The interaction between SP and its preferred receptor NK1R leads to nuclear factor κB (NF-κB) activation and production of proinflammatory cytokines[76]. In IVD, SP has been used as a specific marker for identifying ingrowth of nerves, which may transmit nociceptive signals in degenerative IVDs[69]. SP-treated disc cells showed significant upregulation of IL-1β, IL-6, IL-8, and TNF-α in NP and AF cells. AF and NP cells expressed SP at low levels, but the expression was significantly upregulated after IL-1β or TNF-α treatment. Both SP receptor isoforms are expressed in NP and AF cells[77].

Further studies showed increased SP production in the discs of patients with LBP compared to patients without back pain, leading Richardson et al[71] to suggest that SP plays a central regulatory role in the development of painful disc degeneration. Treatment of IVD cells with NK1R antagonists inhibited IL-1β, IL-6, and IL-8 expression in a dose-dependent manner. This suggests that SP mediates signaling in IVD cells via NK1R. The interaction of SP with its preferred receptor NK1R leads to the activation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 (ERK1/2), suggesting that p38 mitogen-activated protein kinase and ERK1/2 control SP-induced cytokine expression. Inhibition of p38 and ERK1/2 activation reduces SP-induced IL-6 production in human IVD cells[78].

CGRP

CGRP is a 37-amino acid neuropeptide. It is widely produced in the central and peripheral nervous systems. However, it is primarily released from sensory nerves and thus is implicated in pain pathways. CGRP works through the CGRP receptor complex. This complex requires the colocalization of the G-protein component calcitonin-like receptor with a single transmembrane component receptor activity-modifying protein 1 and an intracellular signaling component receptor component protein[79]. The regulation of CGRP synthesis is still unclear. CGRP synthesis is enhanced in tissues undergoing an inflammatory reaction[80].

In a rat model of lumbar IVD injury, CGRP is persistently upregulated in DRG neurons innervating the IVD[4,81]. This may be related to the local release of NGF from disc cells, macrophages, or mast cells. NGF is essential for the growth of sensory nerves and the maintenance of mature nerve function. After treatment with the TRPV1 agonist capsaicin, the sensory neuropeptide in nerve endings is exhausted, and NGF is required to synthesize new peptide[80].

Similar to SP, normal disc cells express low levels of CGRP and its receptor. The expression levels of CGRP and its receptor are significantly increased in degenerative or painful discs[9,74]. In vitro experiments have shown that CGRP can increase the production of inflammatory mediators, such as cyclooxygenase-2, inducible nitric oxide synthase, IL-1β, and IL-6 in human NP cells in a dose-dependent manner. In addition, the CGRP receptor antagonist rimegepant can improve the adverse reaction of CGRP to NP cells, which has been demonstrated both in vitro and in vivo[82]. Data from patients with disc degeneration suggest that NF-κB signaling of peptides, including CGRP, may be involved in the mechanism of peripheral pain[9,74]. Studies have found higher levels of NF-κB and CGRP in disc tissue from patients with degeneration compared to controls[82]. NF-κB activity has been shown to be associated with the degree of disc degeneration. NF-κB may modulate the expression of pain-related neuropeptides in disc cells and the sensory neurons that innervate IVD. This relationship may be a key link in the pain and inflammation cycles that persist in chronic LBP[74].

The release of SP and CGRP from sensory nerves or disc cells into the matrix also upregulate the production of matrix-degrading enzymes. In fact, studies have shown that SP and CGRP can increase the expression of matrix metalloproteinase-3[82,83]. Thus, these neurotransmitters exhibit dual mechanisms of action, including action potential neurotransmission to the central nervous system while inducing or perpetuating peripheral inflammation. These findings provide evidence that SP and CGRP contribute to a degenerative phenotype within the IVD, sensitize ingrowing nerves, and potentially serve as a mechanism for cross-talk between the disc cells and pain fibers to enable transmission of painful stimuli.

Neurotrophins

NGF has many similarities in structure and physiological functions with other neurotrophins (NTs). These NTs include brain-derived neurotrophic factor (BDNF), NT-3, NT-4/5, NT-6, and NT-7[84]. They are secreted proteins that promote the survival and growth of different elements of the peripheral nervous system. NTs can bind to two types of receptors: Trks and p75^NTR, a member of the TNF receptor superfamily. There are three different Trk receptors (TrkA, TrkB, and TrkC), each with its own preference for a specific NT. TrkA has high selectivity towards NGF and NT-3 to a lesser extent, while TrkC binds NT-3 and TrkB BDNF and NT-4/5. On the contrary, p75^NTR is promiscuous, binding all NTs, including NGF, with low affinity[85]. NTs are composed of different subgroups in neuronal and non-neuronal tissues, with different intracellular and extracellular domains. Many receptors bind to several potential ligands, and altered receptor isoforms display partial excitatory or antagonistic effects[85].

The production of NGF is tightly controlled in all areas of the organism innervated by sensory and sympathetic neurons. Normally low basal NGF production is substantially upregulated in the inflammatory response. It is reasonable to assume that mediators regulating local NGF concentrations are released during inflammation due to tissue damage. Many studies have shown that cytokines involved in inflammation, such as IL-1β, TNF-α, and IL-6, are promoters of NGF synthesis in a variety of cell types[86]. NGF is an important cytokine-like neurotrophic mediator. This molecule regulates the survival and differentiation of embryonic sensory and sympathetic neurons from the neuronal crest[86]. In adults, it regulates neuronal regeneration for injury and pain perception and induces neurogenic inflammation by stimulating neuropeptide overexpression and activating immune cells[86,87].

Since TrkA is the main receptor of NGF, it may be considered one of the most important receptors for pain regulation. The expression of TrkA in the basal forebrain of rats is stimulated by NGF itself, suggesting that the upregulation of NGF may lead to amplified pain, accompanied by hyperesthesia and allodynia[88]. Instead, proinflammatory neurotransmitters induce overexpression of NGF, which may lead to chronic self-perpetuating pain sensation mechanisms[85]. In addition, NGF may mediate mechanical and thermal hyperesthesia in systemic applications in animals and humans[84].

NGF and BDNF have been identified in human IVD and have been implicated in mechanisms related to nerve ingrowth and pain perception in IVDD[89,90]. In addition, TrkA and TrkB expression have been demonstrated in both normal and degenerative human IVDs[90]. p75^NTR has been found in the AF superficial layers of rat IVD[91]. It has been reported that a large number of TrkA-positive and p75^NTR-positive neurons colocalize with CGRP-positive sensory neurons in rat DRG[92]. NGF regulates SP in adult sensory neurons through TrkA and p75^NTR[93]. NGF, BDNF, TrkA, and TrkB are expressed in all stages of IVDD[88]. It is generally believed that NGF, TrkA, and p75^NTR are always expressed at elevated levels in painful IVD[8]. The expression of NTs and their receptors in AF and NP cells suggests that these molecules have an autocrine or paracrine role in regulating IVD biology in addition to neurotrophic functions, whose function may be regulated by proinflammatory cytokines, which are known to be upregulated during IVDD. For example, the expression of the BDNF gene in cultured AF cells was significantly positively correlated with IVDD[94].

NGF binding to TrkA initiates complex signal transduction pathways, leading to the activation of transcription factors such as NF-κB and inducing the expression of a large number of proinflammatory cytokines[8]. In addition, receptor proteins, such as TRPV1 and bradykinin receptor, voltage-gated sodium channels (NaV1.7, NaV1.8, and NaV1.9), acid-sensing ion channel proteins (ASIC3), and G-protein-coupled receptors are involved in sensory transmission processes and are also regulated by NGF[5,7]. There is experimental evidence that peripheral inflammation leads to increased BDNF content in DRGs, which correlates with increased and widespread BDNF release in the dorsal horn of the spinal cord[7].

Studies have shown that the level of NGF in IVDs is positively correlated with innervation density[95,96]. NGF from human degenerative NP extraction medium promoted the axon growth of DRG neurons in rats and induced SP expression[97]. It has been proven that painful IVDs have TrkA-expressing nerve fibers[95], and the enhanced expression of NGF activates and sensitizes primary afferent neurons expressing TrkA. According to classical neurotrophic theory, when the NGF produced by the degenerative IVD binds to the receptor TrkA at the nociceptor terminal, the receptor complex is internalized and orthodromically transported to the nucleus of the DRG neurons. Phosphorylation of the NGF-TrkA complex induces the production of SP and CGRP. Subsequently, these neuropeptides are released from activated nociceptors via antidromic transport, leading to neurogenic inflammation of the degenerative IVD[7,84]. Therefore, the NGF-TrkA pathway is critical in inflammatory back pain caused by IVDD.

Immune cells

IVDD is usually related to inflammation, immune cell infiltration, and neovascularization. The pathogenesis of immune cell infiltration into the degenerative IVD is not yet clear, especially since healthy IVDs have immune privileges to a large extent. It is likely that an event secondary to AF injury or tear results in recruitment of immune cells to the painful degenerative discs (Figure 3)[70]. The response of immune cells to acute tissue injury and their role in inflammation suggest that they may play a key role in promoting catabolic remodeling within IVD, thereby enhancing neovascularization and neoinnervation[98].

Figure 3 Painful intervertebral discs are always structurally ruptured. A: Lumbar T2-weighted magnetic resonance imaging showed disc degeneration at L3/4 and L4/5 Levels (black disc), with a high-signal zone (arrow) behind each annulus fibrosus; B: Discography showed that both discs were painful discs (pain reproduction during injection of contrast agent), and the two-dimensional reconstruction of the CT scan after discography showed that the contrast agent leaked into the spinal canal from behind the L3/4 and4/5 intervertebral discs, indicating a tear in the posterior annulus fibrosus of these two intervertebral discs.

In painful IVDs, repeated stimulation of nociceptive fibers can induce antidromic transport, followed by the release of SP or CGRP at nociceptors. These neuropeptides modulate the immune response through receptor binding[96,99-101], activating lymphocytes, mast cells, or macrophages, which in turn lead to further upregulation of inflammation and nociceptive sensations[102]. In addition, because NGF receptors TrkA and p75^NTR are present on the surface of immune cells, NGF is able to induce immune cells to synthesize neuropeptides with immunomodulatory functions, such as CGRP in monocytes and B cells[84]. Furthermore, immune cells express neuropeptides such as SP, which further recruit leucocytes to the peripheral terminals of nociceptors, releasing neuroactive mediators that cause neuropathic pain[103]. Thus, nociceptive neurons and the immune system interact to perpetuate DLBP.

Mast cells

Mast cells are the first responders to tissue damage. Extensive infiltration of mast cells in painful IVDs has been found[70,98]. The mechanism of mast cell infiltration into degenerative IVD is not yet clear, but it may be related to the upregulation of mast cell chemoattractant stem cell factor by degenerative IVD cells[98]. The role of mast cells in DLBP and IVDD may be related to the mediators and bioactive substances released during degranulation, including histamine, proteases, TNF-α, IL-6, NGF, VEGF, SP, etc.[70,95,98,104]. Sensory nerve fibers are usually in close contact with mast cells. Mast cells are particularly relevant to the nervous system in neurogenic inflammation[67].

Neuropeptide SP released from sensory nerve endings acts on mast cells via Mas-related G-protein coupled receptor member B2, inducing mast cell degranulation and promoting proinflammatory effects of subsequent mediators such as histamine[99]. Histamine released from mast cells, in turn, stimulates the release of neuropeptides that act on histamine receptors on sensory nerve endings, thereby establishing a bidirectional loop between mast cells and sensory nerves[105]. In addition, SP induces mast cells to release VEGF, which promotes vascular endothelial cell proliferation and vascularization and promotes inflammatory processes. However, VGEF can also stimulate neurite growth in vitro, promote Schwann cell migration and survival, and may play a role in axon guidance[106,107]. In addition, mast cells promote matrix remodeling by releasing chemokines such as CCL2/monocyte chemotactic protein-1, which are responsible for recruiting other types of cells, such as macrophages[98]. Since DLBP is considered a chronic pain state, it is reasonable to think that mast cells may play an indispensable role in the degeneration and sensitization of IVD.

Macrophages

Macrophage infiltration has been identified in herniated[108,109] and painful IVDs[70], or animal models of IVD injury[10,110]. In humans, levels of macrophage markers were positively associated with degeneration within the NP and endplate, and in cadaveric specimens, macrophage markers were more strongly associated with unhealthy areas of disc structural damage[111]. In addition, the prevalence of CCR7+ M1 macrophages (proinflammatory) and CD163+ M2 macrophages (anti-inflammatory) increases with age and degeneration[111]. A mouse model of disc injury also supports the association between macrophage infiltration and IVDD[112]. Macrophages in injured IVDs produce inflammatory cytokines TNF-α and IL-1β, and macrophage-derived inflammatory factors further regulate the production of NGF and VGEF in IVDs[110,113]. Furthermore, when CD14⁺ cells (a monocyte marker expressed mainly by macrophages) were isolated from degenerative human IVD tissues and stimulated with TNF-α and IL-1β, production of CGRP and NGF increased[71]. Thus, the influx of macrophages into the degenerative IVD can initiate and/or maintain neurogenic inflammation, exacerbate inflammation and degeneration and promote painful stimulation[114].

Lymphocytes

The immune privilege of the NP tissue occurs through vascular isolation and the biochemical phenotype of Fas ligands leading to apoptosis of infiltrating T lymphocytes[30]. T lymphocytes and B lymphocytes have been identified in herniated human disc tissue[115-117] and animal models of disc injury[118,119]. Detection of autoantibodies against degenerative or injured IVD and induction of T lymphocytes into IL-4-producing CD4⁺ Th2 cells further support the hypothesis that contact of NP tissue with systemic circulation in IVD herniation or injury leads to lymphocyte activation and subsequent autoimmune responses[120].

Populations of different types of immune cells express neuronal receptors and respond to neuropeptides and neurotransmitters, suggesting that peripheral neurons are part of a local effector system involved in inflammatory responses to tissue irritation and injury[86]. Thus, the nervous system communicates with the immune system through neuropeptides that are involved in IVD neurogenic inflammation. It is becoming increasingly clear that the nervous system plays a key role in regulating tissue immunity. Given its rapid response and reflex circuits, the ability of the nervous system to sense environmental stimuli and transmit signals to the immune system is essential for an efficient and effective anticipatory immune response. However, our current understanding of the complex dialogue between the nervous system and the immune system is not advanced enough. Addressing these gaps in knowledge will inform the development of new targeted therapeutic strategies to treat chronic inflammation and DLBP in degenerative IVDs[99].

TRP channels

TRP channels are a cation-selective transmembrane receptor superfamily. More than 50 TRP channels have been found in many species, with 28 TRP channels found in mammals[121]. The TRP channel consists of six transmembrane domains, intracellular N-terminal and C-terminal, and a pore domain between segments 5 and 6, which is particularly permeable to Ca²⁺ ions. There are seven families of mammalian TRPs, including TRPC, TRPV, TRPM, TRPA, TRPP, TRPML, and TRPN. In yeast, an eighth TRP family was recently discovered and named TRPY[122]. TRP channels act as cellular sensors that sense a variety of stimuli, including temperature, membrane voltage, oxidative stress, mechanical stimulation, pH, and endogenous and exogenous ligands, illustrating their versatility[123]. Therefore, TRP channels regulate various functions of excitable and non-excitable cells mainly by mediating Ca²⁺ homeostasis. Dysregulation of TRP channels is associated with many disorders, including cardiovascular disease, muscular dystrophy, and hyperalgesia[121]. However, the importance of expression, physiological function, and regulation of TRP channels in IVD cells has been largely unexplored.

TRPV1

The release of neuropeptides from sensory nerves is triggered by an increase in cytoplasmic Ca²⁺ concentration. Cationic channels expressed on sensory nerve endings include some TRP channels involved in neuropeptide release. TRPV1, also known as the capsaicin receptor, is one of six members of the TRPV family (TRPV1-6). TRPV1 is a nociceptive cation channel that is sensitive to high temperature (> 43 °C), and capsaicin is its natural agonist[121]. Most TRPV1-expressing neurons innervating the IVD coexpress CGRP[36]. When TRPV1 is activated by capsaicin and other vanilloids, heat stimuli, and protons, Ca²⁺ influx is initiated, and neuropeptides are released[124].

Interestingly, TRPV1 is also expressed in non-neuronal cells such as chondrocytes[125] and IVD cells[126,127], but its role in relation to inflammation and pain in these tissues is unclear. One possibility is that it works by mediating acidic microenvironments[128]. IVD is composed of avascular tissue, and its metabolism is mainly through glycolysis. TRPV1 is activated by an acidic pH, which is an important factor in IVD homeostasis[128]. Lactic acid builds up in degenerative IVD due to impaired liquid transport, alters cellular repair capacity, and promotes pain[129]. As a presumed sensor of acidic pH, overstimulated or upregulated TRPV1 may be involved in the IVD cell response to acidity. Thus, inhibition or desensitization of TRPV1 may be beneficial for pH-induced hyperalgesia.

Increased expression and activity of TRPV1 in DRGs after inflammatory stimulation may lead to chronic inflammatory pain in rats, including thermal hyperalgesia and mechanical allodynia[130]. In addition, TRPV1 responds to proinflammatory agents, such as TNF-α, which are known to be highly expressed in painful IVD tissues. Therefore, the inflammatory microenvironment associated with IVDD may increase the likelihood of channel opening[131]. Immune cells such as macrophages, lymphocytes, and mast cells also express TRPV1, and activation of TRPV1 directly affects the function of these cells to increase the expression of inflammatory genes that may be involved in neurogenic inflammation[124].

TRPV4

There is evidence that another member of the TRPV family, TRPV4, is a major osmolarity-regulated ion channel in disc tissue. In bovine disc cells, decreased osmolarity activates TRPV4 expression and induces TRPV4-mediated Ca²⁺ signaling and proinflammatory cytokine gene expression[132]. In addition, cytokine IL-1β rather than TNF-α stimulation significantly induced TRPV4 gene expression in human IVD cells[126].

TRPV2

TRPV2 was originally described as a noxious heat sensor, but it now appears to have nothing to do with temperature sensing and instead has more complex physiological characteristics[131]. Cytokine TNF-α stimulation can significantly induce the expression of the TRPV2 gene in human IVD cells[126]. TRPV2 expression was upregulated in DRG induced by peripheral inflammation[133] and may be involved in the release of CGRP[134].

TRPA1

TRPA1 is a ligand-gated non-selective Ca²⁺ channel, which in contrast to TRPV1 responds to cold thermal sensation (< 17 °C). TRPA1 is located in approximately 60%-75% of sensory C-fibers, which are also positive for TRPV1[124]. TRPA1 is the only known member of the TRPA family and may be involved in the development of chronic pain and mechanical hyperalgesia. TRPA1 is widely expressed in sensory neurons, such as nociceptive DRG neurons, and in non-neuronal cells, including epithelial cells, keratinocytes, and chondrocytes[121].

Both neuronal and non-neuronal TRPA1 may be involved in the occurrence of pain. TRPA1 mediated mechanical hyperalgesia in mice after plantar injection of TNF-α[135], and mechanical hyperalgesia was alleviated after application of the TRPA1 antagonist HC-030031[136]. TRPA1 is expressed in degenerative IVD but not in healthy mature IVD tissues, and its expression in IVD cells is upregulated by proinflammatory cytokines[126]. The expression and activity of TRPA1 in sensory and non-neuronal cells are regulated not only by proinflammatory cytokines but also by neuropeptides and reactive oxygen species, all of which are generally elevated in degenerative IVD[121]. There is a growing hypothesis that TRPA1 is involved in neurogenic inflammation. TRPA1 activation leads to the release of neuropeptides from sensory neurons in a Ca²⁺-dependent manner, followed by signs of neurogenic inflammation such as edema and leukocyte infiltration[118]. These data suggest that blocking TRPA1 may help reduce chronic pain and inflammation associated with IVDD.

A recent study found that 26 of the 28 currently known TRP channels in mammals are expressed in IVD at the mRNA level. This study supports the importance of TRP channels in IVD homeostasis and pathology and their potential as pharmacological targets for the treatment of IVDD and LBP. However, the exact function and activation of the prominent TRP channels will be determined in future studies[137].

CONCLUSION

According to our search, the term neurogenic inflammation has barely appeared in the literature in the field of IVD pathology research. In fact, the role of neurogenic inflammation in IVDD and DLBP has been widely involved, but no studies have summarized and generalized it. Data from animal models of IVDD have provided important insights. IVD injury is characterized by an early transient inflammatory response to the initial event with increased production of proinflammatory molecules, including IL-1β, IL-6, IL-8, TNF-α, and NGF[4,81,138,139], followed by a delayed phase that occurs simultaneously with the morphological and biochemical features of progressive IVDD[140,141]. The initial event of increased expression of inflammatory mediators after acute IVD injury is unknown.

It has been shown that IVDs in rats can heal rapidly after a single-stab injury, and the fibrous cap covers the wound track. This process corresponds to the subside of the inflammatory response after the nociceptive stimulus has disappeared[138,139]. Neurogenic inflammation is responsible for this phenomenon. When nociceptors distributed in the outer AF are activated by noxious stimuli (IVD injury), nociceptors can release neuropeptides from their peripheral terminals. These released neuropeptides promote the production of inflammatory mediators by adjacent IVD cells and vascular tissue[142]. Persistent inflammation caused by repeated IVD injury[139] suggests that DRG neurons synthesize and release SP and CGRP via antidromic transport[81,102]. A recent study showed that neurogenic inflammation can aggravate the inflammatory response of injured IVD in an animal model of IVDD. Through antidromic transport, neurogenic inflammation can spread to neighboring healthy discs, triggering chronic progressive degeneration of neighboring discs. The degeneration of adjacent healthy IVDs without puncture indicates that neurogenic inflammation is the root cause of healthy IVDs[143].

Neurogenic inflammation in the IVDs may involve complex communication between IVD cells and infiltrating immune cells and afferent nerve endings. The initial release of neuropeptides SP and CGRP by nociceptive nerve fibers can trigger a variety of processes, including vascular events, immune cell infiltration, IVD cells and immune cell release mediators, and sensitization of primary afferent neurons (Figure 4). The upregulation of NGF is believed to play an important role in the development and maintenance of neurogenic inflammation.

Figure 4 Schematic of the major relationship between disc degeneration and neurogenic inflammation. Intervertebral disc injury or degeneration produces a large number of inflammatory mediators, including histamine, 5-HT, bradykinin, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), nerve growth factor (NGF), H⁺, and prostaglandins. Each of these mediators sensitizes (lowers threshold) or stimulates the terminals of nociceptors by interacting with cell surface receptors expressed by the neurons innervating disc. Activation of nociceptors not only triggers an orthodromic action potential that transmits afferent information to the dorsal horn of the spinal cord but also invades local collaterals and terminals (antidromic activity) unaffected by the original insult, thus initiating the process of neurogenic inflammation. This is an efferent function of nociceptors in which neuropeptides, particularly substance P (SP) and calcitonin gene-related peptides (CGRP), are released from the peripheral terminals to induce vasodilation and plasma extravasation as well as the activation of non-neuronal cells, including disc cells and immune cells. These cells in turn release inflammatory mediators that aggravate the inflammatory response of the disc. TrkA: Tyrosine receptor kinase A.

Emerging evidence highlights the potential role of TRP channels in IVDD, DLBP, and peripheral signal amplification associated with neurogenic inflammation. It is conceivable that some of the effective drugs currently used to treat various inflammatory diseases, especially osteoarthritis, may be beneficial to the IVD-sensory conduction process[102,144]. Pharmacological interventions targeting IVDD-related neurogenic inflammation may provide new research avenues and potential new strategies for the clinical treatment of DLBP[145].