Neuroimmune Mechanisms Underlying Neuropathic Pain: The Potential Role of TNF-α-Necroptosis Pathway

The neuroimmune mechanism underlying neuropathic pain has been extensively studied. Tumor necrosis factor-alpha (TNF-α), a key pro-inflammatory cytokine that drives cytokine storm and stimulates a cascade of other cytokines in pain-related pathways, induces and modulates neuropathic pain by facilitating peripheral (primary afferents) and central (spinal cord) sensitization. Functionally, TNF-α controls the balance between cell survival and death by inducing an inflammatory response and two programmed cell death mechanisms (apoptosis and necroptosis). Necroptosis, a novel form of programmed cell death, is receiving increasing attraction and may trigger neuroinflammation to promote neuropathic pain. Chronic pain is often accompanied by adverse pain-associated emotional reactions and cognitive disorders. Overproduction of TNF-α in supraspinal structures such as the anterior cingulate cortex (ACC) and hippocampus plays an important role in pain-associated emotional disorders and memory deficits and also participates in the modulation of pain transduction. At present, studies reporting on the role of the TNF-α–necroptosis pathway in pain-related disorders are lacking. This review indicates the important research prospects of this pathway in pain modulation based on its role in anxiety, depression and memory deficits associated with other neurodegenerative diseases. In addition, we have summarized studies related to the underlying mechanisms of neuropathic pain mediated by TNF-α and discussed the role of the TNF-α–necroptosis pathway in detail, which may represent an avenue for future therapeutic intervention.


Inflammatory Response, Apoptosis and Necroptosis Induced by TNF-α
TNF-α is important for mammalian immunity and cellular homeostasis. The role of TNF-α as a master regulator in balancing cell survival and death has been extensively studied in various cell types and tissues. As shown in Figure 1, TNF-α induces an inflammatory response and two programmed cell death mechanisms, namely, apoptosis and necroptosis, based on different pathological conditions [54][55][56][57][58]. Receptor interacting protein kinase 1 (RIPK1) and TNF receptor-associated death domain (TRADD) regulate TNF-dependent signalling, which controls the balance between cell death and survival [59].

Cell Survival and Inflammatory Response
When TNF-α binds to TNFR1 on the membrane surface, the conformation of TNFR1 changes, and TNFR1 complex I is rapidly formed via the recruitment of various proteins, including TRADD, RIPK1, TNF receptor-associated factor 2 (TRAF2) and cellular inhibitor of apoptosis protein 1 and 2 (cIAP1/2). TRADD is very important for the recruitment of TRAF2 and ubiquitination of RIPK1 and complex I. The TGF-activated kinase 1 (TAK1) binding protein (TAB) complex and IκB kinase (IKK) complex consisting of IKK1 can prevent cell death, whereas cIAP1/2 can prevent TNFR1-mediatednecroptosis and promote ubiquitination of RIPK1 [60,61]. Ubiquitination of RIPK1 activates the NF-κB signalling pathway through TAK and IKK complexes, promotes cell survival and induces an inflammatory response [62].

Figure 1.
Tumor necrosis factor-alpha (TNF-α)/TNF receptor 1 (TNFR1)-mediated inflammatory response and cell death pathways. The binding of TNF-α to TNFR1 triggers inflammatory responses, apoptosis and necroptosis. Ubiquitination of receptor interacting protein kinase 1 (RIPK1) promotes cell survival and induces an inflammatory response by activating the NF-κB, p38 MAPK, JNK and ERK signalling pathways. If caspase-8 is present in cells, deubiquitination of RIPK1 results in the formation of either complex IIa or complex IIb, leading to RIPK1-independent apoptosis (RIA) or RIPK1-dependent apoptosis (RDA), respectively. If caspase-8 is absent, necroptosis is initiated, in which RIPk1, receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) protein play a key role.

Apoptosis
Deubiquitination of RIPK1 results in the formation of either complex IIa or complex IIb. If caspase-8 is present in cells, TNFR1 complex I recruits TRADD and caspase-8 to form complex IIa (composed of TRADD, FADD and caspase-8), activating a caspase cascade and leading to RIPK1-independent apoptosis (RIA). When receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) protein are fully expressed and caspase-8 is present, ubiquitination of RIPK1 is inhibited, and it interacts with RIPK3 to form complex IIb (mainly composed of FADD, caspase-8, RIPK1 and RIPK3), which activates caspase-8 and triggers RIPK1-dependent apoptosis (RDA) [63,64].

Necroptosis
Stimulation of the Fas/TNFR family can not only trigger a canonical 'extrinsic' apoptotic pathway but also activate necroptosis inhibited by necrostatin-1 (Nec-1), a specific and potent small-molecule substance, in the absence of intracellular apoptotic signalling [65]. When caspase-8 is inactive or inhibited, necroptosis is initiated via complex IIb. RIPK1 recruits RIPK3 and induces auto-and trans-phosphorylation, with consequent Tumor necrosis factor-alpha (TNF-α)/TNF receptor 1 (TNFR1)-mediated inflammatory response and cell death pathways. The binding of TNF-α to TNFR1 triggers inflammatory responses, apoptosis and necroptosis. Ubiquitination of receptor interacting protein kinase 1 (RIPK1) promotes cell survival and induces an inflammatory response by activating the NF-κB, p38 MAPK, JNK and ERK signalling pathways. If caspase-8 is present in cells, deubiquitination of RIPK1 results in the formation of either complex IIa or complex IIb, leading to RIPK1-independent apoptosis (RIA) or RIPK1-dependent apoptosis (RDA), respectively. If caspase-8 is absent, necroptosis is initiated, in which RIPk1, receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) protein play a key role.

Necroptosis
Stimulation of the Fas/TNFR family can not only trigger a canonical 'extrinsic' apoptotic pathway but also activate necroptosis inhibited by necrostatin-1 (Nec-1), a specific and potent small-molecule substance, in the absence of intracellular apoptotic signalling [65]. When caspase-8 is inactive or inhibited, necroptosis is initiated via complex IIb. RIPK1 recruits RIPK3 and induces auto-and trans-phosphorylation, with consequent oligomerisation of the phosphorylated RIPK3. After phosphorylation, RIPK1 and RIPK3 form a necrosome (a multiprotein complex resembling amyloids) with MLKL. RIPK3 recruits MLKL and phosphorylates it. Subsequently, MLKL oligomerises and migrates to the cell membrane from the cytoplasm, which results in membrane permeabilization owing to the binding of MLKL to phosphatidylinositol lipids and cardiolipin, thus leading to cell death [66,67].
oligomerisation of the phosphorylated RIPK3. After phosphorylation, RIPK1 and RIPK3 form a necrosome (a multiprotein complex resembling amyloids) with MLKL. RIPK3 recruits MLKL and phosphorylates it. Subsequently, MLKL oligomerises and migrates to the cell membrane from the cytoplasm, which results in membrane permeabilization owing to the binding of MLKL to phosphatidylinositol lipids and cardiolipin, thus leading to cell death [66,67].
TTX-S Nav1.7 (SCN9A) has been intensively studied in the sensory system. In humans, the loss of function of Nav1.7 leads to a complete inability to sense pain [95,96], and mutations inNav1.7 lead to the functional absence of nociceptors [97]. However, the gain of function of Nav1.7 results in paroxysmal extreme pain disorder [98]. In rodents, deletion or blockage of Nav1.7 in mouse DRG attenuates nerve injury-, inflammation-, burn injury-and paclitaxel (a chemotherapeutic drug)-induced chronic pain [99][100][101][102][103]. Previous studies have shown that TNF-α/NF-κB signalling induces hypersensitivity in DRG via nuclear transcription [104]. In addition, a recent study showed that NF-κB p-p65 non-transcriptionally gates Nav1.7 channels in the membrane of neurons in rat DRG, and TNF-α contributes to the protein-protein interaction between p-p65 and Nav1.7 within several minutes [105], revealing the possible mechanism underlying the rapid regulation of sodium currents by TNF-α [87].

TNF-α Induces Spinal Neuronal Excitation and Inhibition Imbalance Andneuroinflammation
In the spinal cord, TNF-α enhances excitatory synaptic transmission and increases AMPA-and NMDA-evoked excitatory postsynaptic currents (EPSCs) to induce pain hypersensitivity [110]. After nerve injury, the upregulation of TNF-α may activate NF-κB, p38 MAPK and JNK via TNFR1 on neurons and glial cells to induce long-term potentiation (LTP) of C-fibre-evoked field potentials in the spinal dorsal horn [111]. In addition, TNF-α can reduce inhibitory synaptic transmission, indicating that disinhibition of synaptic transmission is mediated by TNF-α/TNFR1 followed by the activation of the p38 MAPK pathway in GABAergic neurons of the spinal cord [78]. In addition to the neuronal excitation and inhibition imbalance, TNF-α-induced neuroinflammation and spinal microglial activation contribute to neuropathic pain by initiating the release of other proinflammatory cytokines and promoting crosstalk between neurons and glial cells to affect synaptic signalling and pain transmission [21,22,112]. In the spinal cord, activation of TNF-α after spinal cord injury and noxious stimulation promotes NF-κB, extracellular signal-regulated kinase (ERK), JNK and caspase-8 pathways, thereby initiating inflammatory and apoptotic processes to affect the development and maintenance of neuropathic pain [113]. Furthermore, microgliaderived TNF-α elevates the expression of cyclooxygenase 2 and prostaglandin I2 (PGI2) synthase in spinal endothelial cells, which promotes neuropathic pain via the neuronal PGI2 receptor [114], suggesting that the glial-endothelial cell interaction of the neurovascular unit via transient TNF-α is responsible for the generation of neuropathic pain.

Supraspinal TNF-α Mediates Neuropathic Pain, Pain-Associated Aversion, Anxiety, Depression and Memory Deficits
Changes in higher-order functions, such as learning and memory disorders, anxiety or depression, are critical components of pain phenotypes, especially in a chronic pain state [115][116][117][118]. TNF-α plays a key role in supraspinal modulation of pain transduction; for example, intracerebroventricular (ICV) injection of TNF-α induces hyperalgesia [119,120]. In addition, blocking TNF-α in the brain reduces neuropathic pain, pain-associated aversion and memory deficits [20,117,121]. Glial cells are considered a major source of cytokines and chemokines in the brain [122], and the activation of microglia in ACC, hippocampus, prefrontal cortex and other brain regions associated with pain information processing contributes to pain aversion [123], memory deficits [124], anxiety and depression [124] and may be associated with overexpression of TNF-α [20,125]. Reciprocal activation between neurons and microglia facilitates pain transmission [75,121,126].

Necroptosis and Bibliometric Analysis
Necroptosis is characterised by necrotic cell death and autophagy activation In 1988, Laser et al. found that cell necrosis can be regulated and occurs active Subsequently, some studies reported a death mechanism that lacked apoptotic sig however, the morphological features of cell death were similar to those of necros After a few years, Chan et al. reported receptor-interacting protein (RIP)-depend grammed necrosis [143]. In addition, Degterev et al. found that Nec-1 and relate cules can regulate the aforementioned form of cell death and termed it nec [65,144]. Finally, necroptosis was officially named in 2018 [145]. The developmen cess is shown in Figure 3B. Necroptosis results in morphological characteristics si those of necrosis and activates autophagy but appears to be tightly regulated [65,1 The literature published on necroptosis in the field of neuroscience from 2012 was quantitatively and qualitatively analyzed using the Bibliometrix R Packa VOSviewer software [149,150], and the top 10 articles with the highest co-citation the top 10 most cited articles are listed in Tables 1-3, respectively. Most publica Table 1 belong to Q1 in the Journal Citation Reports (JCR) division, reflecting the n of in-depth research on necroptosis to a certain extent. Research into necroptos field of neuroscience has gradually deepened, with studies focusing on various from molecular mechanisms to the pathogenesis of central diseases (Tables 2 and

Necroptosis and Bibliometric Analysis
Necroptosis is characterised by necrotic cell death and autophagy activation [65,146]. In 1988, Laser et al. found that cell necrosis can be regulated and occurs actively [141]. Subsequently, some studies reported a death mechanism that lacked apoptotic signalling; however, the morphological features of cell death were similar to those of necrosis [142]. After a few years, Chan et al. reported receptor-interacting protein (RIP)-dependent programmed necrosis [143]. In addition, Degterev et al. found that Nec-1 and related molecules can regulate the aforementioned form of cell death and termed it necroptosis [65,144]. Finally, necroptosis was officially named in 2018 [145]. The developmental process is shown in Figure 3B. Necroptosis results in morphological characteristics similar to those of necrosis and activates autophagy but appears to be tightly regulated [65,147,148].
The literature published on necroptosis in the field of neuroscience from 2012 to 2021 was quantitatively and qualitatively analyzed using the Bibliometrix R Package and VOSviewer software [149,150], and the top 10 articles with the highest co-citation rate and the top 10 most cited articles are listed in Tables 1-3, respectively. Most publications in Table 1 belong to Q1 in the Journal Citation Reports (JCR) division, reflecting the necessity of in-depth research on necroptosis to a certain extent. Research into necroptosis in the field of neuroscience has gradually deepened, with studies focusing on various aspects from molecular mechanisms to the pathogenesis of central diseases (Tables 2 and 3).  Keyword co-occurrence analysis performed using VOSviewer revealed four categories of keywords related to necroptosis, which are shown in red, blue, green and yellow in Figure 4. The red clusters are largest, and 'necroptosis' constitutes the largest node. In addition, the occurrence of terms such as 'inflammation', 'oxidative stress', 'activation' and 'neurodegeneration' suggests that necroptosis activation is closely related to neuroinflammation and neurodegeneration.
Keyword co-occurrence analysis performed using VOSviewer revealed four categories of keywords related to necroptosis, which are shown in red, blue, green and yellow in Figure 4. The red clusters are largest, and 'necroptosis' constitutes the largest node. In addition, the occurrence of terms such as 'inflammation', 'oxidative stress', 'activation' and 'neurodegeneration' suggests that necroptosis activation is closely related to neuroinflammation and neurodegeneration.

Role of Necroptosis in Chronic Pain
Necroptosis, a mode of programmed cell death similar to necrosis and conventional apoptosis, is usually accompanied by plasma membrane rupture, organelle swelling and inflammatory cell infiltration [61,147]. RIP3, the core regulatory protein of necroptosis, activates inflammasome 3 and caspase-1, thus promoting the secretion of the pro-inflammatory cytokines TNF-α and IL-1β [172]. Therefore, it may be a trigger for neuroinflammation [55, 61,[173][174][175] and promotes neuropathic pain via activated microglia [176]. A study on rat models of paclitaxel (PTX)-induced hyperalgesia reported that the necroptosisrelated proteins RIP3/MLKL regulated neuronal necroptosis and increased the levels of pro-inflammatory cytokines in DRG [177]. In another study on rat models, peripheral nerve injury induced by CCI or SNI increased the expression of TNF-α, RIP1 and/or RIP3 [176,178] in the spinal cord, whereas Nec-1, an effective inhibitor of RIP1 and RIPmediated necroptosis [65,159], significantly reduced the levels of spinal pro-inflammatory cytokines and RIP1/RIP3 and alleviated neuropathic hyperalgesia and allodynia [176,178].
Neuroinflammation owing to abnormally elevated TNF-α levels in the primary sensory afferent, spinal cord and ACC following peripheral nerve injury contributes to neuropathic pain [20,21,71]. Peri-sciatic administration of exogenous TNF-α without any nerve injury induces mechanical allodynia by activating the NF-kappaB pathway via an autocrine mechanism [71]. The TNF-α/TNFR1-necroptosis pathway may be a new and important target for research into chronic pain (Figure 2).

TNF-α/Necroptosis in Pain-Associated Anxiety and Depression
Chronic pain is often accompanied by adverse pain-associated emotional reactions such as anxiety and depression [179,180]. Several studies have been reported on emotional problems and chronic pain [181][182][183]. An increase in the levels of proinflammatory cytokines [184,185] and a decrease in the levels of neurotrophins [186,187] are related to emotional disorders. ACC, the first-order cortical region that responds to painful stimuli [188], plays an important role in pain information processing [189][190][191], including the processing of pain affection [192][193][194][195]. Abnormal expression of TNF-α, neuronal hyperexcitability and microglial activation in ACC contribute to inflammatory and neuropathic pain and pain aversion [20,196]. Studies have shown that pharmacologically blocking neuroinflammation and activation of glial cells of ACC reduces chronic pain and prevents the occurrence of accompanying emotional disorders or memory deficits caused by complete Freund's adjuvant (CFA), the chemotherapeutic drug oxaliplatin or peripheral nerve injury [20,197,198].
Earlier studies have confirmed that in patients with severe depression, the loss of glial cells and reduction of neuronal size occur in the deeper cortical layers in ACC and the dorsolateral prefrontal cortex [199,200]. RIPK1, the key protein initiating RIPK3/MLKLdependent necroptosis [201], promotes ischaemia-induced neuronal and astrocytic cell death [202]. A study reported that depression induced by chronic unpredictable mild stress (CUMS) led to anxiety-like behaviour but did not damage spatial learning and memory, which was accompanied by the expression of RIPK3/MLKL and activation of necroptosis [203]. Pharmacological or genetic regulation of necroptosis alleviates depressive or anxiety-like behaviour and improves hippocampal function and neuroinflammation [204]. Recently, the relationship between oligodendrocytes and emotional disorders has been receiving increasing attention [205][206][207][208][209][210]. In addition to the activation of microglia and impairment of astrocytic function, a reduction in the number or density of oligodendrocytes is one of the most prominent observations in depression [211]. Inflammatory cytokines from oligodendrocytes have been implicated in the pathological process of depression [212,213]. Inhibiting the activity of oligodendrocytes using a chemogenetic approach leads to depression-like behavior and increases TNF-α-induced oligodendrocyte necroptosis through interaction with TNFR1 [214]. Although studies reporting on the role of the TNF-α-necroptosis pathway in pain-associated mood disorders are lacking, research prospects of this pathway in the field of neuroscience can be predicted based on the above mentioned studies.

TNF-α/Necroptosis in Pain-Associated Memory Deficits
Depression is closely related to a decline in cognitive abilities such as concentration and memory difficulties [215] with a decreased volume of the hippocampal brain region [216]. These cognitive impairments are commonly related to chronic pain [217,218]. Decreased hippocampal volume can be observed in patients with chronic pathological pain such as chronic back pain, complex regional pain syndrome [219] and knee osteoarthritis [220]. Clinical studies have shown that chronic pain accompanied by a reduction in the hippocampal volume can significantly reduce the learning ability of the body [221,222], resulting in short-and long-term memory defects [223]. At present, the widely accepted theory for the mechanism of pain-impaired memory is the alteration of hippocampal synaptic plasticity (long-term potentiation [LTP]), which is considered the molecular mechanism underlying learning and memory [224,225] or that underlying the effects of pain on memory [117,219]. In addition, morphological and biochemical changes in the hippocampal region underlie cognitive impairment in neuropathic pain [226].
Upregulation of TNF-α [117,227,228], a decrease in the expression of brain-derived neurotrophic factor (BDNF) and microglial activation [226,227] in the hippocampus may be the basis of chronic pain and memory defects. Blocking nerve injury-induced hippocampal TNF-α via oral administration of magnesium L-threonine, a new method of preventing neuropathic pain caused by chemotherapy [229] or using nanocurcumin can improve pain and memory impairment [117,230]. At present, whether TNF-α mediates pain-associated cognitive deficits by activating necroptosis remains unclear; however, its role has been reported in other neurodegenerative diseases. For example, necroptosis mediates TNF-α-induced toxicity of hippocampal neurons [231], leading to memory impairment in AD [159,232,233]. Inhibiting necroptosis and abnormally high expression of TNF-α in the hippocampus can reduce cell death and improve cognitive ability [159,[234][235][236].

Conclusions
The neuroimmune mechanisms underlying neuropathic pain are complex and involve many factors, including inflammatory and anti-inflammatory imbalances that in the most severe form is called cytokine storm. For example, spinal formyl peptide receptor type 2 (FPR2/ALX), a member of the formyl peptide receptors family, plays an analgesic role by reducing cytokines and BDNF [237]. This review focuses on the potential mechanisms of proinflammatory cytokine TNF-α-mediated neuropathic pain and discusses the role of TNF-α-necroptosis pathway in detail. As shown in Figure 2, TNF-α, as a crucial driver, can regulate cation channels to sensitize primary afferents in the peripheral nervous system, affect excitatory and inhibitory synaptic transmissions in CNS and evoke positive feedback between TNF-α and microglial activation to induce neuroinflammation, thus facilitating pain transmission, adverse pain-associated emotional reactions and cognitive deficits. TNFα-triggered necroptosis, a novel form of programmed cell death, may be one of the key factors for inducing neuroimmune responses in neuropathic pain. It not only contributes to allodynia and hyperalgesia but also mediates aversion, anxiety, depression and learning and memory deficits associated with chronic pain. Therefore, understanding the important role of the TNF-α-necroptosis pathway in neuropathic pain may offer novel strategies for the treatment of neurological diseases.