Disruption of Brain TNF Homeostasis Elicits Maladaptive Alterations Producing Chronic Pain

Disruption of Brain TNF Homeostasis Elicits Maladaptive Alterations Producing Chronic Pain

We have determined that enhanced expression of the pro-inflammatory cytokine Tumor Necrosis Factor-α (TNF) by both neurons and glial cells of the brain direct the process involved with the development and also the maintenance of neuropathic pain. However, TNF is not only a pro-inflammatory cytokine but is also a potent neuromodulator. TNF specially affects the nervous system (neurotropic activity), and therefore, the effects of TNF on neurons are among the many pleotropic functions of TNF, both beneficial as well as detrimental. As a damaging neurotropic action, TNF mediates the etiology of neuropathic pain through its activity on nerve terminals, such as inhibiting the release of norepinephrine (NE), and enhancing activity (supersensitization) of the presynaptic α2-adrenergic inhibitory autoreceptor, which is a primary regulator of NE release. In addition, the α2-adrenergic receptor on neurons and glia regulates TNF production from these cells, and this receptor is similarly regulated by TNF, with a feed-forward response to enhance TNF levels. This report provides an overview of the evidence supporting the regulation of brain TNF for treating neuropathic pain.

Neuropathic pain is a chronic pain syndrome

Pain is a normal protective mechanism that alerts an animal (human) to damage or potential damage. Nociception refers to the series of neural events that culminate in the activation of neurons in the brain that are associated with the conscious perception of pain.1 Whereas acute pain is a normal (shorter) vital sensation that warns us of possible injury, chronic pain is a pathological condition that lasts more than three months. Neuropathic pain, a category of chronic pain, is a disease syndrome caused by injury to peripheral nerves, spinal cord, or the brain. Neuropathic pain results from abnormal operation of the pain sensory system following a primary lesion, or dysfunction in the nervous system.2,3

This smoldering disease is not only endemic, but it is also escalating. A 2011 report indicated that over 1.5 billion people suffer from chronic pain with 3-4.5% of the global population suffering specifically from neuropathic pain, and the incidence is increasing complementary to age.4 Neuropathic pain, similar to the other chronic pain syndromes, is costly with total annual health care costs (combined medical and economic costs) ranging from $560-$635 billion (in 2010 dollars) in the United States.5 Standard analgesics that are used to treat acute pain are relatively ineffective for neuropathic pain. Despite the availability of many drugs that are somewhat effective (anti-epileptic drugs, antidepressants), they are often not used due to unwanted side-effects. Therefore, neuropathic pain patients largely remain under- or untreated. More effective treatment is limited by lack of knowledge of the pathophysiology of chronic pain, which is distinct when compared to the pathophysiology of acute pain. Insults such as peripheral nerve injury may lead to chronic pain, which is elicited by maladaptive changes in the CNS, including at integration centers within the brain.

Neuropathic pain is often observed in amputees and nerve crush-injury patients, where it may manifest as a component of syndromes such as Reflex Sympathetic Dystrophy or causalgia, currently designated Complex Regional Pain Syndrome I.6  Sustained activation of peripheral nociceptive afferents (afferent barrage) 7 in response to tissue injury, or due to spontaneous ectopic discharge by injured primary nerve afferents, both by unmyelinated, small C-fiber afferents and myelinated A-fiber afferents8 are implicated in the initiation and maintenance of neuropathic pain.8,9 Neuroplastic changes that are secondary to the sustained afferent barrage may culminate in hyperexcitability of neurons in the CNS.10 This hyperexcitability may be due to a loss of inhibition of nociceptive neurons by neurotransmitters, and or loss of  norepinephrine (NE) in spinal and supraspinal structures. The spatial summation of noxious inputs into the CNS results in an enlargement of nociceptor receptive fields, enabling the activation of more pain receptors, during chronic pain.11

The resultant state of central sensitization or increased responsiveness of neurons in the CNS to stimuli, is a dysfunctional adaptation of the pain sensory system.10  This dysfunction can manifest as spontaneous unevoked pain, as an increased responsiveness to stimuli that are normally painful (hyperalgesia), as well as sensation of pain in response to non-painful stimuli (allodynia).2 Accordingly, clinical manifestations associated with the onset, development and maintenance of neuropathic pain reflect development of a maladaptive central component that we propose occurs at higher centers of the brain, and in particular, at the hippocampus, a brain region involved with memory processing.

The role of TNF in neuropathic pain

Substantial evidence supports the involvement of TNF in the pathogenesis of neuropathic pain, both in its development and its persistence. TNF, as a pro-inflammatory cytokine, is a proximal mediator that orchestrates the inflammatory response that ensues in the lesion at a site where the injury occurs. In support, application of TNF to the sciatic nerve elicits increased spontaneous firing of nociceptive afferents,12 and endoneurial injection of TNF into the sciatic nerve produces allodynia and hyperalgesia, similar to that observed by experimental nerve injury.13

Peripheral nerve injury induces neuroinflammation at sites distant from the site of injury; elevated levels of cytokines in the brain and in the spinal cord are also associated with increased pain. At the same time following peripheral injury (also possibly following brain injury), as a pleotropic neurotropic protein, the subsequent enhanced TNF production at higher centers of the brain, however, is what establishes a central component which is necessary for the formation of a persistent, chronic pain pathology. Intracerebroventricular (icv) microinjection of TNF alone induces hyperalgesia in naïve rats, which demonstrates that a site(s) within the brain mediates nociceptive effects of TNF.14,15 In the absence of a peripheral injury, genetically enhancing TNF production solely in a specific brain region, the hippocampus, produces the symptoms associated with chronic pain pathology.16 Consequently, enhanced TNF production in the brain is a pathology on its own that can be elicited by various distinct insults. Increases in the levels of TNF in the brain were shown to be fundamental in the development of neuropathic (chronic) pain following peripheral nerve damage mediated by chronic constriction injury (CCI) to the sciatic nerve.15,17,18 Furthermore, peripheral administration of TNF communicates to the brain as demonstrated by development of dose-dependent thermal hyperalgesia with intra-peritoneal administration of TNF.19,20 Consequently, it is now apparent that a peripheral inflammatory reaction with its associated enhanced TNF levels will subsequently enhance TNF production in the brain, and this enhanced expression of TNF in the brain can establish a central component necessary for the formation of a persistent chronic pain pathology.

Indeed, TNF mediates hyperalgesia following peripheral administration of substances such as lipopolysaccharide, a bacterial cell wall component that activates immune effector cells, which stimulate TNF production in the periphery.21 During systemic febrile illness (e.g., rheumatoid arthritis or microbial infections) inflammatory/immune cytokine cascades are activated, and patients can experience symptoms similar to that observed following experimental nerve injury,13 such as the onset of nondescript pain symptoms (e.g., polyarthralgias, myalgias, as well as hyperesthesia).22  The majority of the literature supports a pro-nociceptive role for TNF, which demonstrates the “Double-Edge-Sword” response of pleotropic TNF; that is, its protective properties at lower physiologic levels and destructive properties at higher, non-homeostatic levels.23 Clearly the cellular environment influences the behavioral response that is elicited by TNF.

Blocking TNF activity is anti-nociceptive experimentally and analgesic clinically. Therapeutically applied anti-TNF antibodies can significantly reduce joint pain in rheumatoid arthritis patients.24 Patients with severe, chronic, treatment-resistant low back or neck discogenic pain that received etanercept, an anti-TNF fusion protein, by perispinal injection reported sustained and significant reduction of chronic pain.25 Experimentally, administration of a TNF binding protein prevents the hyperalgesia of peripheral lipopolysaccharide administration,21 and pre-operative administration of thalidomide (an inhibitor of TNF synthesis)26,27 as well as metalloprotease inhibitors (limit TNF cleavage from cell surfaces)28 attenuates hyperalgesia. TNF immunoreactivity is increased in the injured sciatic nerve by CCI, concurrent with the manifestation of hyperalgesia at day 5 post-ligature placement.27 Pre-operative administration of thalidomide reduces this increase in immunoreactive TNF in the injured nerve, and also lessens the development of hyperalgesia. However, this treatment is ineffective when initiated after hyperalgesia has manifested, indicating that increases in TNF at the site of nerve injury are required for the initiation, but not for the ultimate development and the maintenance of neuropathic pain.

Due to the characteristic increase in TNF along the neuroaxis following peripheral nerve injury, TNF has been investigated as a potential biochemical marker of pain. Levels of TNF in the synovial fluid of patients with palpable temporomandibular joint pain are significantly higher than in that of patients not experiencing pain.29 Blister fluid of patients with complex regional pain syndrome (CRPS), a neuroinflammatory disorder initiated by a peripheral injury that includes damage to a nerve, showed increased TNF and decreased anti-inflammatory cytokine IL-1 receptor antagonist, which occurred bilaterally, when compared to non-CRPS patients.30 Likewise, mechanical hyperalgesia in CRPS patients was associated with increased plasma levels of soluble TNF receptor 1 (sTNF-R1) and TNF.31 Additionally, increases in TNF immunoreactivity are observed in the spinal cord during experimental mononeuropathy.32 Bilateral increases in TNF immunoreactivity are demonstrated in the dorsal horn of the lumbar spinal cord at day 6 post-ligature placement in the CCI sciatic nerve injury model, concurrent with the manifestation of  hyperalgesia that is characteristic of this neuropathic pain model.32  Since the increased TNF immunoreactivity is restricted to the lumbar segments (area of sciatic innervation) of the spinal cord during CCI, it is likely that a neuron-mediated process is responsible rather than a generalized inflammatory response within the gray matter. Consequently, it is now apparent that the enhanced TNF levels along the neuroaxis, and in particular the enhanced TNF production in the brain, establishes a central component necessary for the formation of a persistent chronic pain pathology.

Chronic pain is mediated by aberrant neurophysiology along the neuroaxis

Modification in sympathetic activity is often observed during the onset and development of neuropathic pain. Sympathetic responses are part of a feedback loop between the periphery and the CNS that is regulated by descending noradrenergic pathways emanating from the brain. Drug action on brain noradrenergic synapses (e.g., clonidine, amitriptyline) may prove efficacious by alleviating sympathetic symptoms.33,34 Our findings show that the initial afferent barrage associated with onset and development of neuropathic pain is concurrent with an induction of TNF synthesis in the brain, specifically in the locus coeruleus and the hippocampus.17,35 This elevated brain TNF synthesis mediates an increased TNF release at noradrenergic nerve terminals and glial cells throughout the brain.

Subsequent to injury or trauma, chronic pain that is disproportionate to the insult may develop. Even when normal healing of the inciting injury occurs, excruciating and overwhelming pain may continue to be perceived due to the development of chronic pain. In response to an injury, activation of the inflammatory response initiates increased production of TNF, the first cytokine to appear in the pro-inflammatory cytokine cascade.36 TNF is not only a proximal mediator, but therefore, is a key mediator involved in neuropathic pain.  Consequent to the injury, a neuroinflammatory response ensues and spreads along the neuroaxis (peripheral nervous system (PNS) to CNS) during the onset and development of neuropathic pain pathogenesis and pathophysiology, which may explain the chronicity and spread of symptoms in these patients.37 The transfer of pain to sites distant to that of the initial site of injury (as often occurs with CRPS) may exist as a result of transport of cytokines, such as TNF, IL-1β, and IL-6, as well as by microglial and astrocytic activation. Furthermore, evidence for aberrant neurophysiology along the neuroaxis during chronic pain in response to a peripheral nerve injury confirms neuroplasticity at both the cellular and molecular levels.

Peripheral nerve injury induces neuroinflammation at sites distant from the site of injury. This spread of neuroinflammation was demonstrated in the thalamus following peripheral nerve injury using radiolabeled PK11195, a ligand for the peripheral benzodiazepine binding site that is absent in normal brain parenchyma but strongly expressed by activated microglia in the vicinity of injured neurons, for imaging of activated microglia and brain macrophages.38 Seven patients with peripheral nerve injury or spinal root lesions underwent [11C](R)-PK11195 PET imaging. All patients showed increased binding of [11C](R)-PK11195 in the thalamus contralateral to the side of injury. Interestingly, this contralateral, thalamic microglial activation was evident up to 20 years after the injury, indicating persistence in the targeted migration of neuroinflammation from the site of peripheral nerve injury, and consequently, the persistence of neuropathic pain. This trafficking of inflammation progresses from the injured neurons to the first order synapse that is located in the spinal cord and continues progressing trans-synaptically to the second order synapse located in the brain.38 Activated microglia participate in synapse removal from injured neurons, the persistence of which may contribute to pathologic neuroplasticity and central sensitization during neuropathic pain.39

Release of signaling molecules such as cytokines including TNF and IL-1β and chemokines (for example, monocyte chemoattractant protein (MCP) 1), from first order nociceptive neuron terminals initiates neuroinflammation at secondary sites (for example, at the spinal level) due to stimulation of respective receptors on immune cells, thereby triggering their activation and migration.40,41 Neuroimmune activation via neuronal projections to second order synapses where these neurons terminate in the thalamus may account for the spread of glial activation at supraspinal sites.37 The spread of neuroinflammatory signals was also demonstrated by showing that unilateral, peripheral nerve CCI increased levels of TNF in the contralateral hippocampus, which was associated with the development of hyperalgesia and allodynia.18,42 A transient breakdown in both the blood-spinal cord barrier and blood-brain barrier (BBB) occurs following damage or injury to peripheral nerves.43 This may explain the leukocyte infiltration into the CNS that contributes to the remote neuroinflammation following nerve injuries.44 This emigration of leukocytes from the periphery into the CNS communicates that there is peripheral nerve and tissue damage, which may be transmitted between leukocytes and neurons by neurotransmitters and cytokines, diffusible molecules produced by both cell types.45 This exposure of neuronal axons and soma to inflammatory cells and mediators contributes to neuronal sensitization that enhances pain signal transmission.46 Therefore, neuroimmune signaling is able to influence nociceptive processing at sites distant from the initial injury.

Central sensitization

Central sensitization refers to the neuroplastic changes in the CNS (spinal cord and brain) that are associated with the progression of acute pain-associated peripheral signs and symptoms to that of a chronic central pain state which is both mediated and perpetuated by neuroinflammatory dysregulation. The development of central sensitization involves a complex set of neuroinflammatory responses, most likely including the release of pro-inflammatory cytokines from neurons, glial cells, and immune-effector cells such as macrophages.37,47 Whereas the role of pro-inflammatory cytokines in peripheral nociceptor sensitization is well-documented, their role in central sensitization is becoming recognized.48,49

Using an ex vivo system, spinal cord slices exposed to TNF, IL-1β, or IL-6 demonstrated a positive influence on central sensitization through enhancement of excitatory neurotransmission and suppression of inhibitory neurotransmission as measured via patch-clamp recording.50 Specifically, application of TNF and IL-1β to spinal cord slices increased the frequency of spontaneous excitatory post-synaptic currents (sEPSC), suggesting presynaptic enhancement of glutamate release by these cytokines. IL-6 and IL-1β when added to spinal cord slices inhibited the frequency of inhibitory post-synaptic currents, suggesting suppression of inhibitory neurotransmission.50 In the CCI sciatic nerve neuropathic pain model, enhanced expression of TNF in hippocampal neurons is associated with greater inhibition of central NE release that is proposed to contribute to central sensitization through lack of engagement of spinal-mediated inhibitory synaptic transmission.15,17,51 In chronic pain conditions, microglia and astrocytes in the spinal cord and brain have enhanced pro-inflammatory cytokine production that facilitates pain via glia-neuron interactions.52,53 Through the release of pro-inflammatory cytokines such as TNF, long-term synaptic plasticity is induced by cAMP-response element binding (CREB)-mediated gene transcription, which favors excitatory over inhibitory synaptic transmission, as shown in the spinal cord.50

We postulate that the transition from acute pain to chronic pain is mediated by neuroplastic changes that generate a maladaptive central component, which is regulated by elevated levels of TNF in the brain.  TNF mediates the development and persistence of neuropathic pain through its activity on noradrenergic neurons, such as the inhibition of NE release, and the supersensitization of the presynaptic α2-adrenergic inhibitory autoreceptor, which is a primary regulator of NE release.  Thus, during neuropathic pain activation of α2-adrenergic autoreceptors elicits greater inhibition of NE release.  Importantly, these events occur in the hippocampus, a region of the brain replete with noradrenergic nerve terminals originating from cell bodies in the locus coeruleus. In addition, the hippocampus is implicated in the clinical manifestations, such as motivational/affective aspects, of pain.54,55  In fact, the hippocampus is well-positioned to participate in the pain experience as it receives information from the spinal ascending pathways, sensory association cortex and motor association cortex, and it modulates subsequent nociceptive processing as a result of activation of descending pain pathways.

These descending pathways that originate in the hippocampus/locus coeruleus normally modify nociception responses at the level of the spinal cord by dampening incoming (first order neuron) pain signal transmission, thereby allowing an anti-nociceptive response. Consequently, the TNF-induced reduction of NE release in the hippocampus mediates descending noradrenergic ‘disinhibition’ (loss of inhibition) with blockade of anti-nociceptive response. Hence, NE is a principal determinant of pain perception and is a primary neurotransmitter mediating the effects of the descending inhibitory pathway.  Activation of the presynaptic α2-adrenergic receptor inhibits NE release from noradrenergic nerve terminals, which is simultaneous to α2-adrenergic receptor induction of neuron and macrophage TNF production.56,57 

Since pathologically high levels of TNF are elicited during persistent pain, α2-adrenergic receptor mediated inhibition of NE release antagonizes the induction of TNF production due to decreased activation of α2-adrenergic receptors.  It is our contention that the α2-adrenergic receptor regulation of NE release and simultaneous control of brain-TNF production directs the dissipation of chronic pain and subsequent resolution of a peripheral lesion due to the effect on the descending pathway. The imbalance of this dynamic interplay between NE levels and TNF production is fundamental to the pathology of the persistent nature of chronic pain.  Our results show that blockade of TNF activity (microinfusion of TNF antibodies into the brain, right lateral cerebral ventricle) during development of the central component of pain produces a prevention or alleviation of hyperalgesia.15 In addition, we have also discovered that a supersensitization of the presynaptic α2-adrenergic receptor that inhibits NE release from noradrenergic nerve terminals occurs during the onset and maintenance (persistence) of neuropathic pain.17,58 Therefore, pain pathogenesis of “central sensitization” involves the alteration of CNS response thresholds that would be involved with neurotransmitter release.59 Although adaptive changes in the spinal cord have been associated with the pathogenesis of a central component of pain, we have revealed the imperative involvement of higher brain centers.  Therefore, we predict that an increase in presynaptic inhibitory α2-adrenergic autoreceptor sensitivity, or an increase of receptor numbers, would decrease NE release and noradrenergic tone facilitating the neuropathology of pain syndromes.

The hippocampus is a principal pain integration site

The hippocampus, a brain region rich in noradrenergic nerve terminals, has extensive α2-adrenergic autoreceptor regulation of NE release, which is typically documented as inhibition of NE release.  The functioning of the hippocampus is associated with memory formation, and this region has been implicated in the motivational and affective aspects of pain.60 Pyramidal neurons of the hippocampus are capable of encoding the intensity of noxious stimuli by the amplitude of their responses. Although non-nociceptive stimuli may depress the hippocampal population spike,61  the effect is inconsistent, and lasts only for the duration of stimulation.  In contrast, nociceptive effects may last for several minutes after the cessation of the stimulus.  This suggests that the hippocampus differentially processes nociceptive versus non-nociceptive stimuli.62  Septal hippocampal neurons, which receive  direct projections from the spinal cord, are responsive to noxious stimuli.63  Hence, these neurons respond to peripheral nociceptive stimuli during pain.63  Septal hippocampal neurons encode the intensity of noxious stimuli, since the amplitude of their recorded electrical responses depends on intensity of mechanical stimuli, or temperature of thermal stimuli.  An ability to encode noxious stimuli occurs in the thalamus and cerebral cortex,64,65 both regions of which have extensive projections to and from the hippocampus, and are integral to integration and processing of nociceptive input.

A major function conducted by the hippocampus is incorporation of novel data into memory patterns. The hippocampus participates in an integral role with different brain regions to process pain signals.  Certain regions of the hippocampus such as the dentate gyrus serve to integrate information regarding new stimuli with other neural systems that also process pain-related information.66 Single-cell recordings of evoked potentials demonstrate that nociceptive stimuli can modify neuronal discharge rate in all hippocampal regions, resulting in a depression of pyramidal cell population spikes.62,67-69  Synaptic plasticity has been described in the hippocampus in the form of long-term potentiation or depression of activity.70  Spike amplitude depression in CA1 population might indicate a decrease in the excitability of CA1 pyramidal neurons.62  Thus, the hippocampus is an integral brain region participating in the experience and expression of pain.

TNF is a neuromodulator of NE release

Regional localization of cytokines and cytokine receptors in the CNS has prompted investigators to examine their regulatory role in neurotransmission within specific brain sites.71,72 Inflammatory cytokines have profound effects on serotonin and NE neuronal systems of the brain,73-76 providing  a definitive link between the immune system and the nervous system. These profound effects show that inflammatory responses in the brain dictate neuron functioning both physiologically and psychologically. Additionally, it shows that neuron functioning is quite similar to cells of the immune system in that neurons employ similar mediators as do cells of the immune system to direct synaptic plasticity. TNF along with classical neurotransmitters regulate neuro-secretory function that includes neurotransmission and neurotransmitter release.75,77,78  TNF serves as a neuromodulator in the CNS specifically by regulating NE release from nerve terminals.75,76,79 TNF inhibits stimulated NE release, as observed in cultured sympathetic neurons78 and isolated brain slices.73,75 This inhibition occurs in functional association with presynaptic α2-adrenergic receptor autoregulation of NE release, since blockade of the presynaptic α2-adrenergic receptor facilitates TNF-induced inhibition of depolarization-elicited NE release.17,73 Additionally, the extent of activation or repression of TNF-mediated NE release depends upon the degree of activation of the α2-adrenergic receptor as well as its coupling to second messenger Gαi/s proteins (Figure 1).

Disruption of TNF homeostasis elicits pain Figure 1Figure 1. Diagram of a varicosity along an adrenergic neuron.  (A) The presynaptic cell, post-synaptic cell, and synaptic cleft are depicted. The alpha symbols represents the α2-adrenergic receptor; the black circles represent NE containing vesicles; the blue arrow represents the amount of NE released, which is depicted in the size of the arrows; the red circles represent NE, and the pink arrow represents reuptake of NE into the nerve terminal. (B) When an action potential depolarizes the axon, the NE containing vesicles fuse with the presynaptic membrane, and NE is released into the synaptic cleft. Once in the synaptic cleft, NE can bind to and activate receptors on the post-synaptic cell, it can be taken back up into the nerve terminal through uptake-1 sites, or it can bind to and activate the presynaptic α2-adrenergic receptor. (C) Upon activation, the presynaptic α2-adrenergic receptor inhibits further release of NE; therefore, it is defined as an autoinhibitory receptor.77 This receptor mediated event is due to preferential coupling of this presynaptic receptor to Gαi-proteins. (D) Activation of the α2-adrenergic receptor also regulates the production of TNF (by neurons), observed as a decrease in TNF production in naïve animals. Additionally, TNF upregulates Gαi protein expression.80,81 TNF also regulates NE release; in functional association with the α2-adrenergic receptor, TNF inhibits NE release.73 Since TNF is inhibitory to NE release, it would follow that a transient reduction in TNF upon activation of the α2-adrenergic receptor by NE would support an increase in NE release, thus maintaining homeostasis of NE release. (E) Perturbations, such as elevated TNF, would disrupt the system’s natural balance. Elevations in TNF levels in the brain remodels α2-adrenergic-regulation of NE release, such that the receptor elicits an enhanced inhibition of NE release in the CNS resulting in less bioavailable NE.58 The resultant decrease in activation of the α2-adrenergic receptor, due to decreased available NE culminates in further increases in TNF, thus completing a self-perpetuating cycle in which NE release is kept low and TNF production is kept high.

Within the brain, TNF regulates the release of NE, and this neurotropic function of TNF is integral in the regulation of pain, such that excess and continuous TNF production correlates with the onset and development of chronic pain. When levels of TNF in the brain are elevated, the release of NE in noradrenergic regions is low, thereby preventing activation of the descending inhibitory pathway and facilitating the perception of pain.15,17,35 Likewise when brain TNF levels are low, NE release is potentiated allowing for activation of descending inhibitory pathway, thereby mitigating incoming pain signals, and decreasing perception of pain. However, the degree of NE release is directed by the interactive relationship of the α2-adrenergic receptor with the local concentration of TNF.  Consequently, this relationship is contingent on brain α2-adrenergic receptor coupling to second messenger proteins (such as either Gαi or Gαs).  The preferential coupling of the α2-adrenergic receptor regulates NE release, and is also dependent upon the microenvironment concentration of TNF (Figure 2).

Disruption of TNF homeostasis elicits pain Figure 2Figure 2: Schematic model of postulated interactions between α2-adrenergic responses and neuron sensitivity to TNF, as a result of TNF synthesis and release in the brain. Under normal, physiologic conditions, α2-adrenergic receptors primarily inhibit NE release, and activation of this inhibitory autoreceptor (α2-adrenergic receptor-Gαi) decreases NE release. Therefore, since α2-adrenergic agonists and TNF inhibit NE release, these lower levels of NE favor the left of the model.  However, activation of the α2-adrenergic receptor also results in a decrease in neuron-associated TNF, possibly explaining why α2-adrenergic agonists are analgesic.  Since TNF normally inhibits NE release within the brain, this transient reduction in levels of TNF supports an increase in NE release (right side of the model), thus serving to maintain homeostasis of neurotransmitter levels within the brain. Pronounced decreases in levels of TNF within regions of the brain associated with noradrenergic neuron activity, such as occurs following chronic administration of antidepressant drugs or acute administration of α2-adrenergic agonists, results in a transformation of α2-adrenergic receptor function, such that TNF now facilitates, rather than inhibits NE release in functional association with α2-adrenergic receptor activation. Under these conditions, the α2-adrenergic receptor exists predominantly as a stimulatory autoreceptor (designated as α2-adrenergic receptor-Gαs), and its activation supports an increase in levels of TNF within the brain.  On the other hand, this increased TNF production due to activation of the α2– adrenergic receptor occurs at a time when TNF is beneficial in that it now facilitates NE release.  This system remains in check by virtue of the fact that increased levels of TNF support Gαi protein coupling of the α2-adrenergic receptor. It is reasoned that these events continually occur in equilibrium by which physiologic levels of TNF and normal functioning of the α2-adrenergic receptor are preserved. We propose that perturbations of this system, such as a pathologic increase in TNF during persistent pain may disrupt the system’s natural balance, resulting in a self-perpetuating cycle, such that increased levels of TNF are sustained and NE release remains low, while the α2-adrenergic receptor undergoes a dysfunctional adaptation, favoring one functional form (Gαi coupling) over the other (Gαs coupling) to a disproportionate degree (i.e., the system favors the ‘left’ side of the model). Conversely, tricyclic antidepressants, which provide analgesia from persistent pain, exert their effects by perturbing the system in the opposite fashion; decreasing levels of TNF in the brain, increasing NE release, and returning the α2-adrenergic receptor to a state of functional balance between two operative forms.

This relationship explains how a peripheral nerve injury may lead to the development of chronic pain, which involves a CNS component. We have demonstrated that a reciprocally permissive adaptation occurs in the brain between NE sensitive α2-adrenergic receptors on noradrenergic neurons as they couple to either of the second messenger Gαi/s proteins and the production of TNF and consequent levels of localized TNF. These adaptations when modified by heightened TNF production or on the other hand modified to enhanced α2-adrenergic receptor coupling to Gαi proteins, both of which may occur during the evolution of acute to chronic pain, cause decreased NE release in the brain and thus the development of persistent pain (Figure 2).

Ultimately, the enhanced coupling of α2-adrenergic receptors to Gαi-proteins not only decreases NE release, but also propagates TNF production, events required for the development of chronic pain. As per our model (Figure 2) and as the literature demonstrates, these adaptions are a result of increased localized production of brain TNF, specifically in the hippocampus. 80,81 Therefore, the excess synthesis of hippocampal‑derived TNF during the development and maintenance of chronic pain augments α2-adrenergic receptor coupling to Gαi-proteins, which enhances α2-adrenergic and TNF inhibition of NE release, leading to ongoing or inappropriate perception of pain.  Dysfunctional neurotransmitter release that is mediated by remodeling of hippocampal neuron sensitivity to TNF and α2-adrenergic agonists (including NE) is fundamental in the etiology of neuropathic pain. Thus, an imbalance in the equilibrium between levels of TNF in the brain and the coupling of α2-adrenergic receptors to Gαi/s proteins is a maladaptation that instigates the development and maintains the persistence of neuropathic pain.

Despite the development of new drugs for chronic pain, therapeutic efficacy is still lacking. A greater understanding of TNF activity within the brain may be the key to developing better treatments for chronic pain.  Current pain treatments lack efficacy because of their limited effect on pain integration centers of the brain, where adaptive neuroplastic changes of pain pathophysiology occur. Our data indicate that excess production of TNF by noradrenergic neurons and glial cells attenuates neurotransmission, which is fundamental in the etiology of pain. Therefore, this TNF regulated maladaptive neuroplasticity may be used in the design of novel chronic pain treatment.  A commonly used class of drugs for the treatment of neuropathic pain is the tricyclic antidepressant drugs. However, the analgesic mechanism of antidepressant drug action is currently unresolved. Our previous work shows that antidepressant drugs decrease TNF expression and production in neurons, especially following its enhanced production that is associated with peripheral nerve injury.51,82,83

Our published work and working model (Figure 2) indicate that much of the analgesic effect of antidepressant drugs is mediated at brain integration sites, especially the hippocampus.  In fact, not only is there a decrease of TNF expression and α2-adrenergic inhibition of NE release, but these two factors actually increase NE release in the hippocampus subsequent to antidepressant drug administration. This switch from inhibition to facilitation of NE release is achieved by switching α2-adrenergic coupling from Gαi to Gαs proteins secondary to decreasing TNF production in neurons.81 Under these conditions, the new established levels of TNF serve to alleviate pain, since under this new paradigm (i.e., α2-adrenergic receptor-Gαs protein coupling) TNF enhances NE release.  In addition, simultaneous with the natural dissipation of persistent pain (as occurs in the CCI animal model), a new supportive equilibrium develops between lower levels of TNF and the coupling of α2-adrenergic receptors to Gαs proteins Gαs.

Brain-immune network

Integration and perception of painful stimuli occurs at higher centers of the brain; therefore, a peripheral nerve injury is believed to result in modifications in the brain and spinal cord. We have established how a peripheral nerve injury enhances the production of TNF in the brain by hippocampal neurons and glial cells and how an abundance of TNF dampens NE release from noradrenergic neurons.  NE is a neurotransmitter involved in pain perception, and a reduction in its release in the hippocampus would prevent neurotransmission along the descending inhibitory pathway as information projects to the periphery, thus enhancing pain perception.  This research to elucidate the reciprocally permissive interactive relationship between the hippocampal presynaptic α2-adrenergic receptor and levels of TNF within its vicinity serves as a focal point on the interdependence of the immune and nervous systems.  Adrenergic neurons in the brain through their connections traversing the descending inhibitory pathway direct the activity of peripheral sympathetic neurons that synapse at the site of nerve injury.  At the site of nerve injury the inflammatory response in the lesion is orchestrated by the macrophage, an inflammatory cell that has adrenergic receptors responsive to NE.  In fact, similar to that occurring in the brain, the presence of α2– and β-adrenergic receptors on macrophage regulate TNF production and release.  Therefore, a change in NE release in the brain alters the descending pathway culminating in disparity of NE release at the periphery where macrophage responses occur.  The release of NE in the brain as directed by TNF both directly and indirectly will dictate an inflammatory cell (macrophage) at the peripheral lesion directing its activity as well as the resolution of the injury.51


TNF is synthesized in noradrenergic neurons within distinct brain regions, decreases NE release, and augments pain. The increased expression of TNF in the brain leads to amplified inhibition of NE release in the hippocampus. Our research substantiates that α2-adrenergic autoinhibitory receptor supersensitization during persistent pain is associated with increases in TNF levels within neurons.  A cross-talk occurs between α2-adrenergic receptors and TNF production that dictates changes in neuron activity during chronic pain.  We predict that this newly established interaction is due to an increase in α2-adrenergic receptor coupling to Gαi proteins as opposed to Gαs proteins.

We predict that our findings along with our novel theories will revolutionize the understanding of how the brain is affected during chronic stress (e.g., depression, neuropathic pain). In particular, how the functioning of a receptor (α2-adrenegic) and a protein (TNF) together regulate neurotransmitter release. The presynaptic α2-adrenergic receptors can function in an opposite manner due to a change in α2-adrenergic receptor coupling to Gαs proteins in order to enhance NE release as they are involved in the regulation of chronic pain.  We theorize that specific brain regions which modulate the perception of pain are modified at distinct stages, dependent on both the amount, and the duration of the increase of TNF that is produced in a myriad of cells, but particularly in neurons and glia. Consequently, this continuous increase in TNF production dictates the pathology of chronic stress, resulting in states such as chronic neuropathic pain.

Authors Affiliation

Robert N. Spengler, Ph.D – NanoAxis, LLC, Clarence, New York 14031;
Tracey A. Ignatowski, Ph.D – Department of Pathology and Anatomical Sciences and Program for Neuroscience, School of Medicine and Biomedical Sciences, University at Buffalo, The State University of New York, Buffalo, New York 14214 and NanoAxis, LLC, Clarence, New York 14031

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