Neuropathic Somatic Pain

Overview of Neuropathic Somatic
Pain Mechanisms

Pain problems are a major component of consultative neurology. The perception of pain is complex and is composed of a discriminative component (location, quality, and intensity), an affective component (unpleasantness), and a motivational and emotional component (anxiety, depression, coping maneuvers). Each component has its unique circuitry

Pain itself may be characterized as:

  1. Nociceptive, the physiologic alerting mechanism that protects the organism from further harm
  2. Inflammatory pain that ceases when the inciting injury heals
  3. Neuropathic pain, which is caused by injury to the somatosensory system that often continues after the injury has healed and is destructive to the organism.

Specific modalities of pain are expressed in pain states. Hyperalgesia is defined as heightened sensitivity to a painful stimulus. Allodynia is pain that is elicited from a non-painful stimulus. In neuropathic pain conditions mechanical and heat hyperalgesia are cardinal features. Dynamic mechanical allodynia refers to pain elicited by a moving cutaneous stimulus (a wisp of cotton stroked across the skin) whereas static allodynia refers to pain elicited by pressure at ordinarily non-painful thresholds. Mechano allodynia is carried by A-beta fibers (myelinated 8-12 μ fibers). Thermal hyperalgesia is associated with A-delta fibers that convey cold and polymodal C-fibers that respond to heat, tissue destruction, and chemical stimuli. Mechanical and thermal hyperalgesia (particularly cold) are major modalities of peripheral neuropathic pain. Radicular conditions (disc disease, spinal stenosis, trauma to nerve roots) are primary causes of peripheral neuropathic pain. Pain is a very dynamic and plastic process in that chronic pain afferences actually change the response characteristic of pain transmission neurons (PTNs). In general, they become more responsive. Pain is a very sensitive modality in that only one C-fiber when stimulated during microneurography can convey location, quality, and intensity of a stimulus. It is now clear that immune mechanisms are important at many levels of pain production and maintenance (microglia, astrocytes and satellite cells).

As a general chain of events, a tissue-modifying stimulus triggers the firing of transient receptor potential (TRPV1, TRPVIII, and TRPA1) receptors on primary pain afferents. In turn, they initiate action potentials in C-fibers and A-delta fiber nociceptive neurons (cell bodies are in the dorsal root ganglia) that synapse in different lamina of the dorsal horn. These second order neurons give rise to spinothalamic and other afferents that activate the pain matrix. Specific aspects of inputs into PTNs induce both central and peripheral sensitization of the pain matrix, which modifies anatomic, physiologic and gene expression of pain pathways at all levels.

Some Aspects of the Anatomy of Somatic Neuropathic Pain

The skin is a complex sensory organ that also serves homeostatic and immunologic barrier functions. It is a neuroimmune cutaneous system that signals the sensory modalities of touch, pressure, temperature, and pain. As noted earlier, these primary modalities are modified in pain states (hyperalgesia, allodynia, and hyperpathia). All chronic pain conditions induce plasticity in pain transmission neurons. It is poorly recognized that there is a descending facilitating and inhibitory pain control system, the diffuse nociceptive inhibitory control (DNIC) system in experimental animals and in the patients’ conditioned pain modulation (CPM) system that modifies the transmission and physiology of pain transmission after a painful stimulus. This is a dynamic system that adjusts its sensitivity (thresholds) by complex mechanisms. Sensory transduction occurs following activation of primary intraepidermal nerve terminal C and A-delta nociceptive afferents. Activation of these primary intraepidermal nerve terminal C and A-delta nociceptive afferents is dependent on ligand activation of neuronal and non-neuronal skin cells of the neuroimmune cutaneous system (NICS). The epidermis is primarily composed of keratinocytes, melanocytes, Langerhans, and Merkel cells. These cells express sensor proteins and neuropeptides (substance P and calcitonin gene-related peptide) that are pivotal in nociception and neurogenic inflammation (vasodilation, plasma extravasation, and hypersensitivity). Keratinocytes comprise approximately 85% of dermal cells and form a tight junction with primary nociceptive nerve fibers. They express transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin (TRPA1) receptors. These channels are members of the transient receptor (TRP) superfamily of nonselective cation channels. TRPV1 channels are noxious heat-gated cation channels that are expressed on nociceptive primary afferents and also respond to protons, endogenous lipid ligands that include endocannabinoids, lipoxygenase, lysophosphatidic and linoleic acid metabolites as well as serotonin, bradykinins, prostanoids and reactive oxygen species (ROS) in the microenvironment following injury. These receptors (TRPV1 and TRPA1) are activated following injury or inflammation, depolarize nociceptive primary afferents in the skin whose central terminals project to the dorsal horn (DH) of the spinal cord. The dorsal root ganglia are composed of large diameter neurons that mediate mechanical modalities, small diameter neurons that mediate pain and temperature, as well as satellite cells that are similar to glial cells in the CNS. There are also blood vessels innervated by unmyelinated autonomic fibers in the DRG. Following injury, there is upregulation of various receptors on the neuronal elements of the DRG and under specific conditions cause sprouting of sympathetic fibers from the blood vessels. These fibers may form basket-like terminals around nociceptive and large mechanosensitive neurons. The spinal nerve-blood barrier and that of the Overview of Neuropathic Somatic Pain Mechanisms

DRG is not as tight as the blood-brain barrier. This is important in the pathophysiology of neuropathy caused by chemotherapy (direct toxic injury from cis-platinum and paclitaxel in the DRG) as well as autoimmune attack from activated lymphocytes and other cellular agents. In the skin, keratinocytes, macrophages, TRPV1-expressing nociceptors release nerve growth factor (NGF), prostaglandins (particularly E2), pro-inflammatory cytokines (IL-1, IL-6, and transforming growth factor beta 1 (TGFΒ1) as well as chemokines which sensitize (lower the threshold to fire) of primary afferent nociceptors. In many instances, after the nociceptors are exposed to the above noted “inflammatory soup,” they fire spontaneously which is the origin of spontaneous pain in many chronic neuropathic pain syndromes.

Nociceptive afferents are principally unmyelinated (1μ C-fibers) and thinly myelinated (1-4μ) A-delta fibers. The A-delta fibers signal location, intensity, cold and the lancinating quality of pain (epicritic qualities or fast pain). C-fibers are slowly conducting, transmit a burning quality of pain, and are poorly localizing (second pain).

Peripheral Input and Central Sensitization

The transmission and processing of painful inputs to the pain matrix is critically dependent on the properties of ion channels that are expressed on A-delta and C-fiber afferents. These include voltage-gated ion channels and leak channels that in concert regulate resting membrane potential, set, and maintain the action potentials and firing properties of pain transmission neurons. A major cause of chronic neuropathic and inflammatory peripheral pain is due to dysregulation of ion channel expression caused by tissue and nerve injury that enhances pain transmission and neuronal excitability. Afferent nociceptive signals due to activation of specific receptors and ion channels on peripheral nerve endings of A-delta and C-fibers are propagated and synapse in the extremely complex circuitry of the spinal cord dorsal horn. These afferents release glutamate and substance P that activate second order neurons that ascend to CNS processing ensembles that process pain signals.

Physiological Properties of Pain Transmission Neurons

The physiological properties of pain transmission neurons are critically dependent on the expression density and function of their ion channels that define:

  1. The resting membrane potential
  2. The initiation of the action potential
  3. Depolarization and repolarization kinetics
  4. The refractory period
  5. Transmitter release from their central terminals on second order neurons in the dorsal horn

Ion Channels

The relevant ion channels that determine these properties are:

  1. Voltage-gated sodium, potassium and calcium channels
  2. Leak channels
  3. Ligand gated channels
  4. Transient receptor potential channels

Sodium Channels

Particularly important for stimulus detection, initiation of action potentials in pain afferents and synaptic transmission are:

  1. Nav 1.7 and Nav 1.8 isoforms of voltage-gated sodium channels
  2. N-type calcium channels
  3. Transient receptor potential channels

The sodium channel isoforms Nav 1.7, 1.8, and 1.9 that are essential for the physiologic properties of peripheral nerves do not interfere with CNS or cardiac function and have been extensively studied as targets for pain pharmacology. Mutations in the gene that code for the Nav 1.7 channel have demonstrated its role in human pain. It is expressed in peripheral neurons, the dorsal root ganglia (DRG), trigeminal and nodose ganglia as well as sympathetic ganglia neurons. It is activated by slow depolarizations that are close to resting membrane potential and sets the gain of nociceptor afferents. It is upregulated in inflammatory pain states, accumulates in neuromas, and is essential in ectopic impulse generation under these conditions. Genetic studies and functional profiling of mutant channels (from mutations in the SCN9A gene that encode the Nav 1.7 channel) have demonstrated its function in inherited erythromelalgia. In this condition, patients experience severe pain (burning and aching) from innocuous warm stimuli. The missense mutations were shown to increase the activity of the channels and to increase their response to small depolarizing stimuli. Other gain of function mutations in the Nav 1.7 channel impair channel inactivation and are associated with paroxysmal extreme pain disorder. These patients suffer rectal pain that in later life may be experienced in periorbital and perimandibular areas after a stimulus to the lower body. Autosomal recessive mutations of the Nav 1.7 channel may demonstrate insensitivity to pain. In this condition, patients do not produce functional Nav 1.7 channels. The exact role of the channel and its location in this deficit has not been determined. A polymorphism in the gene has been associated with hyperexcitability of DRG pain transmission neurons.

Gain of function variants of Nav 1.7 channels enhance activity by: (1) impairing slow inactivation or (2) impairment of both fast and slow inactivation, (3) enhancing activation by producing a persistent (non-activating) current. These physiologic changes lower the action potential threshold of DRG pain transmission neurons, increase their firing frequency, and cause abnormal spontaneous firing that is also thought to be a mechanism for evoked and spontaneous pain in patients with peripheral neuropathies

The Nav 1.8 channel has been associated with painful peripheral neuropathies. It is expressed in DRG pain transmission neurons, their axons and in trigeminal and nodose ganglion neurons. It has depolarized voltage dependence, which renders it relatively resistant to inactivation during neuronal depolarization. It is a major component of the action potential upstroke (its inward current) and confers repetitive firing of depolarized neurons. It has a role in both inflammatory and neuropathic pain. Gain of function mutations in Nav 1.8 channels have been associated with approximately 5% of patients with painful neuropathies by causing hyperexcitability and spontaneous discharge of DRG pain transmitting neurons (PTNs).

The Nav 1.9 channel is expressed in DRG pain transmission neurons, the trigeminal ganglion neurons, and nociceptors of the myenteric plexus. It produces a non-inactivating current that is activated at hyperpolarized potentials close to the resting membrane potential. It prolongs and increases small depolarizations that increase DRG PTN excitability. It appears to be important in inflammatory pain asinflammatory mediators increase its current.

The Nav1.3 channel may be important for neuropathic pain, as it has been shown to:

  1. Produce a persistent current that responds to small depolarizations
  2. Is active close to the resting membrane potential
  3. Is positioned to amplify small nociceptive afferences
  4. Is rapidly inactivated which supports repetitive firing

Potassium Channels

Potassium channels regulate resting membrane potential and action potential repolarization in PTNs. They are divided into:

  1. Voltage-gated ion-activated (sodium or calcium activated)
  2. Two-pore channels
  3. Inward rectifying channels
  4. All of the above are important in the neuronal excitability of PTNs.

Decreased voltage-gated potassium channel function causes increased PTN firing and spike duration as well as decreased spike threshold. Experimental evidence in neuropathic pain models support a strong contribution of Kv channels as inhibitory to pain signaling in nociceptive afferents.

Two-pore potassium channels (K2P) support a hyperpolarized resting membrane potential through their effects on leak potassium currents. K2P channel subtypes that include TRESK, TRAAK, TASK and THIK channels are expressed in DRG PTNs. They are important regulators of primary nociceptive afferent fiber excitability often to mechanical and heat stimuli (hallmarks of neuropathic pain).

Calcium and sodium-activated potassium channels

Calcium activated potassium channels (Kca) are a determinant of after-hyperpolarization that follows an action potential and thus neuronal firing frequency and pattern. Kca channels include large (BK), intermediate (IK) and small (SK) channels: all of which when activated limit pain transmission neuronal discharge. There is decreased expression of SK and IK channels in human DRG PTNs after nerve injury that would increase their neuronal firing.

Recent experimental studies demonstrate decreased BK channel expression to brain derived neurotrophic factor (BDNF) – mediated down regulation at the transcriptional level. Microglia are activated after peripheral nerve injury and may be the source of the BDNF. Sodium activated potassium (Kna) channels are also involved in after-hyperpolarization and are important in the regulation of firing rate adaptation.

In summary, voltage-gated, ion-activated or leak potassium currents inhibit afferent pain signaling.

Voltage-gated calcium channels

Neurons express multiple types of voltage-gated calcium channels (Cav channels) that are the primary source of depolarization-induced calcium increase in PTNs. N-type calcium channels are high voltage-activated channels that trigger neurotransmitter release in the dorsal horn.

T type Cav channels regulate afferent pain signaling whose mechanisms include:

  1. The support of rebound burst activity induced by increasing the activity of co-localized Nav channels
  2. An upregulation of Cav 3.2 channels in Aδ fibers (involved in mechano-transduction)
  3. Interaction with the proteins of synaptic release which promote low threshold neurotransmitter release at specific dorsal horn synapses

Several gene families encode calcium-activated chloride currents that regulate neuronal excitability. Experimental studies of DRG neurons demonstrate that these chloride currents may be involved in after-depolarizations following neuronal discharge.

HCN channels

Hyperpolarization activated cyclic nucleotide-gated (HCN) channels are activated and open at negative membrane potentials and are a component of neuronal excitability and rhythm generation. Their four subtypes are expressed in DRG PTNs. Blockade of the channel decreases mechanical allodynia in both inflammatory and neuropathic pain models.

The Role of Endogenous Lipid Mediators in Peripheral Gating of Pain Signals

Nociceptive signals are modified prior to their arrival at the spinal cord dorsal horn. Primary sensory neurons (in the DRG), their terminals in the skin and peripheral tissues as well as adjacent host-defense cells (satellite cells) release a variety of proteins and peptides that effect nociceptive afference to the dorsal horn. Lipid-derived mediators are a major component of this peripheral gating mechanism by their interaction with nociceptor afferents, macrophages, mast cells, and keratinocytes. In general, most nociceptors are polymodal as they can signal different modalities of harmful stimuli. Nociceptor subclasses express distinctive membrane ion channels, receptors, and intracellular signaling proteins. After tissue and nerve terminal damage, these transduction molecules induce hyper-excitability in nociceptive afferents, which is called peripheral sensitization. As noted earlier, the clinical manifestations of this sensitization is mechanical and thermal allodynia in which innocuous mechanical and thermal stimuli are perceived as painful and hyperalgesia in which a mildly noxious stimulus is perceived as very painful. Peripheral sensitization is often accompanied by neurogenic inflammation. This vasodilatory response is caused by the release of substance P and calcitonin gene-related peptide (CGRP) from the activated C-fiber nociceptor primary terminals.

Nociceptors respond to endogenous proalgesic factors that are rapidly released following injury or are produced slowly during inflammatory states, tumor growth, or peripheral neuropathy. The first wave of proalgesic substances that may affect nociceptor terminals after injury are ATP and ADP leaked from damaged cells and bradykinin released from plasma globulin during blood clotting. Both activate excitatory receptors on primary nociceptive afferents. A later group of sensitizing and proinflammatory molecules includes substance P, CGRP, and lipid-derived mediators released by primary afferent nociceptor fibers and host-defense cells. Other proinflammatory mediators are prostaglandin E2 (PGE2) and prostacyclin (PGI2). They activate specific G protein-coupled receptors on nociceptive afferents that increase membrane excitability and amplify the release of SP and CGRP. Essential components of this signaling cascade are the enzymes cyclooxygenase 1 (Cox)-1 and Cox-2 which convert arachidonic acid into PGH2 the common precursor of all prostanoids.

Enzymatic and non-enzymatic conversions of membrane-derived polyunsaturated fatty acid (PUFA) oxidation form other lipid molecules that excite nociceptors and include:

  1. Hydroxylated derivatives of linoleic acid
  2. Hepoxilin A3
  3. PGE2-glycerol ester
  4. Prostamide F2α
  5. Lysophosphatidic acid
  6. Lysophosphatidyl inositol

A clear role of lipid-mediated signaling in the induction and maintenance of neuropathic pain is well established.

Recent experimental evidence demonstrates that bioactive lipids may also decrease and modulate pain initiation. These analgesic lipid mediators include:

  1. Endogenous cannabinoids
  2. Lipid-amide agonists of peroxisome proliferator-activated receptor–α (PPAR-α)
  3. Products of oxidative PUFA metabolism.

Reactive Oxygen Species

Reactive oxygen species (ROS) are chemical species that contain oxygen and include peroxides, superoxides, hydroxyl radicals, and singlet oxygen. They are formed as a natural by-product of the normal metabolism of oxygen. They have major roles in cell signaling and homeostasis. If ROS increase dramatically they may damage cell structures and this process is known as oxidative stress.

Formation and Degradation

The reduction of molecular oxygen (O2) produces superoxide (.O2) which is the precursor of most other
reactive oxygen species.

The hydroxyl radical is extremely reactive and removes electrons from molecules in its vicinity that produces a free radical from the affected molecule. This creates a propagating chain reaction. H2O2 is more damaging than the hydroxyl radical due to its lower reactivity which gives it more time to enter the nucleus of the cell and react with its DNA.

Production of Endogenous ROS

ROS are produced intracellularly primarily by:

  1. NADPH oxidase (NOX) complexes that have 7 isoforms that are located in cell membranes
  2. Mitochondria
  3. Peroxisomes
  4. Endoplasmic reticulum
  5. Production of nitric oxide synthetase (NOS)

As noted, ROS are produced by multiple mechanisms that depend on the cell and tissue type.

The process of oxidative phosphorylation that creates ATP involves the transport of protons (hydrogen ions) across the inner mitochondrial membrane along the electron transport chain (ETC).
Electrons undergo a series of oxidation-reduction reactions that occur in the proteins of the chain such that each acceptor protein along the chain has a greater reduction potential than the previous protein complex. The oxygen molecule is the final destination of electrons passing along the chain. Under normal conditions, oxygen is reduced to water. In approximately 0.1-2% of electrons that pass through the chain, oxygen is prematurely and incompletely reduced to make the superoxide radical. Electron leakage occurs primarily in complexes I and III. The superoxide molecule may inactivate enzymes or cause lipid peroxidation in its hydroperoxyl HO2 form.

If mitochondria are severely damaged from environmental conditions (toxins, chemotherapeutic agents, anoxia) the cell undergoes apoptosis or programmed cell death. The surface of mitochondria contains layered Bcl-2 proteins that detect mitochondrial damage and activate BAX proteins that produce holes in the mitochondrial membrane. Cytochrome C leaks from the ruptured membranes and binds to APAF-1 (apoptotic protease activating factor 1) that is free in the cytoplasm. ATP in the mitochondrion provides the energy for the binding of APAF-1 and cytochrome C to form apoptosomes. Apoptosomes bind and activate caspase-9, also in the cytoplasm, which then cleaves the proteins of the mitochondrial membrane. The resulting protein denaturation ends in phagocytosis of the cell.

The interaction of. O2 with nitric oxide produces NO-peroxynitrate. Under physiologic conditions the antioxidant defense system primarily composed of manganese superoxide dismutase MnSOD, catalase, glutathione thioredoxin and glutathione peroxidase reduce superoxide to water and molecular oxygen. If the antioxidant defense system is over-whelmed by a large increase of ROS, formation of peroxynitrate and other ROS increases and causes oxidative stress. Peroxynitrate causes a bioenergetics failure of mitochondria by altering and disrupting metabolic enzymes, mitochondrial electron transport proteins (thus reducing the production of ATP), ATP synthase and membrane transport proteins. Its damage of MnSOD causes a feed-forward reaction that increases its own production, which further increases superoxide.

ROS and Cellular Respiration

  1. There is leakage and loss of electrons (ē) primarily from mitochondrial complexes I and III
  2. In neuropathic pain models:
    • a. mtROS are elevated in spinal neurons, microglia and astrocytes.
    • b. NOX-1, 2 (derived from NADPH oxidase) are expressed at the cellular membrane level and produce . O2 after phosphorylation of a cytosolic subunit
    • c. NOX-1 derived from NADPH oxidase:
      • i. Translocates PKCΕ to the membrane which enhances transient receptor potential vanilloid (TRPV1) activity in the DRG
    • d. NOX-2 derived from NADPH oxidase:
      • i. Primarily expressed in phagocytic cells (macrophages and microglia) is upregulated after peripheral nerve injury (PNI) and induces . O2 . Its expression may be initiated by Toll-like receptors (TLRs)
      • ii. Decreases TNF but induces IL-1β and demonstrates the expression of the neuronal injury marker ATF3
      • iii. In NOX-2 deficient mice there is decreased expression of Iba on peptidergic axons
        and a decrease of proinflammatory cytokines
      • iv. Increases gene expression of proinflammatory cytokines in the DRG
    • e. NOX-4 (derived from nicotinamide dinucleotide phosphate, NADPH):
      • i. Is expressed by DRG neurons on both myelinated (A-fibers) and C-fibers
      • It is also expressed by microglia, astrocytes and macrophages
      • Its expression in cellular organelles (endoplasmic reticulum, ER) produces the ROS H2 O2
      • NOX-4 may decrease the neuronal proteins MPZ and PMP22 after nerve injury (shown in experimental sciatic nerve injury)

Effects of ROS

  1. Positive effects:
    • a. Induction of host defense genes
    • b. Mobilization of ion transport systems
    • c. Platelets that are involved in wound repair and blood homeostasis release ROS that recruit platelets to the site of injury. A link is established to the adaptive immune system by ROS recruitment of leukocytes
  2. Damaging Effects of ROS:
    • a. Damage of DNA and/or RNA
    • b. Oxidation of polyunsaturated fatty acids in lipids (lipid peroxidation)
    • c. Oxidation of amino acids in proteins
    • d. Mediation of apoptosis
    • e. Oxidative deactivation of specific enzymes via oxidation of cofactors
    • f. Mitochondrial damage
  3. In the pain state – ROS:
    • a. Activate Ca/CaMKII in glutamatergic neurons
    • b. Induce presynaptic inhibition of GABAergic neurons
    • c. H2 O2 increases action potentials of DRG neurons by activating cGK1α that increases neurotransmitter release from A-delta and C-fiber terminals of primary afferent neurons in the DH

Nitro-oxidative Species and Neuropathic Pain

Nitrosylation is the covalent incorporation of a nitric oxide moiety into another molecule. S-nitrosylation is the covalent attachment of NO to a cysteine residue that forms an S-nitrosothiol (SNO). S-nitrosylation is a post-translational protein modification that is a widespread signaling mechanism and is the primary driver of NO bioactivity. S-nitrosylation is targeted, reversible, spatio-temporally restricted and is important for a host of cellular functions. An important function is the allosteric regulation of proteins by both endogenous and exogenous sources of NO. It is the prototype redox-based signaling mechanism.

NO and Nociceptive transmission

NO action in regard to nociceptive transmission is complex and often opposing. In several pain models neuronal NO (neuronal) at high concentrations in the spinal cord increases pain sensitivity while pharmacologic inhibition and genetic deletion decrease it. Expression of neuronal nitric oxide synthase (nNOS) in sensory neurons is upregulated following peripheral nerve injury. Low concentrations of NO in the spinal cord have been shown to attenuate allodynia after nerve injury. NO may increase the anti-nociceptive effects of opioids, NSAIDs and the NO-releasing derivative of gabapentin (NCX8001) at peripheral transduction sites. NO-dependent activation of ATP-sensitive potassium channels may be a component of peripheral analgesia. These KATP channels are expressed in metabolically active tissues and are complex. They are hetero-octamers and have four regulatory SUR subunits (SUR1, SUR2A, or SUR2B) as well as four ATP-sensitive pore forming inwardly rectifying potassium channels (Kir6.x) with subunits (Kir6.1 or Kir6.2). The ratio of cellular ADP/ATP determines their opening that allows them to function as metabolic sensors thus linking cytosolic energetics with cellular functions. In both the central and peripheral nervous system KATP channels are a component of the regulation of neuronal excitability, neurotransmitter release and ligand effects. S-nitrosylation regulates Na+ channels and acid-sensing channels in DRG neurons, the NMDA receptor channel complex. Ca2+ activated K+ channels that are all-important in pathologic pain states. It has been demonstrated that nitric oxide activates ATP-sensitive potassium channels in sensory neurons by direct, S-nitrosylation.

Nitric oxide is a diffusible gas that is synthesized from L-arginine by NOS-1 (neuronal), NOS-2 (inducible), and NOS-3 (endothelial).

NOS-1

  1. Constitutively expressed in the cytosolic compartment of post-synaptic terminals of neurons and stressed Schwann cells
  2. Requires Ca2+ for activation (NMDA activation)
  3. Decreased NOS-1 decreases chemotherapy and peripheral nerve injury pain

NOS-2 (Cytosolic Form)

  1. Expressed in immune cells and glia
  2. Toll-like receptor 2 activation initiates the transcription of NOS-2
  3. Once translocated, NOS-2 is constitutively active; the process does not require Ca2+

NOS-3

  1. Increases in the DRG after administration of Freund’s adjuvant
  2. Requires Ca2+/calmodulin activation

The activation of the NMDA receptor with its associated increased intracellular calcium induces the transcription of nitric oxide synthase.

Peroxynitrate Mechanisms of Disrupting Glutamate Homeostasis

  1. Nitration and phosphorylation of NMDA subunits that increase intracellular calcium concentration
  2. Decrease glutamate transporter (GLT-1) that increases its concentration at the synapse
    Mechanisms of Nitro-oxidative Species that Directly Modulate Neuroexcitability
  3. Nitro-oxidative products decrease GAD-67 GABAergic DH neurons and decrease GABA release
  4. Increase calcium influx and increase synaptic currents
  5. Decreases glutamine synthesis

Mechanisms of Nitro-oxidative Species that Directly
Modulate Neuroexcitability

  1. TRP channel activation initiates the pain signal and concomitantly releases co-localized vasoactive neuropeptides (substance P (SP) and calcitonin gene related peptide (CGRP) to cause neurogenic inflammation
  2. Induce post-translation modifications of proteins and lipids that drive pain
  3. Mitochondrial DNA is a target for oxidation and nitration
  4. Nitro-oxidative species trigger release of proapoptotic factors disrupting organelle dynamics in mitochondria
  5. Toll-like receptors bind to a variety of endogenous danger signals that include those released from nitro-oxidative damaged mitrochondria. They also activate NF-kB and MAPKs pathways
  6. NOX derived ROS (from NADPH-oxidase) are second messengers for NF-kB and p38MAPK
  7. The Toll-like receptor 2-NOX-1 interaction:
    • a. Upregulates adhesion molecules via the chemokine CCL3 which induces transendothelial cell migration
  8. Mitochondrial derived ROS:
    • a. Activate the NLRP3 inflammasome that proteolytically activates the inflammatory cytokine IL-1B

Nitro-oxidative Species Induce Mitochondrial Dysfunction – Mechanisms

  1. Mitochondria are critically involved in energy production (ATP), lipid synthesis, apoptosis and cellular calcium homeostasis
  2. NOX and NOS derived ROS disrupt mitochondrial homeostasis. There is increased metabolism during pain states that has been demonstrated to induce a bioenergetics crisis with consequent degeneration of nociceptive primary afferent fibers
  3. Mitochondria are a target of oxidation and nitration
  4. Peroxidated lipid end-products:
    • a. Form reactive aldehydes that induce covalent modifications (adducts) with an array of mitochondrial proteins that include mitochondrial antioxidants
  5. Nitro-oxidant species:
    • a. Release pro-apoptotic factors
    • b. Nitric oxide decreases fusion and fission of mitochondria
    • Decreased mitochondrial homeostasis is affected by:
      • i. Translocation of Bcl-2 associated x protein from the cytosol to the mitochondrial membrane that activates apoptotic pathways

Nitro-oxidative Species Induce Neuroinflammatory Signaling

  1. In the course of tissue and/or nerve injury all forms of pain are induced that include:
    • a. Nociceptive
    • b. Inflammatory
    • c. Neuropathic
      • i. Neuropathic pain states have a component of inflammatory pain that fades as the neuropathic pain states become predominant
  2. At the site of tissue injury in addition to the release of hydrogen ion, serotonin, bradykinin, prostanoids – glia and immune cells release TNF alpha, IL-1B and BDNF (brain-derived neurotrophic factor)
  3. Mechanisms for increased inflammation-induced neuronal hyperexcitability:
    • a. Increased glutamate release from the terminals of nociceptive primary afferent fibers
      (A-delta and C-fibers)
    • b. Increased AMPA post synaptic primary nociceptive fiber expression
    • c. Phosphorylation of NMDA subunits that increase the receptor permeability to Ca2+ ions
    • d. Down-regulation of astrocyte glutamate transporters
    • e. Decreased GABA and glycine release from inhibitory interneurons
    • f. Decreased K-Cl co-transporter KCC2 on postsynaptic terminals
    • g. Nitro-oxidative species:
      • Regulate the production of proinflammatory mediators:
  4. . NF-kB and p38MAPK are induced and increase the transcription of proinflammatory cytokines
    • a. Degrade NF-kB and p38MAPK phosphatases and thus maintain their concentration
    • b. Increase neuron to glia signals from released metalloproteinases
    • c. Increased TLR signaling:
      • i. Bind DAMP (damage-associated molecular pattern) that includes DNA and N-formyl peptides from damaged mitochondria
      • ii. ROS are second messengers for TLR signaling
      • iii. There is a rapid respiratory burst after activation of TLR2 and 4 from direct interaction of intracellular domain of NOX1,2 and 4 enzymes that is essential for NF-kB and p38MAPK dependent cytokine production
      • iv. Disruption of blood-brain barrier (BBB) tight junctions
      • v. TLR-NOX1 interaction via the chemokine CCL3 upregulates adhesion molecules
      • vi. Increase of lipid rafts by activation of NOX enzymes
      • vii. Increased transcription of TLRs
      • viii. ROS activate the inflammasome (protein complexes that cause proteolytic activation
        of the inflammatory cytokine IL-1B)
      • ix. Mitochondria are a source of ROS that activate inflammasomes (NLRP3). Nitro-oxidative species induce calcium influx that activates NLRP3 inflammasomes through the receptor TRMPM2
      • x. Transcription of NOX/NOS enzymes is upregulated by TLR4 and 9 as well as by
        NF-kB and p38MAPK
      • xi. ATP signaling via P2X7R (released from neurons) activate NOX2 in a calcium-p38
        dependent manner

Type-specific Synaptic Plasticity Induced by ROS

A major component of central sensitization occurs in the spinal cord dorsal horn. Persistent and intense nociceptive afferent input from nociceptive afferents induces maladaptive neuroplasticity in the synapses of pain projecting neurons primarily of the spinothalamic tract. During the course of this maladaptive neuroplasticity, long-term potentiation (LTP) of excitatory postsynaptic currents is seen in spinothalamic pain projecting neurons while long-term depression (LTD) develops in GABAergic interneurons to the same nociceptive input.

Increasing experimental evidence supports a major role of reactive oxygen species in increasing pain transmission following peripheral nerve injury. A major effect of increased levels of reactive oxygen species (ROS) is the down regulation of GABA transmission in the dorsal horn following nerve and spinal cord injury. Lack of GABAergic inhibition of pain projecting neurons is a major factor in enhanced pain transmission in neuropathic pain states.

In the brain the development of LTP or LTD is posited to be caused by the frequency of stimulation. In the spinal cord cell type specific LTP develops in spinothalamic tract pain projecting neurons while LTD occurs in GABAergic dorsal horn neurons from the same nociceptive stimulus. Recent studies support the hypothesis that specific ROS subtypes are instrumental in cell type-specific synaptic plasticity. Superoxide radicals are posited to cause the induction and maintenance of spinothalamic pain projecting neuron LTP and DH GABAergic LTD, while hydroxyl radicals are essential for GABAergic DH LTD induction and maintenance.

ROS ‘Activated’ Receptors

  1. TRPV1 (transient receptor potential family vanilloid 1:
    • a. Expressed as C-fiber primary nociceptive afferents
    • b. Linoleic acid metabolites that are created during production of eicosanoids are endogenous TRPV1 agonists when oxidized
    • c. TRPV1 receptors are:
      • i. Are activated directly by modified proteins and lipids
      • ii. Are activated during thermal and mechanical hyperalgesia which are major components of neuropathic pain
  2. TRPV2
    • a. Is a non-selective calcium permeable cation channel that is part of the Transient Receptor Potential ion channel superfamily of receptors
    • b. It is physiologically activated by heat via free intracellular ADP-ribose acting in concert with free intracellular calcium. Oxidative stress (an accumulation of ROS that overwhelms anti-oxidant mechanisms) induces the enzyme PARP (Poly ADP-ribose polymerase) that activates the channel
  3. TRPM2 is expressed by neurons, monocytes, macrophages, microglia and T cells. It is directly activated by nitro-oxidative species
    • a. In turn, it activates MAPK and nuclear factor K light chain enhancer of activated B cell, (NF-kB) important for the production of proinflammatory cytokines
    • b. The channel is directly activated by H2 O2 and cytosolic ADP-ribose generated from damaged mitochondria
    • c. The channel is vital for activation of spinal microglia and for macrophage infiltration into the spinal cord after peripheral nerve injury
    • It activates ERK/MAPKs and induces nuclear translocation of NF-kB that is critical for the production of proinflammatory cytokines and chemokines
  4. TRPA1:
    • a. Is a member of the transient receptor potential channel family and contains 14N-terminal ankyrin repeats
    • b. It is activated by both reactive and non-reactive compounds
    • c. It is expressed by pepetidergic C-fibers and is activated by modified proteins and lipids
    • d. A missense mutation of TRPA1 causes hereditary episodic pain syndrome

Carbonylation

  1. Carbonylation are reactions that induce carbon monoxide into organic and inorganic substrates
  2. Modifications of the side chains of histidine, cysteine and lysine in proteins to carbonyl derivatives (aldehydes and ketones) are caused by oxidative stress
  3. Nitro-oxidative species induce protein carbonylation and membrane phospholipid peroxidation and nitration. These reactions produce reactive aldehydes exemplified by acrolein that directly activate TRPA1 receptors. After spinal cord injury, acrolein is elevated in both the dorsal root ganglia and the dorsal horn that may contribute to spinal cord injury pain.
  4. At the site of injury the following compounds contribute to neuroexcitability:
    • a. . H2 O2
    • Peroxynitrate
    • Carbonylated proteins
    • Peroxidated and nitrated lipids
    • Reactive aldehydes

Antioxidant Defense

An antioxidant is a molecule that inhibits the oxidation of other molecules. As noted earlier, oxidation is the loss of electrons during a reaction by a molecule, atom or ion. Oxidation occurs when the state of a molecule, atom, or ion is increased (loss of electrons).

Reduction is its opposite (gain of electrons) by a molecule, atom or ion. As noted above, the major producers of ROS during metabolism that occur in components of the pain matrix are:

  1. NADPH oxidase (NOX)
  2. Nitric oxide synthase (NOS)
  3. Mitochondrial metabolism with loss of electrons from complex I and III

The ROS generated by these reactions in the course of energy production are damaging to multiple cellular functions and if not controlled may destroy the cell. The cell has evolved a complex network of antioxidant metabolites and enzymes that prevent oxidative damage to DNA, protein and lipids. In general, these systems prevent the ROS from being formed or assist in their removal prior to their causing cell damage. The major ROS generated that must be controlled are hydrogen peroxide (H2 O2), the superoxide anion (.O2), nitrosative species and the hydroxyl radical.

The hydroxyl radical is very unstable and reacts rapidly and non-specifically with most biological molecules. The hydroxyl radical is derived from hydrogen peroxide by metal-catalyzed redox reactions such as the Fenton reaction. As noted above, ROS induce chemical chain reactions exemplified by lipid peroxidation, the oxidation of DNA, and proteins. Damage to proteins results in enzyme inhibition, denaturation and protein degradation.

The superoxide anion is a by-product of several steps in the mitochondrial electron transport chain. In particular, the reduction of coenzyme Q in complex III forms a highly reactive free radical as an intermediate Q- . This intermediate is unstable and leads to electron transfer directly to oxygen that forms the superoxide anion. Peroxide is produced from the oxidation of reduced flavoproteins in complex I.

Antioxidants are classified as hydrophilic (soluble in water) or lipophlic (soluble in lipids). Water-soluble antioxidants interact with oxidants in the cytosol and the blood plasma. Lipid-soluble antioxidants protect cell membranes from lipid peroxidation. Both species have a spectrum of concentrations in body fluids and tissues. Glutathione and ubiquinone are primarily confined within cells while uric acid is more evenly distributed. Interactions between antioxidants and their metabolites and enzyme systems may be both synergistic or independent. The effect of the antioxidant depends on its concentration, the reactivity of the specific reactive oxygen species and the state of other antioxidants with which it interacts.

  1. Activation of Antioxidant Genes:
    • a. Nuclear factor Nrf2 (erythroid-derived 2) –like 2 is a transcription factor that is encoded by the NFE2L2 gene. It is a basic leucine zipper (bzip) protein that regulates the expression of antioxidant proteins
    • b. It is expressed in neurons, macrophages, astrocytes, Schwann cells and microglia
    • c. Under homeostatic conditions Nrf2:
      • i. Nrf2 is anchored to the cytoplasm by binding to Kelch-like ECH-associated protein I (Keap1):
  2. Keap1 sequesters cystosolic Nrf2 and ubiquinates it for degradation
    • a. Under conditions of oxidative stress, Nrf2 is released and translocates to the nucleus where it binds to antioxidant response element (ARE) to elicit expression of more than 200 antioxidant genes
    • b. The major antioxidants in pain states include:
      • i. Superoxide dismutase (SOD)
      • ii. Cytosolic SOD2 in mitochondria
      • iii. Catalase
      • iv. Glutathione
      • Heme oxygenase
    • c. Another mechanism that contributes to antioxidant defense is the chelation of transition metals. This process prevents them from catalyzing the production of free radicals. The ability to sequester iron is particularly important and is effected by transferrin and ferritin

Glutathione

  1. Glutathione is a cysteine-containing peptide synthesized in cells from its constituent amino acids. Its antioxidant properties derive from the thiol group in its cysteine moiety that is a reducing agent and is reversibly oxidized and reduced. It is maintained in its reduced form intracellularly by glutathione reductase. It has a central role in maintaining intracellular redox potential.

Antioxidant Enzyme Systems

  1. A major defense against oxidative stress is an interacting network of antioxidant enzymes systems:
    • a. Superoxide released from oxidative phosphorylation is converted to hydrogen peroxide
      and then water by further reduction. This pathway is first catalyzed by superoxide dismutase and then requires catalases and peroxidases

Superoxide dismutase, Catalase and Peroxiredoxins

  1. Superoxide dismutases (SODs):
    • a. Are a family of enzymes that catalyze the degradation of the superoxide anion into O2 and H2 O2
    • b. In humans, the copper/zinc SOD is present in the cytosol. Manganese SOD is located in
      mitochondria
  2. Catalase
    • a. Catalyzes the conversion of H2 O2 to water and O2 in concert with a manganese or iron cofactor
  3. Peroxiredoxins:
    • a. These are peroxidases that catalyze the reduction of peroxynitrate, organic hydroperoxides and hydrogen peroxide

Thioredoxin Systems

  1. The thioredoxin system is composed of thioredoxin and thioredoxin reductase
  2. Its active site has two closely located cysteines in its CXXC motif that cycle between an active dithiol form (its reduced state) and an oxidized disulfide form
  3. It is an effective reducing agent and scavenges ROS and maintains other proteins in a reduced state
  4. Active thioredoxin (after being oxidized) is regenerated by thioredoxin reductase utilizing NADPH as an electron donor