Paraesthesias (tingling or ant crawling sensations over the skin), spontaneous ongoing or shooting pain (stimulus independent) and electric shock-like sensations are included in spontaneous positive symptoms; whereas, hyperalgesia and allodynic (further classified according to the static and dynamic nature of the stimulus) pain are stimulus-evoked positive symptoms of neuropathy (Rasmussen et al., 2004). Negative symptoms of neuropathic pain includes hypoesthesia (reduced sensations to non-painful/innocuous stimuli), pallyhypoesthesia (reduced sensations to vibration), hypoalgesia (abridged impression to noxious stimuli) and thermohypoesthesia (abridged impression to cold/warm stimuli) (Jensen, 2006).

Numerous controlled studies have demonstrated potential effectiveness of opioids, tricyclic anti-depressants, gabapentin, pregabalin, phenytoin, lamotrigine, dextromethorphan, tramadol and mexiletine for painful sensory neuropathy (Harden, 1999; Attal, 2001). However, these therapies result in a 30–50% reduction in pain and are often restricted due to significant side effects, with sedation being the most dominant.

PATHOPHYSIOLOGICAL MECHANISMS OF NEUROPATHIC PAIN

Experimental work in rodents has provided a broadened picture of the pathophysiological mechanisms that generate neuropathic pain. It involves both peripheral and central mechanisms. The peripheral sensitization is carried out via unmyelinated C- and thinly myelinated Aδ- primary afferent neurons that normally elicit the pain sensation by responding to noxious stimuli. However, the peripheral nerve lesions sensitize these neurons advancing into spontaneous activity. Moreover, these lesions cause dramatic changes on the cellular and molecular levels triggering the nerve cells (Figure 1) (Baron, 2006).

 

 

An increased mRNA expression for voltage gated sodium channels in the primary afferent neurons is considered to be responsible for an ectopic spontaneous activity after a nerve injury. This phenomenon may result in the clustering of these channels that lowers the action potential threshold leading to hypersensitivity. Hence, sodium channel blockers like lidocaine produce pain relief in neuropathic pain via this mechanism (Lai et al., 2003).

Up regulation of a variety of receptor proteins also result from peripheral nerve injury. They are present at the membrane of the primary afferents and are only slightly uttered in physiological circumstances. Vanilloid receptors (TRPV 1) being one of them are involved in the sensing of noxious heat greater than 43ºC (Patapoutian et al., 2003), whereas, TRPM8 receptors have been identified as cold and menthol-sensitive raised by temperature in the range of  8-28ºC.  TRPM8  receptor  is revealed in small diameter DRG (dorsal root ganglia) neurons (McKemy et al., 2002). A nerve injury may result in either up regulation or gating of this channel leading to peripheral sensitization of C-nociceptors that causes cold hyperalgesia (Wasner et al., 2004).

Acid-sensing ion channels (ASCIs) are believed to be participating in static mechanical hyperalgesia (Price et al., 2001). On the other hand, α₁- and α₂- adrenoceptors located on the cutaneous afferent fibers also play a pronounced function in the hypersensitivity from nerve injury (Baron et al., 1999). Adrenergic sensitivity has been widely expressed in post herpetic neuralgias, complex regional pain syndromes II (CRPS II) and post-traumatic neuralgias; whereas, in polyneuropathies no sensitivity in the primary afferent neurons has been reported (Uphoff and Binder, 2006). Thus, temperature-induced and sympathetically-induced pain could be treated by blocking their respective receptors on nociceptive neurons.

An ectopic activity is also induced by inflammation in both wounded and adjoining normal primary afferent nociceptors triggered by a nerve lesion that release proinflammatory cytokines, especially TNF-α (Sommer, 2003). Deep proximal and paroxysmal pain are pronounced features in the patients diagnosed with peripheral neuropathies e.g., HIV neuropathy. Nerve biopsy specimens of such patient have shown an increased concentration of COX-2 and proinflammatory cytokines  (Table 1) (Lindenlaub and Sommer, 2003).

 

 

CNS constitutes specific anatomical links with the spinal cord, brain stem, thalamus and the cortex. These connections link sensations generated in the high threshold primary afferents with the cortical regions of the central nervous system, that further process it into ultimate painful sensations (Woolf, 2011). The ongoing hyperactivity generated by injured nerves serves as a trigger for central sensitization and simulates activity-dependent synaptic flexibility occurring within the cortex. Diverse synaptic modulators, changes in ion channel kinetics, excitatory amino acid, increased bulk of ionotropic receptors and pre- and post-synaptic activation of kinases are involved in central sensitization.

Most of the patients presenting with peripheral and all with central neuropathy show predominant synaptic facilitation contributing to allodynia and hypersensitivity (Campbell and Meyer, 2006). Peripheral nerve injury causes “pre-synaptic changes” including modifications in the synthesis of neurotransmitters, neuromodulators and in the density of calcium channels (Hendrich et al., 2008). On the contrary, increased receptor density as a result of enhanced synthesis of ion channels and scaffold proteins and phosphorylation of N-methyl-D-aspartate (NMDA) subunits occur because of “post-synaptic changes” (Cheng et al., 2008). These changes also contribute to an abnormal expression of Nav 1.3 (Hains et al., 2004)and the  mitogen-activated  protein  kinase system (MAPK) (Ji and Woolf, 2001). The role of pathologically sensitized C-fibers which sensitizes neuropeptide substance P and spinal dorsal horn via glutamate release cannot be overlooked. The released glutamate shows an excitatory effect by acting upon post-synaptic NMDA leading to central sensitization (Ultenius et al., 2006). An appreciably abundant evidence reveal the involvement of loss of tonic GABA-conciliated inhibition and increased excitatory neurotransmitters resulted in an initiation of central sensitization, ending in peripheral hypersensitivity, that is, allodynia and hyperalgesia (Knabl, Witschi et al., 2008). Once this sensitivity is developed, the usually innocuous tactile stimuli could activate Aδ and Aβ low threshold mechanoceptors (Tal and Bennett, 1994).

ROLE OF GABA IN NEUROPATHIC PAIN

In the brain, γ-amino-butyric acid (GABA) tends to be the most abundant inhibitory neurotransmitter regulating different physiological characters like sleep, anxiety, memory formation, reward, etc (Zeilhofer et al., 2009)and also control the action of excitatory neurons in the CNS, facilitating a uniform flow of information and therefore, maintaining the homeostasis of neural circuits. Melzack and Wall (1967) previously reported the role of inhibitory neurons in the spinal dorsal horn involved in controlling pain transmission from the periphery to higher levels of the brain (Melzack and Wall, 1967). Later on, GABA  was confirmed to be one of the foremost inhibitory neurotransmitters in the spinal dorsal horn (Yaksh, 1989).

After release from the pre-synaptic neurons, GABA act postsynaptically upon 3 major classes of receptors namely; GABA_A and GABA_C receptors that are ligand-gated ion channels and GABA_B that are G protein-coupled channels (Gavande et al., 2011). Chemically ionotropic GABA_A receptors are formed of transmembrane protein complexes and comprise 5 heteropentameric subunits. In the human brain, α₁β₂γ₂ subunit is considered to be the most dominant one (Wafford, 2005). Upon activation by GABA, the membrane permeability to chloride and carbonate ions increases causing a net inward flow of anions and a consequent hyperpolarization. This hyperpolarizing post-synaptic response is termed as inhibitory post-synaptic potential (Semyanov et al., 2003).

Physiologically, GABA-releasing interneurons, imposes a strong inhibitory control over the dorsal horn neurons. The loss of these neurons could further increase the central sensitization, e.g. as reported in partial peripheral nerve injury models, in which the injury resulted in the reduced spinal GABA release along with decreased GABA-synthesizing enzyme glutamic acid decarboxylase in rodents (Moore et al., 2002), advancing into spontaneous  pain   and  hyperexcitability  manifested  as  allodynia and hyperalgesia.

In diseased states, an increased excitation occurs that is attributed to either a massive loss of GABAergic interneurons or deterioration of interneurons as consequence of depletion of their sensory excitatory inputs or receptors. This imbalance could culminate into many neurological and psychiatric disorders including epilepsy, schizophrenia, neuropathic pain, Alzheimer’s (AD) and Parkinson’s disease (PD) (Tyson and Anderson 2014).

Hence, the collaborative function of excitatory and inhibitory neurons plays a vital role in controlling various brain activities.

As discussed earlier, central and peripheral sensitization due to nerve injury could result in complex neuropathic pain. Besides other mechanisms, the loss of GABAergic interneurons is now considered to be one of the major contributor to such persistent pain states (Bráz et al., 2012). Recent evidence suggest that the deletion of particular GABA subunits or pharmacological blockade of GABAergic neurotransmission in the spinal cord resulted in hyperalgesia and allodynia (Gwak et al., 2006; Jergova et al., 2012). Similarly, electrophysiological studies have revealed that GABA_A receptor inhibition could  induce  an  embellished  behavioral  reaction  to an innocuous mechanical stimulus (Hwang and Yaksh, 1997), consistent with the reported high levels of GABAergic receptors in the dorsal horn (Persohn et al., 1991). Moreover, the impaired GABAergic system in the animals presenting with chronic neuropathic pain propose the practical link between such pain and spinal inhibitory neurotransmission (Figure 2) (Zeilhofer, 2008). Therefore, restoration of spinal inhibitory neurotransmission can be a valuable pharmacological advancement in the treatment of neuropathic pain.

 

 

GABA_A receptor  agonists related antinociceptive effect has been attributed to the stimulation or blockade of other neurotransmitters as well (McCarson and Enna, 2014). Furthermore, the role of central GABA in opioid-mediated antinociception is well documented (Ossipov et al., 2010). Thus, GABA receptor agonists may play a vital role in treating acute and chronic pain (McCarson and Enna, 2014).

In this regard, GABA_A receptor agonists muscimol and isoguvacine are reported to reverse nerve injury-induced tactile allodynia (Hwang and Yaksh, 1997). These receptors   are   closely   linked   to   the   large  diameter afferents involved in innocuous sensation (Price et al., 1984; Sivilotti and Woolf, 1994; Reeve et al., 1998; Ataka et al., 2000; Riley et al., 2001; Turner, 2003). Great body of evidence suggests that a decreased spinal GABA plays a vital role in inducing and maintaining neuropathic pain. Behavioral and pharmacological studies have shown that a continuous or single intrathecal dose of GABA to the spinal cord or the implantation of GABA releasing cells decrease the signs of neuropathic pain (Eaton et al., 1998, 1999; Stubley et al., 2001; Malan et al., 2002). Moreover, blocking the spinal GABA_A receptors results in aggravating peripheral nerve injury associated hyperalgesia (Yamamoto and Yaksh, 1993).

On the contrary, benzodiazepines, and positive allosteric modulators at GABA_A receptors are widely used drugs not only in anxiety, convulsions and sleep disorders but also as analgesic if administered intrathecally (Tucker et al., 2004). Despite its analgesic potential, its use in pain relief is restricted due to the myriad effects like sedation. Hence, there is an increased need of research in the field of GABAergic modulators that could play a pronounced role in the attenuation of neuropathic pain.

FLAVONOIDS

Flavonoids are widely distributed in vegetables, grains, fruits, barks, roots, flowers, stems, tea, etc. Chemically, they are polyphenolic compounds comprising of a diphenyl propane skeleton (C-6-C-3-C-6), with two aromatic rings and an oxygen-containing heterocyclic benzopyran ring, labeled as fused aromatic ring A, the benzopyran ring adjacent to A is ring C and a phenyl ring B (Middleton, 1998).

Long before the isolation of flavonoids, the natural products bearing them were known for their healthful effects. The decreased mortality rate associated with  the use of red wine (constitute flavonoids) was observed in Mediterranean populations that produced an inclination in the flavonoid research, that was further confirmed by epidemiological studies, portraying the cardio protective role of dietary flavonoids in coronary heart disease (Formica and Regelson, 1995; Groot and Rauen, 1998).

Similarly, a new substance isolated from oranges in 1930, was thought to be a vitamin and was called vitamin P, which was later on confirmed to be a flavonoid, that is, rutin which led to an increase in the isolation and study of the mechanisms of various individual flavonoids. A long history of plant species exists that constitute flavonoids and exhibit CNS effects and were used as folk medicines in Europe. Feverfew (Tanacetum parthenium L. Asteraceae) was used as prophylactic agent in migraine and Chamomile flowers (Matricaria recutica L. Asteraceae) as a tranquillizer for centuries, with both found to be containing apigenin as an active constituent via bioassay-guided fractionation studies (Jäger et al., 2009). Furthermore, Linden flowers (Tilia sp. Tiliaceae) were known for their sedative effects and Heather (Calluna vulgaris [L] Hull. Ericaceae) as nerve calming remedy, with both shown to have quercetin and kaempferol flavonoids as active components (Aguirre-Hernández et al., 2010).

Flavonoids can be categorized into ten groups (Table 2) (Peterson and Dwyer, 1998). Flavone, isoflavone and flavanol contains a double bond in the ring C, making the fused A-C ring system planar, whereas, the other classes lack this double bond and instead have C₂ and C₃ chiral centers that are located at each end of the plane ring A (Figure 3) (Jager and Saaby, 2011). Besides the isolation of natural flavonoids, various semi-synthetic and synthetic derivatives have been synthesized and screened for their therapeutic potential (Cushnie and Lamb, 2005).

 

 

 

In plants, flavonoids not only provide UV-protection but also aid in the pollination by masking the flowers with attractive colors and patterns. Until now, more than 6000 varieties of flavonoids have been isolated. A variety of interest gasping pharmacological actions for these naturally occurring flavonoid compounds as well as synthetic derivatives have been found (Vidyalakshmi et al., 2010), including both peripheral and central nervous system effects (Figure 4) (Hall et al., 2005). These compounds attribute to a vast range of biological effects, like anticancer (Liu et al., 2010), anti-inflammatory (Wang et al., 2010), antioxidant (Heim et al., 2002), anxiolytic (Ognibene et al., 2008), cardio protective (Yu et al., 2005), antifungal (Ammar et al., 2013), antiviral (Orhan et al., 2010), antimicrobial (Cushnie and Lamb, 2005), neuroprotective (Cho et al., 2013)and  antinociceptive activities (Wang et al., 2014).

 

 

The substitution at 6- or 3’-positions of flavones with an electronegative functional group enhanced the affinity towards the BDZ receptors (Paladini et al., 1999). Furthermore, the impact of ligand binding on the GABA binding was used to determine the GABA ratios. These ratios showed that flavones exhibited significant biological activities at BDZ receptors (Hanrahan et al., 2011). 6-Bromoflavone, 6-bromo-3’-nitroflavone and 6-chloro-3’-nitroflavone with a GABA ratio of 1.6-2.0, 1.38 and 2.0 were reported as full agonist (Marder et al., 1996), partial agonist (Wolfman et al., 1998)and an antagonist (Viola et al., 2000)at these receptors.

Positive ionotropic modulators of GABA_A receptors enhance the chloride ion flux and provide a strong inhibitory effect. Therefore, for the  management  of  CNS – allied diseases, including panic disorders, generalized anxiety, seizure disorders, muscle spasm, neuropathic pain and sleep disturbances; these modulators stand amongst the strongest candidates (Rudolph and Möhler, 2006). Moreover, with the discovery of the fact that flavonoids may act upon novel binding sites other than the classical benzodiazepine binding site, opportunities to search for new therapeutic agents with less adverse effects has been provided (Rudolph and Möhler, 2006). In this regard, 6-Methoxyflavonone has been reported to act as positive allosteric modulator at α1β2γ2L and α2β2γ2L subunits of GABA_A receptors (Hall et al., 2014).

The substitution at 6-position on flavones is related to its effects on the recombinant GABA_A receptors. 6-Hydroxyflavone depicted a significant effect at flumazenil-sensitive BDZ site (Ren et al., 2010). Moreover, 6-methoxyflavanone and 6-methoxyflavone have been recently   reported   to   exhibit   significant   anti-allodynic effects in streptozotocin- and cisplatin-induced neuropathic pain models (Akbar et al., 2016; Shahid et al., 2017). These protective effects against neuropathic pain were attributed to the positive allosteric modulatory effects of these molecules on opioid and GABA_A receptors respectively (Akbar et al., 2016).

Moreover, myrcitin and baicalin produced significant anti allodynic effects in sciatic nerve ligation models (Meotti et al., 2006; Cherng et al., 2014). Rutin and quercetin has been reported to inhibit oxaliplatin-induced chronic painful peripheral neuropathy (Azevedo et al., 2013). Naringin is also reported as exhibiting anti allodynic potential in the sreptozotocin-induced painful diabetic neuropathy (Kandhare et al., 2012).

OTHER NEUROPATHIC PAIN MODULATING MECHANISMS OF FLAVONOIDS

Besides its action on the GABA_A receptors, flavonoids also exert antioxidant and anti-inflammatory effects. Almost all metabolic diseases are the consequence of oxidative stress. In addition to exogenous damage, the normal oxygen metabolism within the cells and tissues produce free radicals and reactive oxygen species that consistently endanger them. Flavonoids are very well known for their antioxidant potential and are proven to exert positive effects in diseases like atherosclerosis, diabetes, Alzheimer’s disease, cancer, etc (De Groot, 1994; Pal and Verma, 2013).

Although the underlying set of events behind the damage caused by the free radicals to the cellular functions is not fully understood, the contribution of lipid per oxidation leading to cellular membrane destruction and activation of inflammatory mediators by the free radicals culminating in ultimate tissue damage, can provide a lot of help in conceiving a pharmacological target. Nature has provided an inborn mechanism against these ROS, comprising of enzymes like superoxidase, glutathione peroxidase and catalase as well as non-enzymatic elements e.g. ascorbic acid, α-tochopherol, etc (Halliwell, 1995). However, these endogenous scavenging compounds are prone to depletion due to an increased oxidative stress caused by many diseased states including conditions that culminate in neuropathic pain e.g. diabetes mellitus (Schreiber et al., 2015).

Epicatechin and rutin are shown to have the ability to be oxidized themselves by the free radicals giving rise to a stable and less reactive species (Hanasaki et al., 1994). Similarly, quercetin inhibits the nitric oxide (NO) induced cell injury. Nitric oxide combines with the free radicals and produces an extremely harmful peroxynitrite that straightly oxidize LDL leading to a permanent destruction of the cell membrane. Hence, quercetin scavenge the free radicals, restraining them from reacting with NO (Shutenko et al., 1999), while silibin react directly with NO (Dehmlow et al., 1996).

Physiologically, the metabolism of xanthine to uric acid is carried out via xanthine dehydrogenase; however, in case of ischemic-reperfusion this enzyme changes into xanthine oxidase that acts as precursor of free radicals. Quercetin, luteolin and silibin flavonoids are known to act as antioxidant via inhibiting xanthine oxidase (Chang et al., 1992; Shoskes, 1998).

Similarly, reperfusion also results in the mobilization of leucocytes causing the consequent release of inflammatory mediators and cytotoxic oxidants provoking the complement system. A number of flavonoids are reportedly involved in the leucocytes immobilization ultimately causing a reduction in the serum complement system and inflammation (Friesenecker and Tsai, 1995; Ferrándiz et al., 1996). A pronounced body of evidence report the involvement of same pathophysiological mechanisms behind both inflammation and neuropathic pain of peripheral origin. Both types of pathologies manifest as allodynia and hyperalgesia (Clatworthy et al., 1995; DeLeo and Yezierski, 2001; Jin et al., 2003). Infiltration of inflammatory cells and their secretary products like arachidonic acid and cytokines (released for nerve regeneration) as a result of peripheral nerve injury is responsible for the generation and maintenance of the ongoing pain (Tracey and Walker, 1995; Cui et al., 2000; Ma and Eisenach, 2003). Cytokines like tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1) and interleukin-6 (IL-6) when injected into the rat paw resulted in the induction of mechanical and thermal hyperalgesia (Cunha et al., 1992; Ferreira et al., 1993). The blockage of TNF–α in the rodent models of painful neuropathy has resulted in the attenuation of hyperalgesia (Sommer et al., 1998). Cytokines once released induce their own production and consequently activate COX–2 dependent prostanoid releases. The role of PGs in inducing inflammation accompanying enhanced sensitivity to pain is well documented. Intrathecal administration of PGs including PGE₂ and PGF_2α induced allodynia in conscious mice (Minami et al., 1992, 1994), whereas intrathecal injection of PGD₂ and PGE₂ resulted in the induction of hyperalgesia (Uda et al., 1990). Moreover, synthesis of PG and nitric oxide (NO) via COX-2 and inducible nitric oxide synthase (iNOS) is enhanced in the microglia as a result of peripheral nerve injury leading to hyper sensitization (Hanisch, 2002). Flavonoids exhibit an in vivo and in vitro anti-inflammatory activity. The in vivo anti-inflammatory is attributed to the inhibition of ecosanoid generating enzymes including COX, LOX and phospholipase A₂ (Kim et al., 2004).

Flavonoids are known to act upon different enzyme systems, e.g. causes the inhibition of arachidonic acid and blocking the inflammatory response as arachidonic acid tends to start it. By this, antithrombotic and anti-inflammatory features are incorporated into the group (Ferrandiz and Alcaraz, 1991). They also cause decrease in the release of peroxidase and inhibition of ROS production by neutrophils. This inhibition is carried out via interference with α₁-antitrypsin activation (Middleton and Kandaswami, 1992). Certain flavonoids are involved in iron chelation that causes lipid peroxidation, thereby abolishing an erratic factor for the free radicals development (Nelson et al., 1992; Ferrali et al., 1997).

 

 

 

 

 

 

 

 

 

Allosteric modulators at GABA_A receptors modify either the efficacy or affinity of agonists like GABA, consequently regulating their activity. Over the last decade, these modulators have been focused extensively due to the advances in the understanding of the functions of GABA_A receptor subtypes. Flavonoids being potent allosteric modulators may prove to be useful tools in the amelioration of such distressing painful conditions like neuropathic pain. However more studies are required to further elucidate the site of action of these bioactive molecules over the GABAA receptors.

 

The author has not declared any conflict of interests.