Effect of Subpressor Dose of Angiotensin II on Pain-Related Behavior in Relation with Neuronal Injury and Activation of Satellite Glial Cells in the Rat Dorsal Root Ganglia

To clarify the role of angiotensin II (Ang II) in the regulation of sensory signaling, we studied the effect of subpressor dose (150 ng/kg/min) of Ang II on pain-related behavior in relation with neuronal injury and activation of satellite glial cells (SGCs) in the dorsal root ganglia (DRGs) after chronic constriction injury (CCI). Systemic continuous delivery of Ang II induced the tactile, heat and cold hyperlagesia, when measured at 7 days ofpost-injury. Blockade of the AT₁ receptor with losartan (2.5 mg/kg/day) prevented tactile hyperalgesia and attenuated cold hyperalgesia, but did not affect the response to noxious heat stimulus. A marked increase of large-sized injured primary afferent neurons, detected by ATF3 immunolabeling, was seen in lower lumbar DRGs on ipsilateral side after Ang II treatment. Subpressor dose of Ang II induced an increase of activated SGCs (detected by GFAP immunolabeling) enveloping large-diameter neurons. Our results suggested that Ang II through the AT₁ receptor activation is an important regulatory factor in neuropathic pain perception and plays an important role in the injury of large-sized primary afferent neurons and activation of SGCs elicited by the CCI.     

ATP receptors in pain sensation: Involvement of spinal microglia and P2X<Subscript>4</Subscript> receptors

There is abundant evidence that extracellular ATP and other nucleotides have an important role in pain signaling at both the periphery and in the CNS. At first, it was thought that ATP was simply involved in acute pain, since ATP is released from damaged cells and excites directly primary sensory neurons by activating their receptors. However, neither blocking P2X/Y receptors pharmacologically nor suppressing the expression of P2X/Y receptors molecularly in sensory neurons or in the spinal cord had an effect on acute physiological pain. The focus of attention now is on the possibility that endogenous ATP and its receptor system might be activated in pathological pain states, particularly in neuropathic pain. Neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. This type of pain can be so severe that even light touching can be intensely painful; unfortunately, this state is generally resistant to currently available treatments. An important advance in our understanding of the mechanisms involved in neuropathic pain has been made by a recent work demonstrating the crucial role of ATP receptors (i.e., P2X₃ and P2X₄ receptors). In this review, we summarize the role of ATP receptors, particularly the P2X₄ receptor, in neuropathic pain. The expression of P2X₄ receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly P2X₄ receptors produce a reduction of the neuropathic pain behaviour. Understanding the key roles of ATP receptors including P2X₄ receptors may lead to new strategies for the management of neuropathic pain.     

Brain Angiotensin II: New Developments, Unanswered Questions and Therapeutic Opportunities

1. There are two Angiotensin II systems in the brain. The discovery of brain Angiotensin II receptors located in neurons inside the blood brain barrier confirmed the existence of an endogenous brain Angiotensin II system, responding to Angiotensin II generated in and/or transported into the brain. In addition, Angiotensin II receptors in circumventricular organs and in cerebrovascular endothelial cells respond to circulating Angiotensin II of peripheral origin. Thus, the brain responds to both circulating and tissue Angiotensin II, and the two systems are integrated. 2. The neuroanatomical location of Angiotensin II receptors and the regulation of the receptor number are most important to determine the level of activation of the brain Angiotensin II systems. 3. Classical, well-defined actions of Angiotensin II in the brain include the regulation of hormone formation and release, the control of the central and peripheral sympathoadrenal systems, and the regulation of water and sodium intake. As a consequence of changes in the hormone, sympathetic and electrolyte systems, feed back mechanisms in turn modulate the activity of the brain Angiotensin II systems. It is reasonable to hypothesize that brain Angiotensin II is involved in the regulation of multiple additional functions in the brain, including brain development, neuronal migration, process of sensory information, cognition, regulation of emotional responses, and cerebral blood flow. 4. Many of the classical and of the hypothetical functions of brain Angiotensin II are mediated by stimulation of Angiotensin II AT₁ receptors. 5. Brain AT₂ receptors are highly expressed during development. In the adult, AT₂ receptors are restricted to areas predominantly involved in the process of sensory information. However, the role of AT₂ receptors remains to be clarified. 6. Subcutaneous or oral administration of a selective and potent non-peptidic AT₁ receptor antagonist with very low affinity for AT₂ receptors and good bioavailability blocked AT₁ receptors not only outside but also inside the blood brain barrier. The blockade of the complete brain Angiotensin II AT₁ system allowed us to further clarify some of the central actions of the peptide and suggested some new potential therapeutic avenues for this class of compounds. 7. Pretreatment with peripherally administered AT₁ antagonists completely prevented the hormonal and sympathoadrenal response to isolation stress. A similar pretreatment prevented the development of stress-induced gastric ulcers. These findings strongly suggest that blockade of brain AT₁ receptors could be considered as a novel therapeutic approach in the treatment of stress-related disorders. 8. Peripheral administration of AT₁ receptor antagonists strongly affected brain circulation and normalized some of the profound alterations in cerebrovascular structure and function characteristic of chronic genetic hypertension. AT₁ receptor antagonists were capable of reversing the pathological cerebrovascular remodeling in hypertension and the shift to the right in the cerebral autoregulation, normalizing cerebrovascular compliance. In addition, AT₁ receptor antagonists normalized the expression of cerebrovascular nitric oxide synthase isoenzymes and reversed the inflammatory reaction characteristic of cerebral vessels in hypertension. As a consequence of the normalization of cerebrovascular compliance and the prevention of inflammation, there was, in genetically hypertensive rats a decreased vulnerability to brain ischemia. After pretreatment with AT₁ antagonists, there was a protection of cerebrovascular flow during experimental stroke, decreased neuronal death, and a substantial reduction in the size of infarct after occlusion of the middle cerebral artery. At least part of the protective effect of AT₁ receptor antagonists was related to the inhibition of the Angiotensin II system, and not to the normalization of blood pressure. These results indicate that treatment with AT₁ receptor antagonists appears to be a major therapeutic avenue for the prevention of ischemia and inflammatory diseases of the brain. 9. Thus, orally administered AT₁ receptor antagonists may be considered as novel therapeutic compounds for the treatment of diseases of the central nervous system when stress, inflammation and ischemia play major roles. 10. Many questions remain. How is brain Angiotensin II formed, metabolized, and distributed? What is the role of brain AT₂ receptors? What are the molecular mechanisms involved in the cerebrovascular remodeling and inflammation which are promoted by AT₁ receptor stimulation? How does Angiotensin II regulate the stress response at higher brain centers? Does the degree of activity of the brain Angiotensin II system predict vulnerability to stress and brain ischemia? We look forward to further studies in this exiting and expanding field.     

Differential Regulation of Glucocorticoid Receptor Expression in Distinct Columns of Periaqueductal Grey in Rats with Behavioural Disability Following Nerve Injury

Neuropathic pain is diagnosed primarily by sensory dysfunction, which includes both spontaneous, and stimulus-evoked pain. Clinical evaluation highlights the disabilities which characterise this condition for most patients. Chronic constriction injury of the sciatic nerve (CCI) evokes sensory dysfunction characteristic of neuropathic pain. Approximately, 30 % of CCI rats show disabilities similar to those identified in clinical evaluation of neuropathic pain patients, these include: altered social behaviours; sleep disturbances; and endocrine dysfunction. The periaqueductal grey (PAG) is a nodal point in the brain circuits which regulate these functions, and undergoes a distinct set of neural and glial adaptations following CCI, in rats with disabilities. CCI increases corticosterone, which through its actions at the glucocorticoid receptor (GR), can trigger cellular adaptation. GR expression in PAG was quantified using qRT-PCR, Western blotting and immunohistochemical analyses and nerve-injured rats, with and without disabilities, were compared. Our data showed that the PAG of disabled rats has significantly increased expression of GR mRNA and protein. Further, this increased protein expression reflects contrasting patterns of change in GR expression in PAG subregions. The dorsolateral PAG had significant increases in the number of GR-immunoreactive (GR-IR) cells and the caudal lateral and ventrolateral PAG each had significant reductions in the number of GR-IR cells. These regional increases and decreases correlated with the degree of disability, as indicated by the degree of change in social behaviours. Our results suggest a role for altered PAG, GR–corticosterone interactions and their resultant cellular consequences in the expression of disabilities in a subpopulation of nerve-injured rats.     

The Contribution of TRPM8 and TRPA1 Channels to Cold Allodynia and Neuropathic Pain

Cold allodynia is a common feature of neuropathic pain however the underlying mechanisms of this enhanced sensitivity to cold are not known. Recently the transient receptor potential (TRP) channels TRPM8 and TRPA1 have been identified and proposed to be molecular sensors for cold. Here we have investigated the expression of TRPM8 and TRPA1 mRNA in the dorsal root ganglia (DRG) and examined the cold sensitivity of peripheral sensory neurons in the chronic construction injury (CCI) model of neuropathic pain in mice.In behavioral experiments, chronic constriction injury (CCI) of the sciatic nerve induced a hypersensitivity to both cold and the TRPM8 agonist menthol that developed 2 days post injury and remained stable for at least 2 weeks. Using quantitative RT-PCR and in situ hybridization we examined the expression of TRPM8 and TRPA1 in DRG. Both channels displayed significantly reduced expression levels after injury with no change in their distribution pattern in identified neuronal subpopulations. Furthermore, in calcium imaging experiments, we detected no alterations in the number of cold or menthol responsive neurons in the DRG, or in the functional properties of cold transduction following injury. Intriguingly however, responses to the TRPA1 agonist mustard oil were strongly reduced.Our results indicate that injured sensory neurons do not develop abnormal cold sensitivity after chronic constriction injury and that alterations in the expression of TRPM8 and TRPA1 are unlikely to contribute directly to the pathogenesis of cold allodynia in this neuropathic pain model.     

Selective type 1 angiotensin II receptor blockade attenuates oxidative stress and regulates angiotensin II receptors in the canine failing heart

Background and objective Angiotensin II type 1 receptor (AT₁R) blockade reduces vascular oxidative stress but whether myocardial oxidative stress represents a mechanism for the beneficial effect of AT₁R blockade in heart failure is unclear. Furthermore, the impact of AT₁R blockade on the expression of angiotensin II receptors in heart failure has not been well documented. Accordingly, we examined the impact of the AT₁R blocker candesartan on hemodynamics, left ventricular (LV) remodeling (echocardiography), oxidative stress, and tissue expression of AT₁Rs and angiotensin II type 2 receptors (AT₂Rs) in a canine model of pacing-induced heart failure. Methods and results Animals were randomized to rapid right ventricular-pacing (250 beats/min for 3 weeks) to severe heart failure and treated with candesartan (10 mg/kg daily, n = 8) or placebo (n = 8) from day 3 onwards, or no pacing (sham, n = 7). Candesartan significantly reduced mean pulmonary arterial and LV diastolic pressure, LV end-diastolic and end-systolic volume and ascites, increased cardiac output, dP/dt, and ejection fraction, while reversing the marked increase in aldehydes, a marker of oxidative stress, observed in the placebo group. Although candesartan did not alter LV AT₁R protein expression compared to placebo or sham, it reversed the decrease in AT₂R protein observed in the placebo group. Conclusion Our results indicate that in the pacing model of heart failure, chronic AT₁R blockade attenuates hemodynamic deterioration and limits LV remodeling and dysfunction, in part by reversing oxidative stress and AT₂R downregulation.     

Involvement of the AT1 receptor in the venoconstriction induced by angiotensin II in both the inferior vena cava and femoral vein

Although angiotensin II-induced venoconstriction has been demonstrated in the rat vena cava and femoral vein, the angiotensin II receptor subtypes (AT₁ or AT₂) that mediate this phenomenon have not been precisely characterized. Therefore, the present study aimed to characterize the pharmacological receptors involved in the angiotensin II-induced constriction of rat venae cavae and femoral veins, as well as the opposing effects exerted by locally produced prostanoids and NO upon induction of these vasomotor responses. The obtained results suggest that both AT₁ and AT₂ angiotensin II receptors are expressed in both veins. Angiotensin II concentration-response curves were shifted toward the right by losartan but not by PD 123319 in both the vena cava and femoral vein. Moreover, it was observed that both 10⁻⁵ M indomethacin and 10⁻⁴ M L-NAME improve the angiotensin II responses in the vena cava and femoral vein. In conclusion, in the rat vena cava and femoral vein, angiotensin II stimulates AT₁ but not AT₂ to induce venoconstriction, which is blunted by vasodilator prostanoids and NO.     

P2X<Subscript>3</Subscript> Receptor Involvement in Pain States

The understanding of how pain is processed at each stage in the peripheral and central nervous system is the precondition to develop new therapies for the selective treatment of pain. In the periphery, ATP can be released from various cells as a consequence of tissue injury or visceral distension and may stimulate the local nociceptors. The highly selective distribution of P2X₃ and P2X_2/3 receptors within the nociceptive system has inspired a variety of approaches to elucidate the potential role of ATP as a pain mediator. Depolarization by ATP of neurons in pain–relevant neuronal structures such as trigeminal ganglion, dorsal root ganglion, and spinal cord dorsal horn neurons are well investigated. P2X receptor-mediated afferent activation appears to have been implicated in visceral and neuropathic pain and even in migraine and cancer pain. This article reviews recently published research describing the role that ATP and P2X receptors may play in pain perception, highlighting the importance of the P2X₃ receptor in different states of pain.     

Silymarin- and melatonin-mediated changes in the expression of selected genes in pesticides-induced Parkinsonism

Expression of angiotensin II (Ang II) and its receptors (AT₁/AT₂) is undetected in the mature microglia in normal brain. We report here that the immunoexpression of Ang II and AT₁/AT₂ was altered in activated microglia notably at 1 week in rats subjected to middle cerebral artery occlusion (MCAO). Immunolabeled activated microglia were widely distributed in the infarcted cerebral tissue after MCAO. By enzyme immunoassay, Ang II protein expression levels of the ischemic tissues were decreased drastically at 12 h after ischemia, then rose rapidly at 3 days and 1 week after MCAO when compared with the control. On the other hand, AT₁ and AT₂ receptor mRNA and protein levels were up-regulated after MCAO, peaking at 12 h, but declined thereafter. Expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) mRNA and protein levels was concomitantly increased. Edaravone significantly suppressed Ang II and AT₁/AT₂ receptor expression as well as that of TNF-α and IL-1β suggesting that microglia-derived Ang II can act through an autocrine manner via its receptor that may be linked partly to the production of proinflammatory cytokines. We conclude that neuroinflammation in MCAO may be attenuated by Edaravone which acts through suppression of expression of Ang II and its receptors and proinflammatory cytokines in activated microglia.     

Analgesic Effect of Piracetam on Peripheral Neuropathic Pain Induced by Chronic Constriction Injury of Sciatic Nerve in Rats

Despite immense advances in the treatment strategies, management of neuropathic pain remains unsatisfactory. Piracetam is a prototype of nootropic drugs, used to improve cognitive impairment. The present study was designed to investigate the effect of piracetam on peripheral neuropathic pain in rats. Neuropathic pain was induced by the chronic constriction injury of the sciatic nerve. Following this, piracetam was intraperitoneally administered for 2 weeks in doses of 50, 100 and 200 mg/kg, and pain was assessed by employing the behavioural tests for thermal hyperalgesia (hot plate and tail flick tests) and cold allodynia (acetone test). After the induction of neuropathic pain, significant development of thermal hyperalgesia and cold allodynia was observed. The administration of piracetam (50 mg/kg) did not have any significant effect on all the behavioural tests. Further, piracetam (100 mg/kg) also had no effect on the hot plate and tail flick tests; however it significantly decreased the paw withdrawal duration in the acetone test. Piracetam in a dose of 200 mg/kg significantly modulated neuropathic pain as observed from the increased hot plate and tail flick latencies, and decreased paw withdrawal duration (in acetone test). Therefore, the present study suggests the potential use of piracetam in the treatment of neuropathic pain, which merits further clinical investigation.     

Fractalkine and minocycline alter neuronal activity in the spinal cord dorsal horn

Fractalkine (FKN) evokes nociceptive behavior in nai ve rats, whereas minocycline attenuates pain acutely after neuronal injury. We show that, in nai ve rats, FKN causes hyperresponsiveness of lumbar wide dynamic range neurons to brush, pressure and pinch applied to the hindpaw. One day after spinal nerve ligation (SNL), minocycline attenuates after-discharge and responses to brush and pressure. In contrast, minocycline does not alter evoked neuronal responses 10 days after SNL or sciatic constriction, but increases spontaneous discharge. We speculate that microglia rapidly alter sensory neuronal activity in nai ve and neuropathic rats acutely, but not chronically, after injury.     

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population^1,2. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie''s (1988) unilateral sciatic nerve chronic constriction injury (CCI)³ (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity.CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs⁴. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter^5,6. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.     

Discovery of tetrahydropyrido[4,3-d]pyrimidine derivatives for the treatment of neuropathic pain

A series of tetrahydropyridopyrimidine derivatives were synthesized and evaluated for neurotoxicity and peripheral analgesic activity followed by assessment of antiallodynic and antihyperalgesic potential in two peripheral neuropathic pain models, the chronic constriction injury (CCI) and partial sciatic nerve ligation (PSNL). Compounds (4b and 4d) exhibiting promising efficacies in four behavioral assays of allodynia and hyperalgesia (spontaneous pain, tactile allodynia, cold allodynia and mechanical hyperalgesia) were quantified for their ED₅₀ values (15.12–65.10 mg/kg). Studies carried out to assess the underlying mechanism revealed that the compounds suppressed the inflammatory component of the neuropathic pain and prevented oxidative and nitrosative stress.     

Losartan, an antagonist of AT1 receptor for angiotensin II, attenuates lipopolysaccharide-induced acute lung injury in rat

Angiotensin II (Ang II) plays an important role in inflammatory process. Acute lung injury (ALI), an inflammatory disorder of the lung, is commonly associated with endotoxemia; however, the mechanism that endotoxin (lipopolysaccharide, LPS) induces the inflammatory response in ALI is not well defined. Here, we showed, in LPS-induced ALI rat model, that Ang II and Ang II type 1 (AT₁) receptor were significantly increased in lung tissues, compared with those in controls. Meanwhile, nuclear factor (NF)-κB-DNA-binding activity, tumor necrosis factor (TNF)-α mRNA, and pneumocytic apoptosis were significantly increased. Moreover, pretreatment of rats with losartan, an antagonist of AT₁ receptor for Ang II, improved the inflammation, reduced the elevation of Ang II and AT₁ receptor, and inhibited NF-κB-DNA-binding activity, expression of TNF-α mRNA, and pneumocytic apoptosis. The data indicate that Ang II may mediate the inflammatory process in LPS-induced ALI through AT₁ receptor, which can be blocked by losartan.     

Impaired Excitatory Drive to Spinal Gabaergic Neurons of Neuropathic Mice

Adequate pain sensitivity requires a delicate balance between excitation and inhibition in the dorsal horn of the spinal cord. This balance is severely impaired in neuropathy leading to enhanced pain sensations (hyperalgesia). The underlying mechanisms remain elusive. Here we explored the hypothesis that the excitatory drive to spinal GABAergic neurons might be impaired in neuropathic animals. Transgenic adult mice expressing EGFP under the promoter for GAD67 underwent either chronic constriction injury of the sciatic nerve or sham surgery. In transverse slices from lumbar spinal cord we performed whole-cell patch-clamp recordings from identified GABAergic neurons in lamina II. In neuropathic animals rates of mEPSC were reduced indicating diminished global excitatory input. This downregulation of excitatory drive required a rise in postsynaptic Ca²⁺. Neither the density and morphology of dendritic spines on GABAergic neurons nor the number of excitatory synapses contacting GABAergic neurons were affected by neuropathy. In contrast, paired-pulse ratio of Aδ- or C-fiber-evoked monosynaptic EPSCs following dorsal root stimulation was increased in neuropathic animals suggesting reduced neurotransmitter release from primary afferents. Our data indicate that peripheral neuropathy triggers Ca²⁺-dependent signaling pathways in spinal GABAergic neurons. This leads to a global downregulation of the excitatory drive to GABAergic neurons. The downregulation involves a presynaptic mechanism and also applies to the excitation of GABAergic neurons by presumably nociceptive Aδ- and C-fibers. This then leads to an inadequately low recruitment of inhibitory interneurons during nociception. We suggest that this previously unrecognized mechanism of impaired spinal inhibition contributes to hyperalgesia in neuropathy.     

Functional characterization and analgesic effects of mixed cannabinoid receptor/T-type channel ligands

Background

Increased neuronal excitability and spontaneous firing are hallmark characteristics of injured sensory neurons. Changes in expression of various voltage-gated Na⁺ channels (VGSCs) have been observed under neuropathic conditions and there is evidence for the involvement of protein kinase C (PKC) in sensory hyperexcitability. Here we demonstrate the contribution of PKC to P2X-evoked VGSC activation in dorsal root ganglion (DRG) neurons in neuropathic conditions.

Results

Using the spinal nerve ligation (SNL) model of neuropathic pain and whole-cell patch clamp recordings of dissociated DRG neurons, we examined changes in excitability of sensory neurons after nerve injury and observed that P2X3 purinoceptor-mediated currents induced by α,β-meATP triggered activation of TTX-sensitive VGSCs in neuropathic nociceptors only. Treatment of neuropathic DRGs with the PKC blocker staurosporine or calphostin C decreased the α,β-meATP-induced Na⁺ channels activity and reversed neuronal hypersensitivity. In current clamp mode, α,β-meATP was able to evoke action-potentials more frequently in neuropathic neurons than in controls. Pretreatment with calphostin C significantly decreased the proportion of sensitized neurons that generated action potentials in response to α,β-meATP. Recordings measuring VGSC activity in neuropathic neurons show significant change in amplitude and voltage dependence of sodium currents. In situ hybridization data indicate a dramatic increase in expression of embryonic Na_v1.3 channels in neuropathic DRG neurons. In a CHO cell line stably expressing the Na_v1.3 subunit, PKC inhibition caused both a significant shift in voltage-dependence of the channel in the depolarizing direction and a decrease in current amplitude.

Conclusion

Neuropathic injury causes primary sensory neurons to become hyperexcitable to ATP-evoked P2X receptor-mediated depolarization, a phenotypic switch sensitive to PKC modulation and mediated by increased activity of TTX-sensitive VGSCs. Upregulation in VGSC activity after injury is likely mediated by increased expression of the Na_v1.3 subunit, and the function of the Na_v1.3 channel is regulated by PKC.     

Endogenous CBS–H<Subscript>2</Subscript>S Pathway Contributes to the Development of CCI-Induced Neuropathic Pain

Studies showed a complex relationship between hydrogen sulfide (H₂S) and neuropathic pain. In this study, the relationship between endogenous CBS–H₂S pathway in L_4–6 spinal cord and neuropathic pain was explored. A total of 163 adult Kunming mice were used in this study. CBS expression and H₂S formation in L_4–6 spinal cord were detected in the development of neuropathic pain firstly. Then, effect of AOAA, an CBS inhibitor, on treatment of neuropathic pain by chronic construction injury surgery (CCI) was detected. Pain thresholds and activation of NF-κB(p65), ERK1/2 and CREB were measured as biomarks of neuropathic pain. Results showed that CCI surgery significantly upregulated protein expression of CBS and H₂S formation. Correlation analysis showed pain thresholds had negative relationships with protein expression of CBS and H₂S formation. Treatment with AOAA, a CBS inhibitor, inhibited CCI-induced upregulation of CBS expression and H₂S formation (P < 0.05). Further, AOAA significantly decreased activation of NF-κB(p65), ERK1/2 and CREB pathway, and reversed CCI-induced allodynia (P < 0.05). This indicated that CBS–H₂S pathway promoted the development of neuropathic pain. CBS–H₂S pathway could be a promising target for treatment of neuropathic pain.     

miR-202 modulates the progression of neuropathic pain through targeting RAP1A

Neuropathic pain is a somatosensory disorder which is caused by disease or nerve injury that affects the nervous system. microRNAs (miRNAs) are proved to play crucial roles in the development of neuropathic pain. However, the role of miR-202 in neuropathic pain is still unknown. Sprague-Dawley rats were used for constructing the neuropathic pain model. The expression of miR-202 was determined by quantitative real-time polymerase chain reaction. Potential target gene for miR-202 was measured using bioinformatics methods and Western blot analysis. In this study, we used rats to establish a neuropathic pain model and measured the effect of miR-202 in neuropathic pain. We demonstrated that miR-202 expression was downregulated in the spinal dorsal horn of bilateral sciatic nerve chronic constriction injury (bCCI) rat. However, miR-202 expression was not changed in the dorsal root ganglion, hippocampus, and anterior cingulated cortex of bCCI rat. We identified that RAP1A was a direct target gene of miR-202 in the PC12 cell. RAP1A expression was upregulated in the spinal dorsal horn of bCCI rat. Overexpression of miR-202 could improve the pain threshold for bCCI rats in both hindpaws, indicating that miR-202 overexpression could lighten the pain threshold for model rats. Moreover, RAP1A overexpression increased the pain threshold effect of miR-202 overexpression treated bCCI rats, indicating that miR-202 could lighten the pain threshold through inhibiting RAP1A expression. These data suggested that miR-202 acted pivotal roles in the development of neuropathic pain partly through targeting RAP1A gene.     

MiR-183-5p Alleviates Chronic Constriction Injury-Induced Neuropathic Pain Through Inhibition of TREK-1

MicroRNAs have been implicated in nerve injury and neuropathic pain. In the previous study we had shown that miR-96 can attenuate neuropathic pain through inhibition of Nav1.3. In this study, we investigated the role of miR-183, a same cluster member of microRNA with miR-96, in neuropathic pain and its potential mechanisms. We found that the expression level of miR-183-5p in dorsal root ganglion was decreased with the development of neuropathic pain induced by chronic constriction sciatic nerve injury (CCI). By contrast, the TREK-1, a K⁺ channel, was increased. Further investigation identified that intrathecal injection of miR-183-5p mimic efficiently ameliorated neuropathic pain and inhibited the expression of TREK-1, a predicted target gene of miR-183-5p. Luciferase assays confirmed the binding of miR-183-5p and TREK-1. In addition, over-expression of TREK-1 blocked the roles of miR-183-5p in neuropathic pain. Our findings suggested that miR-183-5P participated in the regulation of CCI-induced neuropathic pain through inhibiting the expression of TREK-1.     

Intrathecal NGF Administration Reduces Reactive Astrocytosis and Changes Neurotrophin Receptors Expression Pattern in a Rat Model of Neuropathic Pain

Nerve growth factor (NGF), an essential peptide for sensory neurons, seems to have opposite effects when administered peripherally or directly to the central nervous system. We investigated the effects of 7-days intrathecal (i.t.) infusion of NGF on neuronal and glial spinal markers relevant to neuropathic behavior induced by chronic constriction injury (CCI) of the sciatic nerve. Allodynic and hyperalgesic behaviors were investigated by Von Frey and thermal Plantar tests, respectively. NGF-treated animals showed reduced allodynia and thermal hyperalgesia, compared to control animals. We evaluated on lumbar spinal cord the expression of microglial (ED-1), astrocytic (GFAP and S-100β), and C- and Aδ-fibers (SubP, IB-4 and Cb) markers. I.t. NGF treatment reduced reactive astrocytosis and the density of SubP, IB4 and Cb positive fibers in the dorsal horn of injured animals. Morphometric parameters of proximal sciatic nerve stump fibers and cells in DRG were also analyzed in CCI rats: myelin thickness was reduced and DRG neurons and satellite cells appeared hypertrophic. I.t. NGF treatment showed a beneficial effect in reversing these molecular and morphological alterations. Finally, we analyzed by immunohistochemistry the expression pattern of neurotrophin receptors TrkA, pTrkA, TrkB and p75^NTR. Substantial alterations in neurotrophin receptors expression were observed in the spinal cord of CCI and NGF-treated animals. Our results indicate that i.t. NGF administration reverses the neuro-glial morphomolecular changes occurring in neuropathic animals paralleled by alterations in neurotrophin receptors ratio, and suggest that NGF is effective in restoring homeostatic conditions in the spinal cord and maintaining analgesia in neuropathic pain.