Assay of therapeutic ultrasound induced-antinociception in experimental trigeminal neuropathic pain

Trigeminal neuralgia is considered one of the most painful conditions, and pharmacological treatment can be as debilitating as the pathology itself. The aim of this work was to evaluate the effectiveness of pulsed therapeutic ultrasound (TU) on an experimental rat model of trigeminal neuropathic nociception (chronic constriction injury-infraorbital nerve; CCI-ION). To evaluate facial thermonociception, an apparatus that measured the reaction time for head withdrawal was constructed. After surgery, a gradual reduction in reaction time was observed until day 15 post-CCI, when the values became constant. Three ipsilateral applications of TU to post-CCI rats promoted an increase in latency time. This antinociceptive effect was evident even after the first TU application, reaching maximal values at 24 hr. The magnitude of this effect was proportional to ultrasonic wave intensity (0.3 and 0.4 W/cm(2)). Posttreatment with naltrexone (5 mg/kg, s.c.) completely blocked the hypoalgesic effect of TU. Pretreatment with an opioid antagonist was unable to block the antinociceptive effect during the first 8 hr, suggesting that opioids are involved only in the latter phase of the TU effects. Myeloperoxidase (MPO) levels in the infraorbital nerve were not increased by TU use, indicating that TU causes no injury or is at least insufficient to induce neutrophil migration. In conclusion, TU is an effective resource in a model of trigeminal neuropathic pain, with a mechanism involving opioid receptor activation, confirming its potential usefulness in the treatment of trigeminal neuralgia.     

Comparison of sildenafil,fluoxetine and its co-administration against chronic constriction injury induced neuropathic pain in rats: An influential additive effect

ABSTRACT

Current drug treatment available for neuropathic pain (NP) provides meager and partial pain relief due to incomplete efficacy and dose-dependent adverse effect. Hence, combination therapy can provide prolongation in analgesic effect with milder side effects. The present investigation aimed at observing the effects of sildenafil (SD) on Fluoxetine (FLX) in attenuation of chronic constriction injury (CCI) induced NP in rats. CCI was achieved in rats by placing four loose ligations around the sciatic nerve and rats were received respective treatments on SD and FLX till 14 days further behaviors parameters like heat hyperalgesia and allodynia, pin prick and acetone drop test were executed in order to access thermal, mechanical and cold allodynia, respectively, on a predetermined time interval. On the 21st day the animals were sacrificed for determination of total protein, myeloperoxidase activity in the adjoining muscular tissues while glutathione and TNF-α in the sciatic nerve. Co-administration of SD + FLX + CCI gave the pronounced effect that was superior over individual responses of SD and FLX in all behavioral as well as biochemical parameters. It was observed that attenuation in the altered behavioral pattern of CCI induced rats was modified prominently from 3rd day only in a group of rats treated with SD + FLX + CCI. The whole study was finally supported by histopathological results. Finally, it was concluded that SD produces an additive effect when given with FLX in attenuation of NP may be due to elevation in the level of intracellular concentrations of cyclic guanosine monophosphate which further causes downregulation of calcium channel.     

RSEP1 is a novel gene with functional involvement in neuropathic pain behaviour

Neuropathic pain from nerve injury by trauma, disease or surgery often causes prolonged suffering. To explore the molecular mechanisms that underlie neuropathic pain, we used mRNA from the L4--5 segments of the lumbar spinal cord of rats with chronic constriction injury (CCI)-induced neuropathic pain, and differentially screened a cDNA library from the rat brain. A novel gene, termed RSEP1 (Rat Spinal cord Expression Protein 1), was identified. Northern blots revealed that RSEP1 was expressed mainly in the central nervous system including the cerebral cortex, hippocampus, brainstem and spinal cord, as well as in the kidney and ovary. In situ hybridization showed a high level of RSEP1 expression in the CA1, CA3 and dentate gyrus regions of the hippocampus and the small sensory neurons in the dorsal horn, as well as the large neurons in the ventral horn of the spinal cord. Intrathecal injection of RSEP1 antisense oligonucleotide into the spinal cord lumbar enlargement attenuated neuropathic pain behaviours in CCI rats, suggesting a functional involvement of RSEP1 in neuropathic pain.     

Variation in rat sciatic nerve anatomy: implications for a rat model of neuropathic pain

We discovered a variation of rat sciatic nerve anatomy as an incidental finding during the anatomical exploration of the nerve lesion site in a rat neuropathic pain model. To confirm the composition and distribution of rat sciatic nerve, macroscopic anatomical investigation was performed in both left and right sides in 24 adult Sprague-Dawley rats. In all rats, the L4 and L5 spinal nerves were fused tightly to form the sciatic nerve. However, the L6 spinal nerve did not fuse with this nerve completely as a part of the sciatic nerve, but rather sent a thin branch to it in 13 rats (54%), whereas in the remaining 11 rats (46%), L6 ran separately along with the sciatic nerve. Also, the L3 spinal nerve sent a thin branch to the L4 spinal nerve or sciatic nerve in 6 rats (25%). We conclude that the components of sciatic nerve in Sprague-Dawley rats vary from L3 to L6; however, the major components are L4 and L5 macroscopically. This finding is in contrast to the standard textbooks of rat anatomy which describe the sciatic nerve as having major contributions from L4, L5, and L6.     

Antinociceptive effects of galanin in the rat tuberomammillary nucleus and the plasticity of galanin receptor 1 during hyperalgesia

Although the tuberomammillary nucleus (TM) is well defined in terms of anatomy and neurochemistry, little is known about its function in nociceptive modulation. There was an abundance of galanin-immunoreactive fibers in the TM, and galanin has been implicated in pain processing. The present study assessed the role of galanin in the modulation of nociception in the TM of rats. Intra-TM injection of galanin dose-dependently increased the hindpaw withdrawal latency of rats to a noxious thermal stimulus, indicating an antinociceptive role of galanin in the TM. The antinociceptive effect of galanin was blocked by a subsequent intra-TM injection of galantide, a putative galanin receptor antagonist, suggesting that the antinociceptive effect of galanin is mediated by galanin receptors. Moreover, there was abundant galanin receptor 1 (GalR1) in the TM, and the number of GalR1-positive neurons in the ipsilateral TM increased significantly after unilateral loose ligation of the sciatic nerve compared with the contralateral TM or the TM of intact rats. However, the number of GalR1-positive neurons was not significantly altered by carrageenan-induced inflammation, in either the ipsilateral or the contralateral TM. The results suggest that galanin and GalR1 in the TM may play important roles in pain regulation.     

Ca2+/calmodulin-dependent protein kinase II in the spinal cord contributes to neuropathic pain in a rat model of mononeuropathy

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is known to subserve activity-dependent neuronal plasticity in the central nervous system. To examine in vivo the implication of spinal CaMKII activity in the generation and development of neuropathic pain after peripheral nerve injury, we used an animal model of mononeuropathy, the chronic constriction injury (CCI) model, in the rat. We found that, 3 days after CCI, the total CaMKII (tCaMKII) immunoreactivity increased in the superficial laminae of the spinal cord and this increase continued for up to 14 days. The immunoreactivity of phosphorylated CaMKII showed an increase from 1 day after CCI, which preceded the up-regulation of tCaMKII. A non-selective N-methyl-d-aspartate receptor antagonist, MK801, significantly attenuated the increase of tCaMKII and phosphorylated CaMKII. Moreover, intrathecal administration of an inhibitor of CaMKII, KN93, before the CCI surgery attenuated the development of thermal hyperalgesia and mechanical allodynia. In addition, KN93 significantly reduced the nociceptive behavior in phase II of the formalin test. These findings demonstrate that the activity of CaMKII in spinal neurons is elevated after peripheral nerve injury and may be involved in central sensitization. The alteration of CaMKII is considered to be a neuroplastic change that occurs in spinal neurons that contributes to neuropathic pain, suggesting the potential for the development of novel therapeutics for neuropathic pain that target CaMKII.     

Local nitric oxide synthase activity in a model of neuropathic pain

A local inflammatory reaction may play an important role in the development of neuropathic pain following peripheral nerve injury. One important participant in the inflammatory response of injured peripheral nerve may be nitric oxide (NO). In this work, we examined physiological and morphological evidence for nitric oxide synthase (NOS) activation in the chronic constriction injury model of neuropathic pain in rats. Physiological evidence of local NO action was provided by studying NO-mediated changes in local blood flow associated with the injury site. Immunohistochemistry was used to localize isoforms of NOS that might generate NO. Sciatic nerve injury associated with behavioural evidence of neuropathic pain had substantial rises in local blood flow. The NOS inhibitor N^G-nitro-l -arginine methyl ester (L-NAME), but not N^G-nitro-d -arginine methyl ester (D-NAME), reversed the hyperaemia in a dose-dependent fashion proximal to the constriction at 48 h and distally at 14 days post-operation when applied systemically or topically. Aminoguanidine, a NOS inhibitor with relatively greater selectivity for the inducible NOS (iNOS) isoform, reversed nerve hyperaemia distal to the constriction only at 14 days. NOS-like immunoreactivity of the neuronal and endothelial isoforms was identified just proximal to the constriction at 48 h. iNOS-like immunoreactivity was observed at 7 and 14 days at the constriction and distal sites, respectively. This work provides evidence for local NOS expression and NO action in the chronic constriction injury model of neuropathic pain. NO has local physiological actions that include vasodilatation of microvessels and that may be important in the development of pain sensitivity.     

Sympathetic sprouting and changes in nociceptive sensory innervation in the glabrous skin of the rat hind paw following partial peripheral nerve injury

Previous studies have suggested that sympathetic sprouting in the periphery may contribute to the development and persistence of sympathetically maintained pain in animal models of neuropathic pain. In the present study, we examined changes in the cutaneous innervation in rats with a chronic constriction injury to the sciatic nerve. At several periods postinjury, hind paw skin was harvested and processed by using a monoclonal antibody against dopamine-beta-hydroxylase to detect sympathetic fibers and a polyclonal antibody against calcitonin gene-related peptide to identify peptidergic sensory fibers. We observed migration and branching of sympathetic fibers into the upper dermis of the hind paw skin, where they were normally absent. This migration was first detected at 2 weeks, peaked at 4-6 weeks, and lasted for at least 20 weeks postlesion. At 8 weeks postlesion, there was a dramatic increase in the density of peptidergic fibers in the upper dermis. Quantification revealed that densities of peptidergic fibers 8 weeks postlesion were significantly above levels in sham animals. The ectopic sympathetic fibers did not innervate blood vessels but formed a novel association and wrapped around sprouted peptidergic nociceptive fibers. Our data show a long-term sympathetic and sensory innervation change in the rat hind paw skin after the chronic constriction injury. This novel fiber arrangement after nerve lesion may play an important role in the development and persistence of sympathetically maintained neuropathic pain after partial nerve lesions.     

TRPM2 participates the transformation of acute pain to chronic pain during injury‐induced neuropathic pain

Transient receptor melastatin 2 (TRPM2) is a nonselective Ca²⁺‐permeable cation channel highly expressed in brain and other tissues. Studies showed that TRPM2 contributed to the induction of inflammatory cytokine and chemokine of immune cells, resulted in neuropathic pain. However, how TRPM2 regulates neuropathic pain is not clear. The sciatic nerve chronic constriction injury (CCI) rat model was used to induce chronic neuropathic pain. The RNA and protein level of TRPM2 was detected with real‐time PCR and western blot. SiRNA targeting TRPM2 was used to knockdown the expression of TRPM2. Reactive oxygen species (ROS) levels were determined using H2DCFDA assay and NO production was analyzed by measuring the accumulated level of its stable metabolite (nitrite). We found that CCI significantly increased TRPM2 expression in dorsal root ganglion and spinal cord. Knockdown TRPM2 in early phase after CCI alleviated injury‐induced neuropathic pain. Mechanistically, we demonstrated that TRPM2 knockdown drastically inhibited the iNOS expression and NO generation, with decreased ROS generation in CCI rat. TRPM2 participates in the transformation of acute pain to chronic pain during injury‐induced neuropathic pain, which might serve as a potential therapeutic target for neuropathic pain.     

Effects of decompression on neuropathic pain behaviors and skin reinnervation in chronic constriction injury

Decompression is an important therapeutic strategy to relieve neuropathic pain clinically; there is, however, lack of animal models to study its temporal course of neuropathic pain behaviors and its influence on nerve regeneration to sensory targets. To address these issues, we established a model of decompression on rats with chronic constriction injury (CCI) and investigated the effect on skin reinnervation. Animals were divided into a decompression group, in which the ligatures were removed, and a CCI group, in which the ligatures remained at postoperative week 4 (POW 4). At this time point, the skin innervation indexes of protein gene product 9.5 (PGP 9.5), substance P (SP), and calcitonin gene-related peptide (CGRP) were reduced in both groups to similar degrees. Beginning from POW 6, the decompression group exhibited significant reductions of thermal hyperalgesia and mechanical allodynia compared to the CCI group (p<0.001). At POW 8, neuropathic pain behaviors had completely disappeared in the decompression group, and the decompression group had a higher skin innervation index of SP than the CCI group (0.45+/-0.05 vs. 0.16+/-0.03, p<0.001). These indexes were similar in both groups for PGP 9.5 (0.32+/-0.09 vs. 0.14+/-0.04, p=0.11) and CGRP (0.38+/-0.06 vs. 0.21+/-0.07, p=0.09). These findings demonstrate the temporal changes in the disappearance of neuropathic pain behaviors after decompression and suggest that decompression causes different patterns of skin reinnervation for different markers of skin innervation.     

Cellular mechanisms of hyperalgesia and spontaneous pain in a spinalized rat model of peripheral neuropathy: changes in myelinated afferent inputs implicated

Various hypotheses have been proposed to account for the mechanical hyperalgesia and spontaneous pain seen in animal models of peripheral neuropathy. The purpose of the present study was to determine whether there exists a spinal neuronal correlate to these properties. An experimental neuropathy was induced in male Sprague-Dawley rats by placing a 2-mm PE-90 polyethylene cuff around the sciatic nerve. All rats were subsequently confirmed to exhibit mechanical allodynia in the von Frey test. After induction of anaesthesia with pentobarbital and acute spinalization at T9, electrophysiological experiments were performed, recording extracellular single unit activity from ipsi- and contralateral wide dynamic range dorsal horn neurons in spinal segments L1-4. On-going activity was greater in short-term (11-22 days after cuff implantation) and long-term (42-52 days) cuff-implanted rats; 38 spikes/s in short-term versus 19 spikes/s in controls; 29 spikes/s in long-term ipsi- and contralateral neurons. Receptive fields in controls were always restricted, but in almost all cuff-implanted rats extended over the whole hind paw. Responses to noxious mechanical (pinch) and noxious heat stimulation of the cutaneous receptive field in controls consisted of the typical fast initial discharge followed by an afterdischarge. In all neurons from cuff-implanted rats the initial discharge resembled that in controls. However, the afterdischarge, particularly that in response to noxious pinch, was markedly greater in both magnitude and duration. It is suggested that the greater on-going discharge is the cellular correlate of spontaneous pain, and the potentiation of the afterdischarge in response to noxious stimulation is the correlate of hyperalgesia. Given that acutely spinalized rats were tested, only peripheral and/or spinal mechanisms can be considered to explain these data. Considering all the data, it can be concluded that there is a greater change in fibres mediating noxious mechanical than noxious thermal inputs. Among different hypotheses, the one with which the present data are most compatible is that which proposes that chronic nerve injury or inflammation induces phenotypic changes predominantly in myelinated afferents. There may be a redistribution of membrane-bound ion channels, predominantly sodium channels, which leads to ectopic activity and thus spontaneous discharge of dorsal horn neurons. With regard to mechanical stimulation-evoked synaptic input, the central terminals of myelinated afferents expand into regions of the spinal cord which normally receive their predominant input from unmyelinated nociceptive afferents. This may be coupled with a change in these myelinated afferents so that they now synthesize and release peptides, primarily substance P, from their central terminals with the result that the effects of their chemical mediators of synaptic transmission add to the effects of nociceptive inputs leading to exaggerated responses to painful stimuli, thus the basis of clinical hyperalgesia.     

Effect of a graded single constriction of the rat sciatic nerve on pain behavior and expression of immunoreactive NPY and NPY Y1 receptor in DRG neurons and spinal cord

In the present study, the rat sciatic nerve was constricted to varying degrees using only one ligature with a very thin polyethylene sheath placed between nerve and ligature thread. Complete nerve transection was studied for comparison. With a 40-80% constriction of the nerve we observed allodynia to a similar extent as in the so-called Bennett model based on four loose ligatures. We also monitored changes in the expression of neuropeptide Y (NPY) and the NPY Y1 receptor (Y1R) in the lumbar 4-5 dorsal root ganglia (DRG) and dorsal horn and found upregulation of NPY and downregulation of the Y1R in DRG neurons after injury. These results indicate that similar peptide and receptor changes occur in this model as after axotomy and in other nerve injury models, although the immunohistochemical and behavioral changes seem to be dependent on the degree of constriction of the nerve. Thus, it seems relevant to monitor the degree of constriction when evaluating pain and other post-injury events. The possibility that some of the changes in NPY-ergic neurotransmission are related to the generation of allodynia is discussed; as well as the possibility to use this mononeuropathic model based on a single ligature nerve constriction (SLNC) as a complementary approach to other widely used pain models.     

Estrogen affects neuropathic pain through upregulating N-methyl-D-aspartate acid receptor 1 expression in the dorsal root ganglion of rats

Estrogen affects the generation and transmission of neuropathic pain,but the specific regulatory mechanism is still unclear.Activation of the N-methyl-D-aspartate acid receptor 1(NMDAR1) plays an important role in the production and maintenance of hyperalgesia and allodynia.The present study was conducted to determine whether a relationship exists between estrogen and NMDAR1 in peripheral nerve pain.A chronic sciatic nerve constriction injury model of chronic neuropathic pain was established in rats.These rats were then subcutaneously injected with 17β-estradiol,the NMDAR1 antagonist D(-)-2-amino-5-phosphonopentanoic acid(AP-5),or both once daily for 15 days.Compared with injured drug na?ve rats,rats with chronic sciatic nerve injury that were administered estradiol showed a lower paw withdrawal mechanical threshold and a shorter paw withdrawal thermal latency,indicating increased sensitivity to mechanical and thermal pain.Estrogen administration was also associated with increased expression of NMDAR1 immunoreactivity(as assessed by immunohistochemistry) and protein(as determined by western blot assay) in spinal dorsal root ganglia.This 17β-estradiol-induced increase in NMDAR1 expression was blocked by co-administration with AP-5,whereas AP-5 alone did not affect NMDAR1 expression.These results suggest that 17β-estradiol administration significantly reduced mechanical and thermal pain thresholds in rats with chronic constriction of the sciatic nerve,and that the mechanism for this increased sensitivity may be related to the upregulation of NMDAR1 expression in dorsal root ganglia.     

Spinal P2X(7) receptor mediates microglia activation-induced neuropathic pain in the sciatic nerve injury rat model

P2X₇ receptor is an important member of ATP-sensitive ionotropic P2X receptors family, which includes seven receptor subtypes (P2X₁-P2X₇). Recent evidence indicates that P2X₇R participates in the onset and persistence of neuropathic pain. In tetanic stimulation of the sciatic nerve model, P2X₇R was involved in the activation of microglia, but whether this happens in other neuropathic pain models remains unclear. In this study we used immunohistochemistry and Western blot to explore the relationship of P2X₇R expression with microglia activation, and with mechanical allodynia and thermal hypersensitivity in the chronic constriction of the sciatic nerve (CCI) rat model. The results show that following nerve ligature, mechanical allodynia and thermal hypersensitivity were developed within 3 days (d), peaked at 14 d and persisted for 21 d on the injured side. P2X₇R levels in the ipsilateral L4-6 spinal cord were increased markedly after injury and the highest levels were observed on day 14, significant difference was observed at I-IV layers of the dorsal horn. The change in P2X₇R levels in the spinal cord was consistent with the development of mechanical allodynia and thermal hypersensitivity. Intrathecal administration of the P2X₇R antagonist Brilliant Blue G (BBG) reversed CCI-induced mechanical allodynia and thermal hypersensitivity. Double-labeled immunofluorescence showed that P2X₇R expression were restricted to microglia, spinal microglia were activated after nerve injury, which was inhibited by BBG. These results indicated that spinal P2X₇R mediate microglia activation, this process may play an important role in development of mechanical allodynia and thermal hypersensitivity in CCI model.     

Cell death in the superficial dorsal horn in a model of neuropathic pain

The aims of this study were to investigate the occurrence of apoptotic cell death in the dorsal horn of the adult rat spinal cord following chronic constriction injury (CCI) to the sciatic nerve and to correlate this with behavioural responses. Six groups of six rats were used as follows: 1) CCI, 2) CCI, 3) MK801 + CCI, 4) axotomy, 5) sham, and 6) naive. Group 1 animals were behaviourally tested for thermal hyperalgesia 8 days following surgery and sacrificed and the spinal cords removed and frozen. The rest of the groups underwent the same procedure 14 days following surgery. The lumbar region of the spinal cord was cryosectioned and the incidence of apoptotic cells investigated using the TUNEL technique plus Hoechst double labelling. By 8 days post-CCI, hyperalgesia had developed in the ipsilateral paw, which was still present 14 days after the injury compared to the contralateral paw and naive and sham animals. Preemptive MK-801 prevented the onset of hyperalgesia. Significant numbers of apoptotic cells were present in the ipsilateral dorsal horn of the spinal cord 8 and 14 days following CCI compared to the contralateral side and to naive and sham animals. Preemptive treatment with MK-801 reduced the extent of apoptosis resulting from CCI to the level seen in control animals. This study demonstrates that cells undergo apoptosis as a result of CCI simultaneous with the occurrence of hyperalgesia. Furthermore, MK-801 prevents the onset of hyperalgesia and reduces the extent of apoptotic cell death, suggesting, perhaps, that apoptosis contributes to the initiation/maintenance of hyperalgesia.     

Effect of post-injury NMDA antagonist treatment on long-term Fos expression and hyperalgesia in a model of chronic neuropathic pain

Chronic constriction injury (CCI) of the sciatic nerve results in persistent mechanical hyperalgesia together with Fos protein expression in the lumbar spinal cord. We have examined the relationship between mechanical hyperalgesia and Fos expression within the lumbar spinal cord on days 14, 35 and 55 after either CCI or sham operation. To determine the role of NMDA receptor mechanisms in the maintenance of hyperalgesia and Fos expression, the NMDA antagonist MK-801 (0.3 mg kg-1 s.c.) was administered daily on days 28 to 34 after operation. CCI animals developed unilateral hind limb hyperalgesia that persisted unchanged from days 14 to 55 of the study. MK-801 treatment reduced hyperalgesia by 57% (p=0.02) on day 35 in CCI animals but did influence hyperalgesia at day 55. In the spinal cord, Fos positive cells were present bilaterally throughout laminae 3-10 at all time points examined in both CCI and sham group animals. Fos counts ipsilateral to the side of injury in laminae 3-10 correlated significantly with hyperalgesia scores in the CCI but not sham animals. MK-801 treatment resulted in a suppression of Fos expression in ipsilateral laminae 3-4 (p=0.0017) and laminae 5-10 (p=0.0026) of CCI animals on day 35. Fos expression in sham group animals was not inhibited by MK-801 treatment at day 35. These results indicate that Fos expression is maintained by differing mechanisms following nerve injury or sham operation. The functional consequences of Fos expression following nerve injury and sham operation are discussed.     

Molecular mechanisms underlying the effects of acupuncture on neuropathic pain

Acupuncture has been used to treat neuropathic pain for a long time, but its mechanisms of action remain unknown. In this study, we observed the effects of electroacupuncture and manual acu-puncture on neuropathic pain and on ephrin-B/EphB signaling in rats models of chronic constriction injury-induced neuropathic pain. The results showed that manual acupuncture and elec-puncture significantly reduced mechanical hypersensitivity following chronic constriction injury, es-pecially electroacupuncture treatment. Real-time PCR results revealed that ephrin-B1/B3 and EphB1/B2 mRNA expression levels were significantly increased in the spinal dorsal horns of chronic constriction injury rats. Electroacupuncture and manual acupuncture suppressed the high sion of ephrin-B1 mRNA, and elevated EphB3/B4 mRNA expression. Electroacupuncture signifi-cantly enhanced the mRNA expression of ephrin-B3 and EphB3/B6 in the dorsal horns of neuro-pathic pain rats. Western blot results revealed that electroacupuncture in particular, and manual acupuncture, significantly up-regulated ephrin-B3 protein levels in rat spinal dorsal horns. The re-sults of this study suggest that acupuncture could activate ephrin-B/EphB signaling in neuropathic pain rats and improve neurological function.     

Anti-fibroblast growth factor-2 antibodies attenuate mechanical allodynia in a rat model of neuropathic pain

Peripheral nerve injury leads to the activation of spinal cord astrocytes, which contribute to maintaining neuropathic (NP) pain behavior. Fibroblast growth factor-2 (FGF-2), a neurotrophic and gliogenic factor, is upregulated by spinal cord astrocytes in response to ligation of spinal nerves L5 and L6 (spinal nerve ligation [SpNL]). To evaluate the contribution of spinal astroglial FGF-2 to mechanical allodynia following SpNL, neutralizing antibodies to FGF-2 were injected intrathecally. Administration of 18 μg of anti-FGF-2 antibodies attenuated mechanical allodynia at day 21 after SpNL and reduced FGF-2 and glial acidic fibrillary protein mRNA expression and immunoreactivity in the L5 spinal cord segment of rats with SpNL. These results suggest that endogenous astroglial FGF-2 contributes to maintaining NP tactile allodynia associated with reactivity of spinal cord astrocytes and that inhibition of spinal FGF-2 ameliorates NP pain signs.     

miR-384-5p ameliorates neuropathic pain by targeting SCN3A in a rat model of chronic constriction injury

ABSTRACT

Objective: To explore the potential regulation mechanisms of miR-384-5p in Neuropathic pain (NP).

Methods: Rat model of chronic constriction injury (CCI) was established to induce NP in vivo. NP levels were assessed using Withdrawal Threshold (PWT) and Paw Withdrawal Latency (PWL). qPCR and Western blotting were used to determine the relative expression of miR-384-5p and SCN3A. The inflammation response in spinal microglia cells was determined by ELISA assay. Immunofluorescence assay was used to demonstrate the co-localization of miR-384-5p with SCN3A in rat dorsal root ganglions (DRGs). The target genes of miR-384-5p were verified by dual-luciferase report assays.

Results: In the current study, the miR-384-5p expression level was significantly downregulated in CCI rats when comparing to the sham group. In addition, miR-384-5p agomir significantly repressed mechanical allodynia and heat hyperalgesia in CCI rats. Meanwhile, the current study indicated miR‐384‐5p could decrease inflammation progress in spinal microglia cells incubated in lipopolysaccharide. Consistently, overexpression of miR-384-5p obviously depressed inflammation cytokine levels in CCI rats. Dual-luciferase reporter assays indicated that SCN3A is a target gene of miR-384-5p.

Conclusion: miR-384-5p is a negative regulator in the development of neuropathic pain by regulating SCN3A, indicating that miR-384-5p might be a promising therapeutic target in the treatment of neuropathic pain.

Abbreviations: CCI: Chronic constriction injury; ZEB1: Zinc finger E box binding protein-1; MAPK6: Mitogen-activated protein kinase 6; COX-2: cyclooxygenase-2.     

Investigating the possible pain attenuating mechanisms of pregabalin in chronic constriction injury-induced neuropathic pain in rats

Abstract

Aim of the study: The current study was aimed to investigate the neuropathic pain attenuating mechanism of pregabalin using chronic constriction injury (CCI) model in rats.

Material and Methods: The sciatic nerve was ligated by placing four loose ligatures around it to induce neuropathic pain. The pain development in terms of cold allodynia, mechanical hyperalgesia, and heat hyperalgesia was assessed on the 7th and 14th day after surgery, using acetone drop, pinprick, and hot plate tests. On the 14th day after the injury, pain parameters were assessed 30?minutes after administration of pregabalin (30?mg/kg) and sodium nitroprusside (5?mg/kg) in CCI-subjected rats.

Results: CCI led to induction of neuropathic pain, which was more prominent on 14th day in comparison to 7th day. A single administration of pregabalin and sodium nitroprusside on 14th day, markedly reduced pain parameters and increased serum nitrite levels. Pretreatment with L-NAME abolished neuropathic pain attenuating effects of pregabalin suggesting that pregabalin may increase the levels of nitric oxide to mitigate neuropathic pain. Pretreatment with naloxone significantly abrogated pain attenuating effects of pregabalin and sodium nitroprusside in CCI-subjected rats suggesting that pregabalin and nitric oxide-mediated analgesic action are mediated through release of endogenous opioids. Moreover, naloxone failed to modulate pregabalin-induced increase in nitric oxide levels suggesting that the opioid system does not control the nitric oxide levels, and opioids may be downstream modulators of nitric oxide.

Conclusion: Pregabalin may increase the release of nitric oxide, which may increase the release of endogenous opioids to attenuate neuropathic pain in CCI subjected rats.