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.     

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.     

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.     

The role of CaMKII in neuropathic pain and fear memory in chronic constriction injury in rats

Purpose/Aim of the study In this study, we sought to observe the effects of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) on neuropathic pain and fear memory in a rat model of chronic constriction injury (CCI).

Materials and methods Rats were randomly divided into the Sham, Control, CCI and m-AIP groups. In the m-AIP group, an intrathecal injection of m-AIP, the specific antagonist of CaMKII, was given either pretreatment or posttreatment in rats. Mechanical allodynia and thermal hyperalgesia tests were used to test pain behavior, and the passive avoidance test was used to measure fear memory in rats.

Results The right side of hippocampus tissues were taken at varying time points. The expression levels of CaMKII-α, pCaMKII-α, CaMKII-β, pCaMKII-β, NR2A, pNR2A, NR2B and pNR2B were detected by Western blot analysis. Significant pain behaviors and impaired cognitive function were shown after CCI surgery, accompanied by the upregulation of proteins in the hippocampus. Pretreatment with m-AIP appeared to provide a temporary improvement in pain and fear memory and decreased the expression of the above proteins in the hippocampus seven days after surgery. Furthermore, postoperative treatment with m-AIP provided relief for pain behavior and protein expression but did not affect fear memory.

Conclusions These data suggested that CaMKII played an important role in the crosstalk between neuropathic pain and fear memory, indicating that CaMKII may be a potential therapeutic target for neuropathic pain treatment.     

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.     

Electroacupuncture attenuates neuropathic pain after brachial plexus injury

Electroacupuncture has traditionally been used to treat pain, but its effect on pain following brachial plexus injury is still unknown. In this study, rat models of an avulsion injury to the left brachial plexus root(associated with upper-limb chronic neuropathic pain) were given electroacupuncture stimulation at bilateral Quchi(LI11), Hegu(LI04), Zusanli(ST36) and Yanglingquan(GB34). After electroacupuncture therapy, chronic neuropathic pain in the rats' upper limbs was significantly attenuated. Immunofluorescence staining showed that the expression of β-endorphins in the arcuate nucleus was significantly increased after therapy. Thus, experimental findings indicate that electroacupuncture can attenuate neuropathic pain after brachial plexus injury through upregulating β-endorphin expression.     

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.     

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.     

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.     

Preemptive analgesic effects of low-dose ketamine on growth-associated protein expression in dorsal root ganglion of chronic constriction injury model rats★

BACKGROUND： Ketamine is a noncompetitive N-methyl-D-aspartate （NMDA） receptor antagonists and plays an important role in the treatment of pain. OBJECTIVE： To analyze the preemptive analgesic effects of different doses of ketamine on growth-associated protein-43 （GAP43） expression in dorsal root ganglion in a rat model of chronic sciatic nerve constricted injury, and to study the differences between high-dose and low-dose ketamine DESIGN： Randomized controlled animal study. SETTING： Medical College of Shantou University. MATERIALS： Thirty-five adult male Sprague Dawley rats were provided by the Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine. Ketamine hydrochloride injection was provided by Hengrui Pharmaceutical Co., Ltd., Jiangsu. METHODS： This study was performed at the Immunological Laboratory, Medical College of Shantou University from September to December 2006. Model of chronic sciatic nerve constricted injury： after anesthesia, the right sciatic nerve was exposed and ligated l-cm distal to the ischiadic tuberosity with a No. 3-0 cat gut suture. Grouping and intervention： 35 rats were randomly divided into 4 groups： normal control group （n = 5）, chronic constriction injury （CCI） group （n = 10）, low-dose ketamine group （n = 10）, and high-dose ketamine group （n = 10）. Rats in the normal control group did not undergo any surgery or drug intervention. Rats in the CCI group received intraperitoneal injection of saline （1 mL）, and their sciatic nerves were ligated after 10 minutes. Rats in the low-dose ketamine group underwent intraperitoneal injection of ketamine （25 mg/kg） 10 minutes prior to ligation of sciatic nerve; while, rats in the high-dose ketamine group were given intraperitoneal injection of ketamine （50 mg/kg） 10 minutes prior to ligation of sciatic nerve. On the third and the seventh days after surgery, dorsal root ganglion were resected from the sciatic nerve and cut into sections. MAIN OUTCOME MEASURES：     

Delayed reinnervation by nonpeptidergic nociceptive afferents of the glabrous skin of the rat hindpaw in a neuropathic pain model

Painful peripheral neuropathies have been associated with a reorganization of skin innervation. However, the detailed changes in skin innervation by the different afferent fiber types following a neuropathic nerve injury have never been characterized in an animal model of neuropathic pain. Our objective was to thoroughly characterize such changes in the thick skin of the foot in a well-established rat model of neuropathic pain, namely, the chronic constriction injury (CCI) of the sciatic nerve. We used the immunofluorescence detection of calcitonin gene-related peptide (CGRP), purinergic receptor P2X3, and NF200 as markers of peptidergic nociceptive fibers, nonpeptidergic nociceptive C fibers, and myelinated afferents, respectively. We observed that CCI-operated animals developed significant mechanical allodynia and hyperalgesia as well as thermal hyperalgesia. At 3 days following nerve injury, all CCI-operated animals had a significant decrease in the density of fibers immunoreactive (IR) for CGRP, P2X3, and NF200 within the upper dermis. A recovery of CGRP-IR fibers occurred within 4 weeks of nerve injury and sprouting above control levels was observed at 16 weeks. However, the myelinated (NF200-IR) fibers never recovered to control levels within a period of 16 weeks following nerve injury. Interestingly, the P2X3-IR fibers took considerably more time to recover than the CGRP-IR fibers following the initial loss. A loss in P2X3-IR fibers persisted to 16 weeks and recovered to levels above that of control at 1.5 years following nerve injury. Further studies are required to clarify the relevance of these innervation changes for neuropathic pain generation and maintenance.     

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.     

Different pathophysiology underlying animal models of fibromyalgia and neuropathic pain: comparison of reserpine-induced myalgia and chronic constriction injury rats

The reserpine-induced myalgia (RIM) rat manifests fibromyalgia-like chronic pain symptoms. The present study explored the pathophysiology underlying the pain symptoms in the RIM rat and the chronic constriction injury (CCI) rat, an animal model of neuropathic pain as a reference. Nerve tissue samples were collected from the nociception-tested animals for pathological examinations. Additionally, the therapeutic efficacy of a sodium channel blocker mexiletine was assessed in both rats. A slight vacuolization in the substantia nigra (SN) occurred in some of the RIM rats without any other histopathological changes in the brain or peripheral neurons. All the RIM rats, with or without vacuolization, showed hypersensitivity to tactile, muscle pressure, and cold stimuli. In the CCI rat, neurodegenerative changes were apparent in the sciatic nerve and the spinal cord only. CCI rats displayed muscle hyperalgesia in addition to tactile and cold allodynia. Pharmacotherapy with mexiletine did not attenuate the pain in the RIM rat, although it was effective in the CCI rat. Taken together, it is not likely that pain symptoms in RIM rats are caused by degenerative changes at the level of primary afferents and spinal cord, as is the case for CCI rats. The significance of the vacuolization in the SN is less clear at present because of the minor extent of the change and the lack of correlation with nociceptive sensitivity. The pain symptoms in RIM rats could be associated with dysfunction of biogenic amines-mediated CNS pain control even without apparent pathologies in the nervous system.     

Cold intolerance and neuropathic pain after peripheral nerve injury in upper extremity

Cold intolerance and pain can be a substantial problem in patients with peripheral nerve injury. We aimed at investigating the relationships among sensory recovery, cold intolerance, and neuropathic pain in patients affected by upper limb peripheral nerve injury (Sunderland type V) treated with microsurgical repair, followed by early sensory re‐education. In a cross‐sectional clinical study, 100 patients (male/female 81/19; age 40.5 ± 14.8 years and follow‐up 17 ± 5 months, mean ± SD), with microsurgical nerve repair and reconstruction in the upper extremity and subsequent early sensory re‐education, were evaluated, using Cold Intolerance Symptoms Severity questionnaire‐Italian version (CISS‐it, cut‐off pathology >30/100 points), CISS questionnaire‐12 item version (CISS‐12, 0‐46 points‐grouping: healthy that means no cold intolerance [0‐14], mild [15‐24], moderate [25‐34], severe [35‐42], very severe [43‐46] cold intolerance), probability of neuropathic pain (DouleurNeuropathique‐4; [DN4] 4/10), deep and superficial sensibility, tactile threshold (monofilaments), and two‐point discrimination (cutoff S2; Medical Research Council scale for sensory function; [MRC‐scale]). A high CISS score is associated with possible neuropathic pain (DN4 ≥ 4). Both a low CISS‐it score (ie, < 30) and DN4 < 4 is associated with good sensory recovery (MRC ≥ 2). In conclusion patients affected by upper limb peripheral nerve injuries with higher CISS scores more often suffer from cold intolerance and neuropathic pain, and the better their sensory recovery is, the less likely they are to suffer from cold intolerance and neuropathic pain.     

A subpopulation of rats show social and sleep-waking changes typical of chronic neuropathic pain following peripheral nerve injury

Neuropathic conditions for which treatment is sought, the so-called chronic pain syndrome, are characterized usually by complex behavioural disturbances as well as pain. In this study we evaluated whether social behavioural and sleep disruptions occurred after nerve injury. Before and after chronic constriction of the sciatic nerve, resident-intruder and sleep-wake cycles, as well as mechanical and thermal allodynia/hyperalgesia, were quantified. Sciatic nerve injury in all animals reduced withdrawal thresholds to tactile and thermal (cold) stimuli. Resident-intruder and sleep-waking behaviours were altered in some but not all animals. One group (30%, 'persistent change') had enduring reductions in dominant behaviour to an intruder and decreased slow-wave sleep and increased wakefulness during both light and dark cycles. Another group (25%, 'recovery') had a transient reduction in dominant behaviours and decreased slow-wave sleep and increased wakefulness during only the light cycle. In a third group (45%, 'no effect') resident-intruder and sleep-waking behaviours remained normal. Our finding that the degree of 'pain' as inferred from the allodynia/hyperalgesia was identical in all animals suggests that the alterations to resident-intruder and sleep-wake cycles were independent of the level of sensory disturbance. An absence of correlation between intensity of sensory disturbances and measures of disability (loss of sleep, familial/social problems) is also characteristic of human neuropathic pain. These data indicate that: (i) in a subpopulation of animals sciatic injury results in two of the major complex behavioural changes which are characteristic of neuropathic pain in humans; (ii) testing only for allodynia and hyperalgesia is not sufficient to detect this subpopulation.     

Neuropeptide expression following constriction or section of the inferior alveolar nerve in the ferret

Some of the sensory abnormalities that follow peripheral nerve injury may result from the development of ectopic discharge from the damaged axons. Previous studies in our laboratory have shown that, following tight ligation of the inferior alveolar nerve (IAN), there is a close association between the time-course of this neural activity and the accumulation of neuropeptides at the injury site. In this study we investigated whether the type of injury has any effect on the time-course or level of neuropeptide expression. In 36 adult ferrets, the IAN was either loosely constricted or sectioned, and the animals left to recover for 3 days, 3 weeks, or 3 months. The tissue was processed using indirect immunofluorescence and image analysis was used to quantify levels of substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, enkephalin, galanin, and neuropeptide Y. Immunoreactivity to all of the neuropeptides was present within the injured nerve 3 days after both types of injury, and decreased to lower levels by 3 weeks and 3 months. Comparisons between the levels of neuropeptide immunoreactivity in each group revealed that the pattern of accumulation was similar following loose constriction or section, and also similar to that found in our previous study on tight ligation. For each injury the time-course of neuropeptide expression was similar to that of the spontaneous activity we had previously recorded. These data support the suggestion that neuropeptide accumulation may be linked to the development of ectopic neural activity but indicate that the type of injury has little effect on the extent of expression.     

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.     

Adoptive transfer of peripheral immune cells potentiates allodynia in a graded chronic constriction injury model of neuropathic pain

Recent evidence demonstrates that peripheral immune cells contribute to the nociceptive hypersensitivity associated with neuropathic pain by infiltrating the central nervous system (CNS). We have recently developed a rat model of graded chronic constriction injury (CCI) by varying the exposure of the sciatic nerve and control non-nerve tissue to surgical placement of chromic gut. We demonstrate that splenocytes can contribute significantly to CCI-induced allodynia, as adoptive transfer of these cells from high pain donors to low pain recipients potentiates allodynia (P < 0.001). The phenomenon was replicated with peripheral blood mononuclear cells (P < 0.001). Adoptive transfer of allodynia was not achieved in sham recipients, indicating that peripheral immune cells are only capable of potentiating existing allodynia, rather than establishing allodynia. As adoptively transferred cells were found by flow cytometry to migrate to the spleen (P < 0.05) and potentiation of allodynia was prevented in splenectomised low pain recipients, adoptive transfer of high pain splenocytes may induce the migration of host-derived immune cells from the spleen to the CNS as observed by flow cytometry (P < 0.05). Importantly, intrathecal transfer of CD45⁺ cells prepared from spinal cords of high pain donors into low pain recipients led to potentiated allodynia (P < 0.001), confirming that infiltrating immune cells are not passive bystanders, but actively contribute to nociceptive hypersensitivity in the lumbar spinal cord.     

Expression and effect of sodium-potassium-chloride cotransporter on dorsal root ganglion neurons in a rat model of chronic constriction injury

Sodium-potassium-chloride cotransporter 1 (NKCC1) and potassium-chloride cotransporter 2 (KCC2) are associated with the transmission of peripheral pain.We investigated whether the increase of NKCC1 and KCC2 is associated with peripheral pain transmission in dorsal root ganglion neurons.To this aim,rats with persistent hyperalgesia were randomly divided into four groups.Rats in the control group received no treatment,and the rat sciatic nerve was only exposed in the sham group.Rats in the chronic constriction injury group were established into chronic constriction injury models by ligating sciatic nerve and rats were given bumetanide,an inhibitor of NKCC1,based on chronic constriction injury modeling in the chronic constriction injury + bumetanide group.In the experiment measuring thermal withdrawal latency,bumetanide (15 mg/kg) was intravenously administered.In the patch clamp experiment,bumetanide (10 μg/μL) and acutely isolated dorsal root ganglion neurons (on day 14) were incubated for 1 hour,or bumetanide (5 μg/μL) was intrathecally injected.The Hargreaves test was conducted to detect changes in thermal hyperalgesia in rats.We found that the thermal withdrawal latency of rats was significantly decreased on days 7,14,and 21 after model establishment.After intravenous injection of bumetanide,the reduction in thermal retraction latency caused by model establishment was significantly inhibited.Immunohistochemistry and western blot assay results revealed that the immune response and protein expression of NKCC1 in dorsal root ganglion neurons of the chronic constriction injury group increased significantly on days 7,14,and 21 after model establishment.No immune response or protein expression of KCC2 was observed in dorsal root ganglion neurons before and after model establishment.The Cl^– (chloride ion) fluorescent probe technique was used to evaluate the change of Cl^– concentration in dorsal root ganglion neurons of chronic constriction injury model rats.We found that the relative optical density of N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (a Cl^– fluorescent probe whose fluorescence Cenintensity decreases as Cl– concentration increases) in the dorsal root ganglion neurons of the chronic constriction injury group was significantly decreased on days 7 and 14 after model establishment.The whole-cell patch clamp technique revealed that the resting potential and action potential frequency of dorsal root ganglion neurons increased,and the threshold and rheobase of action potentials decreased in the chronic constriction injury group on day 14 after model establishment.After bumetanide administration,the above indicators were significantly suppressed.These results confirm that CCI can induce abnormal overexpression of NKCC1,thereby increasing the Cl^– concentration in dorsal root ganglion neurons;this then enhances the excitability of dorsal root ganglion neurons and ultimately promotes hyperalgesia and allodynia.In addition,bumetanide can achieve analgesic effects.All experiments were approved by the Institutional Ethics Review Board at the First Affiliated Hospital,College of Medicine,Shihezi University,China on February 22,2017 (approval No.A2017-169-01).