Altered physiology of gastrointestinal vagal afferents following neurotrauma

The adaptability of the central nervous system has been revealed in several model systems.Of particular interest to central nervous system-injured individuals is the ability for neural components to be modified for regain of function.In both types of neurotrauma,traumatic brain injury and spinal cord injury,the primary parasympathetic control to the gastrointestinal tract,the vagus nerve,remains anatomically intact.However,individuals with traumatic brain injury or spinal cord injury are highly susceptible to gastrointestinal dysfunctions.Such gastrointestinal dysfunctions attribute to higher morbidity and mortality following traumatic brain injury and spinal cord injury.While the vagal efferent output remains capable of eliciting motor responses following injury,evidence suggests impairment of the vagal afferents.Since sensory input drives motor output,this review will discuss the normal and altered anatomy and physiology of the gastrointestinal vagal afferents to better understand the contributions of vagal afferent plasticity following neurotrauma.

Fine-tuning of cortical progenitor proliferation by thalamic afferents

During cerebral cortical cortex neurogenesis two major types of progenitors generate a variety of morphologically and functionally diverse projection neurons destined for the different cortical layers in non-gyrified mice. Radial glia cells （RGCs） undergo mitosis in the cortical ventricular zone and exhibit an apical-basal cell polarity, whereas non-polar intermediate progenitor cells （IPCs） divide basally in the subventricular zone （Franco and Muller, 2013; Taverna et al., 2014）.

THE ROLE OF NUCLEUS RAPHE MAGNUS IN THE ANTINOCICEPTIVE EFFECT OF MUSCLE SPINDLE AFFERENTS IN THE RAT

Objective To investigate the role of NRM in the antinociceptive effect of muscle spindle afferents,the influence of NRM lesion on the inhibitory effect of muscle spindle afferents on the nociceptive responses of wide dynamic range (WDR) neurons and the effects of the muscle spindle afferents on the NRM neuronal activities were observed. Methods The single units of WDR neurons in the spinal dorsal horn were recorded extracellularly, and the inhibitory effects of activating muscle spindle afferents by intravenous administration of succinylcholine (SCH) on the C fibers evoked responses (C responses) of WDR neurons were tested before and after lesion of NRM.The effects of the muscle spindle afferents activated by administrating SCH on the single NRM neurons were also examined.Results ①It was found that the C responses of WDR neurons were significantly inhibited by intravenously administration of SCH, and the inhibitory effect was reduced after lesion of NRM;②The activities of most of the NRM neurons could be changed significantly by administrating SCH. According to their responses, NRM neurons could be classified into three types:excitatory, inhibitory and non responsive neurons, and the responses were dose dependent. Conclusion These results suggest that the muscle spindle afferents evoked by SCH may activate the NRM neurons, which plays an important role in the antinociception of muscle spindle afferents.

Hyperpolarization-activated cyclic nucleotide-gated cation channel subtypes differentially modulate the excitability of murine small intestinal afferents

AIM： To assess the role of hyperpolarization-activated cyclic nucleotide-gated cation （HCN） channels in regu- lating the excitability of vagal and spinal gut afferents. METHODS： The mechanosensory response of mesen- teric afferent activity was measured in an ex vivo murine jejunum preparation. HCN channel activity was recorded through voltage and current clamp in acutely dissoci- ated dorsal root ganglia （DRG） and nodose ganglia （NG） neurons retrogradely labeled from the small intestine through injection of a fluorescent marker （DiI）. The isoforms of HCN channels expressed in DRG and NG neurons were examined by immunohistochemistry. RESULTS： Ramp distension of the small intestine evok- ed biphasic increases in the afferent nerve activity, re- flecting the activation of low- and high-threshold fibers.HCN blocker CsCl （5 mmol/L） preferentially inhibited the responses of low-threshold fibers to distension and showed no significant effects on the high-threshold re- sponses. The effect of CsCI was mimicked by the more selective HCN blocker ZD7288 （10 ~mol/L）. In 71.4% of DiI labeled DRG neurons （/7 = 20） and 90.9% of DiI labeled NG neurons （n = 10）, an inward current （Ih current） was evoked by hyperpolarization pulses which was fully eliminated by extracellular CsCI. In neurons expressing Ih current, a typical ＂sag＂ was observed upon injection of hyperpolarizing current pulses in cur- rent-clamp recordings. CsCI abolished the sag entirely. In some DiI labeled DRG neurons, the Ih current was potentiated by 8-Br-cAMP, which had no effect on the Ih current of DiI labeled NG neurons. Immunohistochem- istry revealed differential expression of HCN isoforms in vagal and spinal afferents, and HCN2 and HCN3 seemed to be the dominant isoform in DRG and NG, respec- tively.CONCLUSION： HCNs differentially regulate the excit- ability of vagal and spinal afferent of murine small in- testine.

Bidirectional regulation of the brain-gut-microbiota axis following traumatic brain injury

Traumatic brain injury is a prevalent disorder of the central nervous system.In addition to primary brain parenchymal damage,the enduring biological consequences of traumatic brain injury pose long-term risks for patients with traumatic brain injury;however,the underlying pathogenesis remains unclear,and effective intervention methods are lacking.Intestinal dysfunction is a significant consequence of traumatic brain injury.Being the most densely innervated peripheral tissue in the body,the gut possesses multiple pathways for the establishment of a bidirectional“brain-gut axis”with the central nervous system.The gut harbors a vast microbial community,and alterations of the gut niche contribute to the progression of traumatic brain injury and its unfavorable prognosis through neuronal,hormonal,and immune pathways.A comprehensive understanding of microbiota-mediated peripheral neuroimmunomodulation mechanisms is needed to enhance treatment strategies for traumatic brain injury and its associated complications.We comprehensively reviewed alterations in the gut microecological environment following traumatic brain injury,with a specific focus on the complex biological processes of peripheral nerves,immunity,and microbes triggered by traumatic brain injury,encompassing autonomic dysfunction,neuroendocrine disturbances,peripheral immunosuppression,increased intestinal barrier permeability,compromised responses of sensory nerves to microorganisms,and potential effector nuclei in the central nervous system influenced by gut microbiota.Additionally,we reviewed the mechanisms underlying secondary biological injury and the dynamic pathological responses that occur following injury to enhance our current understanding of how peripheral pathways impact the outcome of patients with traumatic brain injury.This review aimed to propose a conceptual model for future risk assessment of central nervous system-related diseases while elucidating novel insights into the bidirectional effects of the“brain-gut-microbiota axis.”

Cortical Organization of Centrifugal Afferents to the Olfactory Bulb: Mono-and Trans-synaptic Tracing with Recombinant Neurotropic Viral Tracers

Sensory processing is strongly modulated by different brain and behavioral states,and this is based on the top-down modulation.In the olfactory system,local neural circuits in the olfactory bulb(OB)are innervated by centrifugal afferents in order to regulate the processing of olfactory information in the OB under different behavioral states.The purpose of the present study was to explore the organization of neural networks in olfactory-related cortices and modulatory nuclei that give rise to direct and indirect innervations to the glomerular layer(GL)of the OB at the whole-brain scale.Injection of different recombinant attenuated neurotropic viruses into the GL showed that it received direct inputs from each layer in the OB,centrifugal inputs from the ipsilateralanterior olfactory nucleus(AON),anterior piriform cortex(Pir),and horizontal limb of diagonal band of Broca(HDB),and various indirect inputs from bilateral cortical neurons in the AON,Pir,amygdala,entorhinal cortex,hippocampus,HDB,dorsal raphe,median raphe and locus coeruleus.These results provide a circuitry basis that will help further understand the mechanism by which olfactory informationprocessing in the OB is regulated.

Analyzing the Prebiotic Potential of Glucosamine for Targeting the Gut Microbiome Health

Recognizing the composition and modulation of the microbiome, a viable therapeutic tool for multi-targeted therapy is a new strategy that has recently been explored. Glucosamine (GS) is being studied for its prebiotic potential in addition to being the most abundant and naturally occurring amino monosaccharide. The current study focuses on glucosamine’s prebiotic potential by assessing the stability of various GS concentrations (1% - 5%) in the gastrointestinal tract (GIT) and its ability to be fermented by the gut microbiota. The results showed that GS stimulated the most growth in L. acidophilus even after a longer incubation time than B. bifidum and L. acidophilus growth was concentration-dependent, with maximum growth at 3% with a simultaneous decrease in pH (5.6 - 1.7). The decrease in GS concentration with time also represented the growth of bacterial species, demonstrating the species’ utilization of GS. Furthermore, at 3%, GS also represented the prebiotic index of 1.9. In addition, the concentration of GS in various simulated GIT fluids was estimated in both fast and fed conditions to examine GS stability at various levels in the gut. The results showed that GS remained unaffected and non-digestible in all of the simulated GIT fluids (salivary, gastric, intestinal, and colonic), but there was a slight decrease in GS concentration (2.8%) in the fasted state of gastric fluid due to low pH levels (1.6). As a result, the findings are conclusive and suggest that GS possesses prebiotic properties.

Hippocampal plasticity after a vagus nerve injury in the rat

Stimulation of the vagus nerve has been previously reported to promote neural plasticity and neurogenesis in the brain. Several studies also revealed plastic changes in the spinal cord after injuries to somatosensory nerves originating from both the brachial and lumbo-sacral plexuses. However, the neurogenic responses of the brain to the injury of the viscerosensory innervation are not as yet well understood. In the present study, we investigated whether cells in the dentate gyrus of the hippocampus respond to a chemical and physical damage to the vagus nerve in the adult rat. Intraperitoneal capsaicin administration was used to damage non-myelinated vagal afferents while subdiaphragmatic vagotomy was used to damage both the myelinated and non-myelinated vagal afferents. The 5-bromo-2-deoxyuridine （BrdU） incorporation together with cell-specific markers was used to study neural proliferation in subgranular zone, granule cell layer, molecular layer and hilus of the dentate gyrus. Microglia activation was determined by quantifying changes in the intensity of fluorescent staining with a primary antibody against ionizing calcium adapter-binding molecule 1. Results revealed that vagotomy decreased BrdU incorporation in the hilus 15 days after injury compared to the capsaicin group. Capsaicin administration decreased BrdU incorporation in the granular cell layer 60 days after the treatment. Capsaicin decreased the number of doublecortin-expressing cells in the dentate gyrus, whereas vagotomy did not alter the expression of doublecortin in the hippocampus. Both the capsaicin- and the vagotomy-induced damage to the vagus nerve decreased microglia activation in the hippocampus at 15 days after the injury. At 30 days post injury, capsaicin-treated and vagotomized rats revealed significantly more activated microglia. Our findings show that damage to the subdiaphragmatic vagus in adult rats is followed by microglia activation and long-lasting changes in the dentate gyrus, leading to alteration of neurogenesis.

Nerve growth factor in muscle afferent neurons of peripheral artery disease and autonomic function

In peripheral artery disease patients,the blood supply directed to the lower limbs is reduced.This results in severe limb ischemia and thereby enhances pain sensitivity in lower limbs.The painful perception is induced and exaggerate during walking,and is relieved by rest.This symptom is termed by intermittent claudication.The limb ischemia also amplifies autonomic responses during exercise.In the process of pain and autonomic responses originating exercising muscle,a number of receptors in afferent nerves sense ischemic changes and send signals to the central nervous system leading to autonomic responses.This review integrates recent study results in terms of perspectives including how nerve growth factor affects muscle sensory nerve receptors in peripheral artery disease and thereby alters responses of sympathetic nerve activity and blood pressure to active muscle.For the sensory nerve receptors,we emphasize the role played by transient receptor potential vanilloid type 1,purinergic P2X purinoceptor 3 and acid sensing ion channel subtype 3 in amplified sympathetic nerve activity responses in peripheral artery disease.

Neuronal Fc gamma receptor I as a novel mediator for IgG immune complex-induced peripheral sensitization

Chronic pain often accompanies immune-related diseases with an elevated level of IgG immune complex （IgG-IC） in the serum and/or the affected tissues though the underlying mechanisms are largely unknown. Fc gamma receptors （FcyRs）, known as the receptors for the Fc domain of immunoglobulin G （IgG）, are typically expressed on immune cells. A general consensus is that the activation of FcyRs by IgG-IC in such immune cells induces the release of proinflammatory cytokines from the immune cells, which may contribute to the IgG-IC-mediated peripheral sensitization. In addition to the immune cells, recent studies have revealed that FcyRI, but not FcyRII and FcyRIII, is also expressed in a subpopulation of primary sensory neurons. Moreover, IgG-IC directly excites the primary sensory neurons through neuronal FcyRI. These findings indicate that neuronal FcyRI provides a novel direct linkage between immunoglobulin and primary sensory neurons, which may be a novel target for the treatment of pain in the immune-related disorders. In this review, we summarize the expression pattern, functions, and the associated cellular signaling of FcyRs in the primary sensory neurons.

Interplay between inflammation,immune system and neuronal pathways:Effect on gastrointestinal motility

Sepsis is a systemic inflammatory response representing the leading cause of death in critically ill patients,mostly due to multiple organ failure.The gastrointestinal tract plays a pivotal role in the pathogenesis of sepsisinduced multiple organ failure through intestinal barrier dysfunction,bacterial translocation and ileus.In this review we address the role of the gastrointestinal tract,the mediators,cell types and transduction pathways involved,based on experimental data obtained from models of inflammation-induced ileus and (preliminary) clinical data.The complex interplay within the gastrointestinal wall between mast cells,residential macrophages and glial cells on the one hand,and neurons and smooth muscle cells on the other hand,involves intracellular signaling pathways,Toll-like receptors and a plethora of neuroactive substances such as nitric oxide,prostaglandins,cytokines,chemokines,growth factors,tryptases and hormones.Multidirectional signaling between the different components in the gastrointestinal wall,the spinal cord and central nervous system impacts inflammation and its consequences.We propose that novel therapeutic strategies should target inflammation on the one hand and gastrointestinal motility,gas-trointestinal sensitivity and even pain signaling on the other hand,for instance by impeding afferent neuronal signaling,by activation of the vagal anti-inflammatory pathway or by the use of pharmacological agents such as ghrelin and ghrelin agonists or drugs interfering with the endocannabinoid system.

Neuromechanism of acupuncture regulating gastrointestinal motility

Acupuncture has been used in China for thousands of years and has become more widely accepted by doctors and patients around the world. A large number of clinical studies and animal experiments have confirmed that acupuncture has a benign adjustment effect on gastrointestinal(GI) movement;however, the mechanism of this effect is unclear, especially in terms of neural mechanisms, and there are still many areas that require further exploration. This article reviews the recent data on the neural mechanism of acupuncture on GI movements. We summarize the neural mechanism of acupuncture on GI movement from four aspects: acupuncture signal transmission, the sympathetic and parasympathetic nervous system, the enteric nervous system, and the central nervous system.

Affer Effects遮罩局部调色技巧

运用影视后期制作软件Affer Effects对风景视频进行局部调色,可采用动态遮罩精确选取调色范围,再利用遮罩和层的混合模式进行调色。

Neuroanatomy of lower gastrointestinal pain disorders

Chronic abdominal pain accompanying intestinal inflammation emerges from the hyperresponsiveness of neuronal,immune and endocrine signaling pathways within the intestines,the peripheral and the central nervous system.In this article we review how the sensory nerve information from the healthy and the hypersensitive bowel is encoded and conveyed to the brain.The gut milieu is continuously monitored by intrinsic enteric afferents,and an extrinsic nervous network comprising vagal,pelvic and splanchnic afferents.The extrinsic afferents convey gut stimuli to second order neurons within the superficial spinal cord layers.These neurons cross the white commissure and ascend in the anterolateral quadrant and in the ipsilateral dorsal column of the dorsal horn to higher brain centers,mostly subserving regulatory functions.Within the supraspinal regions and the brainstem,pathways descend to modulate the sensory input.Because of this multiple level control,only a small proportion of gut signals actually reaches the level of consciousness to induce sensation or pain.In inflammatory bowel disease(IBD)and irritable bowel syndrome(IBS)patients,however,long-term neuroplastic changes have occurred in the brain-gut axis which results in chronic abdominal pain.This sensitization may be driven on the one hand by peripheral mechanisms within the intestinal wall which encompasses an interplay between immunocytes,enterochromaffin cells,resident macrophages,neurons and smooth muscles.On the other hand,neuronal synaptic changes along with increased neurotransmitter release in the spinal cord and brain leads to a state of central wind-up.Also life factors such as but not limited to inflammation and stress contribute to hypersensitivity.All together,the degree to which each of these mechanisms contribute to hypersensitivity in IBD and IBS might be diseaseand even patient-dependent.Mapping of sensitization throughout animal and human studies may significantly improve our understanding of sensitization in IBD and IBS.On the long run,this knowledge can be put forward in potential therapeutic targets for abdominal pain in these conditions.

The existence of propagated sensation along the meridian proved by neuroelectrophysiology

Propagated sensation along the meridian can occur when acupoints are stimulated by acupuncture or electrical impulses. In this study, participants with notable propagated sensation along the me- ridian were given electro-acupuncture at the Jianyu （LI15） acupoint of the large intestine meridian. When participants stated that the sensation reached the back of their hand, reguJar nervous system action discharge was examined using a physiological recording electrode placed on the superficial branch of the radial nerve. The topographical maps of brain-evoked potential in the primary cortical somatosensory area were also detected. When Guangming （GB37） acupoint in the lower limb and Hegu （LI4） acupoint in the upper limb were stimulated, subjects without propagated sensation along the meridian exhibited a high potential reaction in the corresponding area of the brain cortical somatosensory area. For subjects with a notable propagated sensation along the meridian, the re- action area was larger and extended into the face representative area. These electrophysiological measures directly prove the existence of propagated sensation along the meridian, and the periph- eral stimulated site is consistent with the corresponding primary cortical somatosensory area, which presents a high potential reaction.

Gastro-protective action of lafutidine mediated by capsaicin-sensitive afferent neurons without interaction with TRPV1 and involvement of endogenous prostaglandins

AIM： Lafutidine, a histamine H2 receptor antagonist, exhibits gastro-protective action mediated by capsaicinsensitive afferent neurons （CSN）. We compared the effect between lafutidine and capsaicin, with respect to the interaction with endogenous prostaglandins （PG）, nitric oxide （NO） and the afferent neurons, including transient receptor potential vanilloid subtype 1 （TRPV1）. METHODS： Male SD rats and C57BL/6 mice, both wildtype and prostacyclin IP receptor knockout animals, were used after 18 h of fasting. Gastric lesions were induced by the po administration of HCl/ethanol （60% in 150 mmol/L HCl） in a volume of 1 mL for rats or 0.3 mL for mice. RESULTS： Both lafutidine and capsaicin （1-10 mg/kg, po） afforded dose-dependent protection against HCI/ ethanol in rats and mice. The effects were attenuated by both the ablation of CSN and pretreatment with NG-nitro- L-arginine methyl ester, yet only the effect of capsaicin was mitigated by prior administration of capsazepine, the TRPV1 antagonist, as well as indomethacin. Lafutidine protected the stomach against HCl/ethanol in IP receptor knockout mice, similar to wild-type animals, while capsaicin failed to afford protection in the animals lacking IP receptors. Neither of these agents affected the mucosal PGE2 or 6-keto PGF1α contents in rat stomachs. Capsaicin evoked an increase in [Ca^2＋]i in rat TRPV1-transfected HEK293 cells while lafutidine did not. CONCLUSION： These results suggest that although both lafutidine and capsaicin exhibit gastro-protective action mediated by CSN, the mode of their effects differs regarding the dependency on endogenous PGs/IP receptors and TRPV1. It is assumed that lafutidine interacts with CSN at yet unidentified sites other than TRPV1.

Management of afferent loop obstruction: Reoperation or endoscopic and percutaneous interventions?

Afferent loop obstruction is a purely mechanical complication that infrequently occurs following construction of a gastrojejunostomy. The operations most commonly associated with this complication are gastrectomy with Billroth Ⅱ or Roux-en-Y reconstruction, and pancreaticoduodenectomy with conventional loop or Roux-en-Y reconstruction. Etiology of afferent loop obstruction includes:(1) entrapment, compression and kinking by postoperative adhesions;(2) internal herniation, volvulus and intussusception;(3) stenosis due to ulceration at the gastrojejunostomy site and radiation enteritis of the afferent loop;(4) cancer recurrence; and(5) enteroliths, bezoars and foreign bodies. Acute afferent loop obstruction is associated with complete obstruction of the afferent loop and represents a surgical emergency, whereas chronic afferent loop obstruction is associated with partial obstruction. Abdominal multiple detector computed tomography is the diagnostic study of choice. CT appearance of the obstructed afferent loop consists of a C-shaped, fluidfilled tubular mass located in the midline between the abdominal aorta and the superior mesenteric artery with valvulae conniventes projecting into the lumen. The cornerstone of treatment is surgery. Surgery includes:(1) adhesiolysis and reconstruction for benign causes; and(2) by-pass or excision and reconstruction for malignant causes. However, endoscopic enteral stenting, transhepatic percutaneous enteral stenting and direct percutaneous tube enterostomy have the principal role in management of malignant and radiation-induced obstruction. Nevertheless, considerable limitations exist as a former Roux-en-Y reconstruction limits endoscopic access to the afferent loop and percutaneous approaches for enteral stenting and tube enterostomy have only been reported in the literature as isolated cases.

Exposure to blast shock waves via the ear canal induces deficits in vestibular afferent function in rats

The ears are air-filled structures that are directly impacted during blast exposure.In addition to hearing loss and tinnitus,blast victims often complain of vertigo,dizziness and unsteady posture,suggesting that blast exposure induces damage to the vestibular end organs in the inner ear.However,the underlying mechanisms remain to be elucidated.In this report,single vestibular afferent activity and the vestibuloocular reflex(VOR)were investigated before and after exposure to blast shock waves(~20 PSI)delivered into the left external ear canals of anesthetized rats.Single vestibular afferent activity was recorded from the superior branch of the left vestibular nerves of the blast-treated and control rats one day after blast exposure.Blast exposure reduced the spontaneous discharge rates of the otolith and canal afferents.Blast exposure also reduced the sensitivity of irregular canal afferents to sinusoidal head rotation at 0.5e2Hz.Blast exposure,however,resulted in few changes in the VOR responses to sinusoidal head rotation and translation.To the best of our knowledge,this is the first study that reports blast exposure-induced damage to vestibular afferents in an animal model.These results provide insights that may be helpful in developing biomarkers for early diagnosis of blast-induced vestibular deficits in military and civilian populations.

Management of afferent loop obstruction from recurrent metastatic pancreatic cancer using a venting gastrojejunostomy

Pancreatic cancer is an aggressive malignancy poten-tially curable with surgical intervention. Following pan-creaticoduodenectomy for suspected pancreatic head malignancy, patients have a high risk for both immedi-ate and delayed problems due to surgical complica-tions and recurrent disease. We report here a patient with pancreatic cancer treated with pancreaticoduode-nectomy who developed recurrent disease resulting in obstruction of the afferent limb. The patient developed biliary obstruction and cholangitis at presentation. Her biliary tree failed to dilate which precluded safe percu-taneous biliary decompression. During surgical explo-ration, she was found to have a dilated afferent limb at the level of the transverse mesocolon. The patient underwent decompression of the afferent limb as well as the biliary tree using a venting gastrojejunostomy to the blind loop. This represents a novel surgical ap-proach for management of this complicated and diffi-cult problem.

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).