Conditional gene editing animals and viral vectors have been widely applied in the research fields of biology and medicine. Recently, they are also used as the effective approaches to reveal the underlying mechanism of acupuncture from the nervous system to the specific molecules. In order to further understand the application of conditional gene editing animals and viral vectors, in this article, we analyze their characteristics, advantages and recent development in the field of acupuncture research and discuss their potential roles and prospect in the future.

Objective Glial cells are involved in the analgesic effect of electroacupuncture (EA) in rats with chronic neurological pain. The objective of this study was to observe the role of neuronal–glial interaction and glutamate (Glu) transporters in EA-induced acute neck pain relief in rats. Materials and methods Male rats were placed into the following five groups: control, model, EA Futu (LI18), EA Hegu (LI4)-Neiguan (PC6), and EA Zusanli (ST36)-Yanglingquan (GB34). The incisional neck pain model was established by making a longitudinal incision along the midline of the neck. The thermal pain threshold (TPT) was measured using a radiation heat detector. The immunoactivities of glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (Iba-1), neurokinin-1 receptor (NK-1R), Glu aspartate transporter (GLAST), and Glu transporter-1 (GLT-1) in the dorsal horns (DHs) of the cervico-spinal cord (C2–C5) were detected using immunofluorescence histochemistry. The expression levels of GFAP, Iba-1, GLAST, and GLT-1 mRNAs were determined using quantitative real-time polymerase chain reaction (PCR). Results The TPT and levels of mRNAs expression and immunoactivity of GLT-1 and GLAST were significantly decreased, and those of Iba-1 and GFAP were significantly increased in the model group than those of the control group (P < 0.05). The activated microgliacytes were gathered around the NK-1R positive neurons, and co-expression of NK-1R and astrocytes was observed in the model group. EA LI18 significantly increased the TPT and expression of GLAST and GLT-1 mRNAs (P < 0.05) and notably decreased the number of Iba-1 positive cells and Iba-l mRNA expression (P < 0.05), whereas GLAST and GLT-1 antagonists inhibited the analgesic effect of EA LI18. However, these effects, except for the downregulation of Iba-1 mRNA, were not observed in the EA ST36-GB34 group. Fewer NK-1R-positive neurons were visible in the spinal DHs in the EA LI18 group, and the co-expression of NK-1R and astrocytes was also lower than that in the three EA groups. Conclusion Electroacupuncture of LI18 had an analgesic effect in rats with neck incisions, which may be related to its functions in suppressing the neuronal–glial cell interaction through NK-1R and upregulating the expression of GLAST and GLT-1 in the spinal DHs.

We have recently demonstrated that reactive oxygen species (ROS) scavengers ameliorate mechanical allodynia in a rat model of cancer-induced bone pain (CIBP). In the present study, we investigated anti-nociceptive effect of Nox inhibitor apocynin in CIBP in rats. Mechanical allodynia was assessed by Von Frey tests in sham and CIBP group of rats. Western blotting and immunofluorescence technique were conducted to assess the expression levels and cellular localization of Nox2. Results illustrated that after intra-tibial implantation with tumor cells, Nox2 and ROS were both up-regulated in the spinal cord of rats. Injection of apocynin could dose-dependently decrease the abundance of Nox2 and inhibit the development of CIBP. Furthermore, pretreatment with the apocynin could delay the development of CIBP. This study for the first time proved that Nox2 inhibitors could downregulate the production of ROS in CIBP rats, which highlights the fact that Nox inhibitor is an important therapeutic option for CIBP and that, precise targeting inhibitor of different subtypes of Nox enzymes is needed to developed in future.

INTRODUCTION:Cancer-induced bone pain (CIBP) is acknowledged as a multifactorial chronic pain that tortures advanced cancer patients, but existing treatment strategies for CIBP have not been satisfactory yet. Investigators have demonstrated that the activation of α7-nAChRs exerts analgesic effects in some chronic pain models. However, the role of spinal α7-nAChRs in CIBP remains unknown. This study was designed to investigate the role of α7-nAChRs in a well-established CIBP model induced by Walker 256 rat mammary gland carcinoma cells.METHODS:The paw withdrawal threshold (PWT) of the ipsilateral hind paw was measured using von Frey filament. The expressions of spinal α7-nAChRs and NF-κB were measured with Western blotting analysis. Immunofluorescence was employed to detect the expression of α7-nAChRs and co-expressed of α7-nAChRs with NeuN or GFAP or Iba1.RESULTS:Experiment results showed that the expression of spinal α7-nAChRs was significantly downregulated over time in CIBP rats, and in both CIBP rats and sham rats, most of the α7-nAChRs located in neurons. Behavioral data suggested PNU-282,987, a selective α7-nAChRs agonist, dose-dependently produced analgesic effect and positive allosteric modulator could intensify its effects. Further, repeated administration of PNU-282,987 reversed the expression of α7-nAChRs, inhibited the nuclear factor kappa B (NF-κB) signaling pathway, and attenuates CIBP-induced mechanical allodynia state as well.CONCLUSION:These results suggest that the reduced expression of spinal α7-nAChRs contributes to the maintenance of CIBP by upregulating NF-κB expression, which implying a novel pharmacological therapeutic target for the treatment of CIBP.

Paclitaxel-induced neuropathic pain (PINP) is refractory to currently used analgesics. Previous studies show a pivotal role of oxidative stress in PINP. Because the nuclear factor erythroid-2-related factor 2 (Nrf2) has been considered as the critical regulator of endogenous antioxidant defense, we here explored whether activation of Nrf2 could attenuate PINP. A rat model of PINP was established by intraperitoneal injection of paclitaxel (2 mg/kg) every other day with a final cumulative dose of 8 mg/kg. Hind paw withdrawal thresholds (PWTs) in response to von Frey filament stimuli were used to assess mechanical allodynia. We showed that a single dose of Nrf2 activator, oltipraz (10, 50, and 100 mg/kg), dose-dependently attenuated established mechanical allodynia, whereas repeated injection of oltipraz (100 mg· kg −1 · d −1 , i.p. from d 14 to d 18) almost abolished the mechanical allodynia in PINP rats. The antinociceptive effect of oltipraz was blocked by pre-injection of Nrf2 inhibitor trigonelline (20 mg/kg, i.p.). Early treatment with oltipraz (100 mg· kg −1 · d −1 , i.p. from d 0 to d 6) failed to prevent the development of the PINP, but delayed its onset. Western blot and immunofluorescence analysis revealed that the expression levels of Nrf2 and HO-1 were significantly upregulated in the spinal cord of PINP rats. Repeated injection of oltipraz caused further elevation of the expression levels of Nrf2 and HO-1 in the spinal cord of PINP rats, which was reversed by pre-injection of trigonelline. These results demonstrate that oltipraz ameliorates PINP via activating Nrf2/HO-1-signaling pathway in the spinal cord.

Paclitaxel-induced neuropathic pain (PINP) is a dose-limiting side effect and is refractory to widely used analgesic drugs. Previous studies have demonstrated a protective role of peroxisome proliferator-activated receptor gama (PPARγ) in neuropathic pain. However, whether PPARγ activation could alleviate PINP remains to be elucidated. Our previous study has validated the analgesic effect of oltipraz, an nuclear factor erythroid-2 related factor 2 (Nrf2) activator, in a rat model of PINP. In this study, we tested the hypothesis that rosiglitazone, a selective agonist of PPARγ, could attenuate PINP through induction of Nrf2/heme oxygenase-1 (HO-1) signaling pathway. Paclitaxel was injected intraperitoneally on four alternate days to induce neuropathic pain. Paw withdrawal threshold was used to evaluate mechanical allodynia. Western blot and immunofluorescence were used to examine the expression and distribution of PPARγ, Nrf2 and HO-1 in the spinal cord. Our results showed that rosiglitazone attenuated established PINP and delayed the onset of PINP via activation of PPARγ, which were reversed by PPARγ antagonist GW9662. Moreover, rosiglitazone inhibited downregulation of PPARγ in the spinal cord of PINP rats. Furthermore, the analgesic effect of rosiglitazone against PINP was abolished by trigonelline, an Nrf2 inhibitor. Finally, rosiglitazone significantly increased expression of Nrf2 and HO-1 in the spinal cord of PINP rats. Collectively, these results indicated that PPARγ activation might mitigate PINP through activating spinal Nrf2/HO-1 signaling pathway. Our results may provide an alternative option for PINP patients.

Treating Cancer-induced bone pain (CIBP) continues to be a major clinical challenge and underlying mechanisms of CIBP remain unclear. Recently, emerging body of evidence suggested the endocannabinoid system (ECS) may play essential roles in CIBP. Here, we summarized the current understanding of the antinociceptive mechanisms of endocannabinoids in CIBP and discussed the beneficial effects of endocannabinoid for CIBP treatment. Targeting non-selective cannabinoid 1 receptors or selective cannabinoid 2 receptors, and modulation of peripheral AEA and 2-AG, as well as the inhibition the function of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) have produced analgesic effects in animal models of CIBP. Management of ECS therefore appears to be a promising way for the treatment of CIBP in terms of efficacy and safety. Further clinical studies are encouraged to confirm the possible translation to humans of the very promising results already obtained in the preclinical studies.

Cancer-induced bone pain (CIBP) remains a major challenge in patients suffering from bone metastases because of the complex mechanisms and unsatisfactory treatments. Emerging evidence have shown that activation of inflammasomes contribute to the development of inflammatory and neuropathic pain. However, the role of spinal inflammasomes in CIBP remains unclear. In the present study, we explored the specific cellular mechanisms of NLRP3 inflammasome in the process of CIBP in rats. MCC950 is a small molecule inhibitor of the NLRP3 inflammasome that exhibits remarkable activity in inflammatory diseases. Our behavioral results confirmed that both single and persistent treatment with MCC950 markedly attenuated CIBP-related mechanical allodynia. The expression of NLRP3 inflammasome, including NLRP3, ASC, Caspase-1, were significantly increased in a time-dependent manner. Furthermore, spinal IL-1β, cleaved by cysteine-aspartic acid protease, was upregulated in this study. Chronic administration with MCC950 restored the protein expression of NLRP3 inflammasome and significantly suppressed the upregulation of IL-1β. Spinal NLRP3 inflammasome might be a novel therapeutic target for treatment of CIBP.

Ischemia-reperfusion (I/R) injury is accompanied with high morbidity and mortality and has seriously negative social and economic influences. Unfortunately, few effective therapeutic strategies are available to improve its outcome. Berberine is a natural medicine possessing multiple beneficial biological activities. Emerging evidence indicates that berberine has potential protective effects against I/R injury in brain, heart, kidney, liver, intestine and testis. However, up-to-date review focusing on the beneficial role of berberine against I/R injury is not yet available. In this paper, results from animal models and clinical studies are concisely presented and its mechanisms are discussed. We found that berberine ameliorates I/R injury in animal models via its anti-oxidant, anti-apoptotic and anti-inflammatory effects. Moreover, berberine also attenuates I/R injury by suppressing endoplasmic reticulum stress and promoting autophagy. Additionally, regulation of periphery immune system may also contributes to the beneficial effect of berberine against I/R injury. Although clinical evidence is limited, the current studies indicate that berberine may attenuate I/R injury via inhibiting excessive inflammatory response in patients. Collectively, berberine might be used as an alternative therapeutic strategy for the management of I/R injury.

The development of effective therapeutics for cancer-induced bone pain (CIBP) remains a tremendous challenge owing to its unclear mechanisms. Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system (CNS). Emerging studies have shown that disinhibition in the spinal cord dorsal horn may account for the development of chronic pain. However, the role of GABA in the development of CIBP remains elusive. In addition, accumulating evidence has shown that neuroglial cells in the peripheral nervous system, especially astrocytes and microglial cells, played an important role in the maintenance of CIBP. In this study, we investigated the expression of GABA and Gamma-aminobutyric acid transporter-1 (GAT-1), a transporter of GABA. Our results demonstrate that GABA was decreased in CIBP rats as expected. However, the expression of glutamic acid decarboxylase (GAD) 65 was up-regulated on day 21 after surgery, while the expression of GAD 67 remained unchanged after surgery. We also found that the expression of GAT-1 was up-regulated mainly in the astrocytes of the spinal cord. Moreover, we evaluated the analgesic effect of exogenous GABA and the GAT-1 inhibitor. Intrathecal administration of exogenous GABA and NO-711 (a GAT-1 selective inhibitor) significantly reversed CIBP-induced mechanical allodynia in a dose-dependent manner. These results firstly show that neuron-glia interactions, especially on the GABAergic pathway, contribute to the development of CIBP. In conclusion, exogenous GABA and GAT-1 inhibitor might be alternative therapeutic strategies for the treatment of CIBP.

Background: Neurological diseases have become an obvious challenge due to insufficient therapeutic intervention. Therefore, novel drugs for various neurological disorders are in desperate need. Recently, compelling evidence has demonstrated that chemokine receptor CXCR3, which is a G protein-coupled receptor in the CXC chemokine receptor family, may play a pivotal role in the development of neurological diseases. The aim of this review is to provide evidence for the potential of CXCR3 as a therapeutic target for neurological diseases. Methods: English journal articles that focused on the invovlement of CXCR3 in neurological diseases were searched via PubMed up to May 2017. Moreover, reference lists from identified articles were included for overviews. Results: The expression level of CXCR3 in T cells was significantly elevated in several neurological diseases, including multiple sclerosis (MS), glioma, Alzheimer's disease (AD), chronic pain, human T-lymphotropic virus type 1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and bipolar disorder. CXCR3 antagonists showed therapeutic effects in these neurological diseases. Conclusion: These studies provided hard evidence that CXCR3 plays a vital role in the pathogenesis of MS, glioma, AD, chronic pain, HAM/TSP and bipolar disorder. CXCR3 is a crucial molecule in neuroinflammatory and neurodegenerative diseases. It regulates the activation of infiltrating cells and resident immune cells. However, the exact functions of CXCR3 in neurological diseases are inconclusive. Thus, it is important to understand the topic of chemokines and the scope of their activity in neurological diseases.

Cancer-induced bone pain is one of the most severe types of pathological pain, which often occurs in patients with advanced prostate, breast, and lung cancer. It is of great significance to improve the therapies of cancer-induced bone pain due to the opioids' side effects including addiction, sedation, pruritus, and vomiting. Sinomenine, a traditional Chinese medicine, showed obvious analgesic effects on a rat model of chronic inflammatory pain, but has never been proven to treat cancer-induced bone pain. In the present study, we investigated the analgesic effect of sinomenine after tumor cell implantation and specific cellular mechanisms in cancer-induced bone pain. Our results indicated that single administration of sinomenine significantly and dose-dependently alleviated mechanical allodynia in rats with cancer-induced bone pain and the effect lasted for 4h. After tumor cell implantation, the protein levels of phosphorylated-Janus family tyrosine kinase 2 (p-JAK2), phosphorylated-signal transducers and activators of transcription 3 (p-STAT3), phosphorylated-Ca2+/calmodulin-dependent protein kinase II (p-CAMKII), and phosphorylated-cyclic adenosine monophosphate response element-binding protein (p-CREB) were persistently up-regulated in the spinal cord horn. Chronic intraperitoneal treatment with sinomenine markedly suppressed the activation of microglia and effectively inhibited the expression of JAK2/STAT3 and CAMKII/CREB signaling pathways. We are the first to reveal that up-regulation of microglial JAK2/STAT3 pathway are involved in the development and maintenance of cancer-induced bone pain. Moreover, our investigation provides the first evidence that sinomenine alleviates cancer-induced bone pain by inhibiting microglial JAK2/STAT3 and neuronal CAMKII/CREB cascades.

Cancer-induced bone pain (CIBP) is a frequent complication in patients suffering from bone metastases. Previous studies have demonstrated a pivotal role of reactive oxygen species (ROS) in inflammatory and neuropathic pain, and ROS scavengers exhibited potent antinociceptive effect. However, the role of spinal ROS remains unclear. In this study, we investigated the analgesic effect of two ROS scavengers in a well-established CIBP model. Our results found that intraperitoneal injection of N-tert-Butyl-α-phenylnitrone (PBN, 50 and 100mg/kg) and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol, 100 and 200mg/kg) significantly suppressed the established mechanical allodynia in CIBP rats. Moreover, repeated injection of PBN and Tempol showed cumulative analgesic effect without tolerance. However, early treatment with PBN and Tempol failed to prevent the development of CIBP. Naive rats received repetitive injection of PBN and Tempol showed no significant change regarding the nociceptive responses. Finally, PBN and Tempol treatment notably suppressed the activation of spinal microglia in CIBP rats. In conclusion, ROS scavengers attenuated established CIBP by suppressing the activation of microglia in the spinal cord.

Chronic pain remains to be a clinical challenge due to insufficient therapeutic strategies. Minocycline is a member of the tetracycline class of antibiotics, which has been used in clinic for decades. It is frequently reported that minocycline may has many non-antibiotic properties, among which is its anti-nociceptive effect. The results from our lab and others suggest that minocycline exerts strong analgesic effect in animal models of chronic pain including visceral pain, chemotherapy-induced periphery neuropathy, periphery injury induced neuropathic pain, diabetic neuropathic pain, spinal cord injury, inflammatory pain and bone cancer pain. In this review, we summarize the mechanisms underlying the analgesic effect of minocycline in preclinical studies. Due to a good safety record when used chronically, minocycline may become a promising therapeutic strategy for chronic pain in clinic.

Cumulated evidence reveals that glial cells in the spinal cord play an important role in the development of chronic neuropathic pain and are also complicated in the analgesic effect of EA intervention. But the roles of microgliacytes and astrocytes of spinal cord in the process of EA analgesia remain unknown.

OBJECTIVE:To observe the effect of electroacupuncture (EA) on expression of synaptic plasticity-related glutamate N-methyl-D-aspartate (NMDA) receptor subunit NR 1, gamma-aminobutyric acid (GABA) receptor subunits (Aβ 2, B 1), etc. in the amygdala in chronic neuropathic pain negative affection (CNPPNA) rats, so as to reveal its mechanism underlying pain relief. METHODS:Male Wistar rats were randomized into normal control, CNPPNA model, EA, and anesthesia+EA (AEA)groups (n=14 in each group, 8 for quantitative RT-PCR and 6 for immunofluorescence staining). The CNPPNA model was established by ligation of the left sciatic nerve and repeated electrical stimulation of the hindpaw plantar skin in the pain-paired compartment. EA was applied to bilateral "Zusanli"(ST 36)and "Yanglingquan"(GB 34)for 30 min, once daily for 7 days.Thermal pain threshold (paw withdrawal latency, PWL)of the bilateral paws was measured by using a Tail-Flick Unit. The conditioned place aversion (CPA) was determined by using a CPA-paired compartment. The expression levels of GABAAβ 2, GABAB 1, NMDA receptor subunit NR 1, postsynaptic density-95 protein(PSD-95), Piccolo genes in the right amygdala area were determined using quantitative RT-PCR, and the immunoactivity of metabotropic glutamate receptor subunit 1 (mGluR 1) and GABAB 2 in the basolateral amygdala (BLA) nucleus was detected using immunofluorescence staining. RESULTS:After modeling, PWL difference (PWLD) values of the model group were significantly increased (P<0.001),and the time spent in the CPA-paired compartment was considerably decreased compared with the control group (P<0.001).After EA intervention for 3 and 7 days, the PWLD levels of both EA and AEA groups were apparently decreased(P<0.05),and the time spent in the CPA-paired compartment was apparently increased in the EA and AEA groups(P<0.05),suggesting a pain relief and an improvement of the negative affection after EA intervention. Additionally, following EA, the apparently-decreased expression levels of GABAAβ 2,GABAB 1,PSD-95,Piccolo genes and the reduced numbers of GABAB 2 positive cells and NMDA-NR 1 mRNA as well as mGluR 1 positive fiber numbers were remarkably increased in the EA group (P<0.05, P<0.001).The expression levels of Piccolo gene, GABAB 2 and mGluR 1 positive cells/fiber numbers were apparently lower in the AEA group than in the EA group (P<0.001). No significant differences were found between the EA and AEA groups in the PWLD, time spent in the CPA-paired compartment, and the expression levels of NMDA-NR 1, GABAAβ 2, GABAB 1 and PSD-95 genes (P>0.05). CONCLUSIONS:Repeated EA stimulation of ST 36-GB 34 has a role in relieving both sensory and affection dimensions of chronic pain in CNPPNA rats, which Feb be respectively related to its effects in up-regulating the expression of GABAAβ 2, GABAB 1, NMDA-NR 1, PSD-95 and Piccolo genes, and in promoting the expression of mGluR 1 and GABAB 2 proteins and Piccolo gene in the amygdala.

Chronic pain is among the most disabling and costly disorders, with prevalence ranging from 10% to 55%. However, current therapeutic strategies for chronic pain are unsatisfactory due to our poor understanding of its mechanisms. Thus, novel therapeutic targets need to be found in order to improve these patients' quality of life. PI3K and its downstream Akt are widely expressed in the spinal cord, particularly in the laminae I-IV of the dorsal horn, where nociceptive C and Aδ fibers of primary afferents principally terminate. Recent studies have demonstrated their critical roles in the development and maintenance of chronic pain. In this review, we summarized the roles and mechanisms of PI3K/Akt pathway in the progression of chronic pain through sciatic nerve injury, diabetic neuropathy, spinal cord injury, bone cancer, opioid tolerance, or opioid-induced hyperalgesia.

This paper explores the effects of glial cells in pain mechanisms and acupuncture in the treatment of chronic pain.Chronic pain is one of the intractable diseases in clinical practice.In the past,in revealing the underlying mechanisms of chronic pain,neurons in the peripheral and central nervous system have been focused on all along.In recent years,it has been found that the glial cells are also closely associated with the development of pain.In the present paper,the authors introduce the roles of microgliacytes and astrocytes of the spinal dorsal horns (DH) in the development of chronic inflammatory pain and neuropathic pain and their involvement in acupuncture analgesia.Experimental results indicated that when inducing pain relief,acupuncture can inhibit activation of both microglia and astrocytes in the spinal DH,and suppress neuron-microglia-astrocyte cross-talk by down-regulating pro-inflammatory cytokine levels,receptor protein expression,intracellular ERK/MAPK signaling pathway.However,the more detailed mechanisms underlying acupuncture therapy for relieving chronic pain needs being researched further.