Effect of electroacupuncture on interleukin-6 and procalcitonin expression in the astrocytes and neurons of the spinal cord dorsal horn in a rat model of incisional pain.

To observe the effect of electroacupuncture (EA) at "Huantiao" (GB 30) and "Weizhong" (BL 40) on the activation of glial cells, the expression of brain-derived neurotrophic factor (BDNF), excitability and the number of dendritic spines of neurons in the spinal dorsal horn in rats with spared nerve injury (SNI) of sciatic nerve, and to explore the analgesic mechanism of EA on SNI. PartⅠ: Sixty SD rats were randomly divided into a sham operation group, a model group, an EA group and a sham EA group, 15 rats in each group. Except the sham operation group, the SNI rat model was established in the remaining groups. The rats in the sham operation group were only treated with incision without damaging the nerve. The rats in the EA group were treated with EA at "Huantiao" (GB 30) and "Weizhong" (BL 40) on the affected side, continuous wave, frequency of 2 Hz, current intensity of 1 mA, 30 minutes each time, once a day, for 14 days. The rats in the sham EA group were treated with EA at points 0.5 cm next to "Huantiao" (GB 30) and "Weizhong" (BL 40) on the affected side; the manipulation, EA parameters and treatment course were the same as the EA group. The latency of thermal foot contraction reflex and the threshold of mechanical foot contraction reflex were detected 1 day before modeling and 3, 7 and 14 days after modeling. Fourteen days after modeling, Western blot was used to detect the protein expressions of ionized binding adapter junction protein 1 (Iba-1), glial fibrillary acidic protein (GFAP), BDNF and c-Fos in the spinal dorsal horn; the expressions of Iba-1 and c-Fos proteins in the spinal dorsal horn were detected by immunofluorescence staining; immunohistochemical method was used to detect the expression of GFAP protein in the spinal dorsal horn; Golgi staining was used to detect the number of dendritic spines in spinal dorsal horn neurons. PartⅡ: Thirty SD rats were randomly divided into a control group, a BDNF group and a BDNF+anti-TrkB group, 10 rats in each group. The control group was treated with intrathecal injection of 10 μL mixture with 1︰1 of 0.9% sodium chloride solution and dimethyl sulfoxide (DMSO); the BDNF group was treated with intrathecal injection of 10 μg rat recombinant BDNF dissolved in 10 μL mixture with 1︰1 of 0.9% sodium chloride solution and DMSO; the BDNF+anti-TrkB group was treated with intrathecal injection of 10 μg rat recombinant BDNF and 30 μg tyrosine kinase receptor B (TrkB) antibody dissolved in 10 μL mixture with 1︰1 of 0.9% sodium chloride solution and DMSO. The threshold of mechanical foot retraction reflex was detected 1 day before intrathecal injection and 1, 3 and 7 days after injection. Seven days after injection, the expression of c-Fos protein in the spinal dorsal horn was detected by Western blot and immunofluorescence staining. PartⅠ: Compared with the sham operation group, 3, 7 and 14 days after modeling, the latency of thermal foot contraction reflex and the threshold of mechanical foot contraction reflex in the model group were decreased (P<0.05); 7 and 14 days after modeling, compared with the model group, the latency of thermal foot contraction reflex and the threshold of mechanical foot contraction reflex in the EA group were increased (P<0.05). The expressions of Iba-1, GFAP, BDNF, c-Fos proteins and the number of neuronal dendritic spines in the spinal dorsal horn in the model group were higher than those in the sham operation group (P<0.05); the expressions of Iba-1, BDNF, c-Fos proteins and the number of neuronal dendritic spines in the EA group were lower than those in the model group (P<0.05). PartⅡ: 3 and 7 days after intrathecal injection, the threshold of mechanical foot retraction reflex in the BDNF group was lower than that in the control group (P<0.05); the threshold of mechanical foot retraction reflex in the BDNF+anti-TrkB group was higher than that in the BDNF group (P<0.05). The expression of c-Fos protein in spinal dorsal horn in the BDNF group was higher than that in the control group (P<0.05); the expression of c-Fos protein in spinal dorsal horn in the BDNF+anti-TrkB group was lower than that in the BDNF group (P<0.05). The analgesic effect of EA at "Huantiao" (GB 30) and "Weizhong" (BL 40) on SNI rats may be related to inhibiting the activation of microglia in the dorsal horn of the spinal cord, thereby blocking the signal of microglia-BDNF-neuron, and finally reducing the excitability of neurons.

Objective To evaluate the role of Stargazin in trafficking of GluR1-containing AMPA receptors from cytoplasm to cell membrane in the spinal dorsal horn in rats with incisional pain.Methods Forty-five adult male Sprague-Dawley rats,aged 6-8 yr,weighing 280-300 g,were randomly divided into 5 groups (n =9 each):control group (group C),sham operation group (group S),incisional pain + normal saline group (group P),incisional pain + Stargazin small interference RNA (siRNA) group (group Ⅰ),and incisional pain + non-sense siRNA group (group N).In P,I and N groups,normal saline 10 μl,20 μmol/L siRNA and 20 μmol/L non-siRNA 10 μl were injected intrathecally,respectively,twice a day for 3 consecutive days.A 1 cm long incision was made in the plantar surface of right hindpaw.Cumulative pain score (CPS) and paw-withdrawal threshold to yon Frey stimuli (PWT) were measured at 3 h after incision.The animals were sacrificed after pain behavior assessment.Their lumbar segments of the spinal cord (L3-6) were removed.The expression of GluR1 and GluR2 in cell membrane and cytoplasm in spinal cord dorsal horn was determined by Western blot analysis.The co-expression of Stargazin with GluR1 and GluR2 in the spinal dorsal horn was examined by co-immuno-precipitation.Results Compared with group C,the CPS was significantly increased,the PWT was decreased,the expression of GluR1 in cytoplasm was down-regulated and the expression of GluR1 in cell membrane was up-regulated in N and P groups,and the CPS was increasedin group Ⅰ (P ＜ 0.05 or 0.01).Compared with group P,the CPS was significantly decreased,the PWT was increased,the expression of GluR1 in cytoplasm was up-regulated,the expression of GluR1 in cell membrane was down-regulated,and the expression of Stargazin and co-expression of Stargazin with GluR1 and GluR2 in the spinal dorsal horn was down-regulated in group Ⅰ (P ＜ 0.05 or 0.01).Conclusion Stargazin mediates trafficking of GluR1-containing AMPA receptors from cytoplasm to cell membrane in spinal dorsal horn and is involved in the development of hyperalgesia in rats with incisional pain. Key words: Receptors, AMPA; Protein transport; Pain; Spinal cord; Stargazin

HIGH-THRESHOLD MECHANORECEPTORS and their centrally projecting myelinated fibers make up a functionally distinct group of cutaneous sensory units that have been suggested as part of the afferent apparatus for pain resulting from mechanical damage to the skin (3, 17). The argument for their relation to pain was based on two points: 1) the ability of such afferent elements, unique among those with medullated fibers, to provide signals differentiating noxious from innocuous mechanical events affecting the skin; and 2) the well-established correlation between pain and activity in thin myelinated afferent fibers

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. 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). 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. 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.

To observe the effect of electroacupuncture (EA) at "Ciliao" (BL32) and "Huiyang" (BL35) on the expression of extracellular signal-regulated kinase 1/2 (p-ERK1/2) and cellular oncogene fos (c-fos) phosphorylated of spinal dorsal horn in rats with interstitial cystitis (IC). Eighteen female Wistar rats were randomly divided into control, model and EA groups, with 6 rats in each group. The IC model was established by intraperitoneal injection of cyclophosphamide (150 mg/kg). EA (30 Hz, 1 mA) was applied to bilateral BL32 and BL35 for 20 min, once daily for 3 consecutive days. The bladder pain was measured by using a Von Frey at 48 h after modeling and 24 h after EA. The expression levels of p-ERK1/2 and c-fos protein in L6-S1 segment of spinal cord were detected by Western blot, and the expression of p-ERK1/2 and c-fos in the right spinal dorsal horn were displayed by immunofluorescence staining. After modeling, the bladder mechanical pain threshold (PT) was significantly decreased (P<0.05), the protein expression of p-ERK1/2 and c-fos in the spinal cord was increased (P<0.05) and the immunofluorescence surface density of p-ERK1/2 and c-fos in the right dorsal horn of spinal cord was increased (P<0.05) in the model group relevant to the control group. After EA intervention, IC-induced reduction of PT, and increases of the expression of p-ERK1/2 and c-fos as well as immunofluorescence surface density of p-ERK1/2 and c-fos were reversed in the EA group relevant to the model group (P<0.05). EA at BL 32 and BL 35 has an analgesic effect in IC rats, which may be related to its effect in down-regulating the expression of p-ERK1/2 and c-fos in spinal dorsal horn.

To observe the occurrence time of neuralgia and the expression of purinergic ligand-gated ion channel 7 receptor (P2X7R) in the dorsal horn of the spinal cord after intraperitoneal injection of streptozotocin (STZ) in diabetic rats, and to explore the effect of electroacupuncture (EA) and pretreatment of EA on the heat pain threshold and expression of P2X7R in the spinal dorsal horn in rats with diabetic neuropathic pain (DNP), and to explore the possible mechanism of EA for DNP. PartⅠ: Thirty male SD rats were randomly selected from 64 male SD rats as the control group; the remaining rats were given intraperitoneal injection of STZ (10 mg/mL) at a dose of 65 mg/kg to establish the diabetes model, and 30 rats were successfully modeled as the model group. The control group and the model group were divided into three subgroups respectively at 7, 14 and 21 days, with 10 rats in each subgroup. Body mass, fasting blood glucose (FBG) and thermal pain threshold were recorded at 7, 14 and 21 days after injection; the expression of P2X7R in spinal dorsal horn was detected by Western blot. PartⅡ: Eight SD rats were randomly selected from 35 male SD rats as the blank group, and the remaining 27 rats were given intraperitoneal injection of STZ (10 mg/mL) at a dose of 65 mg/kg to establish the diabetes model. The 24 rats with successful diabetes model were randomly divided into a DNP group, an EA group and a pre-EA group, 8 rats in each group. Fifteen to 21 days after STZ injection, the EA group received EA at "Zusanli" (ST 36) and "Kunlun" (BL 60), continuous wave, frequency of 2 Hz, 30 min each time, once a day; the intervention method in the pre-EA group was the same as that in the EA group. The intervention time was 8 to 14 days after STZ injection. The body mass, FBG and thermal pain threshold were recorded before STZ injection and 7, 14 and 21 days after STZ injection; the expression of P2X7R in spinal dorsal horn was detected by Western blot 21 days after injection. PartⅠ: Compared with the control group, in the model group, the body mass was decreased and FBG was increased 7, 14 and 21 days after STZ injection (P<0.01), and the thermal pain threshold was decreased 14 and 21 days after STZ injection (P<0.05), and the expression of P2X7R in spinal dorsal horn was increased 7, 14 and 21 days after STZ injection (P<0.05, P<0.01). PartⅡ: Compared with the blank group, in the DNP group, the body mass was decreased and fasting blood glucose were increased 7, 14 and 21 days after STZ injection (P<0.01). Compared with the DNP group, in the pre-EA group, the heat pain threshold was increased 14 and 21 days after STZ injection (P<0.05), while in the EA group, the heat pain threshold was increased 21 days after STZ injection (P<0.01), and the expression of P2X7R in the dorsal horn in the EA group and the pre-EA group was decreased (P<0.01). The diabetic neuropathic pain is observed 14 days after STZ injection. EA could not only treat but also prevent the occurrence of DNP, and its mechanism may be related to down-regulation of P2X7R expression in the dorsal horn of the spinal cord.

BackgroundOur previous study demonstrated that neuronal G protein-coupled receptor kinase (GRK2) upregulation alleviated chemotherapy-induced peripheral neuropathy (CIPN) in mice, which was characterized by numbness and pain in distal hind limbs. The neuronal GRK2 was identified as a mediator of electroacupuncture (EA) effects on CIPN. Given that spinal insulin-like growth factor 1 (IGF1), a known inducer of GRK2 in the peripheral neurons, decreases after oxaliplatin treatment in mice, this study is designed to investigate whether spinal IGF1 contributes to EA-mediated prevention of cisplatin-induced peripheral neuropathy via neuronal IGF1 receptor (IGF1R).MethodsA total of 133 male C57BL/6 J mice were included in this study and randomly assigned to different experimental groups. The level of Igf1 mRNA was detected by Real-time PCR, the p-IGF1R protein level by Western blot, after EA treatment in cisplatin-treated mice. The cellular distribution of p-IGF1R in the spinal dorsal horn was observed by immunofluorescent staining. To study the role of neuronal IGF1R in EA preventing cisplatin-induced mechanical allodynia, sensory deficit, and microglia activation and neuroinflammation in the spinal cord of mice, the neuronal IGF1R was downregulated by intraspinal injection of an AAV vector delivering IGF1R shRNA with hSyn promotor (AAV-shIGF1R). Finally, the regulatory effect of EA on spinal GRK2 was assessed by Western blot in AAV-shIGF1R mice.ResultsCisplatin treatment induced mechanical allodynia, sensory deficit, and a decrease of p-IGF1R in the spinal dorsal horn of mice. Immunofluorescence showed that p-IGF1R was localized within neurons (~ 82%), a small mount of microglia (~ 12%) and astrocytes (~ 4%). Cisplatin decreased NeuN+p-IGF1R+ neurons in the spinal dorsal horn. EA treatment significantly alleviated cisplatin-induced mechanical allodynia, sensory deficit, and significantly increased the Igf1 mRNA and p-IGF1R level in the spinal cord. Neuronal IGF1R downregulation in the spinal dorsal horn significantly attenuated the preventive effect of EA on cisplatin-induced mechanical allodynia, sensory deficit, and spinal microglial activation and neuroinflammation in mice. Furthermore, neuronal IGF1R downregulation decreased the spinal GRK2 in cisplatin-treated mice after EA treatment. These findings suggest EA significantly alleviated CIPN symptoms by enhancing IGF1/IGF1R signaling and reducing microglial activation and neuroinflammation.ConclusionSpinal dorsal horn IGF1 contributes to the preventive effect of EA treatment against cisplatin-induced peripheral neuropathy through neuronal IGF1R signaling in mice. The enhanced neuronal IGF1/IGF1R signaling in the spinal cord presents a potential strategy for CIPN prevention.

BackgroundAcupuncture has shown to be effective in relieving post-surgical pain. Nonetheless, its underlying mechanisms remain largely unknown. In the present study, we investigated the effect of electroacupuncture (EA) on the expression of GABA, GABA-A receptor (R) and GABA-BR in the spinal cord dorsal horns (DHs), and the involved neural cells in rats with incisional neck pain.Materials and MethodsMale SD rats were randomly divided into control, model, Futu (LI18), Hegu-Neiguan (LI4-PC6), and Zusanli-Yanglingquan (ST36-GB34) groups. The incisional neck pain model was established by making a longitudinal incision and repeated mechanical separation along the thyroid gland region. EA (2Hz/100Hz, 1mA) was applied to LI18, LI4-PC6, ST36-GB34 separately for 30min, once at 4, 24 and 48h after incision. The local thermal pain threshold (TPT) of the focus was measured and the expression of GABA, and GABAR proteins and mRNAs detected by immunofluorescence stain and quantitative RT-PCR, respectively.ResultsThe analgesic effect of LI18 and LI4-PC6 was superior to that of ST36-GB34 in incisional neck pain rats. Moreover, the EA stimulation of LI18 or LI4-PC6 increased the expression of GABA and GABA-Aα2 and GABA-Aβ3, GABA-B1, and GABA-B2 mRNAs in spinal DHs 4h after surgery, while GABA-A and GABA-B antagonists inhibited the analgesic effect of LI18. Immunofluorescence double staining showed that GABA was expressed on astrocytes and neurons, and GABA-B expressed only on neurons.ConclusionEA of both LI18 and LI4-PC6 has a good analgesic effect in incisional neck pain rats, which is closely related to their effects in upregulating the expression of GABA and its receptors in spinal DHs. The effects of LI18 and LI4-PC6 EA are obviously better that those of ST36-GB34 EA, and GABA is expressed on neurons and astrocytes.

Electroacupuncture modifies the expression of c- fos in the spinal cord induced by noxious stimulation

To observe the effect of electroacupuncture(EA) on the pain behavior and prostaglandin E2 (PGE2), calcitonin gene-related peptide (CGRP) and substance P (SP) in the spinal cord dorsal horn and dorsal root ganglia (DRG) of rats with knee osteoarthritis (KOA), so as to explore the mechanisms of EA underlying improvement of chronic pain in KOA rats. Thirty-two female SD rats were randomly divided into blank group, control group, model group and EA group, with 8 rats in each group. Rats in the control group were injected with 50 μL of 0.9% sodium chloride solution into the left knee joint cavity, and rats in the model and EA groups were injected with 50 μL of Monosodium iodoacetate in the left knee joint. EA(2 Hz/100 Hz, ＜2 mA) was applied to left "Yanglingquan"(GB34) and "Neixiyan" (EX-LE4) for 15 min, once daily, 5 days a course with a total of 2 courses. Paw withdrawal latency (PWL) and mechanical pain threshold (PWT) were tested by Plantar Test and Von Frey, separately. After the last pain test, the contents of PGE2, CGRP and SP in the left lumbar (L) 3-L5 DRG and L3-L5 spinal dorsal horn were detected by ELISA. Compared with the blank group, the PWL and PWT of the rats in the model group were significantly decreased (P<0.01), and the contents of PGE2, CGRP and SP in the DRG and spinal dorsal horn were significantly increased (P<0.01, P<0.05). There were no statistically significant differences in PWL, PWT, contents of PGE2, CGRP and SP in DRG and spinal dorsal horn between the blank group and the control group (P>0.05). Compared with the model group, the PWL and PWT of rats in the EA group were significantly increased (P<0.01), and the contents of PGE2, CGRP and SP in the DRG and spinal dorsal horn were significantly decreased (P<0.01, P<0.05). EA of GB34 and EX-LE4 can reduce the levels of pain-related factors PGE2, CGRP and SP in the DRG and spinal dorsal horn, thereby relieving spinal hyperalgesia in rats with KOA.

Changes in expression of nociceptin/orphanin FQ and its receptor in spinal dorsal horn during electroacupuncture treatment for peripheral inflammatory pain in rats

Objective To evaluate the changes in the expression of neuroligin1 in excitatory postsynaptic membrane of the spinal dorsal horn in a rat model of incisional pain. Methods Forty-eight pathogen-free healthy adult male Sprague-Dawley rats, aged 6-8 weeks, weighing 280-320 g, were divided into control group(group C, n=12)and incisional pain group(group I, n=36)using a random number table.A 1 cm long incision was made in the plantar surface of the right hindpaw in group I. Cumulative pain score(CPS)was assessed and mechanical paw withdrawal threshold to von Frey stimuli was measured at 3 h and 1 and 3 days after operation(T1, 2, 3). The animals were then sacrificed and their lumbar segments(L3-6)of the spinal cord were removed for detection of the expression of neuroligin1, postsynaptic density-95 protein(PSD-95), glutamate receptor 1(GluR1)and GluR2 in the postsynaptic membrane of spinal dorsal horn(by Western blot)and co-expression of neuroligin1 with PSD-95 in spinal dorsal horn(by co-immuno-precipitation). Results Compared with group C, CPS was significantly increased and mechanical paw withdrawal threshold was decreased at T1-3, and the expression of neuroligin1 and GluR1 in the postsynaptic membrane of spinal dorsal horn at T1, 2 and co-expression of neuroligin1 with PSD-95 at T1 were up-regulated in group I(P<0.01 or 0.05). Conclusion The development and maintenance of incisional pain is related to the signaling pathway regulated by neuroligin1 in excitatory postsynaptic membrane of the spinal dorsal horn of rats. Key words: Cell Adhesion Molecules; Pain; Spinal cord; Receptors, AMPA

The effect of electroacupuncture on pain behaviors and noxious stimulus-evoked Fos expression in a rat model of neuropathic pain

Objective To observe the effect of intrathecal clonidine plus morphine on expression of protein kinase A (PKA) catalytic subunit in the spinal dorsal horn in a rat model of incisional pain. Methods Eighty male Sprague-Dawley rats were divided randomly into five groups: sham group, control group, pre-incisional morphine 2.5 μg group, pro-incisional clonidine 5 μg group and preincisional morphine 2.5 μg plus clonidine 5 lag group (n=16). lntrathecal catheter and the model of incisional pain were pro-duced according to Yaksh and Brennan's described method respectively. Changes of pain behavior were assessed by mechanical with-drawal threshold (MWT) and thermal withdrawal latency(TWL). The expressions of PKA catalytic subunit in the spinal dorsal horn were assessed by immunohistochemical method and western blotting analysis. Results Compared with sham group, MWT and TWL in control group were decreased significantly at 2 h after incision (P<0.01) and the number of positive cells and protein expression of PKA catalytic subunit in the spinal dorsal horn were increased significantly in control group (P<0.01). Compared with control group, MWT and TWL in pre-incision morphine 2 μg plus clonidine 5 lag group were increased significantly at 2 h after incision (P<0.01) and the number of positive cells and protein expression of PKA catalytic subunit in the spinal dorsal horn were decreased significantly in pre-incision morphine 2 μg plus clonidine 5 μg group (P<0.01). However, MWT, TWL and the number of positive cells and pro-tein expression of PKA catalytic subunit in the spinal dorsal horn changed with no statistical significance in pre-incisional morphine 2.5 μg group and pre-incisional clonidine 5 μg group compared with control group. Conclusion lntrathecal clonidine significantly enhances the antinociceptive effect of intrathecal morphine in a rat model of incisional pain, which might be associated with inhibi-tion of the increased expression of PKA catalytic subunit in spinal cord. Key words: Pain; Clonidine; Morphine; Protein kinase A; Spinal cord

Effects of electroacupuncture on expression of c-fos protein in the spinal dorsal horn of rats with chronic visceral hyperalgesia