The mechanism of AMPA receptor subunit GluR1 in electroacupuncture treatment of acute spinal cord injury in rats.

Glutamate excitotoxicity plays a role in spinal cord injury (SCI). This study aimed to explore whether electroacupuncture (EA) improved the functional recovery of spinal cord anterior horn neurons of rats with acute SCI by regulating the GluR1 AMPA subunit in the SCI area. Eighty Sprague-Dawley rats were randomly divided into 5 groups: sham operation, model, AMPA antagonist (DNQX), EA and DNQX + EA group (n = 16/group). The models were obtained by using the modified Allen's impact method. DNQX was given by intrathecal injection 0.5 h after modeling. EA was performed at the "Dazhui" and "Mingmen" acupoints for 30 min at 0.5, 12, and 24 h. The BBB scores were evaluated before modeling and at 6, 24, and 48 h after modeling. Histopathological changes were evaluated. GluR1 expression was evaluated through immunofluorescence and western blot. Compared to the sham group, the BBB scores at 6, 24, and 48 h in the model group were all lower. The BBB scores and histopathological changes in the EA, DNQX and DNQX + EA group were between that of the sham and model group. GluR1 expression in the model group was higher than the sham group. Compared with the model group, the expression of GluR1 protein in the EA, DNQX, and DNQX + EA group was decreased, but similar among the three treatment groups, supporting the histopathological observations. In conclusion, these findings indicated that EA treatment might inhibit GluR1 expression, thus contributing to prevention of secondary nerve injury after primary acute SCI.

The mechanism of AMPA receptor subunit GluR1 in electroacupuncture treatment of acute spinal cord injury in rats.

Glutamate excitotoxicity plays a role in spinal cord injury (SCI). This study aimed to explore whether electroacupuncture (EA) improved the functional recovery of spinal cord anterior horn neurons of rats with acute SCI by regulating the GluR1 AMPA subunit in the SCI area. Eighty Sprague-Dawley rats were randomly divided into 5 groups: sham operation, model, AMPA antagonist (DNQX), EA and DNQX+EA group (n=16/group). The models were obtained by using the modified Allen's impact method. DNQX was given by intrathecal injection 0.5 h after modeling. EA was performed at the "Dazhui" and "Mingmen" acupoints for 30 min at 0.5, 12, and 24 h. The BBB scores were evaluated before modeling and at 6, 24, and 48 h after modeling. Histopathological changes were evaluated. GluR1 expression was evaluated through immunofluorescence and western blot. Compared to the sham group, the BBB scores at 6, 24, and 48 h in the model group were all lower. The BBB scores and histopathological changes in the EA, DNQX and DNQX+EA group were between that of the sham and model group. GluR1 expression in the model group was higher than the sham group. Compared with the model group, the expression of GluR1 protein in the EA, DNQX, and DNQX+EA group was decreased, but similar among the three treatment groups, supporting the histopathological observations. In conclusion, these findings indicated that EA treatment might inhibit GluR1 expression, thus contributing to prevention of secondary nerve injury after primary acute SCI.

[Influence of electroacupuncture on the expression of AMPA receptor subunit GluR1 in the spinal injured area of the rats with acute spinal cord injury].

OBJECTIVE: To explore the influence of electroacupuncture (EA) on the expression of AMPA receptor subunit GluR1 in the rats with acute spinal cord injury (SCI) and explore the potential effect mechanism of EA in treatment of acute SCI. METHODS: A total of 80 SD rats were randomly divided into five groups, i.e. a sham-operation group, a model group, an AMPA antagonist (DNQX) group, an EA group and a DNQX+EA group, 16 rats in each group. The modified Allen's impacting method was adopted to prepare the rat model of acute SCI at T10. In the DNQX group, the intrathecal injection of 10 µL DNQX solution with a concentration of 1 nmol/µL was administered in 0.5 h after modeling success. In the EA group, EA (disperse-dense wave, 2 Hz/100 Hz in frequency, 0.5 mA in output current) was given at "Dazhui" (GV 14) and "Mingmen" (GV 4) in 0.5 h, 12 h and 24 h after modeling success for 30 min and totally 3 times. In the DNQX + EA group, the interventions in the above two groups were managed. The Basso, Beattie and Bresnahan locomotor rating score (BBB) was applied to evaluate the changes of locomotor function in the rats before modeling and in 6 h, 24 h and 48 h after modeling successively. Using the hematoxylin-eosin (HE) staining, the histopathological changes in the spinal anterior horn were observed in the spinal injured area. The immunofluorescence method was adopted to determine the number of GluR1 positive neuron of the spinal anterior horn. The Western blot method was used to determine the protein expression of GluR1 in the injured area. RESULTS: Compared to the sham-operation group in 6 h, 24 h and 48 h after modeling, the BBB scores were all significantly decreased in the model group (P<0.001) at the corresponding points. The BBB score was increased in each of intervention groups, but without statistical difference as compared with the model group (P>0.05). In the model group, it was found that the boundary between gray matter and white matter in the spinal anterior horn was blurred, the interstitial space enlarged, the neuron volume obviously shrunken, the cytoplasm decreased, the red stain deepened and some neuron nuclei fixed and shrunk. In the EA group, the morphology of the spinal anterior horn in the injured area was improved obviously, which was similar in the DNQX group and the DNQX + EA group. Compared with the sham-operation group, the GluR1 protein expression in the spinal injury area was increased (P<0.001) and the number of GluR1 positive neurons elevated (P<0.001) in the spinal anterior horn in the model group. Compared with the model group, in the EA group, the DNQX group and the DNQX + EA group, GluR1 protein expression was decreased (P<0.05, P<0.01) and the number of GluR1 positive neurons in the spinal anterior horn reduced (P<0.001). CONCLUSION: The intervention with EA at "Dazhui" and "Mingmen" promotes the repair of the injured nerve in the spinal anterior horn probably through inhibiting GluR1 expression in the spinal injured area in the rats with acute SCI.

[Influence of electroacupuncture of "Dazhui" (EX-B2) and "Mingmen" (GV4) on NR2B expression in anterior horns of spinal cord in rats with acute spinal cord injury].

OBJECTIVE: To observe the influence of eletroacupuncture (EA) at "Dazhui" (EX-B2) and "Mingmen" (GV4) on expression of NR2B subunit of N-methyl-D-aspartate receptor (NMDA) in the injured anterior horn (AH) area of rats with acute spinal cord injury (SCI), so as to explore its mechanisms underlying improvement of neural repair. METHODS: A total of 96 male Sprague-Dawley (SD) rats were randomly and equally divided into four groups: sham operation (sham), model, medication (Methyl-prednisone, MP) and EA (n＝24 in each group). The acute SCI model was established by using a MASCIS spinal cord impactor. EA (2 Hz, 0.5 mA) was applied to EX-B2 and GV4 for 30 min, once at 0.5 h, 12 and 24 h after SCI. Rats of the medication group were treated by tail intravenous injection of MP 30 mg/kg within 15 min (impact therapy) and 5.4 mg•kg－1•h－1 (maintaining treatment) 45 min thereafter for 23 h. The Basso, Beattie and Bresnahan (BBB) rating scale (0 to 21 points) was used to assess changes of locomotor function 6, 24 and 48 h after SCI. Histopathological changes of the injured spinal cord AH region were observed after sectioning and hematoxylin-eosin (H.E.) staining, and the expression levels of NR2B mRNA and protein of AH were measured by quantitative real-time PCR, Western blot and immunofluorescence, respectively. RESULTS: After SCI, the BBB scores at 6, 24 and 48 h were significantly decreased in the model group compared with those of the sham group (P<0.001), but had no remarkable changes in both medication and EA groups after the intervention in comparison with those of the model group (P>0.05). After modeling, the histopathological changes (blurred border of the grey-white matter, cellular karyopyknosis, deepening of the cytoplasmic red stain, and rupture, dissolution and disordered arrangement of myelinated nerve fibers) in the injury area of the spinal cord in the model group were apparent, the number of NR2B positive neurons and the relative expression levels of NR2B mRNA and protein were significantly increased in the model group relevant to the sham group (P<0.05). In contrast to the model group, the injured severity of the spinal cord AH region was relatively milder, and the expression levels of NR2B mRNA and protein were considerably down-regulated in both EA and MP groups (P<0.05). However, there were no significant differences between the EA and MP groups in the expression levels of NR2B mRNA and protein (P>0.05). CONCLUSION: EA at EX-B2 and GV4 may inhibit the expression of NR2B mRNA and protein in acute SCI rats, which may contribute to its action in promoting nerve repair of the injured ventricolumna area of the thoracic spinal cord.

Environmental inhibitors of the expression of cytochrome P450 17A1 in mammals.

Cytochrome P450 17A1 (CYP17A1; EC: 1.14.14.19) is a critically important bifunctional enzyme with nicotinamide adenine dinucleotide phosphate (NADPH) as its cofactor that catalyzes the formation of all endogenous androgens. Its hydroxylase activity catalyzes the 17α-hydroxylation of pregnenolone (PREG)/progesterone (P4) to 17α-OH-pregnenolone/17α-OH-progesterone, and its 17,20-lyase activity converts 17α-OH-pregnenolone/17α-OH-progesterone to dehydroepiandrosterone/androstenedione. Androgens are required for male reproductive development, so androgen deficiency resulting from CYP17A1 inhibition may lead to reproductive disorders. There has been some advances on the study of environmental chemicals inhibiting mammalian CYP17A1 expression but no related review was available so we think it now necessary to review their characteristics and inhibiting properties.

KCC2-GABAA pathway correlates with the analgesic effect of electro-acupuncture in CCI rats.

Potassium-chloride cotransporter 2 (KCC2) has been indicated to serve a crucial role during chronic neuropathic pain (NP). Following the emergence of NP, γ­aminobutyric acid (GABA) A receptor­mediated signaling may be further impaired by the changes of KCC2 chloride anion gradient. In the present study, the authors investigate the effect of electro-acupuncture (EA) on the behavior and the expression of KCC2 and GABAA receptor γ2 subunit in the spinal cord of chronic constriction injury (CCI) model rats. A total of 60 adult male Sprague­Dawley rats were divided into four groups: Normal group, sham­CCI group, CCI group and CCI+EA group. The effect of EA was assessed via the values of mechanical withdrawal threshold and thermal withdrawal latency, which were significantly improved upon stimulation of the ST­36 and GB­34 acupoints. In addition, a marked reduction in both the mRNA and protein levels of KCC2 and GABAA receptor γ2 subunit was observed in the spinal cord following loose ligation of the sciatic nerve. The reductions in KCC2 and GABAA receptor γ2 subunit expression were reversed by EA treatment. These results support the notion that KCC2 and GABAA receptor γ2 subunit contribute to NP following peripheral nerve injury and extend the understanding of the analgesic effects of EA on NP.

[Effects of electroacupuncture on activation of microglia cells in spinal cord in rats with neuropathic pain].

OBJECTIVE: To observe the effects of electroacupuncture (EA) on the activation of microglia cells in the L4 to L6 spinal cord in rats with neuropathic pain, so as to investigate whether EA could inhibit the activation of spinal microglial cells and regulate the expression of brain-derived neurotrophic factor (BDNF) to achieve the analgesic effect. METHODS: Forty male Sprague Dawley rats were randomly divided into a normal group, a sham-model group, a model group and an EA group, 10 rats in each one. The rats in the normal group received no treatment; the rats in sham-model group were treated with operation to exposure sciatic nerve for 2 to 3 min (no knot); the rats in the remaining groups were treated with model establishment of chronic constrictive injury (CCI). 7 days after model establishment, the rats in the EA group were treated with EA at "Zusanli" (ST 36) and "Yanglingquan" (GB 34), 30 min per time, once a day for consecutive 7 days. Only immobilization was used in the remaining groups the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of affected side feet were respectively measured before model establishment and 3 days, 5 days, 7 days, 10 days, 12 days and 14 days after model establishment; 14 days after model establishment, rats were sacrificed; the immunohistochemical method was used to measure the expression of Iba1 and BDNF in the sample of L4 to L6 spinal cord; real-time fluorescent quantitative PCR was used to measure the expression BDNF mRNA. RESULTS: Compared with the sham-model group, the pain threshold was decreased significantly in the model group (P<0.05), leading to hyperpathia. After EA treatment, compared with the model group, the pain threshold was increased significantly in the EA group (P<0.05). 14 days after operation, the microglia cells in the L4 to L6 spinal cord, expression of BDNF and level of mRNA in the model group were significantly higher than those in the normal group and sham-model group (all P<0.01); those in the EA group were significantly lower than those in the model group (all P<0.01). CONCLUSIONS: The analgesic effect on neuropathic pain is likely to be achieved by EA through inhibiting the activation of spinal microglia cells and down-regulating the expression of BDNF.

The regulatory effect of electro-acupuncture on the expression of NMDA receptors in a SCI rat model.

BACKGROUND: In early spinal cord injury (SCI), glutamate receptors, including N-methyl-d-aspartate (NMDA) receptors (NMDARs), are over-stimulated by excessively released glutamate. The enhanced activity of NMDARs may cause cell death by overloading calcium (Ca2+) into cells based on their high permeability to Ca2+. Studies in SCI animals have shown that treatment with electro-acupuncture (EA) is able to reduce cell death and to improve functional recovery. One possible mechanism of this neuroprotective effect is that EA has regulatory effect on NMDARs. AIMS: To test whether EA could protect the spinal cord after SCI by decreasing the expression levels of NR1 and NR2A. MAIN METHODS: We conducted EA treatment on a rat SCI model produced with a New York University (NYU) Impactor and measured hindlimb locomotor function by Basso, Beattie and Bresnahan Locomotor Rating Scale (BBB Scale). The expression of NR1 and NR2, the subunits of NMDARs, in the injured spinal cord was measured by Immunofluorescence stainings, western blot and real-time quantitative PCR (RT-qPCR). KEY FINDING: Our results showed that two days after the SCI the expression of NR1 and NR2 were dramatically enhanced at both protein and mNRA levels, which were significantly reduced by EA treatment at two specific acupoints, Dazhui (DU14) and Mingmen (DU4). SIGNIFICANCE: EA is a potential therapeutic method for treating early SCI in human.