[Influence of electroacupuncture of "Dazhui"（GV14） and "Mingmen" （GV4） on expressions of NL1 and NF-200 in spinal cord injury rats].

OBJECTIVE: To observe the effect of electroacupuncture(EA) on activities of A2 type astrocytes(A2s)and A1 type astrocytes (A1s) , expressions of neurofilament protein 200 (NF-200, a marker of axon regeneration), nexin 1(NL1, a marker of synaptic regeneration), and regeneration of Nissl bodies in rats with spinal cord injury (SCI), so as to explore its mechanisms underlying improvement of SCI. METHODS: A total of 75 male SD rats were rando-mized into sham operation, model, antibody neutralizing (AN), EA and EA+AN groups, with 15 rats in each group. The SCI model was established by using an infinite field impactor to deliver an about 200 k dyne weight onto the exposed spinal cord after making a dorsal laminectomy at vertebral level T10. EA (2 Hz, 1 mA) was applied to"Dazhui"(GV14) and "Mingmen"(GV4) for 20 min, once daily for 28 days. After modeling, intraspinal injection of neutralizing antibodies IL-1α, TNF-α and complement 1q (C1q, 2 µL) to the injured spinal locus for inhibition of A1 type astrocytes (A1s) was conducted on the 1st, 7th , 14th and 21st day for rats of AN and EA+AN groups. BBB rating scale was used to evaluate hindlimb locomotor function on day 1, 7, 14, 21 and 28 after modeling. The activation of A2s (its specific marker S100a10), astrocyte (its specific marker glial fibrillary acidic protein, GFAP), and A1s (its specific marker C3) in the spinal cord was detected by immunofluorescence, and the protein expressions of NF-200 and NL1 in the spinal cord detected by Western blot and immunohistochemistry, separately, and the neuronal regeneration was observed after Nissl staining. RESULTS: After SCI, the BBB scores at 1 , 7, 14, 21 and 28 day, and the immunoactivity of NL1 and NF-200 were significantly decreased (P<0.01), and the fluorescence intensity of double labelled S100a10 (A2s)/GFAP and C3, and the expression of NF-200 were considerably increased in the model group (P<0.05, P<0.01). In contrast to the model group, the BBB scores at 7, 14, 21 and 28 day, and the immunoactivity of NL1 and NF-200, and the fluorescence intensity of A2s/GFAP in the AN, EA and AN+EA groups, and the expressions of NL1 in the EA and AN+EA groups, and expression of NF-200 protein in the AN+EA group were evidently increased (P<0.05, P<0.01), and the fluorescence intensity of C3 was strikingly decreased in the EA group (P<0.01). The effect of AN+EA was significantly superior to that of single AN and EA in increasing BBB scores at 14, 21 and 28 day, and in up-regulating the immunoactivity of NF-200(P<0.01, P<0.05). Nissl staining showed damaged structure of the gray matter of the spinal cord, atrophy of the Nissl body, and pyknosis of neurons, which was milder in the AN and EA groups, particularly in the AN+EA group. CONCLUSION: EA at GV14 and GV4 may promote activation of A2s and promote regeneration of axons and synapses in SCI model rats.

Electroacupuncture Ameliorates Depressive-Like Behaviors in Poststroke Rats via Activating the tPA/BDNF/TrkB Pathway.

BACKGROUND: Electroacupuncture (EA) is a form of physical therapy that has been widely used in clinical practice in China. Post-stroke depression (PSD) is the most common neuropsychiatric complication after stroke. EA has been shown to have beneficial effects on PSD patients. However, the potential mechanism underlying the protective effects of EA on PSD remains unclear. Here, we investigated whether tissue plasminogen activator (tPA)/brain-derived neurotrophic factor (BDNF)/tyrosine kinase receptor B (TrkB) signaling pathway participates in the therapeutic effects of EA in a rat PSD model. METHODS: Experimental PSD was induced by combining middle cerebral artery occlusion (MCAO) with chronic unpredictable mild stimulation (CUMS) in adult male rats. Bodyweight gain, neurological score, sucrose preference, and open field test were determined at 0, 7, 14, and 35 days after completing MCAO. The protein expressions of tPA, precursor BDNF (proBDNF), mature BDNF (mBDNF), and TrkB were measured by immunofluorescence and Western blot analysis. The tPA inhibitor plasminogen inhibitor-1 (PAI-1) was used to explore whether tPA plays a crucial role in the protective effects of EA on PSD. RESULTS: Compared with the sham rats, the PSD rats showed decreased bodyweight, deteriorated neurological score, and significant depressive-like behaviors. EA remarkably reversed bodyweight loss, neurological deficit, and depressive-like behaviors in PSD rats. Immunofluorescence staining and Western blot analysis showed that PSD-induced decreased expression of tPA, mBDNF, and TrkB were prevented by EA. Furthermore, we found that the effects of EA against PSD-induced depressive-like behaviors were abolished by PAI-1, the specific inhibitor of tPA. CONCLUSION: Our results suggest that the improvement in depressive-like behaviors induced by EA is likely achieved via activation of the tPA/BDNF/TrkB pathway.

[Effect of Electroacupuncture Stimulation at "Dazhui" (GV 14) and "Mingmen"(GV 4) on Cell Apoptosis of Spinal Cord and Expression of JNK Signaling Related Protein in Spinal Cord Injury Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation at "Dazhui" (GV 14) and "Mingmen" (GV 4) of the Governor Vessel at different time-points on spinal cord neuronal apoptosis and the expression of c-Jun N-terminal kinases (JNK) protein in spinal cord injury (SCI) rats, so as to reveal its mechanism underlying improving SCI. METHODS: A total of 108 male SD rats were randomly divided into normal control, SCI model and EA groups which were further divided into 1, 3 and 7 d subgroups (12 rats/subgroup, 6 rats in each subgroup for TUNEL or Western blot, separately). SCI model was established by using the modified Allen's method. EA was applied to GV 14 and GV 4 for 20 min, once daily, for 1, 3 and 7 days, respectively. Basso-Beattie-Bresnahan (BBB) scale was adopted to assess the locomotor function of rats, the TUNEL method was used to examine neuronal apoptosis of injuried spinal cord, and the expression of phosphorylated (p)-c-Jun protein of T9-T11 spinal cord was detected by using Western blot. RESULTS: After modeling, the BBB scores of SCI rats on day 1, 3 and 7 were signi-ficantly decreased (P<0.01), while the numbers of apoptotic neuronal cells and the expression levels of p-c-Jun protein in the spinal cord were considerably increased at the 3 time-points in the model group (P<0.01, P<0.05). Following EA intervention, the decreased BBB scores on day 3 and 7, and the increased numbers of apoptotic neuronal cells on day 1, 3 and 7 and the up-regulated expression levels of p-c-Jun protein on day 3 and 7 were obviously suppressed (P<0.05, P<0.01). CONCLUSIONS: EA intervention can improve the locomotor function of SCI rats, which Feb be related to its effects in reducing neuronal apoptosis and down-regulating p-c-Jun protein in the injuried spinal cord.

Effects of Electroacupuncture at Governor Vessel Acupoints on Neurotrophin-3 in Rats with Experimental Spinal Cord Injury.

In an effort to explore new, noninvasive treatment options for spinal cord injuries (SCI), this study investigated the effects of electroacupuncture (EA) for SCI rat models. SCI was induced by a modified Allen's weight-drop method. We investigated the response of EA at Dazhui (GV 14) and Mingmen (GV 4) acupoints to understand the effects and mechanisms of EA in neuroprotection and neuronal function recovery after SCI. BBB testing was used to detect motor function of rats' hind limbs among groups, and EA was shown to promote the recovery of SCI rats' motor function. Nissl staining showed a restored neural morphology and an increase in the quantity of neurons after EA. Also, the antiapoptosis role was exposed by TUNEL staining. Western blotting analysis was used to determine the protein expression of neurotrophin-3 (NT-3) in spinal cord tissue. Compared to the sham group, the expression levels of NT-3 were significantly decreased and EA was shown to upregulate the expression of NT-3. The present study suggests that the role of EA in neuroprotection and dorsal neuronal function recovery after SCI in rats, especially EA stimulation at GV 14 and GV 4, can greatly promote neuronal function recovery, which may result from upregulating the expression of NT-3.

Electroacupuncture at Dazhui (GV14) and Mingmen (GV4) protects against spinal cord injury: the role of the Wnt/ß-catenin signaling pathway.

Electroacupuncture at Dazhui (GV14) and Mingmen (GV4) on the Governor Vessel has been shown to exhibit curative effects on spinal cord injury; however, the underlying mechanism remains poorly understood. In this study, we established rat models of spinal cord injury using a modified Allen's weight-drop method. Ninety-nine male Sprague-Dawley rats were randomly divided into three equal groups: sham (only laminectomy), SCI (induction of spinal cord injury at T10), and EA (induction of spinal cord injury at T10 and electroacupuncture intervention at GV14 and GV4 for 20 minutes once a day). Rats in the SCI and EA groups were further randomly divided into the following subgroups: 1-day (n = 11), 7-day (n = 11), and 14-day (n = 11). At 1, 7, and 14 days after electroacupuncture treatment, the Basso, Beattie and Bresnahan locomotor rating scale showed obvious improvement in rat hind limb locomotor function, hematoxylin-eosin staining showed that the histological change of injured spinal cord tissue was obviously alleviated, and immunohistochemistry and western blot analysis showed that Wnt1, Wnt3a, ß-catenin immunoreactivity and protein expression in the injured spinal cord tissue were greatly increased compared with the sham and SCI groups. These findings suggest that electroacupuncture at GV14 and GV4 upregulates Wnt1, Wnt3a, and ß-catenin expression in the Wnt/ß-catenin signaling pathway, exhibiting neuroprotective effects against spinal cord injury.

[Effects of electroacupuncture on circadian rhythm of temperature and melatonin in depression rats model induced by chronic stress].

OBJECTIVE: To observe the effects of electroacupuncture (EA) on circadian rhythm of temperature and melatonin (MT) in depression rats model induced by chronic stress, so as to explore the biological mechanism of EA for depression. METHODS: Twenty-four SD rats were randomly divided into a control group, a model group and an EA group, 8 cases in each one. Rats in the control group were treated with normal diet for 21 days without any treatment. In the model and EA group, rat model was established by chronic unpredictable stress combined with solitarily feeding method, and rats in the EA group was treated with EA at "Baihui" (GV 20), "Yintang" (GV 29) 1 h before stress stimulation everyday, 2 Hz in frequency and intensity was favorable with the head of rat slightly shivering. The needles were retained for 20 min, once a day for totally 21 days. After EA treatment, open-field experiment was adopted to observe the behavioral improvement; the rats temperatures were monitored at six time points (2:00, 6:00, 10:00, 14:00, 18:00, 22:00) and orbital blood sampling was collected. The level of serum MT was tested by enzyme linked immunosorbent assay. The circadian rhythm changes of temperature and serum MT in each group were compared. RESULTS: The numbers of horizontal movement and vertical movement in the model group were obviously lower than those in the control group (both P < 0.05), while those in the EA group were significantly improved compared with those in the model group (both P < 0.01). The circadian rhythm of temperature and MT disappeared in the model group, which was improved into normal level after EA treatment. CONCLUSION: The electroacupuncture has regulation effects on circadian rhythm of temperature and melatonin in depression rat model induced by chronic stress.

Progress of animal research on electro-acupuncture treatment for depression(△).

This paper summarized the Chinese literatures in the previous 5 years about the pre-clinical animal researches on the application of electro-acupuncture (EA) treatment for depression, searched in China National Knowledge Infrastructure (CNKI). The efficiency of EA treatment for depression and the mechanism of it were discussed, to shed light on new ideas and new fronts for the further research on depression in clinical or pre-clinical fields.

[Effect of electroacupuncture intervention on behavioral changes and hippocampal excitatory amino acid transporter mRNA expression in depression rats].

OBJECTIVE: To observe the influence of music-electroacupuncture (EA) and pulse EA on locomotor activity and hippocampal neuron numbers and excitatory amino acid transporters (EAATs) mRNA expression in depression rats, so as to explore their mechanisms underlying improvement of depression in acupuncture clinic. METHOD: Sixty SD rats were randomly divided into blank control, depression model, medication, pulse EA and music-EA groups, with 12 rats in each group. Depression model was established by using chronic unpredictable mild stress protocols combined with solitary feeding. Rats of the medication group were treated by oral administration of fluoxetine (a standard antidepressant, 2 mg/kg). EA was applied to "Baihui" (GV 20) and "Yintang" (EX-HN 3) for 20 min, once a day for 21 days. The animals' ethological changes were detected by using field tests for recording the number of crossing squares and the number of rearing. The number of hippocampal neurons were calculated under microscope after Nissl's staining of the hippocampal tissue section. Hippocampal EAAT 1 and EAAT 2 mRNA expression levels were assayed by fluorescent quantitative real-time POR. RESULTS: In comparison with the blank control group, the numbers of crossing and rearing movements, as well as the expression levels of hippocampal EAAT 1 and EAAT 2 mRNA were significantly lower in the model group (P < 0.01), while compared with the model group, the numbers of both crossing and rearing movements of the medication, pulse EA and music-EA groups were obviously increased (P < 0.01). Correspondingly, the expression levels of hippocampal EAAT 1 and EAAT 2 mRNA were considerably upregulated in the pulse EA and music-EA groups in comparison with the model group (P < 0.01). No significant differences were found among the medication, pulse EA and music-EA groups (P > 0.05). Results of Nissl's staining displayed that in the model group, the hippocampal neurons were loose in arrangement and some of them were absent, but in the medication, pulse EA and music-EA groups, the neurons were clear in shape and lined in order. CONCLUSION: Both pulse EA and music-EA can effectively improve the locomotor activity and hippocampal neuronal structure in depression rats which may be closely associated with their effects in upregulating hippocampal EAAT 1 and EAAT 2 mRNA expression.

Long-Term Stimulation with Electroacupuncture at DU20 and ST36 Rescues Hippocampal Neuron through Attenuating Cerebral Blood Flow in Spontaneously Hypertensive Rats.

This study was designed to investigate the effect of long-term electroacupuncture at Baihui (DU20) and Zusanli (ST36) on cerebral microvessels and neurons in CA1 region of hippocampus in spontaneously hypertensive rats (SHR). A total of 45 male Wistar rats and 45 SHR were randomly grouped, with or without electroacupuncture (EA) at DU20 and ST36, once every other day for a period of 8 weeks. The mean arterial pressure (MAP) was measured once every 2 weeks. Cerebral blood flow (CBF) and the number of open microvessels in hippocampal CA1 region were detected by Laser Doppler and immunohistochemistry, respectively. Nissl staining and Western blotting were performed, respectively, to determine hippocampus morphology and proteins that were implicated in the concerning signaling pathways. The results showed that the MAP in SHR increased linearly over the observation period and was significantly reduced following electroacupuncture as compared with sham control SHR rats, while no difference was observed in Wistar rats between EA and sham control. The CBF, learning and memory capacity, and capillary rarefaction of SHR were improved by EA. The upregulation of angiotensin II type I receptor (AT1R), endothelin receptor (ETAR), and endothelin-1 (ET-1) in SHR rats was attenuated by electroacupuncture, suggesting an implication of AT1R, ETAR, and ET-1 pathway in the effect of EA.