Roles of CatSper channels in the pathogenesis of asthenozoospermia and the therapeutic effects of acupuncture-like treatment on asthenozoospermia.

Rationale: Idiopathic asthenozoospermia (iAZS) is one of the major causes of male infertility and has no effective therapeutic treatment. Understanding the potential mechanisms that cause it may be helpful in seeking novel targets and treatment strategies for overcoming the problem of low sperm motility in iAZS individuals. Methods: Computer-assisted semen analysis (CASA) was utilized to assess the sperm motility. RT-qPCR, Western blot, immunofluorescence staining, and calcium imaging analysis were performed to examine the expression and function of CatSper channels. Hyperactivation and acrosome reaction were used to evaluate the functional characteristics of epididymal sperm. In vivo fertility assay was applied to determine the fertility of rats. CatSper1 knockdown and overexpression experiments were performed to confirm the roles of CatSper channels in the pathogenesis of iAZS and the therapeutic effects of electroacupuncture (EA) treatment on AZS model rats. Results: Here, we reported a functional down-regulation of CatSper channel from CatSper1 to CatSper 4 in the sperm of both iAZS patients and ornidazole (ORN)-induced AZS model rats, and an impaired sperm function characterized by a reduction of protein tyrosine phosphorylation, hyperactivation, and acrosome reaction in the epididymal sperm of AZS rats. Knockdown of CatSper1 in the testis tissues is sufficient to induce AZS in normal rats, and this action was validated by the reversal effects of CatSper1 overexpression. Transcutaneous electrical acupoint stimulation (TEAS) and electroacupuncture (EA) at 2 Hz frequency improve the sperm motility via enhancing the functional expression of CatSper channels in the sperm. Gene silencing CatSper1 in the sperm abolishes the therapeutic effects of 2 Hz-EA treatment on AZS rats. Conclusions: We conclude that a functional down-regulation of CatSper channel in the sperm may be a contributor or a downstream indicator for a portion of AZS, especially iAZS, while 2 Hz-TEAS or EA treatment has a therapeutic effect on iAZS through inducing the functional up-regulation of CatSper channels in the sperm. This study provides a novel mechanism for the pathogenesis of some AZS especially iAZS, and presents a potential therapeutic target of CatSper for iAZS treatment. Acupuncture treatment like TEAS may be used as a promising complementary and alternative medicine (CAM) therapy for male infertility caused by iAZS in clinical practice.

Electroacupuncture Modulates Spinal BDNF/TrκB Signaling Pathway and Ameliorates the Sensitization of Dorsal Horn WDR Neurons in Spared Nerve Injury Rats.

Neuropathic pain is more complex and severely affects the quality of patients' life. However, the therapeutic strategy for neuropathic pain in the clinic is still limited. Previously we have reported that electroacupuncture (EA) has an attenuating effect on neuropathic pain induced by spared nerve injury (SNI), but its potential mechanisms remain to be further elucidated. In this study, we designed to determine whether BDNF/TrκB signaling cascade in the spinal cord is involved in the inhibitory effect of 2 Hz EA on neuropathic pain in SNI rats. The paw withdrawal threshold (PWT) of rats was used to detect SNI-induced mechanical hypersensitivity. The expression of BDNF/TrκB cascade in the spinal cord was evaluated by qRT-PCR and Western blot assay. The C-fiber-evoked discharges of wide dynamic range (WDR) neurons in spinal dorsal horn were applied to indicate the noxious response of WDR neurons. The results showed that 2 Hz EA significantly down-regulated the levels of BDNF and TrκB mRNA and protein expression in the spinal cord of SNI rats, along with ameliorating mechanical hypersensitivity. In addition, intrathecal injection of 100 ng BDNF, not only inhibited the analgesic effect of 2 Hz EA on pain hypersensitivity, but also reversed the decrease of BDNF and TrκB expression induced by 2 Hz EA. Moreover, 2 Hz EA obviously reduced the increase of C-fiber-evoked discharges of dorsal horn WDR neurons by SNI, but exogenous BDNF (100 ng) effectively reversed the inhibitory effect of 2 Hz EA on SNI rats, resulting in a remarkable improvement of excitability of dorsal horn WDR neurons in SNI rats. Taken together, these data suggested that 2 Hz EA alleviates mechanical hypersensitivity by blocking the spinal BDNF/TrκB signaling pathway-mediated central sensitization in SNI rats. Therefore, targeting BDNF/TrκB cascade in the spinal cord may be a potential mechanism of EA against neuropathic pain.

Responses of Primary Afferent Fibers to Acupuncture-Like Peripheral Stimulation at Different Frequencies: Characterization by Single-Unit Recording in Rats.

The pain-relieving effect of acupuncture is known to involve primary afferent nerves (PANs) via their roles in signal transmission to the CNS. Using single-unit recording in rats, we characterized the generation and transmission of electrical signals in Aß and Aδ fibers induced by acupuncture-like stimuli. Acupuncture-like signals were elicited in PANs using three techniques: manual acupuncture (MAc), emulated acupuncture (EAc), and electro-acupuncture (EA)-like peripheral electrical stimulation (PES). The discharges evoked by MAc and EAc were mostly in a burst pattern with average intra-burst and inter-burst firing rates of 90 Hz and 2 Hz, respectively. The frequency of discharges in PANs was correlated with the frequency of PES. The highest discharge frequency was 246 Hz in Aß fibers and 180 Hz in Aδ fibers. Therefore, EA in a dense-disperse mode (at alternating frequency between 2 Hz and 15 Hz or between 2 Hz and 100 Hz) best mimics MAc. Frequencies of EA output >250 Hz appear to be obsolete for pain relief.

Electroacupuncture Treatment Alleviates the Remifentanil-Induced Hyperalgesia by Regulating the Activities of the Ventral Posterior Lateral Nucleus of the Thalamus Neurons in Rats.

Mechanisms underlying remifentanil- (RF-) induced hyperalgesia, a phenomenon that is generally named as opioid-induced hyperalgesia (OIH), still remain elusive. The ventral posterior lateral nucleus (VPL) of the thalamus, a key relay station for the transmission of nociceptive information to the cerebral cortex, is activated by RF infusion. Electroacupuncture (EA) is an effective method for the treatment of pain. This study aimed to explore the role of VPL in the development of OIH and the effect of EA treatment on OIH in rats. RF was administered to rats via the tail vein for OIH induction. Paw withdrawal threshold (PWT) in response to mechanical stimuli and paw withdrawal latency (PWL) to thermal stimulation were tested in rats for the assessment of mechanical allodynia and thermal hyperalgesia, respectively. Spontaneous neuronal activity and local field potential (LFP) in VPL were recorded in freely moving rats using the in vivo multichannel recording technique. EA at 2 Hz frequency (pulse width 0.6 ms, 1-3 mA) was applied to the bilateral acupoints "Zusanli" (ST.36) and "Sanyinjiao" (SP.6) in rats. The results showed that both the PWT and PWL were significantly decreased after RF infusion to rats. Meanwhile, both the spontaneous neuronal firing rate and the theta band oscillation in VPL LFP were increased on day 3 post-RF infusion, indicating that the VPL may promote the development of RF-induced hyperalgesia by regulating the pain-related cortical activity. Moreover, 2 Hz-EA reversed the RF-induced decrease both in PWT and PWL of rats and also abrogated the RF-induced augmentation of the spontaneous neuronal activity and the power spectral density (PSD) of the theta band oscillation in VPL LFP. These results suggested that 2 Hz-EA attenuates the remifentanil-induced hyperalgesia via reducing the excitability of VPL neurons and the low-frequency (theta band) oscillation in VPL LFP.

[Experimental Study for the Treatment of Asthenozoospermia by Electroacupuncture in Rats].

OBJECTIVE: To investigate the effects of different electroacupuncture (EA) parameters for the treatment of asthenozoospermia in rats. METHODS: One hundred and five male Sprague-Dawley rats were randomly divided into 2 Hz-EA treatment daily in 3 d group (n=9), sham-EA group (n=10), model group (n=10); 2 Hz-EA treatment every other day in 5 d group, sham-EA group, model group (8 rats in each group); 2 Hz-EA treatment every other day in 9 d group, sham-EA group, model group (10 rats in each group); 100 Hz-EA treatment every other day in 9 d group (n=7), sham-EA group (n=8), model group (n=7). Asthenozoospermia model was established by intragastric administration of ornidazole (ORN,400 mg·kg-1·d-1) once daily till the end of treatment. EA treatments (2 Hz or 100 Hz) were applied to "Shenshu" (BL 23,bilateral), "Zusanli" (ST 36, bilateral) for 30 min, intensity of 1-2-3 mA (increasing 1 mA per 10 min), once a day or once every other day for 3 times or 5 times. Sham-EA groups were treated with similar procedure except that the output leads of the stimulator were disconnected. The sperm density, viability, motility, the number of grade A sperm, and grade A+B sperm were examined by computer-assisted sperm analysis. RESULTS: (1) 2 Hz-EA treatment daily in 3 d:compared with the model group and the sham-EA group, 2 Hz-EA treatment once daily had no significant effect on all of the sperm motility indexes in the asthenozoospermic rats (P>0.05). (2) 2 Hz-EA treatment every other day in 5 d:compared with the model group, EA treatment could increase the sperm motility (P<0.05), the number of grade A sperm (P<0.05), and the number of grade A+B sperm (P<0.05) in the asthenozoospermic rats. However, compared with the sham-EA group, EA treatment could only improve the number of grade A+B sperm (P<0.05). (3) 2 Hz-EA treatment every other day in 9 d:compared with both the model group and the sham-EA group, EA treatment could markedly improve the sperm viability (P<0.001), the sperm motility (P<0.001), the number of grade A sperm (P<0.001), and the number of grade A+B sperm (P<0.001) in the asthenozoospermic rats. (4) 100 Hz-EA treatment every other day in 9 days:compared with both the model group and the sham-EA group, all of the sperm indexes in the asthenozoospermic rats including the sperm viability (P<0.001 vs. the model group, P<0.05 vs. the sham-EA group), the sperm motility (P<0.001 vs. the model group, P<0.01 vs. the sham-EA group), the number of grade A sperm (P<0.01) and the number of grade A+B sperm (P<0.01) also could be improved after EA treatment. Unexpectedly,none of the EA treatment had significant influence on the sperm density in the asthenozoospermic rats. CONCLUSIONS: Both 2 Hz-EA and 100 Hz-EA treatment once every other day for 5 times in 9 d had a therapeutic effect on asthenozoospermia by improving the sperm viability and the sperm motility in the rats.

Use of electroacupuncture and transcutaneous electrical acupoint stimulation in reproductive medicine: a group consensus.

With the rapid development of assisted reproductive technology, various reproductive disorders have been effectively addressed. Acupuncture-like therapies, including electroacupuncture (EA) and transcutaneous electrical acupoint stimulation (TEAS), become more popular world-wide. Increasing evidence has demonstrated that EA and TEAS are effective in treating gynecological disorders, especially infertility. This present paper describes how to select acupoints for the treatment of infertility from the view of theories of traditional Chinese medicine and how to determine critical parameters of electric pulses of EA/TEAS based on results from animal and clinical studies. It summarizes the principles of clinical application of EA/TEAS in treating various kinds of reproductive disorders, such as polycystic ovary syndrome (PCOS), pain induced by oocyte retrieval, diminished ovarian reserve, embryo transfer, and oligospermia/ asthenospermia. The possible underlying mechanisms mediating the therapeutic effects of EA/TEAS in reproductive medicine are also examined.

[Transcutaneous electrical acupoint stimulation for asthenozoospermia].

OBJECTIVE: To study the effect of transcutaneous electrical acupoint stimulation (TEAS) in the treatment of asthenozoospermia. METHODS: We randomly divided 72 asthenozoospermia patients into a 2 Hz TEAS (n = 29), a 100 Hz TEAS (n = 20), and a blank control group (n = 23), those in the former two groups treated by 30 minutes of TEAS at 2 Hz and 100 Hz respectively, applied to the acupoints of bilateral Shenshu, left Zusanli, and Guanyuan, once a day for 60 days, while those in the blank control group left untreated. Using computer-assisted sperm analysis (CASA), we examined sperm concentration and motility as well as the percentages of grade a and grade a+b sperm in different groups of the patients. RESULTS: Compared with the baseline, 2 Hz TEAS significantly increased sperm motility (ï¼»12.76 ± 1.39ï¼½ vs ï¼»18.89 ± 2.46ï¼½%, P<0.05) and the percentage of grade a+b sperm ( ï¼»10.68 ± 1.22ï¼½ vs ï¼»16.32 ± 2.10ï¼½%, P<0.05) in the asthenozoospermic patients, while 100 Hz TEAS improved not only sperm motility (ï¼»12.32 ± 2.21ï¼½ vs ï¼»23.81 ± 3.42ï¼½%, P<0.01) and the percentage of grade a+b sperm (ï¼»10.45 ± 1.98ï¼½ vs ï¼»20.25 ± 2.82 ï¼½%, P<0.01), but also the percentage of grade a sperm (ï¼»6.44 ± 1.16ï¼½ vs ï¼»13.31 ± 2.30ï¼½%, P<0.05). Moreover, in comparison with the blank control group, 2 Hz TEAS also remarkably increased sperm motility (ï¼»9.57 ± 1.60ï¼½ vs ï¼»18.89 ± 2.46ï¼½%, P<0.05) and the percentage of grade a+b sperm (ï¼»7.81 ± 1.31ï¼½ vs ï¼»16.32 ± 2.10ï¼½%, P<0.05) in the asthenozoosperma patients, while 100 Hz TEAS improved not only sperm motility (ï¼»9.57 ± 1.60ï¼½ vs ï¼»23.81 ± 3.42ï¼½%, P<0.01) and the percentage of grade a+b sperm (ï¼»7.81 ± 1.31ï¼½ vs ï¼»20.25 ± 2.82ï¼½%, P<0.01) but also the percentage of grade a sperm (ï¼»4.87 ± 1.01ï¼½ vs ï¼»13.31 ± 2.30ï¼½%, P<0.01). Meanwhile, the rate of clinical effectiveness was significantly higher in the 100 Hz TEASthan in the blank control group either in intention-to-treat (ITT) analysis (100% vs 18.18%) orper-protocol (PP) analysis (90% vs 0%), and so was it than in the 2 Hz TEAS group based on the data of ITT (100% vs 33.33%). CONCLUSIONS: Both 2 Hz and 100 Hz TEAS are effective for the treatment of asthenozoospermia by improving sperm motility and vitality.

Shp-1 dephosphorylates TRPV1 in dorsal root ganglion neurons and alleviates CFA-induced inflammatory pain in rats.

Transient receptor potential vanilloid 1 (TRPV1) receptors are expressed in nociceptive neurons of rat dorsal root ganglions (DRGs) and mediate inflammatory pain. Nonspecific inhibition of protein-tyrosine phosphatases (PTPs) increases the tyrosine phosphorylation of TRPV1 and sensitizes TRPV1. However, less is known about tyrosine phosphorylation's implication in inflammatory pain, compared with that of serine/threonine phosphorylation. Src homology 2 domain-containing tyrosine phosphatase 1 (Shp-1) is a key phosphatase dephosphorylating TRPV1. In this study, we reported that Shp-1 colocalized with and bound to TRPV1 in nociceptive DRG neurons. Shp-1 inhibitors, including sodium stibogluconate and PTP inhibitor III, sensitized TRPV1 in cultured DRG neurons. In naive rats, intrathecal injection of Shp-1 inhibitors increased both TRPV1 and tyrosine-phosphorylated TRPV1 in DRGs and induced thermal hyperalgesia, which was abolished by pretreatment with TRPV1 antagonists capsazepine, BCTC, or AMG9810. Complete Freund's adjuvant (CFA)-induced inflammatory pain in rats significantly increased the expression of Shp-1, TRPV1, and tyrosine-phosphorylated TRPV1, as well as the colocalization of Shp-1 and TRPV1 in DRGs. Intrathecal injection of sodium stibogluconate aggravated CFA-induced inflammatory pain, whereas Shp-1 overexpression in DRG neurons alleviated it. These results suggested that Shp-1 dephosphorylated and inhibited TRPV1 in DRG neurons, contributing to maintain thermal nociceptive thresholds in normal rats, and as a compensatory mechanism, Shp-1 increased in DRGs of rats with CFA-induced inflammatory pain, which was involved in protecting against excessive thermal hyperalgesia.

Down-regulation of CatSper1 channel in epididymal spermatozoa contributes to the pathogenesis of asthenozoospermia, whereas up-regulation of the channel by Sheng-Jing-San treatment improves the sperm motility of asthenozoospermia in rats.

OBJECTIVE: To determine the expression of CatSper1 channel in epididymal spermatozoa in a rat model of asthenozoospermia, induced by cyclophosphamide (CP), and further examine the effects of soluble granules of Sheng-Jing-San (SJS), a traditional Chinese medicine recipe, on CatSper1 expression and sperm motility in the CP-induced asthenozoospermic rats. DESIGN: Placebo-controlled, randomized trial. SETTING: Neuroscience Research Institute, Peking University, China. ANIMAL(S): Sexually mature male Sprague-Dawley rats (n = 60). INTERVENTION(S): In the CP group, CP at the dose of 35 mg/kg intraperitoneally injected into rats once a day for 7 days; in the normal saline (NS) group, 0.9% saline solution was injected as control. MAIN OUTCOME MEASURE(S): Sperm motility and count were evaluated by computer-assisted sperm assay (CASA); protein and mRNA expression of CatSper1 channel in epididymal spermatozoa was determined by Western blotting and quantitative real-time RT-PCR, respectively. RESULT(S): The rats were randomly divided into five groups with 12 rats in each group: CP, normal saline (NS), CP + SJS, CP + NS, and treatment naïve. In the CP + SJS group, after the last injection of CP, SJS at a dose of 30 mg/kg was intragastrically administrated to rats once a day for 14 days; in CP + NS group, saline solution instead of SJS was administrated as control. In the treatment naïve group, rats were normally fed for 21 days as controls. We found a statistically significant reduction of the CatSper1 channel, which is associated with an impairment of sperm motility in the epididymal spermatozoa of CP-induced asthenozoospermic rats. Soluble granules of SJS could dramatically restore the CP-induced down-regulation of CatSper1 in epididymal spermatozoa, which greatly improved the sperm motility in the asthenozoospermic rats. CONCLUSION(S): Down-regulation of the CatSper1 channel in epididymal spermatozoa likely contributes to the pathogenesis of asthenozoospermia, whereas up-regulation of the channel by SJS improves sperm motility and thus can be used as an effective therapeutic strategy for the treatment of male infertility diagnosed with asthenozoospermia.

Anterior cingulate cortex is crucial for contra- but not ipsi-lateral electro-acupuncture in the formalin-induced inflammatory pain model of rats.

Acupuncture and electro-acupuncture (EA) are now widely used to treat disorders like pain. We and others have shown previously that current frequency, intensity and treatment duration all significantly influence the anti-nociceptive effects of EA. There is evidence that stimulating sites also affect the antinociception, with EA applied ipsilaterally to the pain site being more effective under some pain states but contralateral EA under others. It was recently reported that local adenosine A1 receptors were responsible for ipsilateral acupuncture, but what mechanisms specifically mediate the anti-nociceptive effects of contralateral acupuncture or EA remains unclear. In the present study, we applied 100 Hz EA on the ipsi- or contra-lateral side of rats with inflammatory pain induced by intra-plantar injection of formalin, and reported distinct anti-nociceptive effects and mechanisms between them. Both ipsi- and contra-lateral EA reduced the paw lifting time in the second phase of the formalin test and attenuated formalin-induced conditioned place aversion. Contralateral EA had an additional effect of reducing paw licking time, suggesting a supraspinal mechanism. Lesions of rostral anterior cingulate cortex (ACC) completely abolished the anti-nociceptive effects of contra- but not ipsi-lateral EA. These findings were not lateralized effects, since injection of formalin into the left or right hind paws produced similar results. Overall, these results demonstrated distinct anti-nociceptive effects and mechanisms between different stimulating sides and implied the necessity of finding the best stimulating protocols for different pain states.

The Neuroscience Research Institute at Peking University: a place for the solution of pain and drug abuse.

Neuroscience research in China has undergone rapid expansion since 1980. The Neuroscience Research Institute of Peking University, one of the most active neuroscience research groups in China, was founded in 1987. Currently, the institute is overseeing four research areas, i.e., (1) pain and analgesia, (2) drug abuse and acupuncture treatment for drug addiction, (3) the mechanism of neurological degenerative disorders, and (4) the role of neuroglia in central nervous system injury. The institute is simultaneously investigating both theoretical and clinical studies. Acupuncture remains the core of research, while pain and drug abuse form the two disciplines.

Long-term synaptic plasticity in the spinal dorsal horn and its modulation by electroacupuncture in rats with neuropathic pain.

Our previous study has reported that electroacupuncture (EA) at low frequency of 2 Hz had greater and more prolonged analgesic effects on mechanical allodynia and thermal hyperalgesia than that EA at high frequency of 100 Hz in rats with neuropathic pain. However, how EA at different frequencies produces distinct analgesic effects on neuropathic pain is unclear. Neuronal plastic changes in spinal cord might contribute to the development and maintenance of neuropathic pain. In the present study, we investigated changes of spinal synaptic plasticity in the development of neuropathic pain and its modulation by EA in rats with neuropathic pain. Field potentials of spinal dorsal horn neurons were recorded extracellularly in sham-operated rats and in rats with spinal nerve ligation (SNL). We found for the first time that the threshold for inducing long-term potentiation (LTP) of C-fiber-evoked potentials in dorsal horn was significantly lower in SNL rats than that in sham-operated rats. The threshold for evoking the C-fiber-evoked field potentials was also significantly lower, and the amplitude of the field potentials was higher in SNL rats as compared with those in the control rats. EA at low frequency of 2 Hz applied on acupoints ST 36 and SP 6, which was effective in treatment of neuropathic pain, induced long-term depression (LTD) of the C-fiber-evoked potentials in SNL rats. This effect could be blocked by N-methyl-d-aspartic acid (NMDA) receptor antagonist MK-801 and by opioid receptor antagonist naloxone. In contrast, EA at high frequency of 100 Hz, which was not effective in treatment of neuropathic pain, induced LTP in SNL rats but LTD in sham-operated rats. Unlike the 2 Hz EA-induced LTD in SNL rats, the 100 Hz EA-induced LTD in sham-operated rats was dependent on the endogenous GABAergic and serotonergic inhibitory system. Results from our present study suggest that (1) hyperexcitability in the spinal nociceptive synaptic transmission may occur after nerve injury, which may contribute to the development of neuropathic pain; (2) EA at low or high frequency has a different effect on modulating spinal synaptic plasticities in rats with neuropathic pain. The different modulation on spinal LTD or LTP by low- or high-frequency EA may be a potential mechanism of different analgesic effects of EA on neuropathic pain. LTD of synaptic strength in the spinal dorsal horn in SNL rats may contribute to the long-lasting analgesic effects of EA at 2 Hz.