Involvement of spinal muscarinic and serotonergic receptors in the anti-allodynic effect of electroacupuncture in rats with oxaliplatin-induced neuropathic pain.

This study was performed to investigate whether the spinal cholinergic and serotonergic analgesic systems mediate the relieving effect of electroacupuncture (EA) on oxaliplatin-induced neuropathic cold allodynia in rats. The cold allodynia induced by an oxaliplatin injection (6 mg/kg, i.p.) was evaluated by immersing the rat's tail into cold water (4℃) and measuring the withdrawal latency. EA stimulation (2 Hz, 0.3-ms pulse duration, 0.2~0.3 mA) at the acupoint ST36, GV3, or LI11 all showed a significant anti-allodynic effect, which was stronger at ST36. The analgesic effect of EA at ST36 was blocked by intraperitoneal injection of muscarinic acetylcholine receptor antagonist (atropine, 1 mg/kg), but not by nicotinic (mecamylamine, 2 mg/kg) receptor antagonist. Furthermore, intrathecal administration of M2 (methoctramine, 10 µg) and M3 (4-DAMP, 10 µg) receptor antagonist, but not M1 (pirenzepine, 10 µg) receptor antagonist, blocked the effect. Also, spinal administration of 5-HT3 (MDL-72222, 12 µg) receptor antagonist, but not 5-HT1A (NAN-190, 15 µg) or 5-HT2A (ketanserin, 30 µg) receptor antagonist, prevented the anti-allodynic effect of EA. These results suggest that EA may have a signifi cant analgesic action against oxaliplatin-induced neuropathic pain, which is mediated by spinal cholinergic (M2, M3) and serotonergic (5-HT3) receptors.

Expression levels of the hypothalamic AMPK gene determines the responsiveness of the rats to electroacupuncture-induced analgesia.

BACKGROUND: Although electroacupuncture (EA) relieves various types of pain, individual differences in the sensitivity to EA analgesia have been reported, causing experimental and clinical difficulties. Our functional genomic study using cDNA microarray identified that 5'-AMP-activated protein kinase (AMPK), a well-known factor in the regulation of energy homeostasis, is the most highly expressed gene in the hypothalamus of the rats that were sensitive to EA analgesia ("responder"), as compared to the rats that were insensitive to EA analgesia ("non-responder"). In this study, we investigated the causal relationship between the hypothalamic AMPK and the individual variation in EA analgesia. METHODS: Sprague-Dawley (SD) rats were divided into the responder and the non-responder groups, based on EA-induced analgesic effects in the tail flick latency (TFL) test, which measures the latency of the tail flick response elicited by radiant heat applied to the tail. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to quantify the expression levels of AMPK mRNA in the hypothalamus of the responder and non-responder rats. Further, we examined whether viral manipulation of the AMPK expression in the hypothalamus modulates EA analgesia in rats. RESULTS: The real-time RT-PCR analysis showed that mRNA expression levels of AMPK in the hypothalamus of the responder rats are significantly higher than those of the non-responder rats, validating the previous microarray results. Microinjection of dominant negative (DN) AMPK adenovirus, which inhibits AMPK activity, into the rat hypothalamus significantly attenuates EA analgesia (p < 0.05), whereas wild type (WT) AMPK virus did not affect EA analgesia (p > 0.05). CONCLUSIONS: The present results demonstrated that levels of AMPK gene expression in the rat hypothalamus determine the individual differences in the sensitivity to EA analgesia. Thus, our findings provide a clinically useful evidence for the application of acupuncture or EA for analgesia.

Effects of electroacupuncture on oxaliplatin-induced neuropathic cold hypersensitivity in rats.

This study investigated whether and how electroacupuncture (EA) attenuates cold hypersensitivity (allodynia) in a rat model of oxaliplatin-induced neuropathic pain. Cold allodynia [evaluated by immersing the tail into cold water (4 °C) and measuring the withdrawal latency] was induced 3 days after an oxaliplatin administration (6 mg/kg, i.p.). EA stimulation (2/100 Hz, 0.3-ms pulse duration, 0.2-0.3 mA) was delivered to ST36 acupoint or non-acupoint for 20 min. Low-frequency (2 Hz) EA at ST36 relieved cold allodynia more effectively than high-frequency EA at ST36 or low-frequency EA at non-acupoint. Naloxone (opioid antagonist, 2 mg/kg, i.p.) completely blocked such EA-induced anti-allodynia, whereas phentolamine (α-adrenergic antagonist, 2 mg/kg, i.p.) did not. Moreover, plasma ß-endorphin levels significantly increased right after the end of EA and subsequently decreased. These results indicate that low-frequency EA at ST36 in rats has a marked relieving effect on oxaliplatin-induced cold allodynia that is mediated by the endogenous opioid, but not noradrenergic, system.

Effect of bee venom acupuncture on oxaliplatin-induced cold allodynia in rats.

Oxaliplatin, a chemotherapy drug, often leads to neuropathic cold allodynia after a single administration. Bee venom acupuncture (BVA) has been used in Korea to relieve various pain symptoms and is shown to have a potent antiallodynic effect in nerve-injured rats. We examined whether BVA relieves oxaliplatin-induced cold allodynia and which endogenous analgesic system is implicated. The cold allodynia induced by an oxaliplatin injection (6 mg/kg, i.p.) was evaluated by immersing the rat's tail into cold water (4°C) and measuring the withdrawal latency. BVA (1.0 mg/kg, s.c.) at Yaoyangguan (GV3), Quchi (LI11), or Zusanli (ST36) acupoints significantly reduced cold allodynia with the longest effect being shown in the GV3 group. Conversely, a high dose of BVA (2.5 mg/kg) at GV3 did not show a significant antiallodynic effect. Phentolamine ( α -adrenergic antagonist, 2 mg/kg, i.p.) partially blocked the relieving effect of BVA on allodynia, whereas naloxone (opioid antagonist, 2 mg/kg, i.p.) did not. We further confirmed that an intrathecal administration of idazoxan ( α 2-adrenergic antagonist, 50 µ g) blocked the BVA-induced anti-allodynic effect. These results indicate that BVA alleviates oxaliplatin-induced cold allodynia in rats, at least partly, through activation of the noradrenergic system. Thus, BVA might be a potential therapeutic option in oxaliplatin-induced neuropathy.

The endogenous CCK mediation of electroacupuncture stimulation-induced satiety in rats.

A major satiety hormone, cholecystokinin (CCK) is well known to be released by electroacupuncture (EA) stimulation at certain body sites which elicits profound psychophysiological responses. Previous clinical and animal studies have shown that EA stimulation reduces food intake and body weight in both normal and obese subjects. The aim of the present study was to elucidate the satiety effect of EA stimulation and its mechanism related to CCK in rats. Here we show that EA stimulation at "Zusanli" (ST36) acupoint significantly reduced 30-min and 60-min food intake in 48-h fasted Sprague-Dawley rats, and such effect was reversed by a lorglumide (CCK-1 receptor antagonist, 10mg/kg, i.p.) pretreatment. The ST36 EA stimulation-induced satiety was not observed in CCK-1 receptor knockout, Otsuka Long-Evans Tokushima Fatty rats, but in their controls, Long-Evans Tokushima Otsuka rats. Subdiaphragmatic vagotomy also blocked the satiety effect of ST36 EA stimulation in Sprague-Dawley rats. These results suggest that ST36 EA stimulation elicits satiety in rats and this is mediated by the endogenous CCK signaling pathway.

The maintenance of individual differences in the sensitivity of acute and neuropathic pain behaviors to electroacupuncture in rats.

"Responder" Sprague-Dawley (SD) rats that were sensitive to electroacupuncture (EA) in an acute thermal pain test (i.e. tail flick latency [TFL] test) maintained sensitivity to EA in the warm allodynia test after peripheral nerve injury. Similarly, the "non-responder" SD rats that were insensitive to EA in the TFL test were also insensitive to EA in the allodynia test. The EA-induced analgesic effects in the TFL test were significantly higher in CCK-A receptor deficient, Otsuka Long-Evans Tokushima Fatty (OLETF) rats than in their littermates, Long-Evans Tokushima Otsuka (LETO) rats. Similarly, the anti-allodynic effects of EA were significantly greater in OLETF rats than in LETO rats. These results suggest that the individual differences in the sensitivity of acute pain behavior to EA were maintained in neuropathic pain behavior following peripheral nerve injury, and that CCK-A receptor expression plays an important role in mediating this phenomenon.

The association of serum leptin with the reduction of food intake and body weight during electroacupuncture in rats.

Previous studies indicate that acupuncture or electroacupuncture (EA) treatment reduces body weight and food intake in rats by increasing the level of anoretic peptides and decreasing that of orexigenic peptides in the hypothalamus. Considering a well-established role of leptin as a major regulator for feeding behavior in the hypothalamus, we hypothesized that EA might exert its effect via increasing serum leptin levels. In this study, we tested our hypothesis by evaluating the effects of EA on food intake and body weight, as well as on serum leptin levels in rats. Rats were randomly divided into 3 groups: AL (fed ad libitum with no treatments), Holder (fed ad libitum with daily holder restraint) and EA (fed ad libitum with daily holder restraint and 100 Hz EA stimulation) groups. During the four-week experimental period, daily food intake and body weight were measured. At the end of the experiment, levels of serum leptin and corticosterone, and plasma epinephrine (Epi) and norepinephrine (NE) were determined. Here we demonstrate that EA treatment indeed led to reduction of food intake and body weight, and to an increase of serum leptin levels. The level of Epi, NE, and corticosterone increased in the Holder group, but such increase in the level of aforementioned stress hormones was not observed in the EA group. Overall, our results suggest that EA treatment reduces food intake and body weight in rats possibly through increasing leptin levels, and that this effect of EA is not due to the stress caused by the daily holder restraint.

The difference in mRNA expressions of hypothalamic CCK and CCK-A and -B receptors between responder and non-responder rats to high frequency electroacupuncture analgesia.

The present study was performed to determine whether the expression levels of the hypothalamic cholecystokinin (CCK) and its receptors are associated with the responsiveness to high frequency electroacupuncture (EA) analgesia in rats. EA stimulation (100 Hz, 0.5 ms pulse width, 0.2-0.3 mA) was delivered to the Zusanli (ST36) acupoint of male Sprague-Dawley rats for 20 min without anesthetics or holder restraint. The analgesic effect of EA was quantified using a tail flick latency test, and subsequently animals were allocated to responder or non-responder groups. The hypothalamus of rats in each group was dissected and RNA was purified. The mRNA expressions of CCK, and CCK-A and -B receptor were determined by real-time RT-PCR. CCK mRNA levels were not significantly different in the two groups, whereas both CCK-A and -B receptors were significantly more expressed in non-responders. These results suggest that the level of CCK receptor mRNA expression in the hypothalamus, rather than CCK mRNA, has an important relationship with the individual variations to high frequency EA analgesia in rats.

Enhancement of electroacupuncture-induced analgesic effect in cholecystokinin-A receptor deficient rats.

Previously, we have showed that the cholecystokinin (CCK)-A receptor expression in hypothalamus is closely related with the responsiveness of electroacupuncture (EA)-mediated analgesic effects in rats. In order to confirm this observation more directly in vivo, the EA-mediated analgesic effects are compared between Otsuka Long-Evans Tokushima Fatty (OLETF) rats, the natural knockout rats with the homozygously disrupted CCK-A receptor gene, with Long-Evans Tokushima Otsuka (LETO) rats. They were stimulated at the zusanli (ST36) acupoint without using anesthetics or holders. The tail flick latency (TFL) test was performed to quantify analgesic effects and then the mean TFL increase ratios were calculated. OLETF rats showed a mean increase of 53% and LETO rats showed a mean increase of 31% of TFL. Our results suggest that the analgesic effect of acupuncture is closely related with the amount of CCK-A receptor expression.

The association of cholecystokinin-A receptor expression with the responsiveness of electroacupuncture analgesic effects in rat.

The purpose of this study is to determine whether the level of cholecystokinin (CCK) receptor expression causes the differences between the responder and non-responder to electroacupuncture mediated analgesic effects. Male Sprague-Dawley rats were stimulated at the Zusanli (ST36) acupoint in the absence of any anesthetics and holders. The tail flick latency test was performed to quantify analgesic effects and then the responder and non-responder groups were classified. The hypothalamus of each group was dissected and RNA was purified. The amount of mRNA expression of CCK-A and CCK-B receptors was determined by reverse transcription-polymerase chain reaction. The results show that CCK-A receptors are significantly more expressed in non-responders than responders, whereas CCK-B receptor expression is similar in both groups.