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Effect of oxytocin on acupuncture analgesia in the rat
Neuropeptides Neuropeptides 41 (2007) 285–292 www.elsevier.com/locate/npep
Effect of oxytocin on acupuncture analgesia in the rat Jun Yang a
a,*
, Yu Yang a,1, Jian-Min Chen a, Wen-Yan Liu b, Cheng-Hai Wang c, Bao-Cheng Lin c
Institute for Pharmaceuticals and Medical Science, Guangdong Bangmin Pharmaceutical Co. Ltd., Jianghai Distract, Jiangmen, Guangdong 529080, China b Department of Physiology, Jining Medical College, Jining, Shangdong 272113, China c Department of Neurobiology, Second Military Medical University, Shanghai 200433, China Received 20 January 2007; accepted 28 May 2007 Available online 30 July 2007
Abstract Oxytocin has been demonstrated to be involved in pain modulation. Acupuncture analgesia is a very useful clinical tool for pain relief, which has over 2500-year history in China. The present study investigated the role of oxytocin in acupuncture analgesia in the rat through oxytocin administration and measurement. Central administration of oxytocin (intraventricular injection or intrathecal injection) enhanced acupuncture analgesia, while central administration of anti-oxytocin serum weakened acupuncture analgesia in a dose-dependent manner. However, intravenous injection of oxytocin or anti-oxytocin serum did not influence acupuncture analgesia. Electrical acupuncture of ‘‘Zusanli’’ (St. 36) reduced oxytocin concentration in the hypothalamic supraoptic nucleus, and elevated oxytocin concentration in the hypothalamic suprachiasmatic nucleus, hypothalamic ventromedial nucleus, thalamic ventral nucleus, periaqueductal gray, raphe magnus nucleus, caudate nucleus, thoracic spinal cord and lumbar spinal cord, but did not alter oxytocin concentration in the hypothalamic paraventricular nucleus, anterior pituitary, posterior pituitary and plasma. The data suggested that oxytocin in central nervous system rather than in peripheral organs is involved in acupuncture analgesia. 2007 Elsevier Ltd. All rights reserved. Keywords: Oxytocin; Anti-oxytocin serum; Acupuncture analgesia; Hypothalamic supraoptic nucleus; Central nervous system; Peripheral organ
1. Introduction Oxytocin (OXT), a nonapeptide posterior pituitary hormone, is mainly synthesized in the hypothalamic paraventricular nucleus (PVN) and hypothalamic supraAbbreviations: OXT, oxytocin; AOXTS, anti-oxytocin serum; icv, intraventricular injection; ith, intrathecal injection; iv, intravenous injection; ACSF, artificial cerebrospinal fluid; PVN, hypothalamic paraventricular nucleus; SON, hypothalamic supraoptic nucleus; SCN, hypothalamic suprachiasmatic nucleus; HVN, hypothalamic ventromedial nucleus; TVN, thalamic ventral nucleus; PAG, periaqueductal gray; RMN, raphe magnus nucleus; CdN, caudate nucleus. * Corresponding author. Present address: 10-6655 Fielding Avenue, Montreal, Que., Canada H4V 1N4. Tel.: +1 514 481 0631. E-mail address: [email protected] (J. Yang). 1 ` cole secondaire Saint-Luc, Montre`al Volunteer, a student from E (Que`bec), Canada H3X 2H4. 0143-4179/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.npep.2007.05.004
optic nucleus (SON) (Buijs, 1978b). The remarkable functions of OXT include uterine contraction during parturition, milk-ejection reflex during lactation, cardiovascular regulation, sex activity, learning and memory (McEwen, 2004). ‘‘MIANSHU’’, a traditional Chinese medical book published 2500 years ago, has recorded that acupuncture analgesia is a very useful clinical tool for pain relief, and acupuncture analgesia has been using in treating diseases and killing pain (Zhang, 1990). ‘‘Zusanli’’ point (St. 36) is one of the 365 classical acupuncture points, located on the leg portion of the stomach meridian (Zhang, 1990). Although ‘‘Zusanli’’ point (St. 36) is no special in dispelling some kind pain (Toda et al., 1980), it is one of the most frequently used acupuncture points and the most intensively studied single point analgesia (Guowei et al.,
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1979; Li et al., 1985; Liu et al., 1990; Lee et al., 2004). We have been using ‘‘Zusanli’’ points (St. 36) to study the acupuncture analgesia since 1975, and we found that the analgesic effect of electrical acupuncture ‘‘Zusanli’’ point (St. 36) depended on not only the animal strains but also the characters of the stimulated current (Lin et al., 1979; Chen and Zhu, 1978). PVN and SON, the most sources of OXT (McEwen, 2004), play an import role in acupuncture analgesia (Lin et al., 1979; Chen et al., 1991; Yang and Lin, 1992). OXT is related to pain modulation at different levels (Arletti et al., 1993; Brown and Perkowski, 1998; Ge et al., 2002; Lundeberg et al., 1994; Madrazo et al., 1987; Robinson et al., 2002; Uvnas-Moberg et al., 1998; Yang, 1994; Yu et al., 2003; Yang et al., 2007a). OXT in the PVN is not related in pain modulation (Yang et al., 2006e,f). However, it has not been proven whether OXT in the other nervous regions, for example OXT in the SON, influences acupuncture analgesia. Electrophysiological and histological studies have found that SON widely connects with the other brain regions such as the periaqueductal gray (PAG), raphe magnus nucleus (RMN), caudate nucleus (CdN) through the nerve fibers including OXT-containing fibers; and some of the other brain regions, which are related to acupuncture analgesia (Zhang, 1990), are connected with SON (Buijs, 1978a,b; Hawthorn et al., 1985; Armstrong and Stern, 1997). This anatomical structure of SON may be biological basic of OXT regulating acupuncture analgesia. For answering this question, first, it needs to study OXT role in acupuncture analgesia. The present work employed the methods of the brain ventricle, spinal cord or venous injection and OXT concentration measurement to investigate OXT effect on acupuncture analgesia in the rat.
2. Materials and methods 2.1. Animals Adult male Sprague–Dawley rats weighing 180–220 g were used in all experiments (Second Military Medical University, Shanghai, China and Nanfang Medical University, Guangzhou, China). Animals were housed in a colony room under controlled temperature, humidity and a 12 h light/dark cycle (light on at 6:00 AM), with food and water available ad libitum. All procedures were conducted according to the guidelines of the International Association for the Study of Pain (Zimmermann, 1983).
Rabbit anti-rat OXT serum (AOXTS) was made by Department of Neurobiology, Second Military Medical University, Shanghai, China (Song et al., 1987). The specificity of the antiserum was over 99.99% cross-reactivity with synthesized OXT and less than 0.01% cross-reactivity with arginine vasopressin (AVP), vasotocin, lysinevasopressin, vasoactive intestinal peptide, neurotensin, Leucine-enkephalin, Methionin-enkephalin, b-endorphin and dynorphinA1 13. The dilution of AOXTS was more than 1: 80,000 for radioimmunoassay. 2.3. Surgery 2.3.1. Intraventricular (icv) injection preparation With Pellegrino L.J. rat brain atlas (1979) as reference, we used the stereotaxic apparatus (Jiangwan I-C, Shanghai, China) to implant a stainless steel guide cannula of 0.5 mm outer diameter into the right lateral ventricle (AP 0.3 mm, LR 0.5 mm, H 3.0 mm) for icv injection under pentobarbital sodium (35 mg/kg, intraperitoneal injection) anesthesia. The guide cannula was fixed to the skull by dental acrylic. 2.3.2. Intrathecal (ith) injection preparation Under pentobarbital sodium (35 mg/kg, intraperitoneal injection) anesthesia, the rat was implanted with a chronic intrathecal catheter (PE-10, 12 cm in length, 0.6 mm outer diameter) extending into the lumbar enlargement of the spinal cord for ith injection (Yaksh and Rudy, 1976). All operated animals needed at least 14 days for recovery after the surgery. 2.4. Nociceptive test 2.4.1. Tail-flick test to noxious potassium iontophoresis All animals were tested under the condition of free activity in a clear acrylic cage (30 cm in diameter, 25 cm in height, open top). The potassium iontophoresis inducing tail-flick served as pain stimulus. The small wet cotton with the saturated solution of potassium iontophoresis was set on the tail skin. The cotton was exposed to direct electrical current, and the anode led the potassium iontophoresis to permeate the tail skin. If the current was strong enough, the permeated potassium iontophoresis resulted in the animal feeling pain stimulation. The intensity of current at the moment of the response was recorded as the pain threshold, which was expressed as mA (WQ-9E Pain Threshold Measurer, Shanghai, China).
2.2. Materials OXT was obtained from Peninsula Laboratories, San Carlos, CA, USA; 125Iodine was from Amersham Pharmacia, Buckinghamshire, UK; the other chemicals were from Sigma Co., St. Louis, MO, USA.
2.4.2. Hot plate test to noxious heat Rats were brought to the testing room and allowed to acclimatize for 10 min before the test began. Pain reflexes in response to a thermal stimulus were measured using RCY-2 Hot Plate Analgesia Meter (Shanghai, China).
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The hot plate surface was heated to a constant temperature of 55 C, as measured by a built-in digital thermometer with an accuracy of 0.1 C and verified by a surface thermometer. Animals were placed on the hot plate (30 cm in diameter), which was surrounded by a clear acrylic cage (25 cm tall, open top), and the Start/Stop button on the timer was activated. The latency, which was expressed as second, to respond with either a hindpaw lick, hindpaw flick, or jump (which ever comes first) was measured to the nearest 0.1 s by deactivating the timer when the response was observed. The rat was immediately removed from the hot plate and returned to its home cage. If a rat did not respond within 30 s, the test was terminated and the animal was removed from the hot plate. All nociceptive tests were carried out in from 8:00 to 10:00 AM. Animals were not habituated to the apparatus prior to testing. The duration between consecutive stimuli was 10 min, and the pain stimulus was terminated at once when the rat showed response to the stimulus. Each animal was tested only once. 2.5. Electrical acupuncture The stainless needles (0.2 mm in diameter and 3 mm in long), which were fixed by the adhesive tape, were placed at the bilateral points of ‘‘Zusanli’’ (St. 36). The stimulated electrical current was passed the bilateral points with alternating polarities and a dense-disperse wave (JSD-731-C electro-stimulator, f1 = 10 Hz, f2 = 20 Hz) for 30 min. The intensity was adjusted until the animal appeared comfortably and its local muscle contractions were seen (v = 2–3 V). 2.6. Sample preparation 2.6.1. Brain nuclei After the treatment, the animals were decapitated. The brains were quickly taken out and put into 80 C physiological saline for heating 2 min, and then fixed in 4% paraformaldehyde physiological saline at 4 C for 3 days. Coronary sections, 400 lm thick, were cut on a cryostat and both sides of nuclei were punched out using special needles (Schelling et al., 1982). All of the same nuclei from three rats in a same group were combined into one sample. The sample was homogenized in 0.5 ml of 0.1 M acetic acid. Two hours later, the same volume of 0.1 M sodium hydroxide was mixed. The 50 ll fluid was taken out for measuring the total protein concentration with Lowry’s method (Lowry et al., 1951). 2.6.2. Other tissues The pituitary and spinal cord were taken out and put into the boiling physiological saline for 5 min. After weighing, the tissue was homogenized in 1.0 ml of 0.1 M acetic acid. Two hours later, the same volume of 0.1 M sodium hydroxide was mixed.
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2.6.3. Plasma The blood was collected using the EDTA Æ Na2 treated tube and kept at 4 C during the treatment. Using the centrifugation at 10,000g for 20 min, the supernatant was withdrawn and stored at 80 C for assay. 2.7. OXT concentration measurement OXT concentration was determined by radioimmunoassay with specific AOXTS (Song et al., 1987). The peptide was labeled 125Iodine using chloramines-T method, and the iodinated OXT was purified by Sephadex G-50. The assay sensitivity of OXT was 1.0 pg/tube and intra- and inter-assay coefficients of variation were less than 5.0% and 8.0%, respectively. 2.8. OXT administration 2.8.1. icv injection A stainless steel needle with 0.3 mm diameter was directly inserted into the guide cannula, 1 mm beyond the tip. After the basic pain threshold measurement, 10 ll serum (AOXTS or normal serum) or solution, which OXT was dissolved in artificial cerebrospinal fluid (ACSF, containing 0.1 M NaCl, 1.0 mM KH2PO4, 4.0 mM KCl, 2.0 mM MgSO4, 2.0 mM CaCl2, 2.1 mM NaHCO3 and 8.0 mM Glucose), was gently injected into the lateral ventricule over 10 min. Only normal serum or ACSF was given in control group. 2.8.2. ith injection After the basic pain threshold measurement, 10 ll serum (AOXTS or normal serum) or solution, which OXT was dissolved in ACSF, was gently injected into the lumbar enlargement of the spinal cord through the chronic intrathecal catheter over 10 min (Yang, 1994). Only normal serum or ACSF was given in control group. 2.8.3. Intravenous (iv) injection After the basic pain threshold measurement, 200 ll serum (AOXTS or normal serum) or solution, which OXT was dissolved in physiological saline, was gently injected into the rat-tail vein. Only normal serum or physiological saline was given in control group. Pain threshold measurements were started 10 min after the administration. 2.9. Statistical analysis Data were expressed as mean ± standard error of the mean (SEM) and were analyzed between groups via analysis of variance (ANOVA). P < 0.05 was considered statistically significant.
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3. Results 3.1. Tail-flick test to noxious potassium iontophoresis 3.1.1. OXT in the central nerve system effects on acupuncture analgesia OXT in the brain and spinal cord enhanced acupuncture analgesia. Electrical acupuncture bilateral ‘‘Zusanli’’ points (St. 36) 30 min increased pain threshold to
100–110%; and the recover time, which this pain threshold recovered to the normal level after stopping acupuncture, was 50 min in the control group (icv or ith) (Fig. 1A and C). Administration of 12.5 ng OXT (icv or ith) enhanced acupuncture analgesia to 110–130% and the recover time was 50 min; 50 ng OXT (icv or ith) enhanced acupuncture analgesia to 180–200% (P < 0.001) and the recover time was 60 min; and 200 ng OXT (icv or ith) enhanced acupuncture analgesia
Fig. 1. Effect of oxytocin and anti-oxytocin serum on acupuncture analgesia in the tail-flick test. icv, intraventricular injection; ith, intraventricular injection; iv, intravenous injection. Acupuncture referred the animals which ‘‘Zusanli’’ points (St. 36) were given electrical acupuncture (densedisperse wave, f1 = 10 Hz, f2 = 20 Hz, 2–3 V) for 30 min. Change of pain threshold (%) indicates (present pain threshold – pain threshold before acupuncture)/pain threshold before acupuncture · 100. The data were expressed as mean ± SEM. n means the animal number in each group.
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to 280–300% (P < 0.001) and the recover time was 80 min (Fig. 1A and C). AOXTS in the brain and spinal cord weakened acupuncture analgesia. Electrical acupuncture bilateral ‘‘Zusanli’’ points (St. 36) 30 min increased the pain threshold to 100–110%, and the recover time was 50 min in the control group (icv or ith) (Fig. 1B and D). Treatment with 2.5 ll AOXTS (icv or ith) weakened acupuncture analgesia to 80–90% and the recover time was 50 min; 5 ll AOXTS (icv or ith) weakened acupuncture analgesia to 60–70% (P < 0.01) and the recover time
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was 40 min; 10 ll AOXTS weakened acupuncture analgesia to 40–50% (P < 0.001) and the recover time was 30 min (Fig. 1B and D).
3.1.2. OXT in the blood effects on acupuncture analgesia Venous administration of OXT (5, 10 and 20 lg) (iv) or AOXTS (50, 100 and 200 ll) (iv) could not influence acupuncture analgesia, which results were similar as those in the control group (iv) (all P > 0.05) (Fig. 1E and F).
Fig. 2. Effect of oxytocin and anti-oxytocin serum on acupuncture analgesia in the hot plate test. icv, intraventricular injection; ith, intraventricular injection; iv, intravenous injection. Acupuncture referred the animals which ‘‘Zusanli’’ points (St. 36) were given electrical acupuncture (densedisperse wave, f1 = 10 Hz, f2 = 20 Hz, 2–3 V) for 30 min. Change of latency (%) indicates (present latency – latency before acupuncture)/latency before acupuncture · 100. The data were expressed as mean ± SEM. n means the animal number in each group.
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3.1.3. Hot plate test to noxious heat Similar as the results of the tail-flick test to noxious potassium iontophoresis, central administration of OXT (icv and ith) enhanced acupuncture analgesia (Fig. 2A and C), and AOXTS (icv and ith) weakened acupuncture analgesia (Fig. 2B and D), but intravenous injection of OXT or AOXTS (iv) did not influence acupuncture analgesia (Fig. 2E and F). 3.1.4. Acupuncture influences OXT concentration Acupuncture changed OXT concentration in the brain and spinal cord, but did not alter OXT concentration in the pituitary and plasma. Electrical acupuncture of ‘‘Zusanli’’ (St. 36) points 30 min reduced OXT concentration in the SON, and elevated OXT concentration
Fig. 3. Effect of electrical acupuncture on oxytocin concentration in rat brain nuclei. SON, hypothalamic supraoptic nucleus; PVN, hypothalamic paraventricular nucleus; SCN, hypothalamic suprachiasmatic nucleus; HVN, hypothalamic ventromedial nucleus; TVN, thalamic ventral nucleus; PAG, periaqueductal gray; RMN, raphes magnus nucleus; CdN, caudate nucleus. Acupuncture referred the animals which ‘‘Zusanli’’ points (St. 36) were given electrical acupuncture (dense-disperse wave, f1 = 10 Hz, f2 = 20 Hz, 2–3 V). The data were expressed as mean ± SEM. n means the animal number in each group.
Table 1 Effect of acupuncture on oxytocin (OXT) concentration
Anterior pituitary (ng/mg) Posterior pituitary (ng/mg) Thoracic spinal cord (pg/mg) Lumbar spinal cord (pg/mg) Serum (pg/ml)
Control
Acupuncture
(n = 8)
10 min (n = 8)
30 min (n = 9)
136.6 ± 12.7
125.3 ± 19.8
131.5 ± 14.9
1412.8 ± 216.4
1455.3 ± 179.4
1758.3 ± 336.5
2.0 ± 0.5
4.1 ± 1.1*
4.7 ± 0.7**
1.9 ± .0.4
6.3 ± 1.2**
8.2 ± 1.9***
13.4 ± 3.1
15.5 ± 4.3
18.0 ± 4.8
All values are showed as mean ± SEM. n indicates the animal number in each group. * P < 0.05. ** P < 0.01. *** P < 0.001.
in the hypothalamic suprachiasmatic nucleus (SCN), hypothalamic ventromedial nucleus (HVN), thalamic ventral nucleus (TVN), periaqueductal gray (PAG), raphe magnus nucleus (RMN), caudate nucleus (CdN), thoracic spinal cord and lumbar spinal cord, but did not alter the OXT concentration in the PVN, anterior pituitary, posterior pituitary and plasma (Fig. 3 and Table 1).
4. Discussion Having been used in many laboratories, classical hot plate/tail-plate test to noxious heat, and hind paw withdrawal to noxious heating are very important to study the hyperalgesia/allodynia in animals with persistent/ chronic pain. However, these tests have some problems in the special research for pain. For example, the latency (pain threshold) shows instability after the animal is tested many times, especially in the experiments that the duration between consecutive stimuli is short. On the other hand, it may cause the animal stress when the animal is removed from the hot plate after the test (Zhang, 1990). Many methods for pain threshold measurement, which included the hot plate test to noxious heat and tail-flick test to noxious potassium iontophoresis, have been compared in our laboratory since we studied pain modulation and acupuncture analgesia in 1954. We found that the tail-flick test to noxious potassium iontophoresis is good, relatively, to keep the basic pain threshold stability in studying pain modulation and acupuncture analgesia, and is easy, specially, to control the animal extra-stimulation under the condition of free activity (Chen and Zhu, 1978). Of course, it is better that the tail-plate test to noxious potassium iontophoresis can combine with classical methods for pain threshold measurement including the hot plate test to noxious heat in one experiment for pain research. It has been demonstrated that central OXT is involved in pain modulation (Brown and Perkowski, 1998; Madrazo et al., 1987; Robinson et al., 2002; Yang, 1994; Yu et al., 2003; Yang et al., 2007a). Treatment with OXT (icv) had an analgesic effect in a patient with intractable cancer pain (Madrazo et al., 1987). Mice lacking OXT reduced stress-induced antinociception following both cold-swim and restraint stress (Robinson et al., 2002). At a spinal cord level, OXT can regulate antinociception in the dog (Brown and Perkowski, 1998) and rat (Yu et al., 2003). OXT is also involved in low back pain in human (Yang, 1994). The present study showed that central administration of OXT (icv or ith) enhanced acupuncture analgesia, while AOXTS (icv or ith) weakened acupuncture analgesia. The data suggested that OXT in the brain and spinal cord played a role in acupuncture analgesia.
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The present study showed that OXT (0.2 mg or less/ rat) or anti-OXT serum (200 ll or less/rat) (iv), one time administration, did not influence acupuncture analgesia. The data suggested that OXT in the blood did not relate to acupuncture analgesia. Some researches reported that intravenous or intraperitoneal injection of OXT shows analgesic effect (Arletti et al., 1993; Lundeberg et al., 1994; Uvnas-Moberg et al., 1998). However, this analgesic effect of OXT (intravenous or intraperitoneal injection) needs a very high dose (10–200 mg OXT/rat) and a very long period (60 days) to treat the animal (Arletti et al., 1993; Lundeberg et al., 1994; Uvnas-Moberg et al., 1998). It is a pharmacological action, because OXT can be penetrated the blood-brain barrier if OXT concentration is high enough in the blood. This point can be proven by the result that intraventricular injection of OXT antagonist prevents the analgesic effect of OXT, which was given by the intraperitoneal injection (Arletti et al., 1993; Lundeberg et al., 1994; Uvnas-Moberg et al., 1998). The present study also showed that electrical acupuncture of ‘‘Zusanli’’ (St. 36) reduced OXT concentration in the SON, and elevated OXT concentration in the SCN, HVN, TVN, PAG, RMN, CdN, thoracic spinal cord and lumbar spinal cord, but did not alter the OXT concentration in the PVN, anterior pituitary, posterior pituitary and plasma. The data indicated that OXT in central nervous system rather than in peripheral organs was related in acupuncture analgesia. In according to the anatomical connection between SON and other nervous structures such as PAG, RMN, CdN and spinal cord (Buijs, 1978a,b; Hawthorn et al., 1985; Armstrong and Stern, 1997), the mechanism of OXT enhancing acupuncture analgesia may be proposed as follows: (1) Acupuncture causes SON releasing OXT; (2) then this OXT is transported to the other brain nuclei (such as PAG, RMN and CdN) and spinal cord; (3) in the relative nervous regions OXT influences the antinociceptive system including the endogenous opiate peptide system, to be involved in the process of pain modulation. The part of this propose is proven by our previous work, i.e. OXT can induce the spinal cord releasing the endogenous opiate peptides and the analgesic effect of OXT can be attenuated by naloxone – an opiate receptor antagonist in the spinal cord (Yang, 1994). AVP is a very important regulator in PVN regulating acupuncture analgesia (Yang, 1992; Yang et al., 2006d,e,f; Yang et al., 2007e). AVP in PVN, which is transported to the other brain nuclei such as PAG (Yang et al., 2006c,h; Yang et al., 2007b,c,d), RMN (Yang et al., 2006a) and CdN (Yang et al., 2006b), is involved in antinociception and acupuncture analgesia (Yang et al., 2006g). OXT and AVP are very similar in their source, synthesis and structure (McEwen, 2004). The present study showed that electrical acupuncture
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decreased OXT concentration in the SON, not in the PVN. In contrast, our previous work showed that electrical acupuncture decreased AVP concentration in the PVN, not in the SON (Yang, 1992; Yang et al., 2006e,f). The data suggested that OXT in the SON rather than in the PVN is involved in acupuncture analgesia, and AVP in the PVN rather than in the SON is related to acupuncture analgesia. Why are the roles of OXT and AVP in SON and PVN regulating acupuncture analgesia different? It is a very interesting topic that needs to be further investigated. In conclusion, our present studies proved that OXT (icv or ith) enhanced acupuncture analgesia, and antiOXT serum (icv or ith) weakened acupuncture analgesia in a dose-dependent manner, but both OXT and antiOXT serum (iv) did not influence acupuncture analgesia; acupuncture changed OXT concentration in the brain nuclei and spinal cord, not in the pituitary and plasma. The data suggested that OXT in central nervous system rather than in peripheral organs played an important role in acupuncture analgesia in the rat. Acknowledgements This work was supported by Guangdong Bangmin Pharmaceutical Co. Ltd. and a grant from the National Science Foundation of China.
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Yang, J., Chen, J.M., Liu, W.Y., Song, C.Y., Wang, C.H., Lin, B.C., 2006c. Through V2, not V1 receptor relating to endogenous opiate peptides, arginine vasopressin in periaqueductal gray regulates antinociception in the rat. Regul. Peptides 137, 156–161. Yang, J., Chen, J.M., Song, C.Y., Liu, W.Y., Wang, G., Wang, C.H., Lin, B.C., 2006d. Through the central V2, not V1 receptor influences the endogenous opiate peptide system, arginine vasopressin, not oxytocin in the hypothalamic paraventricular nucleus involves in the antinociception in the rat. Brain Res. 1069, 127– 138. Yang, J., Lin, B.C., 1992. Hypothalamic paraventricular nucleus plays a role in acupuncture analgesia through the central nervous system in the rat. Acupunct. Electrother. Res. 17, 209–220. Yang, J., Liu, W.Y., Song, C.Y., Lin, B.C., 2006e. Through central arginine vasopressin, not oxytocin and endogenous opiate peptides, glutamate sodium induces hypothalamic paraventricular nucleus enhancing acupuncture analgesia in the rat. Neurosci. Res. 54, 49– 56. Yang, J., Liu, W.Y., Song, C.Y., Lin, B.C., 2006f. Only arginine vasopressin, not oxytocin and endogenous opiate peptides, in hypothalamic paraventricular nucleus play a role in acupuncture analgesia in the rat. Brain Res. Bull. 68, 453–458. Yang, J., Song, C.Y., Liu, W.Y., Lin, B.C., 2006g. Only through the brain nuclei, arginine vasopressin regulates antinociception in the rat. Peptides 27, 3341–3346. Yang, J., Yang, Y., Chen, J.M., Liu, W.Y., Wang, C.H., Lin, B.C., 2007a. Oxytocin enhances antinociception in the rat. Peptides doi:10.1016/j.peptides.2007.03.003. Yang, J., Yang, Y., Xu, H.T., Chen, J.M., Gen, W., Liu, W.Y., Lin, B.C., 2007b. Periaqueductal gray knockdown of V2, not V1a and V1b receptor influences nociception in the rat. Neurosci. Res. 57, 104–111. Yang, J., Yang, Y., Xu, H.T., Chen, J.M., Liu, W.Y., Lin, B.C., 2006h. Arginine vasopressin enhances periaqueductal gray synthesis and secretion of enkephalin and endorphin in the rat. Brain Res. Bull. 71, 193–199. Yang, J., Yang, Y., Xu, H.T., Chen, J.M., Liu, W.Y., Lin, B.C., 2007c. Arginine vasopressin in periaqueductal gray, which relates to antoniciception, comes from hypothalamic paraventricular nucleus in the rat. Neurosci. Lett. 412, 154–158. Yang, J., Yang, Y., Xu, H.T., Chen, J.M., Liu, W.Y., Lin, B.C., 2007d. Arginine vasopressin induces periaquedictal gray release of enkephalin and endorphin relating to pain modulation in the rat. Regul. Peptides 142, 29–36. Yang, J., Yang, Y., Xu, H.T., Chen, J.M., Liu, W.Y., Lin, B.C., 2007e. Arginine vasopressin is an important regulator in antinociceptive modulation of hypothalamic paraventricular nucleus in the rat. Neuropeptides 41, 165–176. Yu, S.Q., Lundeberg, T., Yu, L.C., 2003. Involvement of oxytocin in spinal antinociception in rats with inflammation. Brain Res. 983, 12–22. Zhang, E.Q., 1990. Chinese acupuncture and moxibustion. Publishing House of Shanghai College of Traditional Chinese Medicine, Shanghai. Zimmermann, M., 1983. Ethical guidelines for investigations of experimental pain in conscious animal. Pain 16, 109– 110.
Effect of oxytocin on acupuncture analgesia in the rat Jun Yang a
a,*
, Yu Yang a,1, Jian-Min Chen a, Wen-Yan Liu b, Cheng-Hai Wang c, Bao-Cheng Lin c
Institute for Pharmaceuticals and Medical Science, Guangdong Bangmin Pharmaceutical Co. Ltd., Jianghai Distract, Jiangmen, Guangdong 529080, China b Department of Physiology, Jining Medical College, Jining, Shangdong 272113, China c Department of Neurobiology, Second Military Medical University, Shanghai 200433, China Received 20 January 2007; accepted 28 May 2007 Available online 30 July 2007
Abstract Oxytocin has been demonstrated to be involved in pain modulation. Acupuncture analgesia is a very useful clinical tool for pain relief, which has over 2500-year history in China. The present study investigated the role of oxytocin in acupuncture analgesia in the rat through oxytocin administration and measurement. Central administration of oxytocin (intraventricular injection or intrathecal injection) enhanced acupuncture analgesia, while central administration of anti-oxytocin serum weakened acupuncture analgesia in a dose-dependent manner. However, intravenous injection of oxytocin or anti-oxytocin serum did not influence acupuncture analgesia. Electrical acupuncture of ‘‘Zusanli’’ (St. 36) reduced oxytocin concentration in the hypothalamic supraoptic nucleus, and elevated oxytocin concentration in the hypothalamic suprachiasmatic nucleus, hypothalamic ventromedial nucleus, thalamic ventral nucleus, periaqueductal gray, raphe magnus nucleus, caudate nucleus, thoracic spinal cord and lumbar spinal cord, but did not alter oxytocin concentration in the hypothalamic paraventricular nucleus, anterior pituitary, posterior pituitary and plasma. The data suggested that oxytocin in central nervous system rather than in peripheral organs is involved in acupuncture analgesia. 2007 Elsevier Ltd. All rights reserved. Keywords: Oxytocin; Anti-oxytocin serum; Acupuncture analgesia; Hypothalamic supraoptic nucleus; Central nervous system; Peripheral organ
1. Introduction Oxytocin (OXT), a nonapeptide posterior pituitary hormone, is mainly synthesized in the hypothalamic paraventricular nucleus (PVN) and hypothalamic supraAbbreviations: OXT, oxytocin; AOXTS, anti-oxytocin serum; icv, intraventricular injection; ith, intrathecal injection; iv, intravenous injection; ACSF, artificial cerebrospinal fluid; PVN, hypothalamic paraventricular nucleus; SON, hypothalamic supraoptic nucleus; SCN, hypothalamic suprachiasmatic nucleus; HVN, hypothalamic ventromedial nucleus; TVN, thalamic ventral nucleus; PAG, periaqueductal gray; RMN, raphe magnus nucleus; CdN, caudate nucleus. * Corresponding author. Present address: 10-6655 Fielding Avenue, Montreal, Que., Canada H4V 1N4. Tel.: +1 514 481 0631. E-mail address: [email protected] (J. Yang). 1 ` cole secondaire Saint-Luc, Montre`al Volunteer, a student from E (Que`bec), Canada H3X 2H4. 0143-4179/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.npep.2007.05.004
optic nucleus (SON) (Buijs, 1978b). The remarkable functions of OXT include uterine contraction during parturition, milk-ejection reflex during lactation, cardiovascular regulation, sex activity, learning and memory (McEwen, 2004). ‘‘MIANSHU’’, a traditional Chinese medical book published 2500 years ago, has recorded that acupuncture analgesia is a very useful clinical tool for pain relief, and acupuncture analgesia has been using in treating diseases and killing pain (Zhang, 1990). ‘‘Zusanli’’ point (St. 36) is one of the 365 classical acupuncture points, located on the leg portion of the stomach meridian (Zhang, 1990). Although ‘‘Zusanli’’ point (St. 36) is no special in dispelling some kind pain (Toda et al., 1980), it is one of the most frequently used acupuncture points and the most intensively studied single point analgesia (Guowei et al.,
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1979; Li et al., 1985; Liu et al., 1990; Lee et al., 2004). We have been using ‘‘Zusanli’’ points (St. 36) to study the acupuncture analgesia since 1975, and we found that the analgesic effect of electrical acupuncture ‘‘Zusanli’’ point (St. 36) depended on not only the animal strains but also the characters of the stimulated current (Lin et al., 1979; Chen and Zhu, 1978). PVN and SON, the most sources of OXT (McEwen, 2004), play an import role in acupuncture analgesia (Lin et al., 1979; Chen et al., 1991; Yang and Lin, 1992). OXT is related to pain modulation at different levels (Arletti et al., 1993; Brown and Perkowski, 1998; Ge et al., 2002; Lundeberg et al., 1994; Madrazo et al., 1987; Robinson et al., 2002; Uvnas-Moberg et al., 1998; Yang, 1994; Yu et al., 2003; Yang et al., 2007a). OXT in the PVN is not related in pain modulation (Yang et al., 2006e,f). However, it has not been proven whether OXT in the other nervous regions, for example OXT in the SON, influences acupuncture analgesia. Electrophysiological and histological studies have found that SON widely connects with the other brain regions such as the periaqueductal gray (PAG), raphe magnus nucleus (RMN), caudate nucleus (CdN) through the nerve fibers including OXT-containing fibers; and some of the other brain regions, which are related to acupuncture analgesia (Zhang, 1990), are connected with SON (Buijs, 1978a,b; Hawthorn et al., 1985; Armstrong and Stern, 1997). This anatomical structure of SON may be biological basic of OXT regulating acupuncture analgesia. For answering this question, first, it needs to study OXT role in acupuncture analgesia. The present work employed the methods of the brain ventricle, spinal cord or venous injection and OXT concentration measurement to investigate OXT effect on acupuncture analgesia in the rat.
2. Materials and methods 2.1. Animals Adult male Sprague–Dawley rats weighing 180–220 g were used in all experiments (Second Military Medical University, Shanghai, China and Nanfang Medical University, Guangzhou, China). Animals were housed in a colony room under controlled temperature, humidity and a 12 h light/dark cycle (light on at 6:00 AM), with food and water available ad libitum. All procedures were conducted according to the guidelines of the International Association for the Study of Pain (Zimmermann, 1983).
Rabbit anti-rat OXT serum (AOXTS) was made by Department of Neurobiology, Second Military Medical University, Shanghai, China (Song et al., 1987). The specificity of the antiserum was over 99.99% cross-reactivity with synthesized OXT and less than 0.01% cross-reactivity with arginine vasopressin (AVP), vasotocin, lysinevasopressin, vasoactive intestinal peptide, neurotensin, Leucine-enkephalin, Methionin-enkephalin, b-endorphin and dynorphinA1 13. The dilution of AOXTS was more than 1: 80,000 for radioimmunoassay. 2.3. Surgery 2.3.1. Intraventricular (icv) injection preparation With Pellegrino L.J. rat brain atlas (1979) as reference, we used the stereotaxic apparatus (Jiangwan I-C, Shanghai, China) to implant a stainless steel guide cannula of 0.5 mm outer diameter into the right lateral ventricle (AP 0.3 mm, LR 0.5 mm, H 3.0 mm) for icv injection under pentobarbital sodium (35 mg/kg, intraperitoneal injection) anesthesia. The guide cannula was fixed to the skull by dental acrylic. 2.3.2. Intrathecal (ith) injection preparation Under pentobarbital sodium (35 mg/kg, intraperitoneal injection) anesthesia, the rat was implanted with a chronic intrathecal catheter (PE-10, 12 cm in length, 0.6 mm outer diameter) extending into the lumbar enlargement of the spinal cord for ith injection (Yaksh and Rudy, 1976). All operated animals needed at least 14 days for recovery after the surgery. 2.4. Nociceptive test 2.4.1. Tail-flick test to noxious potassium iontophoresis All animals were tested under the condition of free activity in a clear acrylic cage (30 cm in diameter, 25 cm in height, open top). The potassium iontophoresis inducing tail-flick served as pain stimulus. The small wet cotton with the saturated solution of potassium iontophoresis was set on the tail skin. The cotton was exposed to direct electrical current, and the anode led the potassium iontophoresis to permeate the tail skin. If the current was strong enough, the permeated potassium iontophoresis resulted in the animal feeling pain stimulation. The intensity of current at the moment of the response was recorded as the pain threshold, which was expressed as mA (WQ-9E Pain Threshold Measurer, Shanghai, China).
2.2. Materials OXT was obtained from Peninsula Laboratories, San Carlos, CA, USA; 125Iodine was from Amersham Pharmacia, Buckinghamshire, UK; the other chemicals were from Sigma Co., St. Louis, MO, USA.
2.4.2. Hot plate test to noxious heat Rats were brought to the testing room and allowed to acclimatize for 10 min before the test began. Pain reflexes in response to a thermal stimulus were measured using RCY-2 Hot Plate Analgesia Meter (Shanghai, China).
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The hot plate surface was heated to a constant temperature of 55 C, as measured by a built-in digital thermometer with an accuracy of 0.1 C and verified by a surface thermometer. Animals were placed on the hot plate (30 cm in diameter), which was surrounded by a clear acrylic cage (25 cm tall, open top), and the Start/Stop button on the timer was activated. The latency, which was expressed as second, to respond with either a hindpaw lick, hindpaw flick, or jump (which ever comes first) was measured to the nearest 0.1 s by deactivating the timer when the response was observed. The rat was immediately removed from the hot plate and returned to its home cage. If a rat did not respond within 30 s, the test was terminated and the animal was removed from the hot plate. All nociceptive tests were carried out in from 8:00 to 10:00 AM. Animals were not habituated to the apparatus prior to testing. The duration between consecutive stimuli was 10 min, and the pain stimulus was terminated at once when the rat showed response to the stimulus. Each animal was tested only once. 2.5. Electrical acupuncture The stainless needles (0.2 mm in diameter and 3 mm in long), which were fixed by the adhesive tape, were placed at the bilateral points of ‘‘Zusanli’’ (St. 36). The stimulated electrical current was passed the bilateral points with alternating polarities and a dense-disperse wave (JSD-731-C electro-stimulator, f1 = 10 Hz, f2 = 20 Hz) for 30 min. The intensity was adjusted until the animal appeared comfortably and its local muscle contractions were seen (v = 2–3 V). 2.6. Sample preparation 2.6.1. Brain nuclei After the treatment, the animals were decapitated. The brains were quickly taken out and put into 80 C physiological saline for heating 2 min, and then fixed in 4% paraformaldehyde physiological saline at 4 C for 3 days. Coronary sections, 400 lm thick, were cut on a cryostat and both sides of nuclei were punched out using special needles (Schelling et al., 1982). All of the same nuclei from three rats in a same group were combined into one sample. The sample was homogenized in 0.5 ml of 0.1 M acetic acid. Two hours later, the same volume of 0.1 M sodium hydroxide was mixed. The 50 ll fluid was taken out for measuring the total protein concentration with Lowry’s method (Lowry et al., 1951). 2.6.2. Other tissues The pituitary and spinal cord were taken out and put into the boiling physiological saline for 5 min. After weighing, the tissue was homogenized in 1.0 ml of 0.1 M acetic acid. Two hours later, the same volume of 0.1 M sodium hydroxide was mixed.
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2.6.3. Plasma The blood was collected using the EDTA Æ Na2 treated tube and kept at 4 C during the treatment. Using the centrifugation at 10,000g for 20 min, the supernatant was withdrawn and stored at 80 C for assay. 2.7. OXT concentration measurement OXT concentration was determined by radioimmunoassay with specific AOXTS (Song et al., 1987). The peptide was labeled 125Iodine using chloramines-T method, and the iodinated OXT was purified by Sephadex G-50. The assay sensitivity of OXT was 1.0 pg/tube and intra- and inter-assay coefficients of variation were less than 5.0% and 8.0%, respectively. 2.8. OXT administration 2.8.1. icv injection A stainless steel needle with 0.3 mm diameter was directly inserted into the guide cannula, 1 mm beyond the tip. After the basic pain threshold measurement, 10 ll serum (AOXTS or normal serum) or solution, which OXT was dissolved in artificial cerebrospinal fluid (ACSF, containing 0.1 M NaCl, 1.0 mM KH2PO4, 4.0 mM KCl, 2.0 mM MgSO4, 2.0 mM CaCl2, 2.1 mM NaHCO3 and 8.0 mM Glucose), was gently injected into the lateral ventricule over 10 min. Only normal serum or ACSF was given in control group. 2.8.2. ith injection After the basic pain threshold measurement, 10 ll serum (AOXTS or normal serum) or solution, which OXT was dissolved in ACSF, was gently injected into the lumbar enlargement of the spinal cord through the chronic intrathecal catheter over 10 min (Yang, 1994). Only normal serum or ACSF was given in control group. 2.8.3. Intravenous (iv) injection After the basic pain threshold measurement, 200 ll serum (AOXTS or normal serum) or solution, which OXT was dissolved in physiological saline, was gently injected into the rat-tail vein. Only normal serum or physiological saline was given in control group. Pain threshold measurements were started 10 min after the administration. 2.9. Statistical analysis Data were expressed as mean ± standard error of the mean (SEM) and were analyzed between groups via analysis of variance (ANOVA). P < 0.05 was considered statistically significant.
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3. Results 3.1. Tail-flick test to noxious potassium iontophoresis 3.1.1. OXT in the central nerve system effects on acupuncture analgesia OXT in the brain and spinal cord enhanced acupuncture analgesia. Electrical acupuncture bilateral ‘‘Zusanli’’ points (St. 36) 30 min increased pain threshold to
100–110%; and the recover time, which this pain threshold recovered to the normal level after stopping acupuncture, was 50 min in the control group (icv or ith) (Fig. 1A and C). Administration of 12.5 ng OXT (icv or ith) enhanced acupuncture analgesia to 110–130% and the recover time was 50 min; 50 ng OXT (icv or ith) enhanced acupuncture analgesia to 180–200% (P < 0.001) and the recover time was 60 min; and 200 ng OXT (icv or ith) enhanced acupuncture analgesia
Fig. 1. Effect of oxytocin and anti-oxytocin serum on acupuncture analgesia in the tail-flick test. icv, intraventricular injection; ith, intraventricular injection; iv, intravenous injection. Acupuncture referred the animals which ‘‘Zusanli’’ points (St. 36) were given electrical acupuncture (densedisperse wave, f1 = 10 Hz, f2 = 20 Hz, 2–3 V) for 30 min. Change of pain threshold (%) indicates (present pain threshold – pain threshold before acupuncture)/pain threshold before acupuncture · 100. The data were expressed as mean ± SEM. n means the animal number in each group.
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to 280–300% (P < 0.001) and the recover time was 80 min (Fig. 1A and C). AOXTS in the brain and spinal cord weakened acupuncture analgesia. Electrical acupuncture bilateral ‘‘Zusanli’’ points (St. 36) 30 min increased the pain threshold to 100–110%, and the recover time was 50 min in the control group (icv or ith) (Fig. 1B and D). Treatment with 2.5 ll AOXTS (icv or ith) weakened acupuncture analgesia to 80–90% and the recover time was 50 min; 5 ll AOXTS (icv or ith) weakened acupuncture analgesia to 60–70% (P < 0.01) and the recover time
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was 40 min; 10 ll AOXTS weakened acupuncture analgesia to 40–50% (P < 0.001) and the recover time was 30 min (Fig. 1B and D).
3.1.2. OXT in the blood effects on acupuncture analgesia Venous administration of OXT (5, 10 and 20 lg) (iv) or AOXTS (50, 100 and 200 ll) (iv) could not influence acupuncture analgesia, which results were similar as those in the control group (iv) (all P > 0.05) (Fig. 1E and F).
Fig. 2. Effect of oxytocin and anti-oxytocin serum on acupuncture analgesia in the hot plate test. icv, intraventricular injection; ith, intraventricular injection; iv, intravenous injection. Acupuncture referred the animals which ‘‘Zusanli’’ points (St. 36) were given electrical acupuncture (densedisperse wave, f1 = 10 Hz, f2 = 20 Hz, 2–3 V) for 30 min. Change of latency (%) indicates (present latency – latency before acupuncture)/latency before acupuncture · 100. The data were expressed as mean ± SEM. n means the animal number in each group.
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3.1.3. Hot plate test to noxious heat Similar as the results of the tail-flick test to noxious potassium iontophoresis, central administration of OXT (icv and ith) enhanced acupuncture analgesia (Fig. 2A and C), and AOXTS (icv and ith) weakened acupuncture analgesia (Fig. 2B and D), but intravenous injection of OXT or AOXTS (iv) did not influence acupuncture analgesia (Fig. 2E and F). 3.1.4. Acupuncture influences OXT concentration Acupuncture changed OXT concentration in the brain and spinal cord, but did not alter OXT concentration in the pituitary and plasma. Electrical acupuncture of ‘‘Zusanli’’ (St. 36) points 30 min reduced OXT concentration in the SON, and elevated OXT concentration
Fig. 3. Effect of electrical acupuncture on oxytocin concentration in rat brain nuclei. SON, hypothalamic supraoptic nucleus; PVN, hypothalamic paraventricular nucleus; SCN, hypothalamic suprachiasmatic nucleus; HVN, hypothalamic ventromedial nucleus; TVN, thalamic ventral nucleus; PAG, periaqueductal gray; RMN, raphes magnus nucleus; CdN, caudate nucleus. Acupuncture referred the animals which ‘‘Zusanli’’ points (St. 36) were given electrical acupuncture (dense-disperse wave, f1 = 10 Hz, f2 = 20 Hz, 2–3 V). The data were expressed as mean ± SEM. n means the animal number in each group.
Table 1 Effect of acupuncture on oxytocin (OXT) concentration
Anterior pituitary (ng/mg) Posterior pituitary (ng/mg) Thoracic spinal cord (pg/mg) Lumbar spinal cord (pg/mg) Serum (pg/ml)
Control
Acupuncture
(n = 8)
10 min (n = 8)
30 min (n = 9)
136.6 ± 12.7
125.3 ± 19.8
131.5 ± 14.9
1412.8 ± 216.4
1455.3 ± 179.4
1758.3 ± 336.5
2.0 ± 0.5
4.1 ± 1.1*
4.7 ± 0.7**
1.9 ± .0.4
6.3 ± 1.2**
8.2 ± 1.9***
13.4 ± 3.1
15.5 ± 4.3
18.0 ± 4.8
All values are showed as mean ± SEM. n indicates the animal number in each group. * P < 0.05. ** P < 0.01. *** P < 0.001.
in the hypothalamic suprachiasmatic nucleus (SCN), hypothalamic ventromedial nucleus (HVN), thalamic ventral nucleus (TVN), periaqueductal gray (PAG), raphe magnus nucleus (RMN), caudate nucleus (CdN), thoracic spinal cord and lumbar spinal cord, but did not alter the OXT concentration in the PVN, anterior pituitary, posterior pituitary and plasma (Fig. 3 and Table 1).
4. Discussion Having been used in many laboratories, classical hot plate/tail-plate test to noxious heat, and hind paw withdrawal to noxious heating are very important to study the hyperalgesia/allodynia in animals with persistent/ chronic pain. However, these tests have some problems in the special research for pain. For example, the latency (pain threshold) shows instability after the animal is tested many times, especially in the experiments that the duration between consecutive stimuli is short. On the other hand, it may cause the animal stress when the animal is removed from the hot plate after the test (Zhang, 1990). Many methods for pain threshold measurement, which included the hot plate test to noxious heat and tail-flick test to noxious potassium iontophoresis, have been compared in our laboratory since we studied pain modulation and acupuncture analgesia in 1954. We found that the tail-flick test to noxious potassium iontophoresis is good, relatively, to keep the basic pain threshold stability in studying pain modulation and acupuncture analgesia, and is easy, specially, to control the animal extra-stimulation under the condition of free activity (Chen and Zhu, 1978). Of course, it is better that the tail-plate test to noxious potassium iontophoresis can combine with classical methods for pain threshold measurement including the hot plate test to noxious heat in one experiment for pain research. It has been demonstrated that central OXT is involved in pain modulation (Brown and Perkowski, 1998; Madrazo et al., 1987; Robinson et al., 2002; Yang, 1994; Yu et al., 2003; Yang et al., 2007a). Treatment with OXT (icv) had an analgesic effect in a patient with intractable cancer pain (Madrazo et al., 1987). Mice lacking OXT reduced stress-induced antinociception following both cold-swim and restraint stress (Robinson et al., 2002). At a spinal cord level, OXT can regulate antinociception in the dog (Brown and Perkowski, 1998) and rat (Yu et al., 2003). OXT is also involved in low back pain in human (Yang, 1994). The present study showed that central administration of OXT (icv or ith) enhanced acupuncture analgesia, while AOXTS (icv or ith) weakened acupuncture analgesia. The data suggested that OXT in the brain and spinal cord played a role in acupuncture analgesia.
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The present study showed that OXT (0.2 mg or less/ rat) or anti-OXT serum (200 ll or less/rat) (iv), one time administration, did not influence acupuncture analgesia. The data suggested that OXT in the blood did not relate to acupuncture analgesia. Some researches reported that intravenous or intraperitoneal injection of OXT shows analgesic effect (Arletti et al., 1993; Lundeberg et al., 1994; Uvnas-Moberg et al., 1998). However, this analgesic effect of OXT (intravenous or intraperitoneal injection) needs a very high dose (10–200 mg OXT/rat) and a very long period (60 days) to treat the animal (Arletti et al., 1993; Lundeberg et al., 1994; Uvnas-Moberg et al., 1998). It is a pharmacological action, because OXT can be penetrated the blood-brain barrier if OXT concentration is high enough in the blood. This point can be proven by the result that intraventricular injection of OXT antagonist prevents the analgesic effect of OXT, which was given by the intraperitoneal injection (Arletti et al., 1993; Lundeberg et al., 1994; Uvnas-Moberg et al., 1998). The present study also showed that electrical acupuncture of ‘‘Zusanli’’ (St. 36) reduced OXT concentration in the SON, and elevated OXT concentration in the SCN, HVN, TVN, PAG, RMN, CdN, thoracic spinal cord and lumbar spinal cord, but did not alter the OXT concentration in the PVN, anterior pituitary, posterior pituitary and plasma. The data indicated that OXT in central nervous system rather than in peripheral organs was related in acupuncture analgesia. In according to the anatomical connection between SON and other nervous structures such as PAG, RMN, CdN and spinal cord (Buijs, 1978a,b; Hawthorn et al., 1985; Armstrong and Stern, 1997), the mechanism of OXT enhancing acupuncture analgesia may be proposed as follows: (1) Acupuncture causes SON releasing OXT; (2) then this OXT is transported to the other brain nuclei (such as PAG, RMN and CdN) and spinal cord; (3) in the relative nervous regions OXT influences the antinociceptive system including the endogenous opiate peptide system, to be involved in the process of pain modulation. The part of this propose is proven by our previous work, i.e. OXT can induce the spinal cord releasing the endogenous opiate peptides and the analgesic effect of OXT can be attenuated by naloxone – an opiate receptor antagonist in the spinal cord (Yang, 1994). AVP is a very important regulator in PVN regulating acupuncture analgesia (Yang, 1992; Yang et al., 2006d,e,f; Yang et al., 2007e). AVP in PVN, which is transported to the other brain nuclei such as PAG (Yang et al., 2006c,h; Yang et al., 2007b,c,d), RMN (Yang et al., 2006a) and CdN (Yang et al., 2006b), is involved in antinociception and acupuncture analgesia (Yang et al., 2006g). OXT and AVP are very similar in their source, synthesis and structure (McEwen, 2004). The present study showed that electrical acupuncture
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decreased OXT concentration in the SON, not in the PVN. In contrast, our previous work showed that electrical acupuncture decreased AVP concentration in the PVN, not in the SON (Yang, 1992; Yang et al., 2006e,f). The data suggested that OXT in the SON rather than in the PVN is involved in acupuncture analgesia, and AVP in the PVN rather than in the SON is related to acupuncture analgesia. Why are the roles of OXT and AVP in SON and PVN regulating acupuncture analgesia different? It is a very interesting topic that needs to be further investigated. In conclusion, our present studies proved that OXT (icv or ith) enhanced acupuncture analgesia, and antiOXT serum (icv or ith) weakened acupuncture analgesia in a dose-dependent manner, but both OXT and antiOXT serum (iv) did not influence acupuncture analgesia; acupuncture changed OXT concentration in the brain nuclei and spinal cord, not in the pituitary and plasma. The data suggested that OXT in central nervous system rather than in peripheral organs played an important role in acupuncture analgesia in the rat. Acknowledgements This work was supported by Guangdong Bangmin Pharmaceutical Co. Ltd. and a grant from the National Science Foundation of China.
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