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Endogenous opiate peptides in the spinal cord are involved in the analgesia of hypothalamic paraventricular nucleus in the rat
Peptides 30 (2009) 740–744
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Endogenous opiate peptides in the spinal cord are involved in the analgesia of hypothalamic paraventricular nucleus in the rat Jun Yang a,*, Yu Yang a,1, Jiegen Chu a, Gen Wang b, Hongtao Xu c, Wen-Yan Liu d, Cheng-Hai Wang e, Bao-Cheng Lin e a
Jiangsu (Taizhou) Research Institute for Novel Pharmaceuticals, Yangtze River Pharmaceutical Group, 1 Yangtze River Road (South), Taizhou, Jiangsu 225321, China Institute for Nutrisciences and Health, National Research Council Canada, Charlottetown, Prince Edward Island, Canada C1A 5T1 Lady Davis Institute for Medical Research, Jewish General Hospital, McGill University, Montreal, Quebec, Canada H3T 1E2 d Department of Physiology, Jining Medical College, Jining, Shangdong 272113, China e Department of Neurobiology, Second Military Medical University, Shanghai 200433, China b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 September 2008 Received in revised form 26 December 2008 Accepted 5 January 2009 Available online 15 January 2009
Many studies have shown that hypothalamic paraventricular nucleus (PVN) plays a role in pain process, and endogenous opiate peptide system in the spinal cord is involved in nociception. This communication was designed to study the relationship between PVN and endogenous opiate system in the spinal cord in the rat. The results showed that in both the thoracic and the lumber spinal cord, microinjection of 100 ng L-glutamate sodium into PVN could increase leucine-enkephalin (L-Ek), b-endorphin (b-Ep), dynorphinA1–13 (DynA1–13) concentrations and PVN cauterization decreased L-Ek and b-Ep concentrations. Pretreatment of the spinal cord with 5 mg naloxone, an opiate receptor antagonist could partly reverse the analgesia induced by microinjection of 100 ng L-glutamate sodium into PVN. The data suggested that PVN analgesia might be involved in the endogenous opiate peptide system in the spinal cord independently. ß 2009 Elsevier Inc. All rights reserved.
Keywords: Hypothalamic paraventricular nucleus Analgesia Endogenous opiate peptide Arginine vasopressin Spinal cord
1. Introduction The hypothalamic paraventricular nucleus (PVN) is a complex neural structure, which has been implicated in a number of functions including control of pituitary–adrenocortical activity in response to stress, body fluid homeostasis, milk ejection reflex, hormone probation, circadian rhythm, food intake, gastrointestinal and cardiovascular functions, sexual activity, learning and memory [7]. PVN has also been proven to play a role in analgesia [14,17], where arginine vasopressin (AVP) is more important than other neuropeptides [18,19,21,27,28]. Only through the brain, not spinal cord and systemic circulation, AVP is involved in the pain process [13,15,16,20]. The result that the pituitary removal does not influence the analgesia induced by PVN stimulation [14,17] suggests that PVN should be through the central nervous system, for example the spinal cord, to regulate the pain process.
PVN stimulation not only induces the synthesis and section of leucine-enkephalin (L-Ek), b-endorphin (b-Ep) in the periaqueductal gray (PAG) [27], but also modulates nociceptive responses in dorsal horn wide dynamic range neurons [3]. AVP also enhances the PAG synthesis and section of L-Ek, b-Ep and dynorphinA1–13 (DynA1–13) [29,30]. Spinal cord projecting vasopressinergic neurons are found in the PVN [6]. The endogenous opiate peptides in the spinal cord are involved in pain process [11]. Dynorphin mRNA-expressing neurons in the PVN project to the spinal cord [5]. However, it is not clear whether the endogenous opiate peptide system in the spinal cord is involved in PVN pain regulation process. The present study is designed to investigate the relationship between the endogenous opiate peptide system in the spinal cord and PVN pain-regulation process. 2. Materials and methods 2.1. Animals
* Corresponding author. Tel.: +86 523 86978401; fax: +86 523 86911769. E-mail address: [email protected] (J. Yang). 1 Volunteer, a student from E`cole secondaire Saint-Luc, Montreal, Quebec, Canada H3X 2H2. 0196-9781/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2009.01.004
Adult male Sprague–Dawley rats weighing 180–220 g were used in all experiments (Charles River Laboratories, Montreal, Quebec, Canada and Second Military Medical University, Shanghai,
J. Yang et al. / Peptides 30 (2009) 740–744
China). Animals were housed in a colony room under controlled temperature, humidity and a 12 h light/dark cycle (light on at 6:00 a.m.), with food and water available ad libitum. All procedures were conducted according to the guidelines of the International Association for the Study of Pain [33]. 2.2. Materials L-Ek, b-Ep and DynA1–13 were obtained from Peninsula Laboratories, San Carlos, CA, USA; 125Iodine was from Amersham Pharmacia, Buckinghamshire, UK; Naloxone and other chemical reagents were from Sigma Co., St. Louis, MO, USA. Rabbit anti-rat L-Ek, b-Ep or DynA1–13 serum was made by Department of Neurobiology, Second Military Medical University, Shanghai, China [30]. The specificity of each kind of antiserum was more than 99% reactivity with its corresponding antigen and less than 1% reactivity with other similar peptides. The effective dilution of the antiserum was 1:20,000–80,000 for radioimmunoassay. 2.3. Surgery 2.3.1. For PVN microinjection With the Pellegrino L.J. rat brain atlas 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 PVN (AP 0.4 mm, LR 0.3 mm, H 7.8 mm) for nucleus microinjection under pentobarbital sodium (35 mg/kg, intraperitoneal microinjection) anesthesia.
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The cotton was exposed to direct electrical current, and the anode led the potassium iontophoresis to permeate the skin of the tail. If the current was strong enough, the permeated potassium iontophoresis resulted in the animal feeling the pain stimulation. The intensity of current at the moment of the response was recorded as the pain threshold, which was expressed as mA (WQ9E Pain Threshold Measurer, Shanghai, China). The duration between consecutive stimuli is 10 min, and the pain stimulus was terminated at once when the rat showed response to this stimulus. 2.5. Microinjection 2.5.1. For PVN stimulation The animal was gently handled and a stainless steel needle with 0.3 mm diameter for the PVN microinjection was directly inserted into the guide cannula, 1 mm beyond the tip. One microliter of artificial cerebral spinal 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), which100 ng L-glutamate sodium was dissolved, was gently injected into the PVN over 10 min. In control group, no L-glutamate sodium was dissolved in the ACSF. 2.5.2. For spinal cord administration Ten microliters of solution was gently injected into the lumbar enlargement of the spinal cord through the chronic intrathecal catheter over 10 min. 2.6. Prepare of tissue sample
2.3.2. For intrathecal injection (ith) Under pentobarbital sodium (35 mg/kg, intraperitoneal injection) anesthesia, the rat was implanted a chronic intrathecal catheter (PE-10, 12 cm in length, 0.6 cm outer diameter) extending into the lumbar enlargement of the spinal cord for ith. All operations were carried out under aseptic conditions and the animals were allowed to recover for at least 14 days after the surgery.
After the decapitation, the spinal cords were taken out and put into the boiling physiological saline for 5 min. After weighing, the tissues were 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 in the homogenate. Using the centrifugation at 10,000 g for 20 min, the supernatants were withdrawn and stored at 80 8C for assay.
2.3.3. For PVN cauterization The PVN cauterization was carried out using a similar stereotaxic surgery approach above, the electrodes were inserted into both sides of PVN (AP 0.4 mm, LR 0.3 mm, H 7.8 mm), which were passed the 1 mA direct electrical current for 10 s.
The L-Ek, b-Ep and DynA1–13 concentrations were determined with specific rabbit antiserum. The peptides were labeled 125Iodine using the chloramines-T method and iodinated peptides were purified by Sephadex G-50 [31,32]. The assay sensitivities for the LEk, b-Ep or DynA1–13 were 3.0, 1.2 and 6.3 pg/tube and intra- and inter-assay coefficients of variation were less than 5.1% and 8.0%, respectively [10,31,32].
2.4. Nociceptive tests All animals were tested under the condition of free activity in the small cages (30 cm in diameter, 25 cm in height) from 8:00 to 10:00 a.m. Depending on the 20-year experience of studying pain in our laboratory [23], we used the potassium iontophoresis inducing tail-flick served as pain stimulus. The small wet cotton with the potassium iontophoresis was set on the skin of the tail.
2.7. Radioimmunoassay (RIA)
2.8. Histological verification At the end of the experiments, the rats were sacrificed under a high dose of pentobarbital sodium (80 mg/kg, intraperitoneal microinjection), and the histological locations of microinjection and cauterization were ascertained (Fig. 1). The data were rejected
Fig. 1. Histological verification of hypothalamic paraventricular nucleus (PVN). (A) PVN microinjection; (B) PVN cauterization.
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if the histological locations were not accurate, and the number of included/excluded rats was 8/3–5. 2.9. Statistical analysis Data were expressed as mean standard error of the mean (S.E.M.) and were analyzed between groups by the analysis of variance (ANOVA) and x2 test. P < 0.05 was considered statistically significant. 3. Results 3.1. PVN stimulation increases the concentrations of endogenous opiate peptides in the spinal cord Comparing with the control group, microinjection of 100 ng sodium into PVN could increase the L-Ek (Fig. 2A and D), b-Ep (Fig. 2B and E) and DynA1–13 (Fig. 2C and F) concentrations in both thoracic and lumber spinal cord from 10 to 50 min after the microinjection. L-glutamate
3.2. PVN cauterization decreases the concentrations of endogenous opiate peptides in the spinal cord Comparing with the control group, PVN cauterization could decrease the L-Ek (Fig. 3A and D) and b-Ep (Fig. 3B and E) concentrations in both thoracic and lumber spinal cord in 7 days and 10 days after the cauterization, and PVN cauterization did not influence DynA1–13 concentration in both thoracic and lumber spinal cord in 7 days and 10 days after the cauterization significantly (Fig. 3C and F). 3.3. ith of naloxone reverses the analgesia induced by PVN stimulation Fig. 4 showed that microinjection of 100 ng L-glutamate sodium into PVN increased the pain threshold in 40 min after the microinjection, and ith of 5 mg naloxone decreased the pain threshold in 40 min after the administration. Fig. 4 also showed that the spinal cord pretreatment with 5 mg naloxone could partly block the analgesia induced by microinjection of 100 ng L-glutamate sodium into PVN.
4. Discussion Endogenous opiate peptide system includes three series— enkephalin, endorphin and dynorphin [9] which are found in the PVN [7,11]. PVN stimulation increases the pain threshold, and PVN cauterization decreases the pain threshold [14,17]. PVN plays a role in analgesia [2,3]. Considering that the pituitary removal does not influence the analgesia induced by PVN stimulation [14,17], the PVN analgesia might be through the central nervous system, not systemic circulation. Although dynorphin mRNA-expressing neurons in the PVN are found to project to the spinal cord [5], pain stimulation does not change the concentration of L-Ek, b-Ep and DynA1–13 in both the tissue and the pull–push perfuse liquid of PVN [21], and PVN stimulation does not influence the concentration of L-Ek, b-Ep and DynA1–13 in the pull–push perfuse liquid of PVN [18,19]. Intraventricular injection of anti-L-Ek, b-Ep or DynA1–13 serum cannot influence the analgesia induced by the PVN stimulation [18,19,21]. These data suggested that endogenous opiate peptides might be not involved in the PVN pain regulation process. However, results from our current study showed that PVN stimulation increased the concentration of L-Ek, b-Ep and DynA1– 13 in the spinal cord, and PVN cauterization decreased the concentration of L-Ek and b-Ep in the spinal cord; Pretreatment of naloxone, an opiate receptor antagonist, partly reversed the analgesia induced by PVN stimulation. The data suggested that endogenous opiate peptide system in the spinal cord was involved in PVN pain regulation process. How does PVN influence the endogenous opiate peptides in the spinal cord? We guess that it is related with AVP in the PVN, because AVP is a very important bioactive substance in PVN regulating nociception [1,4,7,28]. Pain stimulation and acupuncture can cause PVN synthesis and release of AVP, not oxytocin, LEk, b-Ep and DynA1–13 [18,19,21]. This AVP can be transferred to the other brain nuclei such as the PAG [26,27,29,30]. AVP can enhance PAG synthesis and secretion of endogenous opiate peptides including L-Ek, b-Ep and DynA1–13 to cause analgesia [26,27,29,30]. Since PAG connects to the spinal cord through the afferent and efferent nervous projections [4,8,11], these endogenous opiate peptides in the PAG may be transferred to the spinal cord, so as to participate in the pain process [11,12].
Fig. 2. Effect of PVN stimulation on the concentration of endogenous opiate peptides in the spinal cord. PVN injection denotes the beginning of the PVN injection. PVN stimulation group were injected with 100 ng L-glutamate sodium/1 ml artificial cerebrospinal fluid (ACSF) into the PVN (~, n = 8). Control group was injected with 1 ml ACSF into the PVN (*, n = 8). The data is expressed as mean S.E.M. *P < 0.05, **P < 0.01 and ***P < 0.001 are for the comparison of the concentration of endogenous opiate peptides in the spinal cord from PVN stimulation group and control group.
J. Yang et al. / Peptides 30 (2009) 740–744
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Fig. 3. Effect of PVN cauterization on the concentration of endogenous opiate peptides in the spinal cord. PVN cauterization group was given the bilateral cauterization of PVN (~, n = 8). Control group was given the sham operation and no cauterization of PVN (*, n = 8). The data is expressed as mean S.E.M. *P < 0.05 and **P < 0.01 are for the comparison of the concentrations of endogenous opiate peptides in the spinal cord from PVN cauterization group and control group.
On the other hand, results from our previous experiments also supported this opinion. Oxytocin, which is a regulator of pain process in the spinal cord [12,22,23], can induce the spinal cord secretion of L-Ek, b-Ep and DynA1–13 [12]. Although AVP is similar with oxytocin in the source and structure [7,24,25], AVP in the spinal cord can not influence the pain process, and pain stimulation or acupuncture do not change the AVP concentration in the spinal cord [20]. We presumed that AVP in the PVN did not effect on the spinal cord directly, and it might act on the other brain nuclei, for example PAG, to influence the endogenous opiate peptide system.
Through AVP in the nucleus raphe magnus and caudate nucleus is also involved in the pain process [13,15], it is not clarified that this AVP influences the endogenous opiate peptide system in these brain nuclei. In conclusion, the present study showed that PVN stimulation increased the concentration of endogenous opiate peptides in the spinal cord, and PVN cauterization decreased the concentration of endogenous opiate peptides in the spinal cord; administration of the opiate receptor antagonists in the spinal cord could partly attenuate the analgesia induced by PVN. The data suggested that PVN pain regulation process might be through the endogenous opiate peptide system in spinal cord independently. Acknowledgments This work was supported by Yangtze River Pharmaceutical Group and a grant from the National Science Foundation of China. References
Fig. 4. Effect of intrathecal injection of naloxone on the analgesia induced by PVN stimulation. ith denotes the intrathecal injection of 5 mg naloxone/10 ml artificial cerebrospinal fluid (ACSF) or 10 ml ACSF. PVN injection had injected 100 ng Lglutamate sodium/1 ml ACSF or 1 ml ACSF into the PVN. ACSF + No PVN stimulation group (*, n = 7), had given the intrathecal injection of 10 ml ACSF and then injected 1 ml ACSF into the PVN. ACSF + PVN stimulation group (&, n = 7), had given the intrathecal injection of 10 ml ACSF and then injected 100 ng L-glutamate sodium/ 1 ml ACSF into the PVN. Naloxone + No PVN stimulation group (~, n = 7), had given the intrathecal injection of 5 mg naloxone/10 ml ACSF and then injected 1 ml ACSF into the PVN. Naloxone + PVN stimulation group (, n = 7), had given the intrathecal injection of 5 mg naloxone/10 ml ACSF and then injected 100 ng L-glutamate sodium/1 ml ACSF into the PVN. The data is expressed as mean S.E.M. +P < 0.05, ++ P < 0.01 and +++P < 0.001 are for the comparison of the concentrations of the pain threshold from Naloxone + PVN stimulation group to ACSF + No PVN stimulation group. *P < 0.05 is for the comparison of the pain threshold from Naloxone + PVN stimulation group to Naloxone + No PVN stimulation group. #P < 0.05 is for the comparison of the pain threshold from Naloxone + PVN stimulation group to ACSF + No PVN stimulation group.
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Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Endogenous opiate peptides in the spinal cord are involved in the analgesia of hypothalamic paraventricular nucleus in the rat Jun Yang a,*, Yu Yang a,1, Jiegen Chu a, Gen Wang b, Hongtao Xu c, Wen-Yan Liu d, Cheng-Hai Wang e, Bao-Cheng Lin e a
Jiangsu (Taizhou) Research Institute for Novel Pharmaceuticals, Yangtze River Pharmaceutical Group, 1 Yangtze River Road (South), Taizhou, Jiangsu 225321, China Institute for Nutrisciences and Health, National Research Council Canada, Charlottetown, Prince Edward Island, Canada C1A 5T1 Lady Davis Institute for Medical Research, Jewish General Hospital, McGill University, Montreal, Quebec, Canada H3T 1E2 d Department of Physiology, Jining Medical College, Jining, Shangdong 272113, China e Department of Neurobiology, Second Military Medical University, Shanghai 200433, China b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 September 2008 Received in revised form 26 December 2008 Accepted 5 January 2009 Available online 15 January 2009
Many studies have shown that hypothalamic paraventricular nucleus (PVN) plays a role in pain process, and endogenous opiate peptide system in the spinal cord is involved in nociception. This communication was designed to study the relationship between PVN and endogenous opiate system in the spinal cord in the rat. The results showed that in both the thoracic and the lumber spinal cord, microinjection of 100 ng L-glutamate sodium into PVN could increase leucine-enkephalin (L-Ek), b-endorphin (b-Ep), dynorphinA1–13 (DynA1–13) concentrations and PVN cauterization decreased L-Ek and b-Ep concentrations. Pretreatment of the spinal cord with 5 mg naloxone, an opiate receptor antagonist could partly reverse the analgesia induced by microinjection of 100 ng L-glutamate sodium into PVN. The data suggested that PVN analgesia might be involved in the endogenous opiate peptide system in the spinal cord independently. ß 2009 Elsevier Inc. All rights reserved.
Keywords: Hypothalamic paraventricular nucleus Analgesia Endogenous opiate peptide Arginine vasopressin Spinal cord
1. Introduction The hypothalamic paraventricular nucleus (PVN) is a complex neural structure, which has been implicated in a number of functions including control of pituitary–adrenocortical activity in response to stress, body fluid homeostasis, milk ejection reflex, hormone probation, circadian rhythm, food intake, gastrointestinal and cardiovascular functions, sexual activity, learning and memory [7]. PVN has also been proven to play a role in analgesia [14,17], where arginine vasopressin (AVP) is more important than other neuropeptides [18,19,21,27,28]. Only through the brain, not spinal cord and systemic circulation, AVP is involved in the pain process [13,15,16,20]. The result that the pituitary removal does not influence the analgesia induced by PVN stimulation [14,17] suggests that PVN should be through the central nervous system, for example the spinal cord, to regulate the pain process.
PVN stimulation not only induces the synthesis and section of leucine-enkephalin (L-Ek), b-endorphin (b-Ep) in the periaqueductal gray (PAG) [27], but also modulates nociceptive responses in dorsal horn wide dynamic range neurons [3]. AVP also enhances the PAG synthesis and section of L-Ek, b-Ep and dynorphinA1–13 (DynA1–13) [29,30]. Spinal cord projecting vasopressinergic neurons are found in the PVN [6]. The endogenous opiate peptides in the spinal cord are involved in pain process [11]. Dynorphin mRNA-expressing neurons in the PVN project to the spinal cord [5]. However, it is not clear whether the endogenous opiate peptide system in the spinal cord is involved in PVN pain regulation process. The present study is designed to investigate the relationship between the endogenous opiate peptide system in the spinal cord and PVN pain-regulation process. 2. Materials and methods 2.1. Animals
* Corresponding author. Tel.: +86 523 86978401; fax: +86 523 86911769. E-mail address: [email protected] (J. Yang). 1 Volunteer, a student from E`cole secondaire Saint-Luc, Montreal, Quebec, Canada H3X 2H2. 0196-9781/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2009.01.004
Adult male Sprague–Dawley rats weighing 180–220 g were used in all experiments (Charles River Laboratories, Montreal, Quebec, Canada and Second Military Medical University, Shanghai,
J. Yang et al. / Peptides 30 (2009) 740–744
China). Animals were housed in a colony room under controlled temperature, humidity and a 12 h light/dark cycle (light on at 6:00 a.m.), with food and water available ad libitum. All procedures were conducted according to the guidelines of the International Association for the Study of Pain [33]. 2.2. Materials L-Ek, b-Ep and DynA1–13 were obtained from Peninsula Laboratories, San Carlos, CA, USA; 125Iodine was from Amersham Pharmacia, Buckinghamshire, UK; Naloxone and other chemical reagents were from Sigma Co., St. Louis, MO, USA. Rabbit anti-rat L-Ek, b-Ep or DynA1–13 serum was made by Department of Neurobiology, Second Military Medical University, Shanghai, China [30]. The specificity of each kind of antiserum was more than 99% reactivity with its corresponding antigen and less than 1% reactivity with other similar peptides. The effective dilution of the antiserum was 1:20,000–80,000 for radioimmunoassay. 2.3. Surgery 2.3.1. For PVN microinjection With the Pellegrino L.J. rat brain atlas 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 PVN (AP 0.4 mm, LR 0.3 mm, H 7.8 mm) for nucleus microinjection under pentobarbital sodium (35 mg/kg, intraperitoneal microinjection) anesthesia.
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The cotton was exposed to direct electrical current, and the anode led the potassium iontophoresis to permeate the skin of the tail. If the current was strong enough, the permeated potassium iontophoresis resulted in the animal feeling the pain stimulation. The intensity of current at the moment of the response was recorded as the pain threshold, which was expressed as mA (WQ9E Pain Threshold Measurer, Shanghai, China). The duration between consecutive stimuli is 10 min, and the pain stimulus was terminated at once when the rat showed response to this stimulus. 2.5. Microinjection 2.5.1. For PVN stimulation The animal was gently handled and a stainless steel needle with 0.3 mm diameter for the PVN microinjection was directly inserted into the guide cannula, 1 mm beyond the tip. One microliter of artificial cerebral spinal 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), which100 ng L-glutamate sodium was dissolved, was gently injected into the PVN over 10 min. In control group, no L-glutamate sodium was dissolved in the ACSF. 2.5.2. For spinal cord administration Ten microliters of solution was gently injected into the lumbar enlargement of the spinal cord through the chronic intrathecal catheter over 10 min. 2.6. Prepare of tissue sample
2.3.2. For intrathecal injection (ith) Under pentobarbital sodium (35 mg/kg, intraperitoneal injection) anesthesia, the rat was implanted a chronic intrathecal catheter (PE-10, 12 cm in length, 0.6 cm outer diameter) extending into the lumbar enlargement of the spinal cord for ith. All operations were carried out under aseptic conditions and the animals were allowed to recover for at least 14 days after the surgery.
After the decapitation, the spinal cords were taken out and put into the boiling physiological saline for 5 min. After weighing, the tissues were 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 in the homogenate. Using the centrifugation at 10,000 g for 20 min, the supernatants were withdrawn and stored at 80 8C for assay.
2.3.3. For PVN cauterization The PVN cauterization was carried out using a similar stereotaxic surgery approach above, the electrodes were inserted into both sides of PVN (AP 0.4 mm, LR 0.3 mm, H 7.8 mm), which were passed the 1 mA direct electrical current for 10 s.
The L-Ek, b-Ep and DynA1–13 concentrations were determined with specific rabbit antiserum. The peptides were labeled 125Iodine using the chloramines-T method and iodinated peptides were purified by Sephadex G-50 [31,32]. The assay sensitivities for the LEk, b-Ep or DynA1–13 were 3.0, 1.2 and 6.3 pg/tube and intra- and inter-assay coefficients of variation were less than 5.1% and 8.0%, respectively [10,31,32].
2.4. Nociceptive tests All animals were tested under the condition of free activity in the small cages (30 cm in diameter, 25 cm in height) from 8:00 to 10:00 a.m. Depending on the 20-year experience of studying pain in our laboratory [23], we used the potassium iontophoresis inducing tail-flick served as pain stimulus. The small wet cotton with the potassium iontophoresis was set on the skin of the tail.
2.7. Radioimmunoassay (RIA)
2.8. Histological verification At the end of the experiments, the rats were sacrificed under a high dose of pentobarbital sodium (80 mg/kg, intraperitoneal microinjection), and the histological locations of microinjection and cauterization were ascertained (Fig. 1). The data were rejected
Fig. 1. Histological verification of hypothalamic paraventricular nucleus (PVN). (A) PVN microinjection; (B) PVN cauterization.
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if the histological locations were not accurate, and the number of included/excluded rats was 8/3–5. 2.9. Statistical analysis Data were expressed as mean standard error of the mean (S.E.M.) and were analyzed between groups by the analysis of variance (ANOVA) and x2 test. P < 0.05 was considered statistically significant. 3. Results 3.1. PVN stimulation increases the concentrations of endogenous opiate peptides in the spinal cord Comparing with the control group, microinjection of 100 ng sodium into PVN could increase the L-Ek (Fig. 2A and D), b-Ep (Fig. 2B and E) and DynA1–13 (Fig. 2C and F) concentrations in both thoracic and lumber spinal cord from 10 to 50 min after the microinjection. L-glutamate
3.2. PVN cauterization decreases the concentrations of endogenous opiate peptides in the spinal cord Comparing with the control group, PVN cauterization could decrease the L-Ek (Fig. 3A and D) and b-Ep (Fig. 3B and E) concentrations in both thoracic and lumber spinal cord in 7 days and 10 days after the cauterization, and PVN cauterization did not influence DynA1–13 concentration in both thoracic and lumber spinal cord in 7 days and 10 days after the cauterization significantly (Fig. 3C and F). 3.3. ith of naloxone reverses the analgesia induced by PVN stimulation Fig. 4 showed that microinjection of 100 ng L-glutamate sodium into PVN increased the pain threshold in 40 min after the microinjection, and ith of 5 mg naloxone decreased the pain threshold in 40 min after the administration. Fig. 4 also showed that the spinal cord pretreatment with 5 mg naloxone could partly block the analgesia induced by microinjection of 100 ng L-glutamate sodium into PVN.
4. Discussion Endogenous opiate peptide system includes three series— enkephalin, endorphin and dynorphin [9] which are found in the PVN [7,11]. PVN stimulation increases the pain threshold, and PVN cauterization decreases the pain threshold [14,17]. PVN plays a role in analgesia [2,3]. Considering that the pituitary removal does not influence the analgesia induced by PVN stimulation [14,17], the PVN analgesia might be through the central nervous system, not systemic circulation. Although dynorphin mRNA-expressing neurons in the PVN are found to project to the spinal cord [5], pain stimulation does not change the concentration of L-Ek, b-Ep and DynA1–13 in both the tissue and the pull–push perfuse liquid of PVN [21], and PVN stimulation does not influence the concentration of L-Ek, b-Ep and DynA1–13 in the pull–push perfuse liquid of PVN [18,19]. Intraventricular injection of anti-L-Ek, b-Ep or DynA1–13 serum cannot influence the analgesia induced by the PVN stimulation [18,19,21]. These data suggested that endogenous opiate peptides might be not involved in the PVN pain regulation process. However, results from our current study showed that PVN stimulation increased the concentration of L-Ek, b-Ep and DynA1– 13 in the spinal cord, and PVN cauterization decreased the concentration of L-Ek and b-Ep in the spinal cord; Pretreatment of naloxone, an opiate receptor antagonist, partly reversed the analgesia induced by PVN stimulation. The data suggested that endogenous opiate peptide system in the spinal cord was involved in PVN pain regulation process. How does PVN influence the endogenous opiate peptides in the spinal cord? We guess that it is related with AVP in the PVN, because AVP is a very important bioactive substance in PVN regulating nociception [1,4,7,28]. Pain stimulation and acupuncture can cause PVN synthesis and release of AVP, not oxytocin, LEk, b-Ep and DynA1–13 [18,19,21]. This AVP can be transferred to the other brain nuclei such as the PAG [26,27,29,30]. AVP can enhance PAG synthesis and secretion of endogenous opiate peptides including L-Ek, b-Ep and DynA1–13 to cause analgesia [26,27,29,30]. Since PAG connects to the spinal cord through the afferent and efferent nervous projections [4,8,11], these endogenous opiate peptides in the PAG may be transferred to the spinal cord, so as to participate in the pain process [11,12].
Fig. 2. Effect of PVN stimulation on the concentration of endogenous opiate peptides in the spinal cord. PVN injection denotes the beginning of the PVN injection. PVN stimulation group were injected with 100 ng L-glutamate sodium/1 ml artificial cerebrospinal fluid (ACSF) into the PVN (~, n = 8). Control group was injected with 1 ml ACSF into the PVN (*, n = 8). The data is expressed as mean S.E.M. *P < 0.05, **P < 0.01 and ***P < 0.001 are for the comparison of the concentration of endogenous opiate peptides in the spinal cord from PVN stimulation group and control group.
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Fig. 3. Effect of PVN cauterization on the concentration of endogenous opiate peptides in the spinal cord. PVN cauterization group was given the bilateral cauterization of PVN (~, n = 8). Control group was given the sham operation and no cauterization of PVN (*, n = 8). The data is expressed as mean S.E.M. *P < 0.05 and **P < 0.01 are for the comparison of the concentrations of endogenous opiate peptides in the spinal cord from PVN cauterization group and control group.
On the other hand, results from our previous experiments also supported this opinion. Oxytocin, which is a regulator of pain process in the spinal cord [12,22,23], can induce the spinal cord secretion of L-Ek, b-Ep and DynA1–13 [12]. Although AVP is similar with oxytocin in the source and structure [7,24,25], AVP in the spinal cord can not influence the pain process, and pain stimulation or acupuncture do not change the AVP concentration in the spinal cord [20]. We presumed that AVP in the PVN did not effect on the spinal cord directly, and it might act on the other brain nuclei, for example PAG, to influence the endogenous opiate peptide system.
Through AVP in the nucleus raphe magnus and caudate nucleus is also involved in the pain process [13,15], it is not clarified that this AVP influences the endogenous opiate peptide system in these brain nuclei. In conclusion, the present study showed that PVN stimulation increased the concentration of endogenous opiate peptides in the spinal cord, and PVN cauterization decreased the concentration of endogenous opiate peptides in the spinal cord; administration of the opiate receptor antagonists in the spinal cord could partly attenuate the analgesia induced by PVN. The data suggested that PVN pain regulation process might be through the endogenous opiate peptide system in spinal cord independently. Acknowledgments This work was supported by Yangtze River Pharmaceutical Group and a grant from the National Science Foundation of China. References
Fig. 4. Effect of intrathecal injection of naloxone on the analgesia induced by PVN stimulation. ith denotes the intrathecal injection of 5 mg naloxone/10 ml artificial cerebrospinal fluid (ACSF) or 10 ml ACSF. PVN injection had injected 100 ng Lglutamate sodium/1 ml ACSF or 1 ml ACSF into the PVN. ACSF + No PVN stimulation group (*, n = 7), had given the intrathecal injection of 10 ml ACSF and then injected 1 ml ACSF into the PVN. ACSF + PVN stimulation group (&, n = 7), had given the intrathecal injection of 10 ml ACSF and then injected 100 ng L-glutamate sodium/ 1 ml ACSF into the PVN. Naloxone + No PVN stimulation group (~, n = 7), had given the intrathecal injection of 5 mg naloxone/10 ml ACSF and then injected 1 ml ACSF into the PVN. Naloxone + PVN stimulation group (, n = 7), had given the intrathecal injection of 5 mg naloxone/10 ml ACSF and then injected 100 ng L-glutamate sodium/1 ml ACSF into the PVN. The data is expressed as mean S.E.M. +P < 0.05, ++ P < 0.01 and +++P < 0.001 are for the comparison of the concentrations of the pain threshold from Naloxone + PVN stimulation group to ACSF + No PVN stimulation group. *P < 0.05 is for the comparison of the pain threshold from Naloxone + PVN stimulation group to Naloxone + No PVN stimulation group. #P < 0.05 is for the comparison of the pain threshold from Naloxone + PVN stimulation group to ACSF + No PVN stimulation group.
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