Co-expression of calretinin and parvalbumin in the rat facial nucleus**★

BACKGROUND： Calretinin and parvalbumin are members of the intracellular calcium binding protein family, which transform Ca^2＋ bioinformation into regulation of neuronal and neural network activities. OBJECTIVE： To observe expression and co-expression of calretinin and parvalbumin in rat facial nucleus neurons . DESIGN, TIME AND SETTING： Neuronal morphology experiment was performed at the Research Laboratory of Applied Anatomy, Department Neurobiology and Anatomy, Xiangya Medical College of Central South University from August to October 2007. MATERIALS： Five healthy, adult Sprague Dawley rats were selected. Polyclonal rabbit-anti-parvalbumin and mouse-anti-calretinin were provided by Sigma, USA. METHODS： Rat brains were obtained and cut into coronal slices using a freezing microtome. Slices from the experimental group were immunofluorescent stained with polyclonal rabbit-anti-parvalbumin and mouse-anti-calretinin antibodies. The control group sections were stained with normal rabbit and mouse sera. MAIN OUTCOME MEASURES： Immunofluorescent double-staining was used to detect calretinin and parvalbumin expression. Nissl staining was utilized for facial nucleus localization and neuronal morphology analysis. RESULTS： The majority of facial motor neurons was polygon-shaped, and expressed calretinin and parvalbumin. The calretinin-immunopositive neurons also exhibited parvalbumin immunoreactivity, that is, calretinin and parvalbumin were co-expressed in the same neuron. CONCLUSION： Calretinin and parvalbumin were expressed in facial nucleus neurons, with varied distribution.     

The central versus peripheral antinociceptive effects of μ-opioid receptor agonists in the new model of rat visceral pain

This study describes the antinociceptive effects of μ-opioid agonists, d-Ala(2),N-Me-Phe(4),Gly(5)-ol-enkephalin (DAMGO) and morphine in a model of rat visceral pain in which nociceptive responses were triggered by 2% acetic acid intraperitoneal (i.p.) injections. DAMGO and morphine were administered i.p., to the same site where acetic acid was delivered or intracerebroventricularly (i.c.v.). The antinociceptive actions of i.p. versus i.c.v. administered DAMGO or morphine were evaluated in the late phase of permanent visceral nociceptive responses. Both compounds inhibited the nociceptive responses in a dose-dependent manner and exhibited more potent agonist activity after i.c.v. than i.p. administration. DAMGO and morphine showed comparable ED(50) values after i.p. injections. However, DAMGO was much stronger than morphine after central administration. Co-administration of the peripherally restricted opioid antagonist, naloxone methiodide (NAL-M), significantly attenuated the antinociceptive effects of i.p. DAMGO or morphine. On the other hand, i.c.v. injections of NAL-M partially antagonized the antinociceptive effect of i.p. morphine and failed to affect the antinociceptive action of i.p. DAMGO indicating the partial and pure peripheral antinociceptive effects of morphine and DAMGO, respectively. These results suggest the role of either central or peripheral μ-opioid receptors (MOR) in mediating antinociceptive effects of i.p. μ-opioid agonists in the rat late permanent visceral pain model which closely resembles the clinical situation.     

Proteomic analysis of effects of Chinese herbs to calm the liver and suppress hyperactive yang in a rat migraine model***★

BACKGROUND: The expression of ubiquitin and energy-associated protein can provoke migraines. Studies have suggested that expression is closely linked to “hyperactivity of liver-yang theory” in Traditional Chinese Medicine (TCM), as well as the function of periphery sympathetic nerve medulla. OBJECTIVE: To observe proteomic changes in a rat migraine model with regard to hyperactivity of liver-yang when treated with Chinese herbs to calm the liver and suppress hyperactive yang compound. DESIGN, TIME AND SETTING: A randomized controlled study. This study was performed at the laboratory of Institute of Integrated Traditional Chinese and Western Medicine, Institute of Human Reproduction and Stem Cell Engineering and Key Laboratory of Cancer Proteomics of Ministry of Health, Xiangya Hospital Affiliated to Central South University between September 2006 and July 2007. MATERIALS: Thirty, male, healthy, Sprague-Dawley rats, aged eight weeks, were included in the final analysis. Aconite, to calm the liver and suppress hyperactive yang compound, was provided by the Dispensary of Traditional Chinese medicine, Xiangya Hospital, Central South University. A physiological electronic stimulator, type SDQ-1, was provided by Bengbu Practical Institute of Technology. The left trigeminal ganglion was localized and stimulated for 10 minutes, and the rats were orally administered an aconite concoction to establish a rat migraine model with hyperactivity of liver-yang. METHODS: Rats were randomly divided into a normal control group, model group, and TCM treatment group, with 10 rats in each group. The TCM treatment group was orally treated to calm the liver and suppress the hyperactive yang compound once a day for 28 days. In contrast, the model group and normal group were orally administered the same amount of distilled water once a day for 28 days. MAIN OUTCOME MEASURES: The total proteins from adrenal glands of the three groups were separated by two-dimensional gel electrophoresis (2-DE), and 2-DE images were analyzed by PDQuest 7.0 software. Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS) was used to obtain peptide mass fingerprints of the differential proteins. Databases were searched to identify the proteins. RESULTS: A total of 30 rats were included in the final analysis. Reproducible 2-DE patterns from rat adrenal gland of the three groups were obtained. Compared with the normal group, nine proteins were down-regulated and five proteins were up-regulated in the model group; however, these expressions returned to normal, or near normal levels, in the TCM treatment group. A total of eight differentially expressed proteins were identified: glycogen phosphorylase, ATP synthase D chain, isovaleryl-CoA dehydrogenase, ubiquitin, Annexin-3, Annexin-A1, Peroxirdoxin-II, and heat shock protein-27. CONCLUSION: Liver calming and suppression of the hyperactive yang compound may up-regulate expression of proteins related to energy metabolism and the ubiquitin system. Compounds that are used to treat migraines may contribute to protein functions in the peripheral sympathetic nervous system. Key Words: migraine; liver yang hyperactivity; adrenal glands; pacifying liver     

Proteomic analysis of effects of Chinese herbs to calm the liver and suppress hyperactive yang in a rat migraine model***★

BACKGROUND: The expression of ubiquitin and energy-associated protein can provoke migraines. Studies have suggested that expression is closely linked to “hyperactivity of liver-yang theory” in Traditional Chinese Medicine (TCM), as well as the function of periphery sympathetic nerve medulla. OBJECTIVE: To observe proteomic changes in a rat migraine model with regard to hyperactivity of liver-yang when treated with Chinese herbs to calm the liver and suppress hyperactive yang compound. DESIGN, TIME AND SETTING: A randomized controlled study. This study was performed at the laboratory of Institute of Integrated Traditional Chinese and Western Medicine, Institute of Human Reproduction and Stem Cell Engineering and Key Laboratory of Cancer Proteomics of Ministry of Health, Xiangya Hospital Affiliated to Central South University between September 2006 and July 2007. MATERIALS: Thirty, male, healthy, Sprague-Dawley rats, aged eight weeks, were included in the final analysis. Aconite, to calm the liver and suppress hyperactive yang compound, was provided by the Dispensary of Traditional Chinese medicine, Xiangya Hospital, Central South University. A physiological electronic stimulator, type SDQ-1, was provided by Bengbu Practical Institute of Technology. The left trigeminal ganglion was localized and stimulated for 10 minutes, and the rats were orally administered an aconite concoction to establish a rat migraine model with hyperactivity of liver-yang. METHODS: Rats were randomly divided into a normal control group, model group, and TCM treatment group, with 10 rats in each group. The TCM treatment group was orally treated to calm the liver and suppress the hyperactive yang compound once a day for 28 days. In contrast, the model group and normal group were orally administered the same amount of distilled water once a day for 28 days. MAIN OUTCOME MEASURES: The total proteins from adrenal glands of the three groups were separated by two-dimensional gel electrophoresis (2-DE), and 2-DE images were analyzed by PDQuest 7.0 software. Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS) was used to obtain peptide mass fingerprints of the differential proteins. Databases were searched to identify the proteins. RESULTS: A total of 30 rats were included in the final analysis. Reproducible 2-DE patterns from rat adrenal gland of the three groups were obtained. Compared with the normal group, nine proteins were down-regulated and five proteins were up-regulated in the model group; however, these expressions returned to normal, or near normal levels, in the TCM treatment group. A total of eight differentially expressed proteins were identified: glycogen phosphorylase, ATP synthase D chain, isovaleryl-CoA dehydrogenase, ubiquitin, Annexin-3, Annexin-A1, Peroxirdoxin-II, and heat shock protein-27. CONCLUSION: Liver calming and suppression of the hyperactive yang compound may up-regulate expression of proteins related to energy metabolism and the ubiquitin system. Compounds that are used to treat migraines may contribute to protein functions in the peripheral sympathetic nervous system. Key Words: migraine; liver yang hyperactivity; adrenal glands; pacifying liver     

Interventional effect of laser acupoint radiation on the expression of Nissl body and brain-derived neurotrophic factor in newborn rat models with ischemic/hypoxic cerebral injury

BACKGROUND: Some researches report that He-Ne laser can activate function of erythrocytes and increase content of blood and oxygen via bio-stimulating effect; therefore, it suspects that laser radiation at Baihui and Dazhui can partially increase blood circulation for oxygen-supplying content of brain and improve functional status of neurons. OBJECTIVE: To verify the effects of laser radiation at Baihui and Dazhui on the expression of Nissl body of brain tissue neurons and brain-derived neurotrophic factor (BDNF) in newborn rats with ischemic/hypoxic cerebral injury. DESIGN: Randomized controlled animal study. SETTING: Department of Neurological Histochemistry, Xianning University. MATERIALS: Forty Wistar rats of 7–8 days old, weighing 15–20 g and of both genders, were selected from Wuhan Experimental Animal Center. All the rats were randomly divided into sham operation group (n =8), model group (n =16) and radiation group (n =16). The experimental animals were disposed according to ethical criteria. BDNF kit was provided by Wuhan Boster Bioengineering Co., Ltd. METHODS: The experiment was carried out in the Department of Neurological Histochemistry, Xianning University from April 2005 to October 2006. Rats in the radiation group and model group were performed with ligation of left common carotid artery, recovered at room temperature for 1–6 days, maintained in self-made hypoxic cabin under normal pressure and injected mixture gas (0.05 volume fraction of O2 and 0.92 volume fraction of N2) for 2 hours. In addition, rats in the sham operation group were treated with separation of left common carotid artery but not ligation and hypoxia. Rats in the model group were not given any treatment; while, rats in the radiation group were exposed with He-Ne laser of 63.28 nm in the wave length at Baihui and Dazhui acupoints on the second day after ischemia-hypoxia. The radiation was given for 10 minutes per day and once a day. Ten days were regarded as a course and the rats were exposed for 2 courses in total. At 20 days after routine breeding, left hemisphere tissues of rats in the three groups were collected for staining of Nissl body and immunohistochemistry of BDNF. MAIN OUTCOME MEASURES: Nissl body staining in left hemisphere tissue and expression of immune positive cells of BDNF. RESULTS: All 40 rats were involved in the final analysis. ① Nissl body staining: Neuronal cytoplasm of brain tissue was full of blue granule Nissl bodies in the sham operation group; while, Nissl body in neuronal cytoplasm in the model group was stained slightly and had a certain degree of degeneration; meanwhile, there were a lot of blank area in ischemic region. Nissl body in neuron cytoplasm was gradually recovered in the radiation group and relieved as compared with that in the model group. ② Positive cells of BDNF: Number of immune positive cells of BDNF which were ligated in lateral cerebral hemisphere of rats in the model group was higher than that in the sham operation group (P < 0.05); while, BDNF expression in the radiation group was increased as compared with that in the model group (P < 0.05). CONCLUSION: After laser acupoint radiation, Nissl body is increased and BDNF expression is also increased. This suggests that laser acupoint radiation has neuroprotective effect on brain tissue after ischemia-hypoxia injury.     

Protection of Cactus Polysaccharide against H2O2-induced damage in the rat cerebral cortex and hippocampus *☆ Differences in time of administration

BACKGROUND： Pharmacological research has shown that cactus polysaccharide （CP） has anti-oxidant, anti-inflammatory, anti-tumor, anti-aging, and immune-stimulating activities. It may also provide protective effects against oxidative stress injuries in the rat brain. OBJECTIVE： To validate the effects of CP on H2O2-induced oxidative stress injuries in the rat cerebral cortex and hippocampal slices 30 minutes prior to injury, as well as 30 minutes and 2.5 hours after injury. DESIGN： A randomized controlled experiment. SETTINGS： Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology; Department of Pharmacology, College of Medical Science, Yangtze University. MATERIALS： A total of 50 male Sprague Dawley （SD） rats, normal grade and weighing 200 300 g, were provided by the Laboratory Animal Center of Tongji Medical College, Huazhong University of Science and Technology. The protocol was performed in accordance with ethical guidelines for the use and care of animals. Cactus polysaccharide, a dried needle crystal, was extracted from Opuntia milpa alta at the Chemistry and Environment Engineering School of Yangtze University. The following chemicals and instruments were used： 2,3,5-triphenyl tetrazolium chloride （Sigma, St Louis, Missouri, USA）; lactate dehydrogenase （LDH）, superoxide dismutase （SOD）, glutathione （GSH）, and total antioxidant competence （T-AOC） assays （Jiancheng-Bioeng Institute, Nanjing）; McIllwain tissue chopper （Mickle Laboratory Engineering, USA）; and ELISA reader and Magellan software （TECAN, Austria）. METHODS： This experiment was performed at the Department of Pharmacology, Medical College of Yangtze University, between March and June 2006. All rats were sacrificed after anesthesia. The cerebral cortex and hippocampus were dissected. Several cerebral cortex and hippocampus slices were selected as controls, while other sections were co-incubated with H2O2 for 30 minutes to induce an oxidative stress injury.     

Mechanisms of antinociceptive ettects of ouabain in combination with neostigmine in the rat

BACKGROUND:It has been previously shown that intrathecal administration of either ouabain or neostigmine can produce antinociceptive effeets.Moreover,ouabain and neostigmine are differently associated with acetylcholine.OBJECTIVE:It has been hypothesized that intrathecal administration of ouabain,in combination with neostigmine,can produce antinociceptive synergistic effects.Atropine,as a competitive antagomst,was pre-injected to verify the mechanisms of action.DESIGN,TIME AND SETTING:This study was a randomized,controlled,animal experiment,performed at the State Key Laboratory of Oneology in Southern China between May 2006 and Febrnary 2007.MATERIALS:A total of 102 healthy.adult,Sprague Dawley rats were included.Ouabain and neostigrnine (Sigma,USA),as well as atropine(Tanabe Seiyaku,Japan),were also used.METHODS:Varied doses of ouabain,neostigmine,and a combination of the two were intrathecally injected into rats.Six rats were allotted for each dose group.Intrathecal pretreatment with atropine was tested 10 minutes prior to intrathecal administration of neostjgmine or the combination of ouabain and neostigmine.MAIN OUTCOME MEASURES:Tail-flick tests were performed to measure tail-flick latency(seconds)prior to and after administration.The response in the tail-flick test was expressed as the percentage of maximum possible effect(% MPE),where% MPE=[tail-flick latency after administration(seconds)-mean baseline value for tail-flick latency]/[10 seconds-the mean baseline value for tail-flick latency(seconds)]×100%.RESULTS:Rat spinal intrathecal administration of either ouabain or neostigminc alone produced antinociceptive effects in a dose-dependent manner.Intrathecally administration of neostigmine(0.05,0.1,0.3 μg)in combination with ouabain(1 μg)produced enhanced antinociceptive effects,with a % MPE of 29%,78%,and 95%,respectively(P<0.05).Intrathecally administration of 0.3 μg neostigmine(% MPE:45%),in combination with 1 μg ouabain(% MPE:27%)produced potent antinociceptive effects(% MPE:95%).Intrathecally pre-injected atropine antagonized the antinociceptive effects of neostigmine(3 μg),ora combination of ouabain(1 μg)and neostigmine(0.3 μg)(P<0.01).CONCLUSION:Rat spinal intrathecal administration of either ouabain or neostigmine alone produeed dose-dependent antinociceptive effects.Ouabain enhanced the antinociceptive effects of neostigmine.Atropine antagonized the antinociceptive effects of neostigmine or the combination of ouabain and neostigmine.This occurs possibly due to the fact that atropine is a competitive antagonist of the muscarinic acetylcholine receptors.     

Administrations of thalidomide into the rostral ventromedial medulla produce antinociceptive effects in a rat model of postoperative pain

The rostral ventromedial medulla (RVM) is highly involved in pain signal transmissions. Previous studies have shown that thalidomide is anti‐nociceptive. Thus, we evaluated the neurobiological mechanisms of thalidomide in the RVM in the regulation of postoperative pain. We used a rat model of postoperative pain to investigate the effects of intra‐RVM thalidomide treatments on postoperative pain, and evaluate the role of cannabinoid receptors in the effects of intra‐RVM thalidomide treatments on GABAergic neurotransmission in the RVM neurons. We found intra‐RVM thalidomide treatments reduced incisional surgery induced mechanical allodynia. This phenomenon was associated with attenuation of the frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs) and spontaneous IPSCs (sIPSCs) in RVM neurons. Furthermore, applications of WIN 55,212‐3 mesylate, a non‐selective cannabinoid receptor antagonist reversed the effects of repeated thalidomide treatment on the frequency but not the amplitude of mIPSCs and sIPSCs. Finally, we found that repeated thalidomide treatment robustly enhanced CB2 receptor expression, but slightly reduced CB1 receptor expression, in the RVM. These results suggested that the antinociceptive effects of thalidomide in the RVM likely involve the attenuation of GABA release, which are critically regulated by cannabinoid receptors.     

Effects of chronic ouabain treatment on [H]ouabain binding sites and electrogenic component of membrane potential in cultured rat myotubes

The effects of incubation of cultured rat skeletal myotubes in ouabain were studied on the number of [³H]ouabain binding sites and electrogenic component of membrane potential. Ouabain treatment for 2–6 days increased the number of binding sites, resting membrane potential (E_m) and the ouabain-sensitive component of E_m in the muscle cells. The findings strengthen the idea that Na, K-ATPase has an important role in regulation of E_m in cultured skeletal muscle and suggest that Na-K pump inhibition during development may be a regulatory mechanism for cellular synthesis of Na, K-ATPase.     

Adenosine preconditions against ouabain but not against glutamate on CA1‐evoked potentials in rat hippocampal slices

Hypoxic and ischaemic brain damage are believed to involve excessive release of glutamate, and recent work shows that glutamate‐induced damage in brain slices can be reduced by preconditioning with hypoxia or glutamate itself. Because adenosine is a powerful preconditioning agent, we have investigated whether adenosine could precondition against glutamate in vitro. In rat hippocampal slices, glutamate depolarization reduced the amplitudes of antidromic‐ and orthodromic‐evoked potentials, with only partial recovery. Applying adenosine before these insults failed to increase that recovery. Ouabain also produced depolarization with partial reversibility, but adenosine pretreatment increased the extent of recovery. The preconditioning effect of adenosine on ouabain responses was prevented by blocking receptors for N‐methyl‐d ‐aspartate (NMDA), but not receptors for kainate or α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA), and was blocked by inhibiting nitric oxide synthase. Preconditioning was also abolished by the ATP‐dependent potassium channel blockers, glibenclamide (cytoplasmic) or 5‐hydroxydecanoate (mitochondrial). We conclude that adenosine does not precondition against glutamate in hippocampal slices, but that it does precondition against ouabain with a pharmacology similar to studies in vivo. Ischaemic neuronal damage is a complex of many factors, and because adenosine can precondition against ischaemic neuronal damage, its failure to protect against glutamate highlights limitations of using glutamate alone as a model for ischaemia. Because damage following ischaemia, trauma or excitotoxicity also involves reduced Na⁺,K⁺‐ATPase activity, and adenosine can precondition against ouabain, we propose that ouabain‐induced damage represents an additional or alternative model for the contribution to cell damage of Na⁺,K⁺‐ATPase loss, this being more relevant to the mechanisms of preconditioning.     

Dissociation of antinociceptive and motor effects of supraspinal opioid agonists in the rat

This study compared the antinociceptive and motor effects produced by intracerebroventricular administration of selective mu- (DAMGO) and delta- (DPDPE) opioid receptor agonists in the rat. Changes in nociceptive thresholds were measured using the Randall-Selitto paw-withdrawal test and changes in motor coordination were evaluated using the rotarod treadmill test. Both DAMGO and DPDPE produced statistically significant, dose-dependent increases in mechanical nociceptive thresholds compared to vehicle controls. However, in the motor coordination studies, neither opioid agonist produced statistically significant changes in rotarod performance scores. The dissociation of antinociceptive and motor effects at this supraspinal site differs from the strong association between antinociceptive and motor effects produced by intrathecal administration of the same opioid agonists.     

Morphine effects on spontaneous, nociceptive, antinociceptive and sensory evoked responses of parafasciculus thalami units in morphine naive and morphine dependent rats

Zinc deficiency during the first 3 postnatal weeks retarded the maturation of Purkinje cells. The dendrites of the Purkinje cells of 21-day-old zinc-deficient (ZD) rats were reduced in size and had fewer branches. Somatic processes were found in 24% of the Purkinje cells of ZD animals. Only 3% of the Purkinje cells of normal animals had somatic processes. A basal polysomal mass in the Purkinje cells of 21-day-old ZD rats indicated that zinc deficiency impaired the cytoplasmic maturation of Purkinje cells. The development of the glial envestment of the dendrites and the maturation of climbing fibers also were retarded. Pair-fed controls were studied to control for the effects of inanition in the ZD dams. In the pups of pair-fed dams, undernutrition slightly impaired the growth of the dendrites but produced few qualitative changes in the maturation of the soma and climbing fibers. Somatic processes were found on 10% of the Purkinje cells of pair-fed animals. Thus, the findings in the ZD animals were not only caused by the decreased maternal food consumption but by zinc deficiency. The retarded maturation of Purkinje cells was related to the altered metabolism of Purkinje cells and to effects secondary to decreased numbers of parallel fibers.     

Comparison of the effects of substance P, neurokinin A, physalaemin and eledoisin in facilitating a nociceptive reflex in the rat

The postulated existence of different types of tachykinin receptor in the spinal cord provided the basis for the present study; substance P, neurokinin A, eledoisin and physalaemin were administered intrathecally in the awake, restrained rat to compare their effects on reaction time in the tail-flick test. Each peptide was delivered via a chronically implanted subdural catheter to the lower lumbar vertebral level of the spinal cord. Intrathecal administration of 10 micrograms of substance P (6.5 nmol), eledoisin (8.0 nmol) or physalaemin (7.9 nmol) decreased the reaction time, respectively, to 22.5, 24.3 and 20.8% of the mean preadministration control values at 1 min after injection; similar administration of 6.5 nmol of neurokinin A produced a smaller decrease in reaction time, to only 49.5% of preadministration values. These effects were transient, the reaction times returning to preadministration values within 5 min. Each peptide also produced an initial vocalization followed by increased restlessness. Analysis of the dose-response curves indicated that the rank order of potency of the fitted curves for these peptides was physalaemin greater than or equal to substance P greater than or equal to eledoisin greater than neurokinin A. The results suggest that the receptor involved in facilitation of the tail-flick reflex resembles a substance P receptor rather than a receptor for one of the other endogenous neurokinins and that this receptor may bear some resemblance to the SP-P type postulated to exist in peripheral tissues.     

Localization of the central antinociceptive effects of diclofenac in the rat

The ethacrynic acid-induced writhing response (WR) in the rat was studied after microinjections of diclofenac 0.1 ng−1 μg/0.5 μl (0.32 pmol to 3.2 nmol) into several brain regions involved in control of nociceptive behavior. The WR was inhibited after injections into the periaqueductal grey matter (PAG), ventromedial thalamus (VM), medial preoptic area (MPA) and the nucleus raphe magnus (NRM). Morphine 50 ng/0.5 μl (0.16 nmol) was used as a positive control and vehicle injections were performed as negative reference. After diclofenac, there was a dose-dependent reduction of the WR with a threshold dose of approximately 1–10 ng in all brain areas studied except the nucleus reticularis paragigantocellularis interna (NRPGi). Naloxone 50 ng/0.5 μl (0.15 nmol) administered into the same site 30 min after diclofenac injection, antagonized the diclofenac-induced inhibition of the WR almost completely in PAG and VM. Previous results demonstrate a central, naloxone-reversible component in the analgetic action of diclofenac. A qualitatively similar, centrally induced inhibition of the WR may be elicited after injections into PAG, VM and NRM. Thus, in addition to its peripheral mechanism of action, the non-steroidal anti-inflanmatory drug, diclofenac, has a central mechanism of action directly or indirectly involves a central opioid component.