Antinociceptive effects of eugenol evaluated in a monoiodoacetate-induced osteoarthritis rat model.

The aim of the present study was to evaluate whether eugenol, the main constituent of clove oil, has the capacity to provide analgesia in the monoiodoacetate-induced rat model of osteoarthritis. Animals (n = 6/group) received either eugenol (20 or 40 mg/kg) or a vehicle by gavage. Daily administrations were initiated 2 days post osteoarthritis induction and continued for the duration of the study (4 weeks). Gait analysis was performed using the CatWalk method and secondary mechanical allodynia was assessed with von Frey filaments. Selected spinal cord peptides (substance P, calcitonin gene-related peptide and dynorphin) were quantified by mass spectrometry. Significant changes were identified in dynamic gait parameters (swing speed, swing phase duration and duty cycle) of the affected limb following 40 mg/kg eugenol treatment compared with the vehicle (p < 0.05). Von Frey results revealed significant differences between the 40 mg/kg treatment and the vehicle group during the first and the third week of the study (p < 0.02). Spinal pain-related peptide analysis revealed a decreased content of substance P and CGRP accompanied by an increase of dynorphin in animals treated with 40 mg/kg eugenol. These results suggest a therapeutic potential of eugenol to alleviate osteoarthritis-related pain.

Dynorphin immunoreactive fibers contact GnRH neurons in the human hypothalamus.

Dynorphin, an endogenous opioid peptide, mediates progesterone-negative feedback on gonadotropin-releasing hormone (GnRH) neurons in other species. The role of dynorphin in humans is unclear. The objective of this study was to determine if dynorphin fibers have close contacts with GnRH neurons in humans. Dual-label immunocytochemistry was performed on postmortem human hypothalamic tissue. The majority of GnRH neurons, 87.5%, had close contacts with dynorphin fibers and multiple close contacts were common, 62.5%. There were no regional differences between the hypothalamus and preoptic area in the distribution of close contacts. More close contacts were identified on the GnRH dendrites compared to the cell bodies (P < .001), but this difference was not significant when corrected for length. In conclusion, dynorphin fibers form close contacts with GnRH neurons in humans. This neuroanatomical evidence may suggest that dynorphin has effects on GnRH regulation in humans as seen in other species.

Only arginine vasopressin, not oxytocin and endogenous opiate peptides, in hypothalamic paraventricular nucleus play a role in acupuncture analgesia in the rat.

Our previous study proved that hypothalamic paraventricular nucleus (PVH) plays an important role in acupuncture analgesia. The effect of acupuncture on the concentrations of arginine vasopressin (AVP), oxytocin (OXT), leucine-enkephaline (L-Ek), beta-endorphin (beta-Ep) and dynorphinA(1-13) (DynA(1-13)) was investigated in rat PVH. Electrical acupuncture of "Zusanli" points (St. 36) 30 min increased the AVP, not OXT, L-Ek, beta-Ep and DynA(1-13) concentrations in PVH tissue using micropunch and radioimmunoassay, which showed a negative relationship between the pain threshold and AVP concentrations in PVH tissue. Electrical acupuncture could elevate the AVP concentrations in PVH perfuse liquid during acupuncture, and then reduce the AVP concentrations in PVH perfuse liquid after acupuncture. But no change in OXT, L-Ek, beta-Ep and DynA(1-13) concentrations was detected in PVH perfuse liquid. Electrical acupuncture decreased the number of AVP, not OXT, L-Ek, beta-Ep and DynA(1-13) immunoreactive cells in PVH using immunocytochemistry. The results suggested that only AVP, not OXT and endogenous opiate peptides in PVH involved acupuncture analgesia in the rat.

Reduced kappa-opioid activity in a rat model of cholestasis.

Increased endogenous opioid activity has been implicated in cholestatic pruritus. In the present study, we have further defined the involvement of opioids in cholestasis. Rats underwent either bile duct ligation or a sham operation. Five days after surgery, brains were removed and agonist-stimulated [35S]GTPgammaS binding was measured in ten brain regions. Serum endomorphin-2, leu-enkephalin and dynorphin A levels were measured using ELISA on day five. Microdialysis to the dorsal hypothalamic area was conducted in the same animal before and after cholestasis. Dialysate endomorphin-1, leu-enkephalin and dynorphin A levels also were measured. Delta- and kappa-stimulated binding was significantly decreased in cholestasic animals compared to controls in the dorsal hypothalamic area. The serum dynorphin A level was lower in the cholestasic group than in controls (2.56+/-0.09 and 3.29+/-0.22 ng/ml, respectively, P<0.01). We propose that pruritus in cholestasis may result from an impaired balance between mu- and kappa-opioid systems.

Colocalization of progesterone receptors in parvicellular dynorphin neurons of the ovine preoptic area and hypothalamus.

Recent evidence suggests that the dynorphin-kappa receptor opioid system acts to mediate the inhibitory effect of progesterone (P) on GnRH pulse frequency during the luteal phase of the ovine estrous cycle. It is known that progesterone receptors (PRs) are required for the actions of P on GnRH secretion. Therefore, if P acts directly on dynorphin (DYN) neurons, then these neurons should contain PRs. To test this hypothesis, we used a dual-label immunoperoxidase procedure to visualize PRs and DYN in the preoptic area (POA) and hypothalamus of ovary-intact ewes killed during the luteal phase of the estrous cycle. The PR was colocalized in more than 90% of parvicellular DYN neurons in the POA, anterior hypothalamus (AHA), and arcuate nucleus (ARC). By contrast, none of magnocellular DYN cells of the paraventricular and supraoptic nuclei coexpressed immunoreactive PRs. The high percentage of colocalization of PRs in parvicellular DYN cells of the POA, AHA, and ARC suggests that these cells are prime targets of P. In addition, DYN cells in the ARC, but not the POA or AHA, were found to receive synaptic inputs from DYN-positive axon terminals. This observation raises the possibility that an ultrashort feedback loop controls the release of DYN from ARC neurons.

Altered opioid-mediated control of the spinal release of dynorphin and met-enkephalin in polyarthritic rats.

Previous studies showed that spinal opioidergic neurotransmission is markedly altered in the polyarthritic rat, a model of chronic inflammatory pain. Present investigations aimed at assessing possible changes in opioid-mediated control of the spinal outflow of met-enkephalin (ME) and dynorphin (DYN) in these animals. Intrathecal (i.t.) perfusion under halothane anesthesia showed that polyarthritis was associated with both a 40% decrease in the spinal outflow of ME-like material (MELM) and a 90% increase in that of DYNLM. Local treatment with the mu-opioid agonist DAGO (10 microM i.t.) inhibited equally (-30%) the MELM outflow in polyarthritic and control rats, whereas the delta agonist DTLET (10 microM i.t.) also reduced the peptide outflow in controls (-27%) but enhanced it in polyarthritic animals (+56%). On the other hand, both DAGO (10 microM i.t.) and DTLET (10 microM i.t.) decreased (-40 and -49%) DYNLM outflow in polyarthritic rats, but were inactive in controls. Finally, neither MELM outflow nor that of DYNLM were affected by the kappa-agonist U50488H (10 microM i.t.) in both groups of rats. In all cases, the changes due to active agonists could be prevented by specific antagonists which were inactive on their own except the kappa antagonist nor-binaltorphimine (10 microM i.t.) that decreased (-38%) DYNLM outflow in polyarthritic rats. These data indicate that functional changes in spinal opioid receptors may promote enkephalinergic neurotransmission and reduce dynorphinergic neurotransmission in polyarthritic rats, thereby contributing to the analgesic efficacy of opioids in inflammatory pain.

Differential effects of intrastriatally infused fully and endcap phosphorothioate antisense oligonucleotides on morphology, histochemistry and prodynorphin expression in rat brain.

In the present study, we investigated the selectivity and specificity associated with continuous intrastriatal treatment with antisense oligonucleotides. Rats were given intrastriatal infusions for 72 h with phosphodiester, and fully and endcap phosphorothioated oligonucleotide probes complementary to prodynorphin mRNA. Dynorphin (Dyn) peptide levels were measured by radioimmunoassay. The integrity of three other striatal transmitter systems, the neuropeptide Y (NPY)-ergic interneurons, the cholinergic interneurons and the dopaminergic afferent innervation, was assessed histochemically. The gross morphology of the striatum and the distribution of fluorescently labelled antisense probes were also investigated. Brains infused with phosphodiester probes had tissue Dyn levels not different from control. They also showed little or no change in staining for NPY, acetylcholinesterase (AChE) and tyrosine hydroxylase (TH) and essentially normal striatal gross morphology. In contrast, brains treated with fully phosphorothioated oligonucleotides showed significant decreases in striatal Dyn levels but also severe tissue damage accompanied by massive cell infiltration and decreases in immunoreactivities for the striatal neurochemical markers. Fluorescently labelled phosphorothioate probes were observed widely in the striatum and adjacent structures and, presumably retrogradely transported, in the dopamine cell bodies in the substantia nigra, also revealing the presence of abnormal cellular structures within the striatum. By comparison, endcap probes significantly reduced striatal Dyn levels and showed good tissue penetration without inducing major changes in tissue morphology or histochemistry of non-dynorphinergic systems, except for cell infiltration. The deleterious tissue effects of fully phosphorothioated oligonucleotides and the ineffectiveness of phosphodiester oligonucleotides in inhibiting protein synthesis suggest that, of the probes examined in this study, endcap oligonucleotides are the most useful for in vivo studies in the central nervous system.

Neonatal handling in rats induces long-term effects on dynorphin peptides.

The effects of neonatal handling on the opioid dynorphin peptides in the brain and pituitary gland of Sprague-Dawley rats were investigated. Ten weeks after the neonatal handling, handled rats had higher tissue levels of dynorphin A and B in the hypothalamus, pituitary gland and striatum and slightly higher dynorphin B levels in the hippocampus, medulla oblongata and midbrain as compared with non-handled controls. The results indicate a persistent upregulation of the dynorphin system in certain brain areas after neonatal handling, which could contribute to the behavioural changes in these rats observed later in life. Observation in the open field and the elevated plus-maze tests confirmed behavioural effects of neonatal handling, i.e. showing that handled rats exhibit attenuated fearfulness in novel environments as compared with non-handled rats.

Occurrence of secretogranin II in the prolactin-immunoreactive neurons of the rat lateral hypothalamus: an in situ hybridization and immunocytochemical study.

The occurrence of secretogranin II in a neuron population of the rat lateral hypothalamus specifically detected by an anti-serum to ovine prolactin was examined. As this population was previously reported to synthesize dynorphin, the distribution of neurons recognized by ovine prolactin-, dynorphin B- and secretogranin II anti-sera was investigated on adjacent sections of hypothalami. The prolactin immunoreactive neurons were the only cells in the lateral hypothalamus to be stained by secretogranin II anti-serum. Moreover, coupling immunocytochemical detection and in situ hybridization with an oligonucleotide probe complementary to secretogranin II mRNA showed that these neurons expressed the secretogranin II gene. These new findings should help to study the physiological role of the prolactin immunoreactive neurons of the lateral hypothalamus.

Brain cholecystokinin octopeptide (CCK-8) concentrations: effect of tryptophan and other serotonergic agents.

The effects of 1-week drug treatment on the brain contents of neuropeptides were investigated. The cholecystokinin (CCK) concentrations in the hypothalamus were significantly decreased by tryptophan treatment but not by imipramine and cyproheptadine, which changed the serotonergic function. Proglumide, the CCK antagonist, induced in the hypothalamic and hippocampal-striatal areas an increase in CCK concentration, which was not reversed in the presence of tryptophan. Dynorphin and substance P(SP) concentrations were also modified by proglumide treatment.

Tachykinin systems in the spinal cord and basal ganglia: influence of neonatal capsaicin treatment or dopaminergic intervention on levels of peptides, substance P-encoding mRNAs, and substance P receptor mRNA.

The aim of the study was to test whether the synthesis of substance P (SP) and that of its receptor (also known as NK1 receptor) are coordinately regulated after chronic pharmacologic intervention in two neural systems, the spinal cord and basal ganglia. In one set of experiments, capsaicin was administered subcutaneously during the early postnatal period (day 3 after birth) to induce degeneration of afferent sensory neurons in the spinal cord. In the other set of experiments, interruption of dopaminergic transmission was achieved by two methods: (a) The neurotoxin 6-hydroxydopamine was used to denervate dopaminergic neurons during the early postnatal period, and (b) haloperidol was used in adult animals to block dopaminergic transmission by receptor blockade. The spinal cord, striatum, or both were used for the quantification of tachykinin [SP and neurokinin A (NKA)] and opioid peptides [[Met5]-enkephalin (ME) and dynorphin A (1-8) (DYN)] by radioimmunoassays. The abundance of total SP-encoding preprotachykinin (PPT) mRNA and SP receptor (SPR) mRNA in spinal cord (C5 to T1 segments), striatum, or microdissected substantia nigra was determined by northern blot or solution hybridization analysis. Amines and their acid metabolites were quantified by HPLC. Capsaicin administration (subcutaneously) during the early postnatal period increased latency in a hot-plate test, decreased SP and NKA levels, increased levels of PPT mRNAs, and did not affect SPR mRNA levels in the spinal cord. Intraspinal SP systems may attempt to compensate for the loss of afferent SP input, whereas spinal cord receptor mRNA levels do not appear to be altered.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity of immunoreactive dynorphin B-like material in human, rat, rabbit and guinea-pig heart.

Immunoreactive dynorphin B-like material (ir-dyn B) was detected in acetic acid extracts of human atrial specimens and of rat, rabbit and guinea-pig atria and ventricles by a validated radioimmunoassay. Levels were high in rabbit atrium (66.76 +/- 7.04 pmol/g) but lower and superimposable in human and rat atria (28.18 +/- 3.20 and 30.22 +/- 2.45 pmol/g, respectively). Gel permeation chromatography revealed ir-dyn B eluting close to column exclusion and in forms with an apparently higher molecular weight than authentic dyn B in human and rat samples. In contrast, almost all the immunoreactivity from rabbit and guinea-pig acetic extracts eluted as a single peak in the region of standard dyn B. Reverse-phase high performance liquid chromatography of the pooled gel chromatography fractions of this peak showed up a molecular form with the same retention time as authentic dyn B and a second minor peak of unknown immunoreactive material eluting three fractions earlier. Digestion with carboxypeptidase B excluded the hypothesis that this latter could be dyn B-Arg14. Therefore, it might be a metabolite of endogenous dyn B recognized by the antibody used in this study.

The immunostaining for the hypothalamic vasoactive intestinal peptide, but not for beta-endorphin, dynorphin-A or methionine-enkephalin, is affected by the glucocorticoid milieu in the rat: correlation with the prolactin secretion.

Effects of the glucocorticoid milieu on the basal and ether stress-induced prolactin (PRL) release and on the immunostaining for hypothalamic vasoactive intestinal peptide (VIP), beta-endorphin (beta-EP), dynorphin-A (DYN-A) and methionine-enkephalin (Met-ENK), were examined in separate groups of male rats. After colchicine treatment in intact rats, VIP-containing cell bodies were observed only in the suprachiasmatic nucleus (SCN). Adrenalectomy (ADX), performed 7 days previously, resulted in the additional appearance of VIP-immunoreactive neurons in the parvocellular subdivision of the paraventricular nucleus (PVN), as well as in significantly higher basal and stressed PRL levels than intact values. Treatment of intact rats with a high dose (500 micrograms/kg body weight (s.c.) daily for 7 days) of dexamethasone (DEX), but not with a low dose (50 micrograms/kg) of DEX, significantly reduced both the basal and stressed PRL release. Administration of either the low or high dose of DEX to ADX rats prevented the appearance of the PVN-VIP neurons. In addition, the ADX-induced high basal and stressed PRL levels were restored to intact values by the low dose of DEX, and completely suppressed by the high dose of DEX. The staining of SCN-VIP-, beta-EP-, DYN-A or Met-ENK neurons was not affected by any treatment employed in this study. These results suggest that the appearance of PVN-VIP immunostaining in ADX rats may, at least in part, be responsible for the enhanced PRL secretion observed in this group. However, SCN-VIP-, beta-EP-, DYN-A- or Met-ENK neurons do not seem to play a pivotal role in the glucocorticoid regulation of PRL secretion.

A low hippocampal dynorphin A (1-8) immunoreactivity in spontaneously hypertensive rats.

Dynorphin A (1-8)-like immunoreactivity (DN-LI A(1-8] was determined by radioimmunoassay in the brains of age matched spontaneously hypertensive rats (SHR) and two normotensive control groups consisting of Wistar-Kyoto (WKY) and Sprague-Dawley (SD) strain rats. A significantly lower DN-LI A(1-8) was found in the hippocampus and hypothalamus of the SHR compared with the WKY groups. DN-LI A(1-8) was 24% of control WKY levels in hippocampus and 79% of that in WKY hypothalamus at 16 weeks. Similar lower levels of DN-LI A(1-8) were also observed in SHR at 4, 8, and 12 weeks during the development of hypertension when compared with both WKY and SD groups. We failed to find significant differences in brain stem DN levels between the groups. The relationship between the low hippocampal dynorphin levels in SHR and the hypertension is problematical because the differences were present before (4 wks), during (8 and 12 wks) and after (16 wks) its development.

Gonadal steroids and anterior lobe dynorphin in the male rat.

Immunoreactive (ir)-dynorphin levels were measured, and the species characterized by high performance liquid chromatography (HPLC), in the pituitary and hypothalamus of intact and castrate male rats. On HPLC, ir-dynorphin co-eluted with authentic dynorphin A 1-8, dynorphin A 1-17 and dynorphin 1-32 in the hypothalamus and intermediate lobe; in two different reversed phase (RP)-HPLC systems, anterior lobe ir-dynorphin co-eluted uniquely with dynorphin 32 (4K dynorphin). Anterior lobe levels of total ir-dynorphin were significantly lowered 7 days after castration, while HPLC profiles in all tissues remained unchanged. The change in anterior pituitary ir-dynorphin levels was reversed in a dose-related manner by dihydrotestosterone (15-500 micrograms/100 g b. wt/day); estradiol benzoate (3 micrograms/100 g/day) was without effect. The changes on castration and androgen administration suggest that gonadal steroids play a role in the regulation of dynorphin, as well as gonadotrophins and prolactin, within the anterior pituitary gland.

Immunocytochemical demonstration of dynorphin(PH-8P)-like immunoreactive elements in the human hypothalamus.

PH-8P (dynorphin[1-8])-like immunoreactive neuronal perikarya, processes, and terminals located within the human hypothalamus were investigated by the avidin-biotin peroxidase complex (ABC) immunocytochemical procedure. Immunopositive neurons were distributed throughout the hypothalamus. The distributional pattern was found to be similar to that in other mammalian species by the use of antisera against dynorphin. A large number of immunoreactive neuronal perikarya were detected in the supraoptic nucleus (SON) and the magnocellular portion of the paraventricular nucleus (PVN). Their processes appeared to project to the posterior pituitary via the internal layer of the median eminence and their distribution seemed to be less dense than in other mammalian species. PH-8P and vasopressin were colocalized in the neuronal perikarya in the human SON unlike the colocalization of these peptides in the rat SON and PVN. There were a few immunoreactive terminals in the external layer of the median eminence; their immunoreactive substances may be released into the portal veins to act on anterior pituitary cells. In addition, PH-8P-like immunoreactive neurons in the human hypothalamus may project to the extrahypothalamic area.

Immunocytochemical identification of long ascending, peptidergic lumbar spinal neurons terminating in either the medial or lateral thalamus in the rat.

The peptidergic content of rat spinothalamic tract neurons was investigated by combining the retrograde transport of the fluorescent dye Fluoro-gold with immunocytochemistry for enkephalin, dynorphin or vasoactive intestinal polypeptide. Evidence is presented for the existence of enkephalin and dynorphin in a subpopulation of spinothalamic neurons terminating in the medial thalamus and vasoactive intestinal polypeptide in a subpopulation terminating primarily in the lateral thalamus.

Immunohistochemical distribution of cholecystokinin, dynorphin A and Met-enkephalin neurons in sheep hypothalamus.

The distribution of cholecystokinin (CCK)-, Met-enkephalin (M-ENK)- and dynorphin (DYN)-like immunoreactive perikarya were examined in the sheep hypothalamus using the peroxidase-anti-peroxidase technique. CCK- and DYN-containing neurons were found primarily in the suprachiasmatic nucleus (SCH) and supraoptic nucleus (SO). No CCK- or DYN-containing neurons were found in the paraventricular nucleus (PVN). M-ENK-containing neurons were found mainly in the PVN of the hypothalamus. In addition, M-ENK neurons were found in the dorsomedial (DMH), lateral (LH), anterior (AH) and periventricular hypothalamic areas. The distribution of these neuropeptides may provide a basis for understanding differences in responsiveness to centrally administered peptides.

Deprivation of REM sleep in the rat and the opioid peptides beta-endorphin and dynorphin.

Effects of 'rapid eye movement' sleep deprivation (REMd) on two opioid peptides, beta-endorphin and dynorphin, were studied in rats. Both peptides were measured by radioimmunoassay techniques. The level of beta-endorphin was estimated in the hypothalamus, in the anterior lobe of the pituitary and in the blood. The amount of dynorphin was estimated in the hypothalamus. REMd was induced for 72 h and achieved by two different methods, the platform technique and the pendulum technique. Three control groups were additionally run. As a consequence of REMd, an increase in beta-endorphin level was discovered in the blood plasma, while a small decrease was found in the hypothalamus. No changes could be detected for beta-endorphin levels in the pituitary or for hypothalamic dynorphin concentration. The deprivation effects are interpreted as belonging to a group of changes, all of which point to a small increase in tonic arousal as a result of REMd.