dynorphins and Brain-Ischemia

The goals of the present study were to assess the efficacy and safety of nalmefene (Cervene) in patients with acute (< or =6 hours) ischemic stroke and to investigate the safety of combined recombinant tissue plasminogen activator and nalmefene in a separate subset of patients. Nalmefene, an opioid antagonist with relative kappa receptor selectivity, has shown neuroprotective effects in multiple experimental central nervous system injury and ischemic models. Results from an earlier phase II study in patients with acute ischemic stroke suggested that nalmefene was safe and tolerable and may be effective for patients <70 years old.. This investigation was a phase III, placebo-controlled, double-blind, randomized study of a 24-hour infusion of nalmefene. Patients with acute ischemic stroke who had an onset of symptoms within 6 hours and a baseline score of > or =4 on the NIH Stroke Scale were randomized to receive either 60 mg nalmefene administered as a 10-mg bolus over 15 minutes and then a 50-mg infusion over 23.75 hours or placebo. The primary efficacy outcome was the proportion of patients achieving a score of > or =60 on the Barthel Index and a rating of "moderate disability" or better on the Glasgow Outcome Scale at 12 weeks. Assessments were performed at baseline (predose), hours 12 and 24, days 2 and 7, and week 12.. A total of 368 patients were randomized at 42 centers, including 32 patients treated with recombinant tissue plasminogen activator and study drug. Nalmefene was well tolerated. Overall, there was no significant difference in 3-month functional outcome for nalmefene treatment compared with placebo on any of the planned analyses. A prospective secondary analysis also failed to find a treatment effect in patients <70 years old.. Although nalmefene appears to be safe and well tolerated, this study failed to find any treatment benefit in stroke patients treated within 6 hours.

Topics: Acute Disease; Aged; Aged, 80 and over; Brain Ischemia; Dose-Response Relationship, Drug; Double-Blind Method; Dynorphins; Female; Humans; Male; Middle Aged; Naltrexone; Narcotic Antagonists; Nausea; Neuroprotective Agents; Receptors, Opioid, kappa; Risk Factors; Survival Analysis; Treatment Failure

Using a focal cerebral ischemia model in rats, brain ischemia-induced changes in expression levels of mRNA and protein, and activities of proprotein convertase 2 (PC2) in the cortex were examined. In situ hybridization analyses revealed a transient upregulation of the mRNA level for PC2 at an early reperfusion hour, at which the level of PC2 protein was also high as determined by immunocytochemistry and western blotting. When enzymatic activities of PC2 were analyzed using a synthetic substrate, a significant decrease was observed at early reperfusion hours at which levels of PC2 protein were still high. Also decreased at these reperfusion hours were tissue levels of dynorphin-A(1-8) (DYN-A(1-8)), a PC2 substrate, as determined by radioimmunoassay. Further examination of PC2 protein biosynthesis by metabolic labeling in cultured neuronal cells showed that in ischemic cells, the proteolytic processing of PC2 was greatly attenuated. Finally, in mice, an intracerebroventricular administration of synthetic DYN-A(1-8) significantly reduced the extent of ischemic brain injury. In mice those lack an active PC2, exacerbated brain injury was observed after an otherwise non-lethal focal ischemia. We conclude that brain ischemia attenuates PC2 and PC2-mediated neuropeptide processing. This attenuation may play a role in the pathology of ischemic brain injury.

Topics: Animals; Brain Ischemia; Dynorphins; In Situ Hybridization; Neuropeptides; Peptide Fragments; Proprotein Convertase 2; Rats; Reperfusion; RNA, Messenger; Stroke; Time Factors; Up-Regulation

Different studies have supported neuroprotective effects of Corticotropin-releasing hormone (CRH) against various excitotoxic and oxidative insults in vitro. However, the physiological mechanisms involved in this protection remain largely unknown. The present study was undertaken to determine the impact of CRH administration (at concentrations ranging from 200 fmol to 2 nmol) before and at delayed time intervals following potassium cyanide (KCN)-induced insult in rat primary cortical neurons. A second objective aimed to determine whether kappa and delta opioid receptor (KOR and DOR) blockade, using nor-binaltorphimine and naltrindole respectively (10 microM), could alter CRH-induced cellular protection. Our findings revealed that CRH treatments before or 3 and 8 h following KCN insult conferred significant protection against cortical injury, an effect blocked in cultures treated with alpha-helical CRH (9-41) prior to KCN administration. In addition, KOR and DOR blockade significantly reduced CRH-induced neuronal protection observed 3 but not 8 h post-KCN insult. Using western blotting, we demonstrated increased dynorphin, enkephalin, DOR and KOR protein expression in CRH-treated primary cortical neurons, and immunocytochemistry revealed the presence of opioid peptides and receptors in cortical neurons. These findings suggest protective effects of CRH against KCN-induced neuronal damage, and the contribution of the opioid system in modulating CRH actions.

Topics: Animals; Brain Ischemia; Cell Death; Cells, Cultured; Corticotropin-Releasing Hormone; Dynorphins; Enkephalins; Indoles; Naltrexone; Narcotic Antagonists; Nerve Tissue Proteins; Neurons; Neuroprotective Agents; Potassium Cyanide; Rats; Rats, Sprague-Dawley; Receptors, Corticotropin-Releasing Hormone; Receptors, Opioid, delta; Receptors, Opioid, kappa; RNA, Messenger

Alterations in the opioidergic system have been found in cerebral ischemia. Neuroprotection studies have demonstrated the involvement of the opioidergic system in cerebral ischemia/reperfusion (I/R). However, the neuroprotective mechanisms remain largely unclear. This study was conducted to investigate whether intracerebroventricular administration of opioidergic agonists has a neuroprotective effect against cerebral ischemia in rats and, if this proved to be the case, to determine the potential neuroprotective mechanisms. Using a focal cerebral I/R rat model, we demonstrated that the opioidergic agents, BW373U86 (delta agonist) and Dynorphin A 1-13 (kappa agonist), but not TAPP (mu agonist), attenuated cerebral ischemic injury as manifested in the reduction of cerebral infarction and preservation of neurons. The antagonism assay showed that the neuroprotective effect of Dynorphin A was attenuated by nor-Binaltorphimine (kappa antagonist). Surprisingly, BW373U86-induced neuroprotection was not changed by Naltrindole (delta antagonist). These findings indicate that BW373U86 and Dynorphin A exerted distinct neuroprotection against ischemia via opioid-independent and -dependent mechanisms, respectively. The post-ischemic protection in beneficial treatments was accompanied by alleviations in brain edema, inflammatory cell infiltration, and pro-inflammatory cytokine interleukin 6 (IL-6) expression. In vitro cell study further demonstrated that the opioidergic agonists, delta and kappa, but not mu, attenuated IL-6 production from stimulated glial cells. Our findings indicate that opioidergic agents have a role in post-ischemic progression through both opioid-dependent and -independent mechanisms. In spite of the distinct-involved action mechanism, the potential neuroprotective effect of opioidergic compounds was associated with immune suppression. Taken together, these findings suggest a potential role for opioidergic agents in the therapeutic consideration of neuroinflammatory diseases. However, a better understanding of the mechanisms involved is necessary before this therapeutic potential can be realized.

Topics: Amidines; Analgesics, Opioid; Animals; Benzamides; Brain; Brain Ischemia; Cytoprotection; Disease Models, Animal; Disease Progression; Dynorphins; Encephalitis; Immune Tolerance; Interleukin-6; Male; Narcotic Antagonists; Neuroprotective Agents; Peptide Fragments; Piperazines; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Stroke

To explore changes and role of dynorphinA(1 - 13) on the experimental treatment of hypoxic-ischemic brain injury (HIBI) by intracerebral transplantation of genetically modified myoblasts expressing and secreting brain-derived neurotrophic factor (BDNF) in neonatal rats.. Seven-day-old Sprague-Dawley rats were randomly divided into HIBI + BDNF group (A), HIBI + BDNF + U50, 488H group (B), HIBI group (C) and sham-operated group (D). Pups were intracerebroparenchymally transplanted with either genetically modified myoblasts producing and secreting BDNF (A, B) or their parent cells (C) at 0.8 microl (4 x 10(4)/microl) followed by a cerebroventricular microinjection of opioid kappa receptor agonist U50, 488H (0.5 microg, B) or vehicles (A, C) shortly after HIBI undergone by a permanent ligation of left common carotid artery followed by a 2.5 h inhalation of humidified 8%O(2) + 92%N(2) at 37 degrees C. Water contents of the brain, levels of malondiadehye (MDA) and cell apoptosis were investigated 1 d after the procedure. Contents of dynorphinA(1 - 13)-like immunoreactivity (ir-DynA(1 - 13)) at the left and right cortex and hippocampus were tested by radioimmunoassay 0, 1, 3 d postinjury.. Levels of ir-DynA(1 - 13) in left cortex were markedly increased in group A, B or C when compared to group D different times after the procedure and markedly decreased in group A or B vs group C 1, 3 d after the procedure. Levels of ir-DynA(1 - 13) in left hippocampus were also markedly increased in group A, B and C (0 d), B, C (1 d) and C (3 d) when compared to group D and markedly decreased in group A (1 d) and B (3 d) vs group C, respectively. Water contents, MDA levels and percentage of cell apoptosis were significantly higher in group A, B or C than those in group D and these parameters were obviously lowered in group A compared to group C in the left brain 1 d after the procedure. There were no significant differences of water contents and levels of MDA between group B and C but significantly lowered percentage of cell apoptosis in group B was seen when compared to group C in the left brain.. It is suggested that dynorphinA(1 - 13) participates in the pathophysiological process of HIBI. One of the pathways of the beneficial effects of intracerebral transplantation of genetically modified myoblasts producing BDNF on HIBI lies on the inhibition of the function of dynorphin A(1 - 13) through the binding of kappa opioid receptor.

Topics: Animals; Brain Ischemia; Brain-Derived Neurotrophic Factor; Disease Models, Animal; Dynorphins; Female; Hypoxia-Ischemia, Brain; Male; Malondialdehyde; Oxygen; Peptide Fragments; Rats; Rats, Sprague-Dawley

To study the secondary pathological changes in the central nervous system after injury and the role of dynorphin A1-13 in hypoxi-ischemic brain injury in neonatal rats.. Changes of concentrations of dynorphin A1-13 immunoreactives in some brain areas at different times after brain injury and the effect of injecting 8 microliters dynorphin A1-13 anti-serum into the medulla pool on pathological process of brain injury were observed in perinatal cerebral hypoxia ischemic rat models prepared by permanent ligation of right common carotid artery combined with a temporary systematic hypoxia at 37 degrees C in 7 day old Wistar rats.. Concentrations of dynorphin A1-13 in cortex, hypothalamus and hippocampus increased significantly after the injury. Microinjection of anti-dynorphin A1-13 serum into the medulla pool 1 h preinjury might markedly reduce brain edema and improve the physiological condition.. Dynorphin A1-13 plays a role in the pathophysiological process of brain hypoxia-ischemia and excessive amount of dynorphin A1-13 had some detrimental effect on the process.

Topics: Animals; Animals, Newborn; Brain; Brain Edema; Brain Ischemia; Dynorphins; Female; Hypoxia, Brain; Immune Sera; Male; Peptide Fragments; Rats; Rats, Wistar

We have developed a stroke model involving middle cerebral artery occlusion in the rat which elicits changes in cardiac and autonomic variables that are similar to those observed clinically. It is likely that these neurogenic autonomic responses are mediated by changes in neurotransmitter systems subsequent to the stroke. This possibility was investigated by examining changes in immunohistochemical staining for tyrosine hydroxylase, neuropeptide Y, leu-enkephalin, neurotoxins and dynorphin following middle cerebral artery occlusion in the rat. Computerized image analysis was used to provide semi-quantitative measurements of the changes. The ischemic region was centered primarily in the insular cortex. The results indicate that there are significant increases in immunostaining for tyrosine hydroxylase and neuropeptide Y in the insular cortex within the peri-infarct region. Neuropeptide Y staining was also significantly increased in the basolateral nucleus of the amygdala, ipsilateral to the middle cerebral artery occlusion, which did not appear to be included in the infarct. Leu-enkephalin, neurotensin and dynorphin staining was significantly elevated in the central nucleus of the amygdala ipsilateral to the occlusion of the middle cerebral artery. These neurochemical changes are discussed as possible mechanisms mediating the cardiac and autonomic consequences of stroke or as part of a process to provide neuro-protection following focal cerebral ischemia.

Topics: Amygdala; Animals; Brain Chemistry; Brain Ischemia; Brain Stem; Cerebral Arteries; Cerebral Cortex; Cerebral Infarction; Dynorphins; Enkephalin, Leucine; Image Processing, Computer-Assisted; Immunohistochemistry; Male; Prefrontal Cortex; Rats; Rats, Wistar; Tyrosine 3-Monooxygenase

Perinatal hypoxic-ischemic brain injury was induced in 7- to 8-day-old rats by ligating the left carotid artery with subsequent exposure to 9% oxygen atmosphere for 2.5 h. The animals were killed 7 days later and grouped according to the degree of brain injury sustained after hypoxia-ischemia. Total protein content measured in striatum ipsilateral to the ligation, and dissected from brains showing extensive damage, was reduced to 64% of contralateral tissue. The protein content was not altered in other groups including control animals exposed to air and in sham-operated animals exposed to hypoxic conditions. The concentration of (pg/mg protein) and total (pg/striatum) striatal dynorphin A-like immunoreactivity (DLI) from brains with extensive damage were increased to 481% and 285% of the contralateral side, respectively. Hypoxia-ischemia increased striatal neuropeptide Y-like immunoreactivity (NPYLI) concentration from brains with extensive damage to 157% of contralateral side, but when the results were expressed as total NPYLI content per striatum, NPYLI content in striatum with extensive damage remained unaltered. Substance P-like immunoreactivity (SPLI) concentration and total content per striatum from brains with extensive damage were reduced to 66% and 43% of the contralateral side, respectively. D1 and D2 receptor density in animals killed 10 days after injury was reduced by 24% and 22% of control, respectively, in striatum from brains with extensive damage. These results indicate complex changes in brain neuropeptides following neonatal hypoxia-ischemia. Damage in the substance P system could have functional effects on dopaminergic transmission while the increase in NPYLI and in DLI concentrations may respectively reflect the relative preservation from neuronal damage and possibly an increase in neuropeptide synthesis or decrease in release. The decrease in SPLI concentration and the increase DLI concentration induced by hypoxia-ischemia suggests that these peptides may be present in separate neurons.

Topics: Aging; Animals; Animals, Newborn; Brain; Brain Ischemia; Corpus Striatum; Dynorphins; Female; Functional Laterality; Hypoxia, Brain; Neuropeptide Y; Pregnancy; Rats; Rats, Sprague-Dawley; Receptors, Dopamine; Receptors, Dopamine D1; Receptors, Dopamine D2; Substance P; Substantia Nigra

Thromboembolic phenomena and transient ischaemic attacks (TIA) are considered the basis of ischaemic pathologies. The aim of the present research is to investigate the involvement of k-opioid receptors in cerebral blood flow (CBF) impairment which results in experimental stroke or dietary atherosclerosis in rabbits. CBF measurement showed a significant decrease in rabbits submitted to embolization and/or atherosclerosis. Binding studies showed that massive cerebral ischaemia and atherosclerosis produced a significant increase in the number of k-opioid receptors (Bmax), without changing (KD) affinity values. In conclusion, the results obtained seem to indicate that the increase in k-opioid receptors might play a crucial role in a common cerebral biochemical mechanism both in ischaemic and atherosclerotic pathologies.

Topics: Animals; Arteriosclerosis; Brain Ischemia; Cerebral Cortex; Cerebrovascular Circulation; Diet, Atherogenic; Dynorphins; Electroencephalography; Evoked Potentials, Somatosensory; Kinetics; Lipids; Rabbits; Receptors, Opioid, kappa

Recently it has been suggested that endogenous k-opioid receptors may have a physiopathological role in ischemia induced neurodegeneration. The aim of this research is to show that in experimental thromboembolic (obtained mechanically using microspheres injected in the carotid) and atherosclerotic pathologies (obtained through a special diet) there is a common mechanism which involves mediation by dynorphine and the receptor compartment considered. The results, obtained using receptor binding techniques, showed a statistically significant increase in the number of k-opioid receptors (Bmax) without variations in the affinity (Kd) for the 3H dynorphine. We can therefore support the hypothesis that these changes in the modulation of the dynorphinergenic system may be part of a mechanism causing early cerebrovascular damage which results from embolic insults and is a consequence of such metabolic risk factors as are activated by atherogenesis.

Topics: Animals; Arteriosclerosis; Brain Ischemia; Cerebral Cortex; Diet, Atherogenic; Dynorphins; Electroencephalography; Kinetics; Lipids; Microspheres; Rabbits; Receptors, Opioid; Receptors, Opioid, kappa; Thromboembolism

Dynorphin and catecholamine were measured in ischemic rat produced by four-vessel (2 vertebral arteries and 2 common carotid arteries) occlusion for 10 min. The results showed that: (1) The contents of dynorphine (pg/mg tissue) in cerebral cortex were 5.5 +/- 0.6 (n = 7) in normal rats and decreased to 4.9 +/- 0.5 (n = 9, P less than 0.05) in cerebral ischemic rats; with immediate ip phencyclidine (1-(1-phenylcyclophexyl)piperidine, PCP, 1 mg.kg-1), the contents of dynorphin were increased to 5.3 +/- 0.4 (n = 5, P less than 0.05 vs the ischemic rats). (2) The contents of DOPAC (pg/mg tissue) in cerebral cortex were 38 +/- 6 (n = 7) and increased to 120 +/- 60 (n = 5, P less than 0.05) in 10 min cerebral ischemic rats; with immediately ip PCP (1 mg.kg-1), the contents of DOPAC were decreased to 26 +/- 13 (n = 7, P less than 0.05 vs the ischemic rats). (3) The release of DA (pg/mg tissue) in cortical slices in vitro, in high K+ solution were 24 +/- 3 (n = 5) and significantly increased to 57 +/- 15 (n = 5, P less than 0.05) in ischemic rat brain slices; with immediate ip PCP (1 mg.kg-1), the contents of DA were decreased to 38 +/- 10 (n = 5, P less than 0.05 vs the ischemic rats). These results suggest PCP play an antagonistic role in cerebral ischemic damage of rats.

Topics: 3,4-Dihydroxyphenylacetic Acid; Animals; Brain; Brain Ischemia; Catecholamines; Cerebral Cortex; Dopamine; Dynorphins; Hydroxyindoleacetic Acid; In Vitro Techniques; Male; Phencyclidine; Rats; Rats, Inbred Strains

By occluding the bilateral carotid arteries of rabbits to produce bilateral partial cerebral ischemia, and by using radioimmunoassays to measure the levels of dynorphin A1-13 like immunoreactivity (ir-Dyn A1-13) in plasma and cerebrospinal fluid (CSF), the authors find that the levels of ir-Dyn A1-13 in plasma and CSF have significantly increased (P less than 0.01) after cerebral ischemia appears. The result of the Ligusticum wallichii Franch (Ligusticum) pretreatment to the test-group shows a definite improvement of the changes of ir-Dyn A1-13 levels in plasma and CSF. The severity of brain ischemic damage and neurologic dysfunction in Ligusticum-treated animals is lighter than that of saline-treated group, too. In this study, some new approaches are explored to explain the pathophysiology of cerebral ischemia and the mechanisms by which Ligusticum prevents and treats cerebral ischemia.

Topics: Animals; Brain Ischemia; Drugs, Chinese Herbal; Dynorphins; Female; Male; Peptide Fragments; Rabbits

Levallorphan and dynorphin were effective on the motor dysfunction in the gerbil model of unilateral cerebral ischemia. The effect of opioids, levallorphan (mixed agonist-antagonist), dynorphin (kappa-receptor agonist) and naloxone (mu-receptor antagonist), on neurological impairment was evaluated using the unilateral cerebral ischemia model of gerbil. Motor function was evaluated quantitatively by using the inclined plane method. Both levallorphan-treated group and dynorphin-treated group showed a significant improvement of the motor dysfunction compared with saline-control group. On the other hand, naloxone-treated group did not differ from saline-control group. The beneficial effect of these opioids on motor dysfunction might be mediated by the kappa-opioid receptor. This study also showed the potential usefulness of the inclined plane method for the investigation on the cerebral ischemia.

Topics: Animals; Brain Ischemia; Dynorphins; Female; Kinetics; Levallorphan; Male; Movement Disorders

We previously reported that the opioid peptide dynorphin1-13 improves survival chances in stroked cats. Some evidence also suggests that changes in dopamine and gamma-aminobutyric acid (GABA) uptake may be associated with stroke. In the present study, therefore, we determined binding of the opiate [3H]ethylketocyclazocine (EKC), as well as dopamine and GABA uptake in various brain regions of control, stroked and dynorphin1-13-treated stroked cats. Cats were stroked by middle cerebral artery occlusion. In the EKC binding study, the Kd of the high-affinity site of the occluded cortex was significantly increased, relative to that of both the unoccluded side and control cortex. Dynorphin1-13 treatment reversed this effect, lowering the Kd to control level. In the dopamine uptake study, the Km was decreased and Vmax was increased significantly in unoccluded cortex, compared with that in the occluded cortex or in control cortex. Again, dynorphin1-13 reversed these effects, raising the Km and lowering the Vmax. However, the Km of occluded cortex was also increased so that it became significantly higher than that of control cortex. The Km of unoccluded subcortex in stroked cats treated with dynorphin1-13 was significantly reduced compared with control. In the GABA uptake study, there was no significant change in any parameter. The change in opioid binding observed here and its reversal by dynorphin1-13 are consistent with the notion that the peptide's beneficial effect on stroke is mediated through opiate receptors. Since opioid systems in the brain are known to have association with dopaminergic ones, the change in dopamine uptake could also be the result of an opioid effect.

Topics: Animals; Brain; Brain Ischemia; Cats; Cyclazocine; Disease Models, Animal; Dopamine; Dynorphins; Ethylketocyclazocine; gamma-Aminobutyric Acid; Kinetics; Male; Peptide Fragments; Receptors, Opioid

Topics: Animals; Brain; Brain Ischemia; Cats; Cerebral Cortex; Dopamine; Dynorphins; gamma-Aminobutyric Acid; Male

We studied the effects of acute and long-term, continuous administration of six opioid compounds--naloxone, naltrexone, diprenorphine, leucine enkephalin, dynorphin 1-13, and dynorphin 3-13--on neurologic function, survival, and infarct size in a feline model of acute focal cerebral ischemia. Acutely, naloxone, naltrexone, and diprenorphine significantly improved motor function over baseline scores; the other drugs and saline (control) had no effect. In the long-term condition, no substance administered significantly affected level of consciousness, sensory function, or pupillary reactions. Naloxone, naltrexone, and dynorphin 1-13 significantly prolonged survival (p less than 0.1); the other substances had no effect. Evaluations of cat brains postmortem showed that the infarcts involved the sensory and motor cortex, internal capsule, and caudate nucleus. Infarct size was unaltered by any treatment administered; results among groups were remarkably similar. In evaluations of opiate receptor binding characteristics, high-affinity binding of ekylketocyclozocine was significantly reduced in the right (occluded) side of the cortex. Dynorphin 1-13 given 8 h postocclusion but before sacrifice increased this binding affinity to the previous level in non-occluded cortex. The observed protective effect of dynorphin 1-13 warrants further investigation. Our results support the involvement of endogenous opioid peptides in the pathophysiology of cerebral ischemia and suggest that, administered appropriately, opiate antagonists may be useful in the treatment of focal ischemic neurologic deficits.

Topics: Acute Disease; Animals; Brain Ischemia; Cats; Dynorphins; Enkephalin, Leucine; Male; Naloxone; Naltrexone; Narcotic Antagonists; Peptide Fragments