biphalin and Brain-Ischemia

To meet the challenge of identification of new treatments for stroke, this study was designed to evaluate a potent, nonselective opioid receptor (OR) agonist, biphalin, in comparison to subtype selective OR agonists, as a potential neuroprotective drug candidate using in vitro and in vivo models of ischemic stroke. Our in vitro approach included mouse primary neuronal cells that were challenged with glutamate and hypoxic/aglycemic (H/A) conditions. We observed that 10nM biphalin, exerted a statistically significant neuroprotective effect after glutamate challenge, compared to all selective opioid agonists, according to lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Moreover, 10nM biphalin provided superior neuroprotection after H/A-reoxygenation compared to selective opioid agonists in all cases. Our in vitro investigations were supported by in vivo studies which indicate that the nonselective opioid agonist, biphalin, achieves enhanced neuroprotective potency compared to any of the selective opioid agonists, evidenced by reduced edema and infarct ratios. Reduction of edema and infarction was accompanied by neurological improvement of the animals in two independent behavioral tests. Collectively these data strongly suggest that concurrent agonist stimulation of mu, kappa and delta ORs with biphalin is neuroprotective and superior to neuroprotection by activation of any single OR subtype.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Opioid; Brain Edema; Brain Infarction; Brain Ischemia; Cells, Cultured; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Glutamic Acid; Ischemia; Motor Activity; Neurons; Neuroprotective Agents; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Reperfusion Injury; Severity of Illness Index; Stroke

Approximately 795,000 people experience a new or recurrent stroke in the United States annually. The purpose of this study was to assess the protective effect of a nonselective opioid receptor agonist, biphalin, in brain edema and infarct damage by using both in vitro and in vivo models of stroke. In an in vivo model of ischemia, biphalin significantly decreased edema (66.6 and 58.3%) and infarct (52.2 and 56.4%) ratios in mouse transient (60-min occlusion/24-h reperfusion) and permanent (6 h) middle cerebral artery occlusion models, respectively. Biphalin administration also showed decreased neurodegeneration in hippocampal, cortical, and striatal brain tissue after ischemia, evidenced by reduced Fluoro-Jade C staining. In addition, biphalin improved neurological function after stroke injury evidenced by neurological score and locomotor activity evaluation. Biphalin significantly decreased penumbral expression of Na(+), K(+), 2Cl(-) cotransporter (NKCC) and the translocation of the conventional isoforms of protein kinase C (PKC). It also reversed the activation of PKC-induced cell volume increase during ischemia in primary neuronal cell cultures exposed to 1 h of oxygen glucose deprivation. These data suggest that opioid receptor activation provides neuroprotection during stroke, and a possible explanation of this mechanism could be the inhibition of NKCC function via the regulation of PKC-dependent cell signaling.

Topics: Animals; Brain; Brain Edema; Brain Ischemia; Cell Size; Cells, Cultured; Disease Models, Animal; Drug Evaluation, Preclinical; Enkephalins; Hippocampus; Infarction, Middle Cerebral Artery; Male; Mice; Molecular Targeted Therapy; Motor Activity; Nerve Degeneration; Neuroprotective Agents; Protein Kinase C; Receptors, Opioid; Sodium-Potassium-Chloride Symporters; Stroke