pnu-120596 and Brain-Ischemia

Most survivors of ischemic stroke remain physically disabled and require prolonged rehabilitation. However, some stroke victims achieve a full neurological recovery suggesting that the human brain can defend itself against ischemic injury, but the protective mechanisms are unknown. This study used selective pharmacological agents and a rat model of cerebral ischemic stroke to detect endogenous brain protective mechanisms that require activation of α7 nicotinic acetylcholine receptors (nAChRs). This endogenous protection was found to be (1) limited to less severe injuries; (2) significantly augmented by intranasal administration of a positive allosteric modulator of α7 nAChRs, significantly reducing brain injury and neurological deficits after more severe ischemic injuries; and (3) reduced by inhibition of calcium/calmodulin-dependent kinase-II. The physiological role of α7 nAChRs remains largely unknown. The therapeutic activation of α7 nAChRs after cerebral ischemia may serve as an important physiological responsibility of these ubiquitous receptors and holds a significant translational potential.

Topics: Administration, Intravenous; Allosteric Regulation; alpha7 Nicotinic Acetylcholine Receptor; Animals; Benzylamines; Brain; Brain Ischemia; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Dose-Response Relationship, Drug; Infusions, Intraventricular; Isoxazoles; Male; Organ Culture Techniques; Phenylurea Compounds; Rats; Rats, Sprague-Dawley; Sulfonamides

Activation of α7 nicotinic acetylcholine receptors (nAChRs) can be neuroprotective. However, endogenous choline and ACh have not been regarded as potent neuroprotective agents because physiological levels of choline/ACh do not produce neuroprotective levels of α7 activation. This limitation may be overcome by the use of type-II positive allosteric modulators (PAMs-II) of α7 nAChRs, such as 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)-urea (PNU-120596). This proof-of-concept study presents a novel neuroprotective paradigm that converts endogenous choline/ACh into potent neuroprotective agents in cerebral ischaemia by inhibiting α7 nAChR desensitization using PNU-120596.. An electrophysiological ex vivo cell injury assay (to quantify the susceptibility of hippocampal neurons to acute injury by complete oxygen and glucose deprivation; COGD) and an in vivo middle cerebral artery occlusion model of ischaemia were used in rats.. Choline (20-200 μM) in the presence, but not absence of 1 μM PNU-120596 significantly delayed anoxic depolarization/injury of hippocampal CA1 pyramidal neurons, but not CA1 stratum radiatum interneurons, subjected to COGD in acute hippocampal slices and these effects were blocked by 20 nM methyllycaconitine, a selective α7 antagonist, thus, activation of α7 nAChRs was required. PNU-120596 alone was ineffective ex vivo. In in vivo experiments, both pre- and post-ischaemia treatments with PNU-120596 (30 mg·kg(-1) , s.c. and 1 mg·kg(-1) , i.v., respectively) significantly reduced the cortical/subcortical infarct volume caused by transient focal cerebral ischaemia. PNU-120596 (1 mg·kg(-1) , i.v., 30 min post-ischaemia) remained neuroprotective in rats subjected to a choline-deficient diet for 14 days prior to experiments.. PNU-120596 and possibly other PAMs-II significantly improved neuronal survival in cerebral ischaemia by augmenting neuroprotective effects of endogenous choline/ACh.

Topics: alpha7 Nicotinic Acetylcholine Receptor; Animals; Brain Ischemia; Choline; Hippocampus; In Vitro Techniques; Isoxazoles; Male; Neurons; Neuroprotective Agents; Nicotinic Agonists; Patch-Clamp Techniques; Phenylurea Compounds; Rats; Rats, Sprague-Dawley

In the absence of clinically-efficacious therapies for ischemic stroke there is a critical need for development of new therapeutic concepts and approaches for prevention of brain injury secondary to cerebral ischemia. This study tests the hypothesis that administration of PNU-120596, a type-II positive allosteric modulator (PAM-II) of α7 nicotinic acetylcholine receptors (nAChRs), as long as 6 hours after the onset of focal cerebral ischemia significantly reduces brain injury and neurological deficits in an animal model of ischemic stroke. Focal cerebral ischemia was induced by a transient (90 min) middle cerebral artery occlusion (MCAO). Animals were then subdivided into two groups and injected intravenously (i.v.) 6 hours post-MCAO with either 1 mg/kg PNU-120596 (treated group) or vehicle only (untreated group). Measurements of cerebral infarct volumes and neurological behavioral tests were performed 24 hrs post-MCAO. PNU-120596 significantly reduced cerebral infarct volume and improved neurological function as evidenced by the results of Bederson, rolling cylinder and ladder rung walking tests. These results forecast a high therapeutic potential for PAMs-II as effective recruiters and activators of endogenous α7 nAChR-dependent cholinergic pathways to reduce brain injury and improve neurological function after cerebral ischemic stroke.

Topics: Allosteric Regulation; alpha7 Nicotinic Acetylcholine Receptor; Animals; Behavior, Animal; Brain Ischemia; Disease Models, Animal; Isoxazoles; Male; Motor Activity; Neuroprotective Agents; Phenylurea Compounds; Rats; Rats, Sprague-Dawley; Stroke