α7 nicotinic acetylcholine receptor agonism confers neuroprotection through GSK-3β inhibition in a mouse model of intracerebral hemorrhage.
OBJECTIVE
Perihematomal edema formation and consequent cell death contribute to the delayed brain injury evoked by intracerebral hemorrhage (ICH). We aimed to evaluate the effect of α7 nicotinic acetylcholine receptor (α7nAChR) stimulation on behavior, brain edema, and neuronal apoptosis. Furthermore, we aimed to determine the role of the proapoptotic glycogen synthase kinase-3β (GSK-3β) after experimental ICH.


METHODS
Male CD-1 mice (n=109) were subjected to intracerebral infusion of autologous blood (n=88) or sham surgery (n=21). ICH animals received vehicle administration, 4 or 12 mg/kg of α7nAChR agonist PHA-543613, 12 mg/kg of α7nAChR agonist PNU-282987, 6 mg/kg of α7nAChR antagonist methyllycaconitine (MLA), 15 μg/kg of phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, or PHA-543613 combined with MLA or wortmannin. Behavioral deficits and brain water content were evaluated at 24 and 72 hours after surgery. Western blotting and immunofluorescence staining were used for the quantification and localization of activated Akt (p-Akt), GSK-3β (p-GSK-3β), and cleaved caspase-3 (CC3). Neuronal cell death was quantified through terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL).


RESULTS
α7nAChR stimulation improved neurological outcome and reduced brain edema at 24 and 72 hours after surgery (P<0.05 compared with vehicle). Furthermore, PHA-543613 treatment increased p-Akt and decreased p-GSK-3β and CC3 expressions in the ipsilateral hemisphere (P<0.05, respectively), which was reversed by MLA and wortmannin. P-Akt, p-GSK-3β, and CC3 were generally localized in neurons. PHA-543613 reduced neuronal cell death in the perihematomal area (P<0.05).


CONCLUSIONS
α7nAChR stimulation improved functional and morphological outcomes after experimental ICH in mice. PHA-543613 reduced the expression of proapoptotic GSK-3β through the PI3K-Akt signaling pathway.
Department of Physiology and Pharmacology, Loma Linda University, Loma Linda, CA 92354, USA.
Paul R
PR
Krafft
Orhan
O
Altay
William B
WB
Rolland
Kamil
K
Duris
Tim
T
Lekic
Jiping
J
Tang
John H
JH
Zhang
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001522:Behavior, Animal / Q000187:drug effects; D001549:Benzamides / Q000494:pharmacology*; D015153:Blotting, Western; D001834:Body Water / Q000378:metabolism; D001923:Brain Chemistry / Q000187:drug effects; D001929:Brain Edema / Q000209:etiology / Q000473:pathology; D001643:Bridged Bicyclo Compounds / Q000494:pharmacology*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D053148:Caspase 3 / Q000502:physiology; D061945:Caspase Inhibitors; D005455:Fluorescent Antibody Technique; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors*; D000071679:Glycogen Synthase Kinase 3 beta; D020300:Intracranial Hemorrhages / Q000188:drug therapy* / Q000523:psychology; D008297:Male; D051379:Mice; D009474:Neurons / Q000187:drug effects; D018696:Neuroprotective Agents; D018722:Nicotinic Agonists / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011812:Quinuclidines / Q000494:pharmacology*; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D020521:Stroke / Q000188:drug therapy / Q000523:psychology; D016896:Treatment Outcome; D064569:alpha7 Nicotinic Acetylcholine Receptor
D001549:Benzamides; D001643:Bridged Bicyclo Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D053148:Caspase 3; D061945:Caspase Inhibitors; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D018696:Neuroprotective Agents; D018722:Nicotinic Agonists; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011812:Quinuclidines; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; C579166:Chrna7 protein, mouse; C513163:N-(1-azabicyclo(2.2.2)oct-3-yl)furo(2,3-c)pyridine-5-carboxamide; C498513:PNU-282987; C000605507:Gsk3b protein, mouse