General anesthetics such as sevoflurane interfere with dendritic development and synaptogenesis, resulting in cognitive impairment. The collapsin response mediator protein2 (CRMP2) plays important roles in dendritic development and synaptic plasticity and its phosphorylation is regulated by cycline dependent kinase-5 (Cdk5) and glycogen synthase kinase-3β (GSK-3β). Here we investigated whether Cdk5/CRMP2 or GSK-3β/CRMP2 pathway is involved in sevoflurane-induced developmental neurotoxicity.
Rats at postnatal day 7 (PND7) were i.p. injected with Cdk5 inhibitor roscovitine, GSK-3β inhibitor SB415286 or saline 20 min. before exposure to 2.8% sevoflurane for 4 h. Western-blotting was applied to measure the expression of Cdk5/CRMP2 and GSK-3β/CRMP2 pathway proteins in the hippocampus 6 h after the sevoflurane exposure. When rats grew to adolescence (from PND25), they were tested for open-field and contextual fear conditioning, and then long term potentiation (LTP) from hippocampal slices was recorded, and morphology of pyramidal neuron was examined by Golgi staining and synaptic plasticity-related proteins expression in hippocampus were measured by western-blotting. In another batch of experiment, siRNA-CRMP2 or vehicle control was injected into hippocampus on PND5.
Sevoflurane activated Cdk5/CRMP2 and GSK-3β/CRMP2 pathways in the hippocampus of neonatal rats, reduced dendritic length, branches and the density of dendritic spine in pyramidal neurons. It also reduced the expressions of PSD-95, drebrin and synaptophysin in hippocampus, impaired memory ability of rats and inhibited LTP in hippocampal slices. All the impairment effects by sevoflurane were attenuated by pretreatment with inhibitor of Cdk5 or GSK-3β. Furthermore, rat transfected with siRNA-CRMP2 eliminated the neuroprotective effects of Cdk5 or GSK-3β blocker in neurobehavioral and LTP tests.
Cdk5/CRMP2 and GSK-3β/CRMP2 pathways participate in sevoflurane-induced dendritic development abnormalities and cognitive dysfunction in developing rats.