Cholinergic Agonists Inhibit Microglial Activation And Are Neuroprotective On Delayed Neuronal Death In CA1 Hippocampus In Cerebral Ischemia Rat

Stroke is the most common life-threatening neurological disease, and is the thirdleading cause of death after heart disease and cancer, and in the elderly it is a majorsource of disability leading to institutionalization. While pharmacological therapy toreduce ischemic damage is being pursued, prevention and rehabilitation are still theonly strategies, albeit relatively inefficient; to reduce the disability and lethality of thedisease. Most forms of cerebral ischemia initiate from some sort of vascular problem,and are clinically defined by a loss of function due to the death of neurons. Fordecades, therefore, cerebral ischemia research was mainly focused on blood flow,cerebral vasculature, or neuronal cells. It is a rather recent concept that astrocytes,oligodendrocytes, as well as microglial cells, play significant roles in the demise ofbrain tissue after cerebral ischemia, in addition to protecting brain function andenhancing survival and regeneration under these conditions. Accumulating evidenceover the past two decades has indicated that the brain has an active endogenousimmune system. Chronic inflammation, predominantly mediated by microglial cells,is thought to play an important role in the progressive neuronal death seen in manyneurological diseased states, including Alzheimer's disease (AD), Parkinson's disease (PD) and stroke. The activation of microglia involves a change in morphology, andexpression and release of molecules such as inducible nitric oxide synthase (iNOS)and nitric oxide (NO), cyclooxygenase (COX)-2 and prostaglandins (PG) as well asproinflammatory cytokines and reactive oxygen species (ROS).The brain damage produced by cerebral ischemia maturates over a period ofseveral hours or days. Especially in global ischemia, a delayed hippocampal damageis observed 3 days or so after the insult in CA1 pyramidal neurons, suggesting thatmechanisms that develop slowly after ischemia have a role in ischemic cell death.Recent studies have shown that inflammatory cells infiltrate the ischemic brain area(24), and several proinflammatory genes or mediators, such as iNOS,cyclooxygenase-2, and cytokines are strongly expressed in the ischemic brain.Inflammation is now recognized as a significant contributing mechanism in cerebralischemia because antiinflammatory compounds or inhibitors of iNOS andcyclooxygenase-2 reduce ischemic damage and improve the outcome of animals afterischemic insult. It has been observed that N-cholinergic receptors were expressed onmicroglia and regulated some inflammatory factors released from these cells. Beingsuggested from the above cultures, we studied whether nicotine has neuroprotectiveeffects on delayed neuronal death in cerebral ischemia rat through anti-inflammationparthway.Transient cerebral ischemia was performed by occluding 4 major arteriessuppllying the brain ,i.e.,the bolateral vertibral and common carotid arteries,modifying the original method by Pulsinelli on adult mate wistar rats(body weight200+20g). We found that pretreatment of nicotine 30 minutes before ischemia areneuroprotective against ischemic stroke. However this prevention is not suitable onclinical therapy, it is not humanistic to apply nicotine on healthy people before theyare subject to stroke. Therefore we observed nicotine's neuroprotective effects at different time after ischemia against reperfusion.In sham operated rats, the number ofneurons in the CA1 pyramidal cell layer was 209.5±35个/mm2(n=8), whereas 7 daysafter 15-minute global ischemia,the number was decreased to 11.9% (25±7个/mm2).Nicotine-posttreated rats at 2, 6, 12 hours after ischemia had 54.6%(114.2±17.6个/mm2), 28.1%(58.7±13个/mm2), 20.1%(42±10个/mm2) of the neuron profilesleft in the CA1 pyramidal cell layer respectively. The neuroprotection wasstatistically significant in every animal group(P＜0.05), and the effect of nicotine isdose-dependent. Treatment of the same dose of saline did not provide any protection.Therefore, the earlier posttreatment of nicotine after ischemia, the betterneuroprotection. The neuroprotective effects of nicotine are still statisticallysignificant even applied 12h after ischemia.In order to determine the effect of nicotine on microglia inflammatory reactioninduced by reperfusion after ischemia, we observed the number of microglia in theCA1 pyramidal cell layer 7 days after 15-minute global ischemia. In sham operatedrats, the number of microglia in the CA1 pyramidal cell layer was 26.5±4.5个/mm2(n=8), whereas 7 days after 15-minute global ischemia,the number was increased to10.7 times (283.4±24个/mm2). Nicotine-posttreated rats at 2,6,12 hours afterischemia had 2.4 times(62.9±11.5个/mm2), 3.1 times(82.3±17.6个/mm2), 5.6times(151.3±24.6个/mm2) of the microglia profiles left in the CA1 pyramidal celllayer respectively. The anti-inflammation of nicotine was statistically significant inevery animal group(P＜0.05), and the effect of nicotine is dose-dependent. Treatmentof the same dose of saline did not provide any anti-inflammation effects. Therefore,the earlier posttreatment of nicotine after ischemia, the better anti-inflammationeffects. The anti-inflammation effects of nicotine are still statistically significant evenapplied 12h after ischemia.In order to study whether nicotine has a direct antiinflammtory effects on microglia, we performed the following experiments on cultured microglia in vitro.Microglial cultures were prepared from 10-14 day mixed primary glial culturesobtained from the cerebral cortex of 1-day-old rats, as previously described.Microglial cells, harvested from the mixed primary glial cultures by mild shaking,were resuspended in DMEM supplemented with 10 % fetal calf serum, and plated onuncoated plastic wells at a density of 1.25×105 cells/cm2. Cells were allowed toadhere for 20 min and then washed to remove non-adhering cells, the percents ofOX-42 positive cells is more than 98%. After a 24 h of incubation, the medium wasreplaced with fresh medium containing the substance(s) under study. Microglial cellswere stimulated with different concentrations of nicotine for different periods, anddetermined the microglia viability through MTT method. We found that nicotinedecreased the number of primary cultured microglia in 24 h,48 h, 72 h groupsrespectively, and the most appearent effect concentration is 10μM treated for 48 h.The similar effects of nicotine was observed on microglia cell line(BV-2). We furtherdetermined whether nicotine could decrease the number of survival primary culturedmicroglia pretreated with GM-CSF, microglial cells were prestimulated for 30 minwith different concentrations of nicotine, and then stimulated for 24 h, 48 h, 72 h inthe presence of 5 ng/ml GM-CSF. The results show that nicotine could decrease thenumber of survival primary cultured microglia pretreated with GM-CSF at differenttime points, and the most significant effect concentration is 10μM treated for 48 h.The similar effects of nicotine was observed on microglia cell line(BV-2). Then weobserved that carbachol, another cholinergic agonist, decreased the number ofmicroglia(BV-2), and the number of microglia induced by GM-CSF.At last, we determined whether nicotine decreased the number of microgliathrough inhibiting the proliferation of microglia, indicated by thymidine analog5'-bromo-2'deoxyuridine-5'-monophosphate (BrdU) positive cell proportions. We found that nicotine inhibited the proliferation of micrglia dose-dependently. Wefurther found that there are 30.7±7.9% BrdU positive cells of the DAPI positive cellsin control groups, and 45.8±10.5% BrdU positive cells of the DAPI positive cells inGM-CSF treated groups. Then BrdU positive cells decreased to 31.6±4.0% with10nM nicotine, and to 14.1±5.2% with 10μM nicotine, the most significant dose. Thedata suggested that nicotine could inhibit the proliferation of microglia induced byGM-CSF dose-dependently, which is a inflammtory factor.To conclude, Nicotine could increase the number of neurons left in the CA1pyramidal cell layer after ischemia, and decrease the number of microglia in the CA 1pyramidal cell layer 7 days after 15-minute global ischemia dose-dependently.cholinergic agonists decreased the number of micrglia, and they inhibited thespontaneous proliferation of microglia, as well as the proliferation induced byGM-CSF. All the data above suggests that cholinergic agonists are neuroprotective ondelayed neuronal death in the CA 1 of hippocampus in global ischemia rats and inhibitmicroglia activation even applied 12 hours after ischemia. Our results supplied a newmethod to cure stroke.