The Effect Of Nicotine On The Degeneration Of Dopaminergic Neurons In The Rats Of Parkinson's Disease

Background and PurposeParkinson's disease (PD) is a common chronic neurodegenerative disease in central nervous system (CNS). It is charactered by a selective and progressive loss of dopaminergic neurons in the substantia nigra (SN) and then the reduction of dopamine(DA) in the striatum that lead to motor deficits including tremor, rigidity, akinesia and posture reflex impairment. PD has a high disability rate. However, to date, the etiology and pathogenesis is still unknown. The most commonly used treatment is traditional medicine, concentrating on increasing DA and promoting the function of DA. Although initially effective, these drugs provide only symptomatic relief with serious inevitable side effects and they wouldn't prevent the progressive development of PD. Therefore, the effective treatment that can prevent the loss of DA neurons in PD has become the most imperative of our research currently.Accumulating evidences suggest chronic inflammation (mainly the abnormal activation of microglia) probably play an important role in progressive degeneration of DA neurons in PD. Anti-inflammation treatment may inhibit the development of PD. The research focusing on the anti-inflammation treatment of PD has become the hot spot recently.There is new evidence that nicotine could attenuate acute and chronic inflammation such as septic peritonitis and ulcerative colitis. The anti-inflammatory effect is mediated by an interaction of alpha7 nicotine and cholinergic nicotinic acetylcholine receptor(nAChR) on inflammatory cell. This process is called "cholinergic anti-inflammation pathway". Microglia play a critical role in the inflammation of the brain. Research in vitro has reported that a7-nAChR expressed on the membrane of microglia. Nicotine can inhibit the activation of LPS-induced microglia and the release of inflammatory factors by interacting withα7-nAChR. Epidemiological studies show that smoking has a negative correlation with PD. Cigarette contains numerous chemicals, however, nicotine could be responsible for the apparent effect. As is known to us, smoking is harmful to the health. However, how does nicotine protect the brain? We speculate that nicotine probably inhibit the persistent activation of the microglia in brain and avoid brain injury which is mediated by inflammation. In order to identify the hypothesis, we establish the PD rat model by injecting LPS into the SN. Then we use the PD model to study the neuro-protective mechanism of nicotine.Methods1. Experiments in vivo(1)Establish LPS-induced PD model by injecting stereoscopically LPS into the SN of the rat.(2)Institute rat model exposed by nicotine by intraperitoneal injection.(3)Investigate the change of TH or ox-42 immuno-positive cell in the injected SN different times after LPS injection the by immunohistochemistry staining and explore the effect of nicotine on these changes.(4)RT-PCR detect the expression of TH mRNA in the injected SN different times after LPS injection and explore the effect of nicotine on the TH mRNA eppression.(5)Western-blot detect the expression of TH and iNOS in the injected SN different times after LPS injection and explore the effect of nicotine on the TH and iNOS protein expression.(6)Using nitrate reductase detect the change of NO concentration in the injected SN different times after LPS injection and explore the effect of nicotine on the change. 2. Experiments in invitro.(1)PC12 cell subculturing.(2)Assay the effect of LPS to PC12 cell activity by using MTT.Results1. Normal microglia show smaller cell bodies and thinner and longer processes. Microglia exhibit enlager cell body with shorter and grossus processes 24h after LPS injection.2. Compared with control group, TH immuno-positive neurons,TH mRNA and TH protein decrease 3d after LPS injection in a time-dependant way.3. Compared with PD rats, microglia mostly presence ramified form of smaller cell body with thinner and longer processes in nicotine group. Beides, TH immuno-positive neurons,TH and TH mRNA increase dramatically.4. Expression of iNOS/NO in substantia nigra increased dramatically 14h after LPS injection and reached a peak at 24h. However, the peak of iNOS/NO surprisingly decreased in nicotine group.5. Different concentration LPS didn't produce obvious effect on the activity of PC12 cell.Conclusion1. We could make inflammation-meditated PD model by injecting LPS into substantia nigra in which LPS induce the activation of microglia and the degeneration of dopaminergic neurons.2. Nicotine can resist LPS-induced injury and protect dopamiergic neurons. The protective effect of nicotine is probably related to the inhibition of activation of microglia and iNOS/NO.