Effects Of Acute Nicotine Exposure On The Cell Apoptosis In Adolescent Rats

PurposeNicotine is the main alkaloid in tobacco, which is the mainly harmful bio-activator and is studied at the highest frequency. It is widely known that prenatal nicotine exposure is harmful to fetal brain, and changes synoptic functions, and induces abnormal behaviors, which will extend to the adult. However, there are only a few studies to research whether nicotine exposure will also bring about those similar adverse effects on the adolescent. In fact, nearly all smokers begin to smoke since the adolescent and there are more and more adolescent smokers, who are becoming the center of the next generation smokers. Only in American, 25% of the adolescent are smokers (the total are over three million). Therefore , it is significant for improving total quality of population to study effects and relevant mechanism of nicotine on the adolescent.Because acetylcholine ( Ach) and other transmitters play unique tropic roles, drugs and environmental toxins, which promote and interrupt neural transmitters ' functions, can induce abnormal neural development by disrupting the timing of tropic actions and intensity. As one kind of bioactivator, nicotine disrupts neural transmitters through affecting physical nicotine chloine receptors and finally plays biological role, however, it is not clear about its certain mechanism in different diseases.It is reported that the administration of nicotine inhibits cell proliferation and increases cell apoptosis within the dentate gyrus(DG) of the hippocampus. DG is one site of continuous precursor cell generation throughout the adult life; these cells finally differentiate into granule neurons. So it is very significant tostudy the effects of nicotine on neurons within this site. In addition, hippocam-pal CA1, CA2, CA3 and CA4 are also important functional places, where neuronal stem cells get together and these places are relevant to LTP, and cerebral cortex and thalamus have close relationship with physical functions, so the current experiments focus on the above places.At present, there are many accesses to explain the mechanism about apoptosis , among which the caspase protease family plays critical roles in the apoptosis through death receptors. This access can sequentially activate caspase - 4, caspase - 7, caspase - 8, caspase - 3 and caspase - 6 and eventually induces cell apoptosis. It is found that caspase - 3 knocked - out mouse tend to dead early, which tells us that caspase - 3 plays the important role in neuronal apoptosis. Caspase -3 exists in the type of pro - caspase -3 in normal tissues and is mainly distributed in plasma; pro - caspase - 3 has not the biological function, and only the activated fragments can work. The distribution of caspase - 3 varies in different tissues and the content of pro - caspase - 3 is higher in cells of brain, spinal cord and embryo. The main substrate of caspase -3 is poly( ADP -ribose) polymerase (PARP) , which is relevant with the DNA reparation and the guard of general integrality. At the beginning of apoptosis, 116KD PARP is cut into 31KD and 85 KD fragments between Asp216 and Gly217, which makes it fail to work. As a result, the activity of endonuclease, which is regulated by PRAPis negative regulation and dependent on Ca2 + /Mg2 + , is increased , and then it cuts the DNA among nucleocome,and eventually leads to cell apoptosis.In current studies, we choose hippocampus, cerebral cortex and thalamus as the main research positions, and establish the adolescent rat model of nicotine exposure; we apply HE staining, immunohisto — chemistry, Western — blot and Tunel to observe effects of nicotine on expression and distribution of caspase - 3 and neurons apoptosis in hippocampus, cerebral cortex and thalamus of adolescent rats. This study will help to further study the adverse influences and relevant mechanism of nicotine on adolescent smokers.Methods1. Establishment of rat animal model that are acutely exposed to nicotine during adolescenceMale Wistar rats in PN30 are grouped at random. Nicotine (lmg/kg,i. p. ) is administrated in experiment groups and saline of the same volume is given in control groups.2. Heart perfusion to fix the brain and HE stainingRats in P30 are perfused with 4% formaldehyde, and then paraffin sections and freezing sections are made. HE staining is to confirm several sites and finds whether neurons in experiment groups are changed.3. Identification of neuronsThrough immunohistochemistry of NSE, we can identify neurons.4. Immunohistochemistry (including immune - fluorescent method) Sections are examined by immunohistochemistry to study the expression anddistribution of Caspase - 3, and the results are analyzed by MetaMorph/DPIO/ BX41 system and statistical methods.5. Western - blotCerebral cortex and hippocampus are removed from rats'brain in order to examine the expression of Caspase - 3 antitype and active fragments through western - blot. Gray values are qualified with Band Scan image analysis software.6. In situ Cell Apoptosis Detection (Tunel)Sections are examined by Tunel in order to observe the apoptosis cells in cerebral cortex, thalamus and hippocampus.Results1. Animal model acutely exposed nicotine was established The adolescent rats in PN30 were feed individually; there are no significant difference in weight gain between NIC and CON group.2. Results of HE stainingThe nucleuses of neuron in groups NIC contracted to together and are stained deeply, which tells that they are apoptosis cells.3. Results of identification of neurons NSE( + ) cells are neurons.4. Results of Immunohistochemistrya. results of Immunofluorescence : the expression of Casprase - 3 in NIC group was higher than that in group CON. The law of expression of Caspase -3 in group NIC is that the positive cells in strl8 - strl7 - strl8a - FeAud decrease gradually and then that in SR increase, and that in PCP are nearly distributed everywhere. There are many positive cells in thalamus either group NIC or group CON.b. results of Immunohistochemistry: The positive cells in cerebral cortex, thalamus, hippocampal Dentate Gyrus, CA2 and CA3 in group NIC were significantly more than that in group CON. It was found that the expression of Caspase-3 in cytoplasm was transferred partly to nucleus.5. Results of Western - blotIn cerebral cortex and hippocampus, the expression of Caspase - 3 including antitype and active types in group NIC are higher than that in group CON. Comparison of gray values both Caspase - 3 and ￡ - acting was done through Band Scan analysis soft, and the results were significant.6. Results of In situ cell apoptosis detectionIn cerebral cortex, hippocampus and thalamus, more Tunel ( + ) cells were found in group NIC than group CON. Especially, and vast Tunel( + ) cells are found in thalamus of group NIC.ConclusionsIn the adolescent rats, acute exposition to nicotine increases the expression of Caspase - 3 in cerebral cortex, thalamus, hippocampal Dentate Gyrus, CA2 and CA3, and makes Caspase - 3 distribute both in nucleus and cytoplasm.In the adolescent rats, acute exposition to nicotine can activate the antitype...