Relationship Between The Expression Of Tau Protein And P-tau Protein And Apoptosis In The Hippocampus Of Hypoxic-ischemic Neonatal

ObjectiveEstablishing hypoxic ischemic brain damage (HIBD) model in neonatal rats, toobserve the expression of Tau protein, phosphorylated Tau (p-tau) protein, andapoptosis related proteins Bcl-2and Bax, and nerve cell apoptosis at different timepoints in the hippocampus after HIBD, and to study its correlation to explore therelationship among Tau protein and p-tau protein and brain cells apoptosis after HIBD,and contribute to the analysis of pathogenesis of HIBD, and provide some guidancewith clinical intervention of HIBD.MethodsSeven-day-old newborn clean Sprague Dawley(SD) rats56, were randomlydivided into normal control group8, sham group8, and HIBD group (which wasdivided by death3,6,12,24,48h5subgroups according to the execution time)40.Each subgroup had8animals. HIBD group established animal model by Rice method.To observe the change of animal behavior before and after modeling, and took thebrain at different time points. Sham group just exposed the left carotid artery, noligation and no hypoxia, and was decapitated immediatelyat the end of surgery. Thecontrol group did not perform surgery and hypoxia, and were sacrificed at the sametime with the sham group.Through conventional fixed, paraffin-embedded sections, using hematoxylin-eosin (HE) staining to observe morphological changes in thehippocampus,and immunohistochemical staining to detect the expression of Tauprotein, p-tau protein, Bcl-2and Bax, and in situ nick end labeling (TUNEL) to detectneuronal apoptosis. Data to indicate by x s, SPSS17.0statistical software foranalysis, comparison among groups, if it was in line with the normal distribution andhomogeneity of variance, using one-way ANOVA, there was significant differencebetween the groups who used the least significant difference (LDS) method foepairwise comparison; if it is non-normal distribution or heterogeneity of variance,rank sum test would be used; the correlation analysis of Tau protein, p-tau protein,Bcl-2, Bax and apoptotic cells used Pearson or Spearson correlation analysis,α=0.05was the test standard.Results1Identification of animal models: neonatal rats in HIBD group were foundvarying degrees of abnormal behavior after hypoxia-ischemia, such as head trembling,limb shaking, staggering, difficulty turnover, turning left round, and even convulsions.HE staining showed hippocampal neuronal cells became swelling, degeneration andnecrosis, karyopyknosis, karyorrhexis, disordered arrangement, and the number ofneurons reduced after HIBD.2Immunohistochemical results: Tau protein, p-tau protein, Bcl-2and Baxshowed positive staining in brown or dark brown particles deposition, the coloringsite was located in the cytoplasm of neurons or axons, the former two positiveexpression mainly was seen in the dentate gyrus,and the latter two widely expressedin the hippocampus.①Tau protein: the normal control group and the sham groupboth showed a small amount of Tau protein positive cells, while the expression of Tauprotein increased after HIBD3h, and the number of positive cells increased andreached the peak at HIBD24h. Although the number was slightly smaller after HIBD24h, but still larger than that of the normal control group and sham group;②p-tauprotein: normal control group and the sham group showed a small amount of p-tauprotein positive cells, the number began to increase after HIBD3h, and graduallyincreased, and reached a peak at HIBD12h，and then gradually dropped, at HIBD48hthe level of p-tau protein was basically same with the normal control group and the sham group;③Bcl-2:normal control group and the sham group showed no Bcl-2positive cells, and the expression of Bcl-2increased after HIBD3h, the number of positive cells gradually increased, and reached a peak at HIBD24h, and then gradually decreased, but still larger than that of the normal control group and sham group;④Bax:the expression of Bax in the normal control group and the sham group was low, while began to increase after HIBD3h, and reached a peak at HIBD48h. The positive cells in HIBD group at each time point were more than that in the normal control group and the sham group.3TUNEL detection of apoptotic cells:apoptosis positive cells rarely expressed in the normal control group and the sham group, and began to increase after HIBD3h, and reached a peak at HIBD24h, and then gradually dropped, but still more than that in the normal control group and the sham group.4Correlation analysis of Tau, p-tau, Bcl-2, Bax and apoptotic cells:in the hippocampus of neonatal rat, Tau protein positive cells and Bcl-2, Bax, apoptosis positive cells were highly positive correlated(r1=0.819, P<0.01; r2=0.928, P<0.01; r3=0.771, P<0.01), namely within a certain time(within HIBD24h), with Tau protein positive cells increasing, Bcl-2, Bax and apoptosis positive cells were gradually increased;p-Tau positive cells was positively correlated with Bcl-2positive cells(r1=0.337, P<0.05), and negatively correlated with Bax positive cells and apoptotic cells (r2=-0.380, P<0.05; r3=-0.294, P<0.05), that is, within a certain period of time(within HIBD12h), with p-Tau positive cells increasing, Bcl-2positive cells increased,while Bax,apoptosis positive cells gradually reduced.Conclusions1Tau protein and p-tau protein expressed a certain amount in the hippocampus of normal newborn rat, while both dynamically changed after HIBD. The change was associated with neuronal apoptosis, indicating that both Tau protein and p-tau protein involved in neuronal apoptosis after HIBD.2In the hippocampus of normal newborn rat,the expression of Tau protein was positively correlated with apoptosis related proteins Bcl-2, Bax, and apoptosis positive cells, while the expression of p-tau protein was positively correlated with Bcl-2, and negatively correlated with Bax and apoptosis positive cells, indicating that overexpression of Tau protein was the result of apoptosis after HIBD and a transientincrease of p-tau protein may be make the cells escape apoptosis after HIBD.