Studies Of Adverse Effect On Hippocampus After Whole-brain X-ray Irradiation

Objective To establish a mouse X-ray whole-brain irradiation model (in vivo)or hipppcampus neuron irradiation model (in vitro) to study pathological injury,ultrastructural lesions, neuron apoptosis, DNA double strand breaks, protein expressionof NBS1, ATM, P53, PUMA and cell cycle arrest in hippocampus at different time pointafter exposure to different doses of X-ray irradiation and discussing the mechanism ofdamages in hippocampus induced by ionizing radiation and provide theoretical basis forradiation protection.Methods80adult ICR mice were randomly divided into five groups includingfour X-ray treatment groups and one control group. Mice in four treatment groups wererespectively accepted a single dose (0.4Gy,4Gy,10Gy and18Gy; dosage ratio was200cGy/min) X-ray whole-brain irradiation. Mice in control group were treated as sameas treatment groups but no X-ray irradiation. Whole brain of the mice in all groups wasgotten according to the time4h,12h,24h,48h (four mice in each time point).Hippocampus tissue HE staining technology was used as pathological analysis andelectron microscopy was used to evaluate ultrastructural injury in dentate gyrus ofhippocampus. In situ end labeling (TUNEL) assay was a method to detect apoptosis ofhippocampal neurons. Single cell microgel electrophoresis (SCGE) andImmunocytochemistry (ICC) was performed for DNA double strand breaks (DSBs)analysis. The expression of NBS1,ATM,P53,PUMA proteins was observed byimmunohistochemistry (IHC) and western blot. Moreover, cell cycle arrest was tested byFlow cytometer.Results(1) The results of histopathological examination indicated that X-irradiationcould evoke the obvious histopathological damage of hippocampal cells, and have a gooddose-time-effect relationship. Some of significant severe injury was also showed that in18Gy treatment group, such as cell body shrink, shape irregular, membrane shrinkage, nuclear pyknosis and etc. Cell necrosis was also found in18Gy48h. While in the controlgroups, the hippocampal cells were observed under a regular arrangement, completeshapes, full nucleus, clear nucleolus, non-glial cells infiltration.(2) The results from Electron microscopy showed that the chromatin densebegun to appear at0.4Gy4h in granular cells of dentate gyrus; irregular nuclearmorphology was showed at0.4Gy12h. In the4Gy treatment group, irregular nuclearmorphology was more severe than the group of0.4Gy. Nuclear membrane blurred wasappeared in some of granular cells at4Gy12h. Mitochondrial swelling was observed attime point of24h and48h. In the group of10Gy, many nuclei were become pyknotic at4h and12h, nuclei was seemed to break at the time point of48h. These phenomena weremost serious in the group of18Gy. No injury was observed in the control group,organelles such as mitochondria, endoplasmic reticulum were normal.(3) The results of SCGE showed that in the groups of different X-raydoses(0.4Gy,4Gy,10Gy,18Gy). DNA damage was increased with the X-ray doseincreasing. And in different time point of the same dose, the degree of DNA damage wasincreased at4h,12h or24h to the peak, and then decreased at48h.(4) The results of ICC of γ-H2AX showed that the rate of γ-H2AX positivecells was increased with dose of X-ray irradiation increasing. Compared with thedifferent time point in the same X-ray dose treatment group, the degree of DSBs wassignificantly increased at0.5h, and got its peak at1h, then decreased from4h to24h.The number of γ-H2AX foci had no statistical significance between4Gy group and10Gygroup.(5) The TUNEL results indicated that the rate of apoptotic cells was increasedwith the X-ray dose increasing and time increasing in the same X-ray treatment groupexcept the0.4Gy group. The rate of apoptotic cells in0.4Gy group was increased slightlytill24h and then decreased at48h. Rate of apoptotic cells had a sharp decrease at18Gy48h.(6) The immunohistochemistry staining showed that the number of NBS1,ATM,P53protein positive cells increased with the dose increasing. In same dose groups,the number of positive cells expressing ATM,P53was increased along with the timeextending except the0.4Gy48h and18Gy48h. In different time point of the same dose(0.4~4Gy), the number of positive cells expressing NBS1was increased firstly and, thendecreased. In the10Gy group, the number of NBS1positive cells increased with the time increasing.(7) Western-blot showed that NBS1, ATM, P53, PUMA expression wasincreased with dose of X-ray increasing, except18Gy48h in which ATM, P53, PUMAexpression were decreased. In different time point of the same dose (0.4~4Gy), NBS1expression reached its peak at different time point after different doses X-ray. In the10Gy group, the level of NBS1protein increased with the time increasing.(8) Cell cycle of hippocampus cells could be arrested by different doses X-rayat different time point after the treatment.Conclusions1. Both of the factors (dose and time) could induce histopathological injury ingranular cells of dentate gyrus after X-ray irradiation. The degree of injury had a positiverelationship with dose of X-ray and time.0.4Gy X-ray had no obvious injury at lightmicroscope level.18Gy X-ray at48h may induce granular cell necrosis of dentate gyruscells. Ultrastructural injury induced by X-ray could be found in all treatment groups andat all time point. Even the low level dose of X-ray (0.4Gy) also can induce chromatindense of granular cells.2. DNA damage could be induced by X-ray irradiation in hippocampus afterirradiation. The degree of DNA damage could be increased with the X-ray doseincreasing and time extending. But at24h or48h in all X-ray treatment groups (0.4Gy,4Gy,10Gy,18Gy), the degree of DNA damage began to go down gradually. The reasonof that may be DNA damage repair or much more severe injury of tissues.3. DSBs could be induced by different doses of X-ray in a varying degree in rathippocampal neurons after irradiation. In a certain range, the number of γ-H2AX focivaried with the radiation doses, both of them present a linear relationship, and the extentof DSBs was significantly increased at0.5h, and got its peak at1h, and decreased from4h to24h.4. X-ray irradiation could induce apoptosis of hippocampus neurons. The rate ofapoptotic cells in hippocampus showed good dose-time-effect relationship after X-rayirradiation. On the low dose level (0.4Gy), when the time extended from24h to48h, thephenomena of apoptosis could be improved. However, at18Gy48h the reason of the rate ofapoptosis sharply went down may be due to hippocampus severe injury.5. The levels of NBS1,ATM,P53,PUMA proteins in hippocampus showed gooddose-time-effect dependence after X-ray irradiation (0.4Gy,4Gy,10Gy,18Gy). And the levels of these proteins might be related with different extent of DNA damage. Theresults suggested that these proteins might be involved in DNA damage recognize andrepair.6. Cell cycle of hippocampus cells could be arrested at S or G2/M after X-rayirradiation. The changes of cell cycle were related to X-ray dose and time after thetreatment. And cell cycle changed with the different level expression of ATM, this mightsuggested that ATM was the centre of cell cycle regulation.7. The effect of X-ray irradiation had a good dose-time-effect relationship onhippocampus injury. These results might suggest that different injury in hippocampuscould be induced by X-ray. And the expression of ATM and NBS1could be induced byDNA double strand breaks. ATM and NBS1may be a centre of DNA damage recognize,DNA damage repair and cell cycle regulation.