Effects And Mechanisms Of Neonatal X-Ray Exposure On The Development Of Cerebellum And Prefrontal Cortex In Mice

Background: Ionizing radiation(IR)has the most obvious damage to the body with changes in the nervous system,and neuroinflammation after radiation has been linked to brain damage and cognitive impairment.Previous studies have focused on the effects of radiation on hippocampal development,and exposure to the hippocampal region in neonatal period can lead to cognitive impairment in adulthood.However,the effects of radiation on the development of cerebellar and prefrontal cortex in mice and its mechanism have not been determined.In addition,studies have confirmed that the cerebellar and prefrontal cortex are also closely related to cognition and learning ability.Ionizing radiation can disrupt the brain-cerebellar resting state functional connectivity,thus leading to cognitive dysfunction in the body.However,the effects of neonatal period X-ray exposure(≤2 Gy)on the cerebellum and prefrontal cortex of mice and the mechanism of its occurrence have not been confirmed.The effects of 2 Gy X-ray exposure in neonatal period on cerebellum and prefrontal cortex of mice at different developmental stages and different glial cells and their underlying mechanisms have not been systematically reported.Objective: To investigate changes in cerebellar and prefrontal cortex development in mice induced by 2 Gy X-ray irradiation exposure at different periods.In order to understand the changes in the number of glial cells in different brain regions and the occurrence of neuroinflammation after radiation.,and to analyze its possible mechanism,and explore the target of radiation on cerebellar and prefrontal cortex.It plays a guiding role in radiation protection.Methods: A total of 60 male and female 8-week-old C57BL/6J mice were fed adaptively for 1 week,and then randomly caged together according to a 2:1 male to female ratio.The neonatal mice were randomly divided into two groups: control group and irradiation group(the mice in each group containing 5-6 different pregnant mice).The first day after birth was called Postanal day(PD)1.Animals in the irradiation group received whole-body X-ray irradiation with a distance of 100 cm and a dose of 2 Gy(2Gy/min)at PD 3.The body weight of mice in the two groups was recorded at 1 day after radiation and every 7 days after radiation.Animals in the two groups were divided into 8groups according to different periods,which were the control group and the irradiation group at postpartum day 1,7,21,and 90(PD 3+1,PD 3+7,PD 3+21 and PD 3+90).Euthanasia was performed at different time points for sampling,and HE staining,immunohistochemistry and western blotting were performed on the sampled tissues.Results:(1)Compared with the control group,the body weight of mice in the irradiated group began to decrease at 14 days after exposure and continued to decrease at56 days after exposure.(2)HE staining of cerebellar tissue showed that the thickness of outer cerebellar granulosa cells narrowed from day 1 to day 7 after irradiation(p < 0.01),inflammatory cell infiltration occurred from day 21 to day 90 after irradiation,and the number of inner granulosa cells decreased(p < 0.05).The Purkinje cell layer showed unclear cell boundaries and disordered arrangement at 7 to 90 days after irradiation,and the Purkinje cell migration into the IGL and inflammatory cell infiltration at 21 to 90 days after irradiation.(3)HE staining of the prefrontal cortex showed inflammatory cell infiltration,irregular cell shape,and nuclear shrinkage or dissolution from 7 to 90 days after irradiation.(4)Compared with PD 3+7,the expression of marker protein IBa1 in cerebellar microglia of mice in the control group was increased,and the increased peak was at PD 24.The number of cerebellar microglia of mice in the irradiation group was increased at PD 3+21(p < 0.05),but there was no change at other time points.Compared with PD 3+7,the expression of IBa1 protein in the prefrontal cortex of mice in the control group was increased,and the increase peak was also at PD 24.The number of microglia cells in the prefrontal cortex of mice in the irradiation group was increased from PD3+7to PD 3+90(p < 0.05,p < 0.01).(5)Compared with PD 3+7,the expression of the marker protein GFAP of cerebellar astrocytes in the control group was decreased.Compared with the control group,the number of GFAP positive cells in the cerebellar decreased at PD3+7(p < 0.05),but had no significant change at other time points.Compared with PD3+7,the astrocytes in the prefrontal cortex of mice in the control group showed a timedependent decline(p < 0.05),and the astrocytes in the prefrontal cortex of mice in the irradiation group showed a time-dependent decline.(6)Compared with the control group,the expression of NLRP3 protein in cerebellum tissue of mice in irradiation group was increased in PD 3+7,PD 3+7 and PD 3+21 in caspase-1 protein were increased(p < 0.05),and ASC was not significantly changed.Compared with the control group,NLRP3 and caspase-1 protein expressions in the prefrontal cortex of the irradiation group only increased in PD 3+7,and ASC protein expression decreased in PD 3+21(p < 0.05).(7)Compared with the control group,the expression level of HMGB1 and P-P65/P65 protein in cerebellum of mice in the irradiation group was increased in PD 3+7 and PD 3+21(p< 0.05),while the expression level of HMGB1 and P-P65/P65 protein in the prefrontal cortex was increased only in PD 3+7(p < 0.05).(8)Compared with the control group,the expression level of IL-1β was increased at all time points after PD 3+7(p < 0.05,p <0.01),and the expression level of TNF-α protein was increased at PD 3+7(p < 0.05).Compared with the control group,the expression level of IL-1β protein was increased at all time points after PD 3+7(p < 0.05),and the expression level of TNF-α protein was increased only at PD 3+7(p < 0.05).Conclusion: X-ray exposure at 2 Gy in neonatal period mice,(1)caused continuous pathological changes in cerebellar granulosa cell layer,Purkinje cell layer,and inflammatory cell aggregation in prefrontal cortex.(2)Increased the number of IBa1-positive microglia and decreased the number of GFAP-positive astrocytes in cerebellum and prefrontal cortex of mice.(3)The change of IL-1β was the main inflammatory factor.(4)Promotes cerebellar and PFC neuroinflammation possibly through the HMGB1 / NF-κB/ NLRP3 / IL-1β signaling pathway.(5)The injury of cerebellum and prefrontal cortex occurred in neonatal mice 7-21 days after 2 Gy radiation exposure.