Effects Of Endogenous ROS On The Brain Development Of Mice

Background and objective:Reactive oxygen species are a kind of highly reactive chemical species generated in the process of mitochondrial oxidative metabolism and involved in regulation of many physiological functions within life organism. The aim of this present dissertation was to investigate the effects of ROS on the brain growth and development of the offspring mice, when antioxidants N-acetyl-cysteine was administered by drinking water (6.523 g/L) on maternity mice during gestation and lactation.Materials and methods:2-month old mice with the C57BL/6 background were kept couples feeding by ratio of 1:2 for male and female. Subsequently, these mice were randomly divided into 2 groups:NAC treatment group and normal control group. The offspring mice were selected for the following comparative study at P1, P3, P7, P14, and P21, respectively.(1) At first, we measured the weight of the bodies and brains of NAC mice and their control mice at the above mentioned five time points. The gross profiles of body and brain were observed.(2) We then observed the changes of brain histomorphology in NAC mice and their control mice at 1,3,7,14,21 days old by Nissl staining.(3) We compared proliferation of nerve cells in the brains of NAC mice and control mice at P1, P3, P7, P14 and P21 by using immunohistochemical staining to detect the expression of proliferating cell nuclear antigen (PCNA).(4) To compare the differentiation of neural cells between NAC mice and their control mice during postnatal development, the expressions of neuronal nuclear protein (NeuN), glial fibrillary acidic protein (GFAP), myelin basic protein (MBP) were detected by immunohistochemical staining.(5) At last, we detected the levels of ROS in the brains of P3 NAC mice and their control mice by fluorescence microscope after 4 hours of intraperitoneal injection of DHE.Results:(1) At P1 and P3 days, there was no significant difference of body weight, brain weight and appearance between NAC and control groups, while at P7 and P14 days, the experimental mice showed lighter weight of the body and brain than controls. However, at P21, there was no significant difference between the two groups.(2) Nissl staining showed that, at P1 and P3, there was no significant difference in the anteroposterior diameter, while at P7 and P14, mice of NAC group showed less brain size than control group; at P21, no significant difference was observed between the two groups. Moreover, the area of CA pyramidal cell layer and DG granular cell layer in the brain of the control mice increased gradually from P1 to P7, until P7, the size and shape of hippocampus are similar to those at P14 and P21. There was no significant difference at P1 and P3 of NAC mice to the control group, at P7-P21 the area of the cerebellum of NAC mice was slightly less than that of the control group, but the difference was not significant. However, at P21, the development of cerebellar lobules in NAC mice was still slightly inferior to the control group.(3) The PCNA immunoreactive positive cells were observed in the cerebral cortex, hippocampus and cerebellum at P1 in both groups, and the expression of PCNA gradually reduced after reaching a peak point P3. At P3, the number of PCNA positive cells and staining intensity in the above area of NAC mice was less than in the control group.(4) The immunostaining results showed that NeuN expression was gradually enhanced after birth. At P3, the number of NeuN positive cells and staining intensity of the cerebral cortex and hippocampus were lower in NAC mice than control mice, but the difference in the cerebellum was not obvious. At other time points, there was no significant difference between the two groups. Expression of GFAP in the hippocampal and cerebellar cortex of NAC mice was lower than the control group at P3 and P7, and there was no significant differences at other time points. The cerebellum of normal mice, but not NAC-treated mice, showed a few MBP positive fibers at P3. While the MBP immunoreactivity occurred at the forebrain until P14, and the expression intensity of NAC group was lower than that of the control group.(5) DHE staining results showed the level of ROS in the hippocampus and cerebellum of NAC group mice was significantly lower than that of the control group at P3.Conclusion:Feeding the mother-mice with the antioxidant NAC during gestation and lactation causes the reduced growth in brain size and weight of the offspring mice and a delay in proliferation and differentiation of neural precursor cells into neurons, astrocytes and oligodendrocytes. These results suggest that endogenous ROS is essential for brain development, and that adding antioxidants during gestation and lactation inappropriately may affect the normal brain development.