| Objective To explore the effects of iNO on brain damage and neurobehavior of neonatal HIBD SD rats with neonatal hypoxic-ischemic encephalopathy(HIBD)by establishing the model of neonatal hypoxic-ischemic encephalopathy(HIBD),inhaling nitric oxide(iNO),neurobehavioral evaluation,calculating infarct volume by TTC staining,TUNEL staining in hippocampal CA1 region,and detecting apoptotic cells in hippocampal CA1 region under fluorescence microscope,so as to provide theoretical basis for iNO in clinical treatment of HIBD.Methods 72 postnatal 7-day-old(Postnatal7,P7)SD rats(half male and half female)were randomly divided into 6 groups: control female group,control male group,HIBD female group,HIBD male group,HIBD+iNO female group and HIBD+iNO male group,with 12 rats in each group.SD rats in HIBD group and HIBD+iNO group were made models: P7 SD rats were anesthetized with sevoflurane,cut open the neck skin and exposed the left common carotid artery,and ligated with 5-0 surgical line.It takes no more than 5 minutes for anaesthesia and surgery to produce newborn rats with ischemia.After the operation,the filial mice were put back into the mother’s cage for 1 hour to recover,and the ambient temperature was strictly maintained at 36 ℃.In order to produce hypoxia,the HIBD model was constructed after exposure to low-oxygen gas mixture(8% oxygen,92% nitrogen)for 1.5 hours.The newborn rats in the control group were also anesthetized with sevoflurane,but only cervical skin incision,exposure of left common carotid artery and suture skin incision were performed,without artery ligation or hypoxia.After that,the control group and the HIBD group were fed in the cage,and the newborn rats in the HIBD+iNO group were immediately put into the iNO box with the concentration of 20 ppm for 1.5 hours.At the same time,the gas concentration during the experiment was monitored by the NO concentration tester to reduce the effect of exhaled breath on the experiment.The neurobehavioral development of 6 groups of SD rats was evaluated by cliff turn reflex,negative geotaxis and gait analysis on the 14 th day after birth(P14).Then the offspring were killed and the whole brain tissue was taken.6 rats in each group were selected for TTC staining to calculate the cerebral infarction volume,and the remaining 6 rats in each group were stained with hippocampal TUNEL staining to observe apoptotic cells.Results(1)There was no significant difference in body weight among the six groups of SD offspring at P7(P > 0.05).The body weight and growth rate of P14 in 6 groups were significantly different from those in HIBD group and HIBD+iNO group.There was significant difference between HIBD male group and HIBD+iNO male group(P <0.05).There was no significant difference between HIBD+iNO female group and HIBD female group.The growth rate of HIBD female group(81.17 ±4.78%)was higher than that of HIBD male group(74.25 ±6.21%),the difference was statistically significant(P < 0.05),but there was no significant difference between HIBD+iNO female group and HIBD+iNO male group.In 6 groups,20 eyes were opened,4 eyes were opened and0 eyes were not opened in the control group,1 eye was opened,16 eyes were opened and 7 eyes were not opened in the HIBD group,and 5 eyes were opened,15 eyes were opened and 4 eyes were not opened in the HIBD+iNO group.There was significant difference between control group and HIBD group(P < 0.05),but there was no significant difference between control group and HIBD+iNO group(P > 0.05).There was significant difference between HIBD male group and HIBD+iNO male group(P <0.05).There was no significant difference between HIBD+iNO female group and HIBD female group.There was no significant difference between HIBD female group and HIBD male group,HIBD+iNO female group and HIBD+iNO male group.In group 6,there were 22 germinated teeth and 2 ungerminated teeth in control group,17 germinated teeth and 7 ungerminated teeth in HIBD group,20 germinated teeth and 4ungerminated teeth in HIBD+iNO group.(2)There was no significant difference in cliff rotation,negative geotaxis and gait analysis between the control female group and the control male group at P14(P > 0.05).There was significant difference in cliff rotation reflex between control group and HIBD group at P14(P < 0.05),but there was no significant difference between control group and HIBD+iNO group(P > 0.05).There was significant difference between HIBD group and HIBD+iNO group.There was no significant difference between HIBD female group and HIBD male group,HIBD+iNO female group and HIBD+iNO male group.The results of negative geotaxis and gait analysis at P14 in the control group were significantly different from those in the HIBD group and HIBD+iNO group.There was significant difference between HIBD group and HIBD+iNO group.There was no significant difference between HIBD female group and HIBD male group,HIBD+iNO female group and HIBD+iNO male group.(3)The infarct volume of P14 whole brain tissue stained by TTC in 6 groups was significantly different from that in HIBD group and HIBD+iNO group.There was significant difference between HIBD group and HIBD+iNO group.There was significant difference between HIBD female group and HIBD male group,HIBD+iNO female group and HIBD+iNO male group(P < 0.05),and the infarct volume of male HIBD group after iNO treatment(10.81 ±3.71%)was higher than that of female HIBD group after iNO treatment(6.00 ±1.68%).(4)The number of apoptotic cells stained by TUNEL in hippocampal CA1 region of 6groups at P14 was 6.08 ± 1.62,6.25 ± 1.48,16.58 ± 2.02,20.83 ± 2.12,12.17 ±2.37,15.92 ±1.44 in control female group,HIBD female group,HIBD male group,HIBD+iNO female group and HIBD+iNO male group,respectively.There was no significant difference in the number of apoptotic cells in hippocampal CA1 region TUNEL staining between the control female group and the control male group at P14.There were significant differences between control group and HIBD group and HIBD+iNO group(P < 0.05).There was significant difference between HIBD group and HIBD+iNO group.There was significant difference between HIBD female group and HIBD male group(P < 0.05),and the number of apoptotic cells in control male group after HI injury was significantly higher than that in control female group(10.50± 2.75).There was significant difference between HIBD+iNO female group and HIBD+iNO male group(P < 0.05),but there was no significant difference in the number of apoptotic cells decreased after iNO treatment between HIBD female group and HIBD male group after iNO treatment(P > 0.05).Conclusion The results of this study show that iNO reduces neonatal hypoxic-ischemic brain damage in a gender-dependent manner,and this protection is more significant in men,emphasizing the importance of evaluating male and female neuroprotective agents.In this paper,we selected iNO to study the differences of neonatal SD rats with different genders of HIBD,hoping to promote the further study of iNO in the treatment of HIBD newborns of different genders in clinical work. |
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