Expression of c-Fos protein and nitricoxide synthase in neurons of cerebral cortex from fetal rats in hypoxia and protective role of Angelica sinensis

BACKGROUND： Both c-Fos protein and nitricoxide synthase （NOS） have been used as general indexes in relative research about neurons, but it is lack of reports that c-Fos protein and NOS are applied synchronously to study the neurons of hypoxic fetal rats in uterus. OBJECTIVE： To study the effect of hypoxia in uterus on the expression of c-Fos protein and NOS in neurons of cerebral cortex from fetal rats and whether Angelica sinensis has the protective effect on these neurons in hypoxia. DESIGN： Randomized control experiment.SETTING ： Department of Histology and Embryology, Luzhou Medical College.MATERIALS ： Twelve adult female Wistar rats in oestrum and 1 male Wistar rat with bodymass from 220 to 250 g were chosen. Parenteral solution of Angelica sinensis mainly contained angelica sinensis, 10 mL/ampoule, was provided by Department of Agent of the Second Hospital Affiliated to Hubei Medical University （batch number： 01062310）. METHODS ： This experiment was completed in the Department of Histology and Embryology of Luzhou Medical College from September 2003 to June 2004. ①Twelve adult female Wistar rats in oestrum and 1 male Wistar rat were housed in one rearing cage. Vaginal embolus was performed on conceive female rat at 8： 00 am next day. On the 15^th conceiving day, all conceiving rats were divided randomly into three groups： control group, hypoxia group and Angelica group with 4 in each group. Rats in hypoxia group and Angelica group were modeled with hypotonic hypoxia in uterus. Angelica group： Rats were injected with 8 mL/kg Angelica sinensis injection through caudal veins before hypoxia. Hypoxia group： Rats were injected with the same volume of saline. Control group： Rats were not modeled and fed with normal way. ② Twenty embryos of rats were chosen randomly from each group and then routinely embedded in paraffin. Paraffin sections were cut from the brain of embryos to anterior fontanelle. Double-label staining was used to detect the expression of nNOS and c-Fos in neurons of cerebral cortex from embryos of rats. OLYMPUS Bx-50 microscope was used to observe sections and DP12 digit camera was also used under 400 times to detect types of cells. Under microscope, the number of c-Fos, NOS, c-Fos/NOS positive neurons in cerebral cortex from embryos of rats were counted in 2 fields with magnification of 400 in one section per animal. ③ The data in experiments were analyzed by one-way analysis of variance （ANOVA） followed by q test. MAIN OUTCOME MEASURES： ① Results of immunohistochemical double-label staining of c-Fos/NOS from cerebral cortex; ② Comparison of amount immunohistochemical double-label staining of c-Fos/NOS positive cells from cerebral cortex. RESULTS：① The positive NOS cells and c-Fos/NOS cells in the three groups were mainly distributed in cerebral cortex, but positive c-Fos neurons were not observed. ② Positive NOS cells and c-Fos/NOS cells in hypoxia group were more than those in control group （76.55±12.02, 50.45±10.39; 33.35±7.42, 26.35±6.67, P 〈 0.05）, but those in Angelica group were less than those in hypoxia group （51.70±9.82, 35.65±8.37, P 〈 0.05）. CONCLUSION： Hypoxia can stimulate the increase of expression of c-Fos protein and NOS in neurons of cerebral cortex. However, Angelica sinensis can decrease this expression so as to play a protective role in cerebral neurons of hypoxic fetal rats.

Early application of nerve growth factor affects serum inflammatory cytokine levels in neonatal hypoxic ischemic encephalopathy

BACKGROUND： It has been demonstrated that there are changes of various cytokines, chemokines and adhesion factors in neonatal hypoxic ischemic encephalopathy （HIE）. What are the changes of interleukin-6 and interleukin-18 in serum of HIE neonates.OBJECTIVE： To observe the dynamic changes of interleukin-6 and interleukin-18 in peripheral serum at different time after HIE in neonates, and analyze the possible therapeutic efficacy of early application of NGF.DESIGN： A non-randomized controlled observation synchronically.SETTING： Department of Neonatology, Sun Yat-sen Hospital affiliated to Sun Yat-sen University.PARTICIPANTS： Sixty neonates with HIE were selected from the Department of Neonatology, Sun Yat-sen Hospital affiliated to Sun Yat-sen University from January 2004 to October 2006, including 32 boys and 28 girls, who were all accorded with the diagnostic standards for moderate to severe HIE. The neonates were divided into two groups NGF-treated group （n =30）, HIE group （n =30）. The HIE neonates in the NGF-treated group were given routine treatment and intramuscular injection of NGF within 24 hours after birth. Those in the HIE group were given routine treatments. Meanwhile, 30 apneic normal neonates （17 boys and 13 girls） at the same period were selected as the control group. The gestational age was 37 - 42 weeks in all the three groups, the body mass at birth was 2 500 - 4 000 g. Informed contents were obtained from the relatives of all the enrolled neonates.METHODS： The HIE neonates in the NGF-treated group were given routine treatment and intramuscular injection of NGF （2 000 U） within 24 hours after birth, once a day, 10 days as a course. Those in the HIE group were given routine treatments. Blood samples （3 mL） were drawn from femoral vein in all the neonates 1, 3 and 7 days after birth. The levels of interleukin-6 and interleukin-18 in serum were detected with enzyme-linked immunoabsorbent assay （ELISA）.MAIN OUTCOME MEASURES： Serum levels of interlenkin-6 and interleukin-18 at 1, 3 and 7 days after birth.RESULTS： All the 60 HIE neonates and 30 normal neonates were involved in the final analysis of results.① Serum level of interleukin-6： The serum levels of interlenkin-6 at 1, 3 and 7 days after birth in the HIE group were all lower than those in the control group （P 〈 0.05）, and gradually recovered to the normal level as time prolonged. The serum levels of interleukin-6 at 1, 3 and 7 days after birth in the NGF-treated group were higher than those in the HIE group （P 〈 0.05）. ②Serum level of interleukin-18： The serum levels of interleukin-18 at 1, 3 and 7 days after birth in the HIE group were all higher than those in the control group （P 〈 0.05）, especially that at 3 days. The serum levels of interleukin-18 at 1, 3 and 7 days after birth in the NGF-treated group were lower than those in the HIE group （P 〈 0.05）.CONCLUSION： After hypoxic ischemia, the level of interleukin-6 was decreased and that of interleukin-18 was increased in peripheral serum in HIE neonates, while NGF could balance the levels of interleukin-6 and interleukin-18, adjust the immunological function, and protect the nerve cells.

The relationship between the c-jun mRNA expression and apoptosis of neurons in rat brain following perinatal ischemic-hypoxia

Objective To investigate the relationship between c jun mRNA and apoptosis of neurons following perinatal ischemic hypoxia. Methods We set up a fetal rat model of perinatal ischemic hypoxia by ligating unilateral uterine horn vessel of pregnant Wistar rats (21 day gestation). The contralateral horn vessel was not ligated and the fetuses in these uteri served as controls. Rat pups were delivered by cesarean section at the end of ischemic hypoxic insult and then the rats' brain tissues were collected in different points of time. In situ hybridization and TUNEL methods were used to detect the c jun mRNA expression and neural cells apoptosis separately. Results The expression of c jun mRNA in brain tissues began at 15 minutes, reached the first peak at 1-2 hours after ischemic hypoxic insult and reduced gradually after 4 hours and the second peak at 24th hour after insult, gradually disappeared till 72nd hour. There were very low c jun mRNA expression of hippocampus in the control group 24 h after birth. Meanwhile, we observed that the apoptosis of neuronal cells in cerebral cortex was much more than that in the control group. Conclusion The increases of immediate early gene c jun expression and the cell apoptosis could be induced by perinatal ischemic hypoxia. The higher expression of c jun mRNA might induce the transcription of its target gene, especially, so called “death related genes”, which would be a promoter for cell apoptosis.