The Effect Of Orexin A On The Respiration In Neonatal Rats Exposed To Nicotine Prenatally

PurposeEpidemiological studies suggested that in uterus exposure to maternal smoking is associated with sudden infant death syndrome.As one of the major components in cigarette smoking,nicotine is likely to contribute to the development of central nervous system in fetus.Recent studies showed that neurons containing orexin neuropeptides in hypothalamic area might mediate not only feeding,energy homeostasis,endocrine,wakefulness maintenance,but also the cardiovascular and respiratory activities.The present studies test the hypothesis that orexin A may increase respiratory activity in neonatal rat.Prenatal nicotine exposure might alter the expression of orexin A in hypothalamus and orexin type 1 receptor in the medulla. The effect of orexin A on the regulation of respiratory activity may be changed too. Our aim is to observe the effect of orexin A on the regulation of hypoglossal nerve rootlets via orexin type 1 receptor in the neonatal rat transverse medullar slice,and to observe of orexin effect on respiratory activity or that prenatal nicotine exposure may cause change of orexin neurons and orexin type 1 receptor expression in the central nerve system.Methods1.Nicotine model:The pregnant Sprague-Dawley(SD) rats were divided into 2 groups randomly.Either nicotine(3mg/kg,twice/day) or normal saline solution(as same volume as nicotine group) was administered into the pregnant SD rats hypodermically from the fifth day to the end of gestation.The neonates with prenatal nicotine exposure are as nicotine group,the other neonates with normal saline are as control group.2.Transverse medullar slice:The experiment was performed on neonates(1～3d) who were exposed to nicotine prenatally or not.Thin slices were cut off using a vibratome.A 700～800μm transverse brainstem slice incorporatingâ…«nerve rootlets was made.The slice was then transferred into a recording chamber and perfused with artificial cerebrospinal fluid(ACSF).The ACSF was bubbled with carbogen(95%O₂+5%CO₂) to produce a pH of～7.4.The discharge activity of hypoglossal nerve rootlets was recorded with a glass electrode.The recorded signals were input to a computer via A/D interface and stored for calculating later and archival purpose.3.Drugs perfusion:The experiment was performed under the control condition perfused with ACSF,orexin of different concentration,SB408124 which is an antagonist of orexin type 1 receptor.According to the discharge activity of hypoglossal nerve rootlets,the effect of orexin on the regulation of respiratory activity via OX1R in the medulla of neonatal rats was observed.4.Immunohistochemical technology and ImageMeasure:The different expression of orexin A in the hypothalamus or OX1R in the medulla between the two groups was observed by immunohistochemical technology and calculated by software of ImageMeasure.5.The statistical analysis:It was performed by SPSS version 10.0 statistical package. Statistical comparisons were analyzed by one-way ANOVA,Bonferroni, independent - samples t test and paired-samples t test.A value of P＜0.05 was regarded as statistically significant.Results1.The burst frequency of hypoglossal nerve rootlets from transverse medullar slice of neonatal rats increased quickly during perfusing orexin A,and the response to orexin A dispelled after washing.The increase of burst frequency,duration,burst integral area of hypoglossal nerve rootlets in the neonates was at a dose-dependent manner in response to 50nM,100nM,300nM orexin A.2.The burst frequency,integrated area,and duration of hypoglossal nerve rootlets did all not change significantly,compared to control,during bathing a mixture of 100nM orexin A and 100nM SB408124.3.In prenatal nicotine - exposed neonates,100nM orexin A did not induce increase of the bursts frequency of hypoglossal nerve rootlets as same as control neonates. There was significant difference between the two groups(P＜0.05).But the orexin A-induced integrated area of bursts was significantly increased compared to control(P＜0.05).Orexin A did not induce increase of the duration in nicotine group.The difference between the two groups was insignificant statistically..4.Orexin A immunopositive neurons are distributed in the hypothalamus of both groups.They are mainly in the dorsomedial hypothalamic nucleus and around fornix of hypothalamus.The relative optical density(ROD) of orexin A was larger in the nicotine-exposed neonates than that in the saline neonates(P＜0.05).OX1R immunopositive neurons localized extensively within the neonatal medulla of both groups.They are mainly in the ventralateral area and hypoglossal nucleus.The ROD of OX1R was larger in the nicotine-exposed neonates than that in the saline neonates(P＜0.001).Conclusion1.Orexin A is a neuropeptite,which promotes the respiratory activities of neonatal rats at a dose-dependent manner.2.The excitement of respiratory activity by orexin A is mediated via orexin type 1 receptor.3.Prenatal nicotine exposure attenuates the respiratory excitement of orexin A partly.4.Prenatal nicotine exposure upregulates the expression of orexin A in hypothalamus and OX1R in the medulla of neonates.