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The expression and distribution of orexinA and orexin type 1 receptor in medulla of neonatal and adult SD rats

【摘要】:正Objective:Although the localization of orexin-containing cell bodies is restricted to the hypothalamus,orexin- containing fibers and their receptors are widely distributed in the brain.The present study was to examine the expression of orexinA(OXA) and orexin type 1 receptor(OX1R) in medulla of neonatal and adult SD rats.The differential expression of both groups was observed with maturation.Methods(1) Tissue preparation:neonatal(1-5d,n=5) and adult(6-8w,n=5) SD rats were perfused transcardially via the left ventricle with 4%paraformaldehyde in PBS(pH 7.4) under deep anesthesia. After postfixation,30μm thick sections were cut serially on a freezing microtome(Leica CM 1900).(2) Immunohistochemistry: free-floating sections were washed(5 min,3 times) before incubated with 0.3%Triton-100(30 min),0.3%H_2O_2(10 min), blocking(goat serum,20 min at 37℃),and incubated with anti-OXA or anti-OX1R antibody(rabbit-anti-rat IgG,1:500,1: 100,both Sigma) overnight at 4℃,and were washed(5 min,3 times) before secondary antibody(bio-goat-anti-rabbit IgG, both Huamei,20 min,37℃),ABC complexation(20 min at 37℃).Then the sections were stained in DAB reagent,pigmentation was controlled under microscope and ended in PBS,adhered on glass slices and mounted with neutro-gum.(3) Image measure:expression and distribution of OXA-like-immunoreactivity(OXA-like-ir) and OX1R-like-immunoreactivity (OX1R-like-ir) were observed and calculated the value of relative optical density(MOD) in the medulla by using software of ImageMeasure.4.Data analysis:Average values(means±SD) for the analyzed variables of MOD were calculated. Statistical significance was considered achieved when P0.05.Result:Expression of OXA-like-ir fibers and OX1R-likeir neurons was found throughout the medulla of neonatal and adult rats.OXA-like-ir fibers were obvious in the nucleus tractus solitarius(NTS),hypoglossal nucleus(Ⅻ) and ventrolateral medulla(VLM).OX1R-like-ir positive cells were heavily stained in the hypoglossal nucleus(Ⅻ) and ventrolateral medulla(VLM).In theⅫand VLM,relative optical density of OX1R-like-ir positive cells of neonatal rats was more less than that of adult rats(P0.001).Conclusion:The results show that there are no OXA-like-ir neurons in medulla of neonatal and adult rats,but OXA-like-ir fibers projecting to the NTS,Ⅻand VLM.OX1R-like-ir neurons are also expressed in the(Ⅻ) and VLM.High level expression of OX1R in adult rats may indicate its important role in physiological functions with maturation.Medulla is the cardiovascular and respiratory center.The presence of orexinA-like-ir fibers and OX1R-like-ir neurons in theⅫand VLM both is in support of its proposed role in regulation of cardiovascular and respiratory activities.

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