边缘性维生素A缺乏及干预对幼鼠海马神经元形态与功能的影响

目的了解胚胎期开始的边缘缺乏(MVAD)对幼鼠海马神经元的形态及功能造成的损害,以及从新生儿期开始维生素A干预,能否使这种损害得以恢复。方法 11只健康母鼠于交配前3 w开始喂饲含VA 400 IU/kg的MVAD饲料,随机抽取其子鼠19只于断乳后继续喂饲同样的MVAD饲料为VAD组。另外随机抽取13只子鼠于出生0 d开始喂饲其母鼠含VA 6500 IU/kg的正常饲料,断乳后继续饲正常饲料为干预(VAI)组。随机抽取7只健康母鼠的10只子鼠作为正常对照(control,C)组,其饲料为含VA 6500 IU/kg的正常饲料。各组幼鼠均于7 w龄处死。用高效液相色谱法(HPLC)检测血清VA浓度。对海马切片进行HE染色,了解其神经元形态及细胞核大小的改变。运用电生理技术检测幼鼠离体海马脑片CA1区长时程增强(LTP),并在VAD组人工脑脊液(ACSF)中直接加入视黄酸(RA)后检测LTP。用透射电镜观察强直刺激前后,海马CA1区突触超微结构的变化。结果 MVAD孕鼠交配时血清VA浓度明显低于对照组。VAD组血清VA浓度明显低于C组,VAI组的血清VA浓度低于C组水平,但明显高于VAD组。海马CA1区锥体细胞层神经元细胞核的面积,VAD组明显小于C组,VAI组大于VAD组,与C组比较无明显差别。C组海马CA1区群峰电位增长幅度明显大于VAD组,VAI组小于C组,大于VAD组(P<0.05);VAD组脑片的ASCF中加入RA,其群峰电位增长幅度由(22.86±3.26)%上升到(59.08±7.22)%(P<0.01),与C组无显著差异。强直刺激后的C组与空白对照组(BC)比较,其海马CA1区突触的活性区长度和突触后致密物(PSD)厚度均明显增加,突触界面模拟圆半径明显减小,突触间隙宽度没有明显差异;强直刺激后,C组突触活性区长度明显大于VAD组,与VAD+RA组比较无显著差异,C组和VAD+RA组的突触界面模拟圆半径小于VAD组。结论 MVAD可导致少量CA1区神经元缺失和坏死,并减小了海马神经元细胞核的面积。同时直接影响海马神经元LTP的诱发,以及减小了LTP诱发后突触超微结构变化的程度。新生儿期开始的VAI可使MVAD对海马神经元的影响得到部分恢复。

EFFECTS OF MARGINAL VITAMIN A DEFICIENCY AND VITAMIN A INTERVENTION ON NEURONS AT HIPPOCAMPUS IN YOUNG RATS

Objective To study the impairment to the neurons at hippocampus about morphology and function in young rats with marginal vitamin A(VA) deficiency and the effect of VA intervention.Method Eleven female rats were fed with marginal VA deficient(VAD) feed containing VA 400 IU/kg for 3 w before coitus.Nineten pups were randomly selected to the VAD group which was kept on feeding with VAD feed after ablactation.Thirteen pups were randomly selected to the VAI group,in which mother rats were fed the normal food containing VA 6500IU/kg after delivery and the pups were kept on feeding with the normal feed after ablactation.Eleven pups from 7 female rats fed with normal food were randomly selected to be normal control(C) group,which were kept on feeding with normal food after ablactation.All of the young rats were killed at 7 w old.The serum VA concentrations of the young rats were detected by HPLC.We could observe the morphology and the area of nucleus of the neurons at hippocampus by HE staining.The hippocampal CA1 long-term potentiation(LTP) was detected in vitro in all three groups and in the artificial cerebrospinal fluid of VAD group after adding retinoic acid.The ultrastructure of synapse in hippocampal CA1 region after tetanus stimulation was observed by transmission electron microscope.Results(1)The serum VA concentration of marginal VA deficient dams and pups was decreased.No clinical manifestation of VA deficiency was observed.The serum VA concentration of VAD group was lower than C group(P<0.05).The serum VA concentration of VAI group was lower than C group(P<0.05),but higher than VAD group(P<0.05).(2) A little neuron loss and red neuron were found in the hippocampal CA1 region pyramidal cell layer of VAD group.The area of nucleus in the hippocampal CA1 pyramidal cell layer of VAD group was smaller than that in C group (P<0.05).And that of VAI group was bigger than VAD group(P<0.05),but there had no significant difference with C group (P>0.05).(3) The hippocampal CA1 LTP of VAD group was lower than C group significantly.The LTP of VAI group was lower than C group(P<0.05) and was higher than VAD group(P<0.05).The hippocampal CA1 LTP of VAD group was 22.86%±3.26% and 59.08%±7.22%,before and after adding retinoic acid in the artificial cerebrospinal fluid respectively (P<0.01).(4) Compared with the normal control group,the thickness of postsynaptic densities(PSD),the length of active zones increased and the radii of the modeling circle of synaptic interface decreased significantly in the hippocampal CA1 region of C group(P<0.05).And there were no obvious differences on the width of synaptic cleft between them(P>0.05).The length of active zones of C group was longer than VAD group(P<0.05),and had no conspicuous difference from VAD+RA group(P>0.05).The radii of the modeling circle of synaptic interface of C group and VAD+RA were less than VAD group respectively(P<0.05).But the thickness of PSD and width of synaptic cleft of C group and VAD+RA had no conspicuous difference from VAD group respectively(P>0.05).Conclusion Marginal VA deficiency may influence the size of nucleus of hippocampal neuron and may contribute to a little neuron loss and necrosis.Marginal VA deficiency may directly impair the hippocampal CA1 LTP and decrease the changes of the ultrastructure of synapse in hippocampus after tetanus stimulation.The impairment to the neurons of hippocampus would recover incompletely if VA intervention performed from the day of birth.