Impairment In Motor Coordination Of Offspring Cerebellar Vermis Inflammation Induced By Sleep Deprivation During Pregnancy

Objective:1.To investigate the impaired development of motor coordination in the offspring of C57BL/6J mice during and after sleep deprivation in the third trimester of pregnancy;2.To explore the potential mechanism of activation of cerebellar vermis microglia in offspring induced by sleep deprivation.Methods:C57BL/6J pregnant mice were modeled by multi-platform method and randomly divided into experimental group(sleep deprivation group from the second trimester,MT;Sleep deprivation group from the third trimester,LT)and control group(second trimester,MC;Late pregnancy control group,LC),sleep deprivation for 16h every day until delivery.Body length,tail length and body weight were measured during PND3-PND21.At 3 weeks of age,behavioral experiments(including balance beam experiment,suspension experiment,rotating rod experiment and gait analysis experiment)were conducted to detect the movement and work coordination ability.After the behavioral experiment,the cerebellar vermis tissues of the offspring were taken and the following experiments were carried out.(1)The morphological changes of microglia cells were observed by HE staining.(2)The expressions of microglia-specific markers Iba-1 and CD68 were observed by immunohistochemistry,and the staining intensity,proportion of positive cells and density of positive cells were semi-quantitatively analyzed by Image J software.(3)The expressions of Iba-1,CD68,IL-4,IL-6,IL-10 and TNF-αwere detected by real-time quantitative PCR.SPSS 26.0 software was used for statistical analysis.P<0.05 was considered statistically significantResults:1.Effects of sleep deprivation during pregnancy on offspring physiological parameters(body length,tail length,and weight):The average daily body length increase in MT group was higher than that in MC group(P=0.011).The average daily weight increase in LT group was lower than LC group(P=0.035).2.Effects of sleep deprivation during pregnancy on motor coordination of offspring:(1)Balance beam experiment:the static time(P=0.006)and the total passing time(P=0.017)of the MT group were lower than that of the LT group;The rest time(P=0.003)and total passage time(P=0.026)of the LT group were higher than those of the LC group.(2)Suspension experiment:the suspension rest time(P=0.006)and total suspension time(P=0.005)of MT group were higher than that of MC group;The suspension rest time(P=0.024)and total suspension time(P=0.024)of MT group were higher than those of LT group.The suspension rest time(P=0.041)and total suspension time(P=0.038)of LT group were higher than those of LC group.The proportion of child drops in the total test times of MT group(75%)was higher than that of MC group(25%)and LT group(30%).(3)Rod spinning experiment:The maximum speed(P<0.001)and duration(P<0.001)of MT group were lower than that of MC group.(4)Gait analysis experiment:the gait width of the rear paw of MT group was lower than that of MC group(P=0.014)and LT group(P=0.020).The left hind paw stride length(P=0.046),right front paw stride length(P=0.037),and right hind paw stride length(P=0.043)of LT group offspring were lower than those of LC group offspring.3.Morphological changes of microglia cells in the vermis of offspring:HE staining results showed that,compared with the control and experimental groups,the cerebellar microglia cells in the experimental group were mostly rod-shaped,with enlarged cell bodies and shorter branches,presenting a typical amoebia-like or intermediate activation state.Among them,the offspring of MT group is the most obvious.4.Aggregation of microglia cells in the vermis of the offspring:(1)Semi-quantitative immunohistochemical analysis of Iba-1 showed that the mean gray value of offspring Iba-1 in LT group was higher than that in LC group(P=0.002).Further analysis showed that the average gray value of Iba-1 of male offspring in LT group was higher than that of male offspring in LC group(P=0.043),and the average gray value of Iba-1 of female offspring in LT group was higher than that of female offspring in LC group(P=0.020)..(2)The m RNA expression level of Iba-1 was detected by real-time fluorescence quantitative PCR.The expression level of LT group was higher than that of MT group(P=0.015)and LC group(P=0.017).Further analysis showed that the expression level of LT group male offspring was higher than that of MT group male offspring(P=0.018)and LC group male offspring(P=0.011).5.Activation of microglia cells in the vermis of the offspring:(1)semi-quantitative immunohistochemical analysis of CD68:he density of CD68 positive cells in MT progeny was higher than that in MC progeny and LT progeny(P<0.001).Further analysis showed that the density of CD68-positive cells in the male(female)progeny of MT group was higher than that in male(female)progeny of MC group and male(female)progeny of LT group(P value was less than0.05)..(2)Real-time quantitative PCR was used to detect the expression level of CD68m RNA.The expression level of female progeny in MT group was higher than that in LT group(P=0.028).6.Expression of inflammatory factors in the vermis of the offspring:(1)The m RNA expression level of anti-inflammatory factor:the IL-10expression level of MT group was higher than that of MC group(P=0.029).The expression level of IL-10 in LT group was lower than that in LC group(P<0.001).The expression level of IL-10 in MT group was higher than that in LT group(P<0.001).Further analysis showed that the expression level of IL-10 in male(female)offspring in MT group was higher than that in male(female)offspring in LT group(P value was lower than 0.01).The expression level of IL-10 in the LT group was lower than that in the LC group(P<0.001).(2)proinflammatory factor m RNA expression levels:the levels of IL-6(P=0.001)and TNF-α(P<0.001)in MT group were higher than those in MC group.The expression levels of IL-6(P=0.021)and TNF-α(P<0.001)in LT group were higher than those in LC group.Further analysis showed that the expression level of IL-6 and TNF-αin male progeny in MT group and LT group was higher than that in control group(P value was lower than 0.001).The expression level of TNF-αin the MT group was higher than that in the MC group(P<0.001).7.Correlation analysis between progeny behavioral experimental indexes and expressions of Iba-1,CD68,and inflammatory factors:Spearman correlation analysis showed that the maximum rotational speed of LT group offspring on the rotating rod was positively correlated with the expression level of IL-10(r_s=0.665,P=0.036),while the right hind paw stride length was negatively correlated with the expression level of Iba-1(r_s=-0.713,P=0.021).Conclusions:Sleep deprivation in late pregnancy of C57BL/6J mice may have a greater impact on the weight and development of their offspring,and no adverse impact on the growth and development of their offspring has been found.The motor coordination ability of the offspring of sleep deprivation mothers is impaired,especially when sleep deprivation begins in the middle of pregnancy,the offspring are more likely to show worse motor coordination ability,and the aggregation and/or activation of microglia are more obvious,which aggravates the inflammatory reaction of cerebellar vermis.After sleep deprivation during pregnancy,the inflammatory factors IL-6 and TNF-αin the cerebellar vermis of the offspring.The expression levels of both increased,indicating that inflammatory reactions may be involved in the development process of motor coordination disorders.