| ObjectiveIt is known that infection/immune activation during pregnancy can cause inflammation and activation of maternal immune system,which can lead to a significant increase in morbidity and mortality in pregnant women,resulting in worse outcomes(abortion,monster or stillbirth,etc).In addition,maternal immune activation(MIA)has been shown to cause neurodevelopmental disorders such as autism and schizophrenia-like behavior in offspring.There is currently no drug which could improve maternal fertility and symptoms of mental disorders in offspring induced by maternal immune activation.Nowadays,the effect and safety of acupuncture in clinic has been widely confirmed.However,it is still unknown whether acupuncture has a potent effect on maternal fertility and neurodevelopment in offspring after maternal immune activation.Based on the above theoretical background,this study attempts to explore the effect and possible mechanism of gestational infection/immune activation on pregnant women and their offspring.Methods1.Pregnant C57BL/6J mice at G12.5 were randomly assigned to control,poly(I:C)and electroacupuncture(at least 5～8 in each group).Mice in control group received a single sterile physiological saline injection.Other mice received a single poly(I:C)injection(8mg/kg,i.v.).Mice in electroacupuncture group received electroacupuncture Zusanli bilaterally from G13 to G18.2.The influence of mean body mass,abortion rate,litter size of pregnant women and mean body mass(P0,3,7,21)and neonatal mortality of offspring were observed and recorded.3.Blood from each group of pregnant mice were collected at G18 and brain from each group of offspring were collected at P0 to detect cytokines and chemokines levels(MCP-1,IL-1β,IL-2,IL-6,IL-17A,IL-22,IL-23,TNF-α,IFN-γ,GM-CSF).4.Splenocytes from each group of pregnant mice were isolated at G18 to analyze the CDllb+,CD68+CD86+ and CD68+CD206+ cells.5.It was used to detect the exploration activities in open field test.It was used to detect the spatial learning and memory cognitive ability in Morris water maze.It was used to detect the sensory motion gating ability in pre-pluse inhibition.6.The proliferation of cortical microglia(Iba-1+)in offspring were observed by immunofluorescence.7.The effect of the electroacupuncture was evaluated by the above methods after deleption of the maternal spleic macrophages by clodronate liposomes.8.After intervention with IL-6 neutralizing antibody,the above methods were used to detect the effect of electroacupuncture on pregnant mice and offspring.Results1.The influence of maternal fertility after maternal electroacupuncture(1)Pregnant mice with injection of poly(I:C)exhibited significant weight loss during the perinatal period when compare with saline-injected controls(P<0.01),both EA intervention at G13-18 and IL-6 neutralizing antibody reverse the reduced weight of pregnant mice exposed to MIA(P<0.01).(2)The administration of poly(I:C)result in remarkable increase of abortion at the end of gestation as compare to control mice,which were also ameliorated by electroacupuncture treatment and IL-6 neutralizing antibody treatment.(3)The number of delivery pups from the dams with administration of poly(I:C)was remarkable reduced in compare to dams with vehicle injection,which was reversed by electroacupuncture or IL-6-neutralizing antibody treatment.(4)Injection of poly(I:C)strongly induced increase of the levels of maternal serum of MCP-1 and IL-6 at pregnant mice in compared with controls.Significantly,the elevation was attenuated by electroacupuncture.(5)Splenic macrophage-depleted plus electroacupuncture mice result in remarkable increase of abortion during gestation as compare to control mice,consistent with poly(I:C)mice.Likewise,splenic macrophage-depleted mice showed a significant reduction in the number of delivery pups.(6)The majority of CD11b+cells were remarkable elevated in poly(I:C)-injected mice when compared with controls(P<0.01),but not in poly(I:C)-injected mice with electroacupuncture intervention(P<0.05).The poly(I:C)-injected group showed significantly increased CD68+CD86+M1 cells(P<0.01),compared to controls,and the increasing was diminished after electroacupuncture intervention(P<0.05).Meanwhile,the reduced CD68+CD206+M2 cells in pregnant mice of MIA were also been reversed by electroacupuncture intervention(P<0.05).Furthermore,IL-6 neutralizing antibody treatment reduced the elevated of CD11b+ cells in poly(I:C)-injected mice significantly(P<0.05).Both the increased CD68+CD86+ cells(P<0.05)and decreased of CD68+CD206+ cells(P<0.05)in mice with poly(I:C)also could be attenuated by co-administration of IL-6-neutralizing antibody.Co-administration of IL-6 neutralizing antibody should not be able to further reduced the inflammation response when combine with EA treatment(P>0.05).2.The influence of development of offspring after maternal electroacupuncture(1)The result showed that the mean body mass of all pups was equal to those of controls at P0,P3,P7 and P21(P>0.05).The survival of pups from the dams with administration of poly(I:C)was remarkable reduced in compare to dams with vehicle injection(P<0.01),which was reversed by electroacupuncture(P<0.01)or IL-6-neutralizing antibody treatment(P>0.05).(2)Splenic macrophage-depleted mice plus electroacupuncture result in remarkable decrease of survival during weaning as compare to control mice(P<0.01),consistent with poly(I:C)mice.(3)Injection of poly(I:C)strongly induced increase of the levels of homogenate of newborn pups of MCP-1 and IL-6 at pregnant mice in compared with controls.Significantly,the elevation was attenuated by electroacupuncture(MCP-1,P<0.05;IL-6,P<0.05).(4)Open-field test:The results showed that both electroacupuncture and IL-6-neutralizing antibody treatment group have significantly decreased locomotor activity(P<0.05),but there were no statistically different between two groups(P>0.05).Morris water maze:Results showed that with the increase in training time,the escape latency of offspring mice was reduced.Compared with the control group,escape latency of poly(I:C)group(day3:P<0.05;day4:P<0.01)were significantly shorter in the third and fourth days,and electroacupuncture or IL-6-neutralizing antibody treatment group was significantly shorter in the fourth day compared with the poly(I:C)group(P<0.01).In the probe phase,electroacupuncture(P<0.01)or IL-6-neutralizing antibody treatment(P<0.05)pups spent more time to come across the probe.Likely,electroacupuncture(P<0.01)or IL-6-neutralizing antibody treatment(P<0.05)pups displayed a larger proportion of target quadrant in the total swimming time compared with poly(I:C)group.Pre-pulse Inhibition:There were no statistically different among the four groups in the response of the 70dB background noise(P>0.05).PPI was impaired in poly(I:C)-offspring(P<0.01),compared to controls,which was attenuated by electroacupuncture treatment(P<0.01)and IL-6-neutralizing antibody treatment(P<0.05).(5)The number of Iba-1+cells in poly(I:C)pups(P<0.01)were significantly more than controls in mPFC.However,the number of Iba-1+ cells were decreased in electroacupuncture and IL-6 group compared with the poly(I:C)pups(P<0.01).Conclusion1.Both prenatal electroacupuncture intervention and IL-6-neutralizing antibody treatment could increase the mean body mass of pregnant mice,improve the abortion rate and increase the average litter size.2.Prenatal electroacupuncture can significantly lower MCP-1 and IL-6 in maternal serum and fetal brain.3.Both electroacupuncture intervention and L-6-neutralizing antibody treatment could decrease CD68+CD86+M1 cells and increased of CD68+CD206+M2 cells.4.Both electroacupuncture intervention and L-6-neutralizing antibody treatment could improve poly(I:C)-Induced schizophrenia-like behavior,which appear as the decrease of exploration behavior and the enhancement of spatial learning,memory ability and sensory gating ability.5.Both prenatal electroacupuncture intervention and IL-6-neutralizing antibody treatment could reduce poly(I:C)-induced proliferation of microglia in mPFC.6.The maternal macrophages are crucial to the potent role of electroacupuncture in pregnant mice and offspring. |
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