Supplementary Material for: Sexual dimorphism in the closure of the hippocampal postnatal critical period of synaptic plasticity after intrauterine growth restriction – link to oligodendrocyte and glial dysregulation

Intrauterine growth restriction (IUGR) resulting from hypertensive disease of pregnancy (HDP) leads to sexually dimorphic hippocampal-dependent cognitive and memory impairment in humans. In our translationally-relevant mouse model of IUGR incited by HDP, we have previously shown that the synaptic development in the dorsal hippocampus including GABAergic development, NPTX2+ excitatory synaptic formation, axonal myelination, and perineural net (PNN) formation, were perturbed by IUGR at adolescent equivalence in humans (P40). The persistence of these disturbances through early adulthood and the potential upstream mechanisms are currently unknown. Thus, we hypothesized that NPTX2+ expression, PNN formation, axonal myelination, all events closing synaptic development in the hippocampus, will be persistently perturbed, particularly affecting IUGR female mice through P60 given the fact that they had worse short-term recognition memory in this model. We additionally hypothesized that such sexual dimorphism is linked to persistent glial dysregulation. We induced IUGR by a micro-osmotic pump infusion of a potent vasoconstrictor U-46619, a thromboxane A2-analog (TXA2), in the last week of the C57BL/6 mouse gestation to precipitate HDP. Sham-operated mice were used as controls. At P60, we assessed hippocampal and hemispheric volumes, NPTX2 expression, PNN formation, as well as MBP, Olig2, APC/CC1, and M-NF expression. We also evaluated P60 astrocytic (GFAP) reactivity and microglial (Iba1 and TMEM119) activation using IF-IHC and Imaris morphological analysis plus cytokine profiling using mesoscale discovery platform (MSD). IUGR offspring continued to have smaller hippocampal volumes at P60 not related to changes in hemisphere volume. NPTX2+ puncta counts and volumes were decreased in IUGR hippocampal CA sub-regions of female mice compared to sex-matched shams. Intriguingly, NPTX2+ counts and volumes were concurrently increased in the DG sub-region. PNN volumes were smaller in CA1 and CA3 of IUGR female mice along with PNN intensity in CA3 but they had larger volumes in the CA3 of IUGR male mice. The myelinated axon (MBP+) areas, volumes and lengths were all decreased in the CA1 of IUGR female mice compared to sex-matched shams, which correlated with a decrease in Olig2 nuclear expression. No decrease number in APC/CC1+ mature oligodendrocytes was identified. We noted an increased in M-NF expression in the mossy fibers connecting DG to CA3 only in IUGR female mice. Reactive astrocytes denoted by GFAP areas, volumes, lengths, and numbers of branching were increased in IUGR female CA1 but in IUGR male CA3 compared to sex-matched shams. Lastly, activated microglia was only detected in IUGR female CA1 and CA3 sub-regions. We detected no difference in the cytokine profile between sham and IUGR adult mice of either sex. Collectively our data support a sexually-dimorphic impaired closure of pCP in the hippocampus of young adult IUGR mice with greater effects on females. A potential mechanism supporting such dimorphism may include oligodendrocytes dysfunction in IUGR females limiting myelination allowing axonal overgrowth followed by a reactive glial-mediated synaptic pruning.

由妊娠期高血压疾病（hypertensive disease of pregnancy, HDP）诱发的宫内生长受限（intrauterine growth restriction, IUGR）会导致人类出现性别二态性的海马依赖型认知与记忆损伤。在我们构建的、与临床转化相关的HDP诱发IUGR小鼠模型中，既往研究已证实，在相当于人类青少年期的小鼠出生后第40天（P40），背侧海马的突触发育——包括γ-氨基丁酸能神经元发育、NPTX2阳性兴奋性突触形成、轴突髓鞘化以及神经周围网（perineural net, PNN）形成——均因IUGR而出现紊乱。目前，这些紊乱在成年早期的持续存在情况及其潜在上游调控机制尚不明确。因此，我们提出如下假说：作为海马突触发育闭合相关事件的NPTX2表达、PNN形成以及轴突髓鞘化将持续紊乱，且鉴于本模型中IUGR雌性小鼠的短期识别记忆损伤更为严重，这种紊乱在出生后第60天（P60）的IUGR雌性小鼠中表现尤为显著。我们同时提出另一假说：此类性别二态性与持续存在的胶质细胞功能失调相关。我们通过在C57BL/6小鼠妊娠期最后一周向其体内微量渗透泵输注强效血管收缩剂血栓素A2类似物（thromboxane A2-analog, TXA2）U-46619，以构建HDP诱发的IUGR模型，并以假手术组小鼠作为对照。在小鼠出生后第60天（P60）时，我们检测了海马与大脑半球体积、NPTX2表达、PNN形成情况，以及髓鞘碱性蛋白（myelin basic protein, MBP）、Olig2、APC/CC1和神经丝蛋白M（neurofilament M, M-NF）的表达水平。此外，我们采用免疫荧光组织化学（immunofluorescence immunohistochemistry, IF-IHC）结合Imaris形态学分析评估了星形胶质细胞（胶质纤维酸性蛋白, GFAP）反应性与小胶质细胞（Iba1和TMEM119）活化情况，并通过介观尺度发现平台（mesoscale discovery platform, MSD）进行了细胞因子谱分析。P60阶段的IUGR子代小鼠仍表现出海马体积减小，且该现象与大脑半球体积变化无关。与性别匹配的假手术组相比，IUGR雌性小鼠海马CA亚区的NPTX2阳性斑点计数与体积均有所降低；有趣的是，齿状回（dentate gyrus, DG）亚区的NPTX2阳性斑点计数与体积却同时升高。IUGR雌性小鼠的CA1与CA3区PNN体积更小，且CA3区的PNN强度降低；而IUGR雄性小鼠的CA3区PNN体积则更大。与性别匹配的假手术组相比，IUGR雌性小鼠CA1区的髓鞘化轴突（MBP阳性）面积、体积与长度均有所降低，这与Olig2细胞核表达水平下降相关，但未观察到成熟少突胶质细胞（APC/CC1阳性）数量减少。我们仅在IUGR雌性小鼠中发现，连接DG与CA3的苔藓纤维内的M-NF表达出现升高。反应性星形胶质细胞的相关指标（GFAP阳性区域面积、体积、长度以及分支数量）在IUGR雌性小鼠的CA1区以及IUGR雄性小鼠的CA3区均较性别匹配的假手术组有所升高。最后，活化小胶质细胞仅在IUGR雌性小鼠的CA1与CA3亚区中被检测到。我们未在任意性别的假手术组与IUGR成年小鼠中观察到细胞因子谱的差异。综上，我们的数据证实，年轻成年IUGR小鼠的海马突触发育闭合过程存在性别二态性损伤，且雌性小鼠受影响更为严重。介导此类性别二态性的潜在机制可能包括：IUGR雌性小鼠的少突胶质细胞功能障碍限制了髓鞘形成，进而导致轴突过度生长，随后引发反应性胶质介导的突触修剪。