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）会导致人类出现性二态性的海马依赖性认知与记忆损伤。在本研究此前建立的、可转化应用的HDP诱导IUGR小鼠模型中，我们已证实：在相当于人类青少年阶段的小鼠出生后第40天（P40），海马背侧的突触发育——包括γ-氨基丁酸能（GABAergic）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受到扰动。目前，这些扰动在成年早期的持续状态及其潜在上游调控机制尚不明确。
因此，我们提出如下假说：海马内参与突触发育闭合的所有事件，即NPTX2阳性表达、PNN形成与轴突髓鞘化，将持续受到扰动；结合该模型中雌性小鼠短期识别记忆受损更严重的现象，该扰动尤其会影响直至出生后第60天（P60）的IUGR雌性小鼠。我们进一步推测，这种性二态性与持续的神经胶质功能失调相关。
我们通过在C57BL/6小鼠妊娠最后一周，经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，以诱发HDP并建立IUGR模型，以假手术组小鼠作为对照。于P60时，我们评估了海马与全脑体积、NPTX2表达、PNN形成情况，以及髓鞘碱性蛋白（Myelin Basic Protein, MBP）、少突胶质细胞转录因子Olig2、APC/CC1与神经丝蛋白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 Histochemistry, IF-IHC）结合Imaris形态学分析，检测了P60小鼠的星形胶质细胞（胶质纤维酸性蛋白, GFAP）反应性与小胶质细胞（离子钙接头蛋白分子1, Iba1和跨膜蛋白119, TMEM119）激活状态，并通过中尺度发现平台（Mesoscale Discovery Platform, MSD）开展细胞因子谱分析。
结果显示，P60时IUGR子代小鼠仍表现出更小的海马体积，且该变化与全脑体积改变无关。与同性别假手术组相比，IUGR雌性小鼠海马CA亚区的NPTX2阳性斑点数量与体积均显著降低；有趣的是，其齿状回（Dentate Gyrus, DG）亚区的NPTX2阳性斑点数量与体积却同时升高。IUGR雌性小鼠CA1与CA3区的PNN体积更小，且CA3区的PNN强度降低；而IUGR雄性小鼠CA3区的PNN体积则更大。与同性别假手术组相比，IUGR雌性小鼠CA1区的髓鞘化轴突（MBP阳性）面积、体积与长度均显著降低，该变化与Olig2核表达水平下降相关，但未观察到APC/CC1阳性成熟少突胶质细胞数量减少。仅在IUGR雌性小鼠中，连接DG与CA3的苔藓纤维内的M-NF表达水平出现升高。与同性别假手术组相比，IUGR雌性小鼠CA1区以及IUGR雄性小鼠CA3区中，以GFAP标记的反应性星形胶质细胞的面积、体积、长度与分支数量均显著增加。仅在IUGR雌性小鼠的CA1与CA3亚区中检测到激活的小胶质细胞。两种性别的假手术组与IUGR成年小鼠的细胞因子谱均无显著差异。
综上，本研究数据证实，年轻成年IUGR小鼠的海马突触发育闭合过程存在性二态性受损，且雌性小鼠受影响更为显著。支持该性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细胞介导的突触修剪。