Sex differences in prefrontal cortex microglia morphology: impact of a two-hit model of adversity throughout development

Neuroimmune mechanisms play critical roles in brain development and can be impacted by early life adversity.  Microglia are the resident immune cells in the brain, with both sex-specific and region-specific developmental profiles.  Since early life adversity is associated with several neuropsychiatric disorders with developmental pathogeneses, here we investigated the degree to which maternal separation (MS) impacted microglia over development. Microglia are dynamic cells that alter their morphology in accordance with their activation states and in response to stressors.  While males and females reportedly display different microglial morphology in several brain regions over development and following immune and psychological challenges, little is known about such differences in the prefrontal cortex (PFC), which regulates several early life adversity-attributable disorders.  Additionally, little is known about the potential for early life adversity to prime microglia for later immune challenges.  In the current study, male and female rats were exposed to maternal separation followed by lipopolysaccharide administration in juvenility or adolescence.  The prelimbic and infralimbic PFC were then separately analyzed for microglial density and morphology.  Typically developing males expressed smaller soma and less arborization than females in juvenility, but larger soma than females in adolescence.  MS led to fewer microglia in the infralimbic PFC of adolescent males.   Both MS and lipopolysaccharide administration affected morphological characteristics in juvenile males and females, with MS exposure leading to a greater increase in soma size following lipopolysaccharide.  Interestingly, adolescent rats generally did not display lasting effects of MS nor immediate effects of lipopolysaccharide on PFC microglial morphology.  Taken together, these findings provide insight into how PFC microglia may differentially respond to challenges over development in males and females.

Methods
Microscopy & Image Analysis

Images of PFC sections were taken at 20x magnification (image size: 440µm x 330µm) on a Zeiss Axio Imager M2 microscope system. For each animal, twelve regions of interest per hemisphere, over three sections, were used to determine Iba1-labeled microglia quantification and morphological differences. All analyses were performed in the PFC prelimbic (PL) and infralimbic (IL) areas using ImageJ [1]. The total number of Iba-1 positive cells per image was quantified using “Multi-Point” tool. To determine morphological alterations in microglia, soma size was measured using ImageJ “Freehand line” followed by “Analyze and Measure.” Images were then converted to binary and skeletonized to further identify microglial process complexity and ramification. The “Analyze Skeleton” ImageJ plugin was used to quantify the number of branches, junctions, and end-points, as well as the average branch length, for each identified cell [2]. Images were thresholded, where all foreground cells and respective processes were visible from the background, while adhering to an optimized cutoff range. Fragments of microglial processes were removed via a script using the Python programming language (Python Software Foundation, Fredericksburg, VA). Raw data for each image was organized in descending order by branch number and all fragments after the corresponding cell number for that image were removed. Measures were then summed and normalized to the number of cells per image. All image acquisition and analyses were performed by an experimenter blind to experimental condition.

[1] C.A. Schneider, W.S. Rasband, K.W. Eliceiri, NIH Image to ImageJ: 25 years of image analysis, Nat Methods 9 (2012) 671-675.

[2] I. Arganda-Carreras, R. Fernández-González, A. Muñoz-Barrutia, C. Ortiz-De-Solorzano, 3D reconstruction of histological sections: Application to mammary gland tissue, Microsc Res Tech 73 (2010) 1019-1029.

神经免疫机制在脑发育中发挥关键作用，且可受到早期生活逆境（early life adversity）的影响。小胶质细胞（microglia）是脑内的驻留免疫细胞，兼具性别特异性与区域特异性的发育表型。由于早期生活逆境与多种具有发育病理机制的神经精神疾病相关，本研究探讨了母婴分离（maternal separation, MS）在发育过程中对小胶质细胞的影响程度。小胶质细胞是一类动态细胞，可根据其激活状态及对应激源的响应改变形态。尽管已有研究表明，在发育过程中以及免疫与心理应激后，雌雄个体在多个脑区的小胶质细胞形态存在差异，但目前对于前额叶皮层（prefrontal cortex, PFC）中的此类差异知之甚少——而前额叶皮层正是多种由早期生活逆境引发的疾病的调控脑区。此外，关于早期生活逆境能否使小胶质细胞致敏以应对后续免疫应激的潜在机制，同样缺乏研究。

在本研究中，我们让雌雄大鼠接受母婴分离处理，并在少年期或青春期给予脂多糖（lipopolysaccharide）给药。随后分别对前额叶皮层的 prelimbic 区（PL）与 infralimbic 区（IL）的小胶质细胞密度与形态进行分析。

正常发育的大鼠中，少年期雄性的胞体更小、突起分支更少，而青春期雄性的胞体则大于雌性。母婴分离会导致青春期雄性大鼠的 infralimbic 区前额叶皮层内小胶质细胞数量减少。

母婴分离与脂多糖给药均可影响幼年雌雄大鼠的小胶质细胞形态特征，且母婴分离可使脂多糖给药后的胞体尺寸增幅更大。值得注意的是，青春期大鼠一般未表现出母婴分离的长期效应，也未出现脂多糖给药对前额叶皮层小胶质细胞形态的即时影响。综上，本研究结果为前额叶皮层小胶质细胞如何在发育过程中对雌雄个体产生差异化应激响应提供了新的见解。

方法
显微镜成像与图像分析

本研究使用蔡司Axio Imager M2显微镜系统，以20倍放大倍率拍摄前额叶皮层切片图像（单张图像尺寸：440μm×330μm）。对于每只动物，我们选取3个切片中每个半球的12个感兴趣区域，用于定量分析Iba1标记的小胶质细胞数量及形态差异。所有分析均在前额叶皮层的 prelimbic 区与 infralimbic 区内通过ImageJ软件完成[1]。

使用ImageJ的多点工具（Multi-Point Tool）定量每张图像中Iba1阳性细胞的总数。为分析小胶质细胞的形态变化，我们首先使用ImageJ的自由手绘线工具（Freehand Line Tool）测量胞体尺寸，随后执行分析与测量命令（Analyze and Measure）。随后将图像转换为二值图并进行骨架化处理，以进一步分析小胶质细胞突起的复杂性与分支程度。使用ImageJ的分析骨架插件（Analyze Skeleton Plugin）对每个识别出的细胞的分支数、连接点、末端点数量以及平均分支长度进行定量[2]。

对图像进行阈值化处理，使所有前景细胞及其突起均可与背景区分开，同时需符合优化后的截断值范围。使用Python编程语言（Python Software Foundation, 弗雷德里克斯堡, 弗吉尼亚州）编写脚本去除小胶质细胞突起的碎片。将每张图像的原始数据按分支数降序排列，移除该图像对应细胞数量之后的所有碎片数据。随后将各项测量值求和，并归一化至每张图像中的细胞数量。所有图像采集与分析过程均由对实验分组不知情的实验人员完成。

[1] C.A. Schneider, W.S. Rasband, K.W. Eliceiri, NIH Image to ImageJ: 25 years of image analysis, Nat Methods 9 (2012) 671-675.
[2] I. Arganda-Carreras, R. Fernández-González, A. Muñoz-Barrutia, C. Ortiz-De-Solorzano, 3D reconstruction of histological sections: Application to mammary gland tissue, Microsc Res Tech 73 (2010) 1019-1029.