DOI: 10.5061/dryad.3tx95x6cq
title: Sex differences in prefrontal cortex microglia morphology: impact of a two-hit model of adversity throughout development

description: 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.
description: <i>Microscopy & Image Analysis</i> 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.