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.

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.