Microglia Reaction and Peripheral Immune Cells in Spinal Cord Injury

Loss of Ia/VGluT1 synapses requires a microglia reaction (Iba1⁺, CD11b⁺ cells) and activation of CCR2, which is also associated with infiltration of CD45⁺ CCR2⁺ CD11b⁺ peripheral macrophages and CD45⁺ CCR2⁺ CD3e⁺ T cells (see Introduction). The microglia reaction was analyzed by counting all Iba1⁺ cells in the ventral horn of the spinal cord at 3 d (n = 3), 5 d (n = 3), 7 d (n = 4), 14 d (n = 4), 21 d (n = 4), and 60 d (n = 2) after nerve crush injury at P10. To test for the presence of peripheral immune cells we tested for Iba1⁺, CD11b⁺, and/or CD45⁺ cells at 7, 14, 21, and 60 d after the injury. Transverse sections (50 μm thick) of L4-5 spinal cord were obtained in a sliding freezing microtome, collected free floating, blocked for 1 h with 10% normal donkey serum diluted in PBST, and incubated overnight at room temperature in a mixture containing a goat polyclonal antibody against Iba1 (1:500; Novus Biologicals; RRID:AB_521594) and a rat monoclonal antibody against CD11b (1:50; OX-43; catalog #M1/70.15.11.5.2-s, Developmental Hybridoma Bank) or a rabbit polyclonal antibody against CD45 (1:50; Abcam; RRID:AB_442810). Sections were washed in PBS and incubated for 2 h in donkey anti-goat IgG antibody conjugated to FITC and donkey anti-rat or anti-rabbit IgG conjugated to Cy3 (all secondary antibodies diluted 1:200 in PBST; Jackson ImmunoResearch). The sections were mounted on slides, coverslipped with Vectashield (Vector Laboratories), and imaged throughout with a confocal microscope (model FV1000, Olympus) using 1 μm z-steps and a 20× objective. Overlapping image tiles were captured to sample the whole spinal cord. At least three sections were imaged for each animal. We found no CD45⁺ cells, and therefore quantitative analyses focused on Iba1⁺ microglia. The ventral horn of each hemisection was manually selected by marking a region of interest (ROIs) limited dorsally by a straight horizontal line above the central canal and in all other directions by the border between gray and white matter. All Iba1⁺ cells inside these ROIs were counted using ImageJ software. Because the spinal cord changes in size with maturation, we calculated cell densities, as follows: the number of Iba1⁺ microglia divided by the ventral horn volume calculated from the ROI area multiplied by section thickness (50 μm). Densities ratios were obtained for each section between the side ipsilateral to the injury and the contralateral control side.