Microglial Modulation of Neuronal Integrity

Cortices from P1 rats were finely minced and digested for 30 min at 37 °C in DPBS (Gibco) containing papain (Worthington Biochemicals) and DNase (Sigma-Aldrich). papain was inhibited by the addition of ovomucoid (Worthington Biochemicals). Neurons were plated at a density of 60,000 cells/cm² on poly-D-lysine pre-coated 8-well Permanox chamber slides (Sigma-Aldrich) in Neurobasal/B27 medium (Invitrogen) and cultured for 10 days. Rat BMDMs were cultured in RPMI-1640 with 10% heat-inactivated FBS (Invitrogen), 1% penicillin-streptomycin (Corning), and 10 ng/ml rat M-CSF (#400–28, Peprotec). BMDMs were plated on 25 μg/ml fibrin-coated plates with 20 μg/ml 5B8 or IgG2b for 24 h and were lifted with PBS-EDTA as described^{16 (link)} and added to cortical neuron cultures for two days, fixed with 4% PFA and immunostained with anti-MAP-2 (1:1000; clone AP20, EMD Millipore) and thresholded images were quantified with the NeurphologyJ plug-in in ImageJ. 2.5 × 10¹⁰ GC/ml of AAV1.hSyn.TurboRFP (University of Pennsylvania Vector Core) was used to transduce primary cortical neurons for 8 d prior to the addition of fibrin-stimulated BMDMs for 12 h. RFP images were thresholded and the neurite fragments were analyzed using the ImageJ plugin ‘Analyze Particles’. Quantification was performed by an observer blinded to the experimental treatments.