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Simon Dennis; Aneja, Raj; Lewis, Jesse; Bayir, Hulya; Bell, Michael; Kochanek, Patrick; Clark, Robert
Critical Care Medicine: December 2013
doi: 10.1097/01.ccm.0000439195.54340.48
Oral Abstract Session: Brain Injury, Repair, and Recovery: PDF Only

Introduction: Microglial activation may be robust in the developing brain after traumatic brain injury (TBI). Minocycline (MINO), a second-generation tetracycline antibiotic, has been shown to be neuroprotective in multiple forms of brain injury including TBI, but whether or not this is linked to its effects on inflammation and subsequent secondary brain injury is undetermined. An anti-inflammatory effect of MINO has been linked to high mobility group box-1 (HMGB1), a non-histone DNA-binding protein, that can translocate from the nucleus to cytoplasm and extracellular space following injury and serves as a danger-associated molecular pattern molecule (DAMP). In the present study, we investigated the role of MINO on microglial activation and HMGB1 translocation after severe TBI in postnatal day (PND) 17 rats. Methods: Anesthetized PND 17 Sprague-Dawley rats were subjected to controlled cortical impact (CCI) with velocity 4 m/s, depth 0.4 mm, and duration of 50 ms. Rats were randomized to treatment with MINO (90 mg/kg i.p.) given 10 min and 24 h after injury) or vehicle (n=6/group). Naïve rats were used for comparison to resting microglia (n=3/group). At 7 d rats were sacrificed. We evaluated microglial activation with immunohistochemistry using an ionized calcium-binding adaptor molecule-1 (Iba-1) antibody and HMGB1 translocation with confocal microscopy using an anti-HMGB1 antibody labeled with an Alexa488 fluorophore and co-labeled with 4’,6-diamidino-2-phenylindole (DAPI) to identify nuclei. Results: After TBI, an increase in Iba-1 immunoreactive, “amoeboid” microglia were observed in the ipsilateral cortex, hippocampus, and thalamus consistent with activation, in contrast to naïve rats which showed weakly immunoreactive, ramified microglia. Treatment with MINO reduced the number of activated microglia in the hippocampus (595 ± 110 vs. 2341 ± 647), cortex (1103 ± 181 vs. 3076 ± 502), and thalamus (1348 ± 233 vs. 2679 ± 149; all p<0.05 vs. vehicle). HMGB1 was localized in the nucleus in naïve rats. Following TBI, extranuclear HMGB1 was observed in cells with the morphological appearance of microglia and neurons in the ipsilateral thalamus and hippocampus. MINO appeared to reduce nuclear translocation of HMGB1. Conclusions: Our data suggest that MINO inhibits microglial activation and HMGB1 translocation after TBI in the developing brain. Further study is needed to evaluate the functional and histological effects of MINO treatment, as well as any direct mechanistic link between HMGB1 and microglial activation, after TBI in the developing brain. Support: T32 HD040686

© 2013 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins