Macrophagic and microglial responses after focal traumatic brain injury in the female rat.
BACKGROUND
After central nervous system injury, inflammatory macrophages (M1) predominate over anti-inflammatory macrophages (M2). The temporal profile of M1/M2 phenotypes in macrophages and microglia after traumatic brain injury (TBI) in rats is unknown. We subjected female rats to severe controlled cortical impact (CCI) and examined the postinjury M1/M2 time course in their brains.


METHODS
The motor cortex (2.5 mm left laterally and 1.0 mm anteriorly from the bregma) of anesthetized female Wistar rats (ages 8 to 10 weeks; N = 72) underwent histologically moderate to severe CCI with a 5-mm impactor tip. Separate cohorts of rats had their brains dissociated into cells for flow cytometry, perfusion-fixed for immunohistochemistry (IHC) and ex vivo magnetic resonance imaging or flash-frozen for RNA and protein analysis. For each analytical method used, separate postinjury times were included for 24 hours; 3 or 5 days; or 1, 2, 4 or 8 weeks.


RESULTS
By IHC, we found that the macrophagic and microglial responses peaked at 5 to 7 days post-TBI with characteristics of mixed populations of M1 and M2 phenotypes. Upon flow cytometry examination of immunological cells isolated from brain tissue, we observed that peak M2-associated staining occurred at 5 days post-TBI. Chemokine analysis by multiplex assay showed statistically significant increases in macrophage inflammatory protein 1α and keratinocyte chemoattractant/growth-related oncogene on the ipsilateral side within the first 24 hours after injury relative to controls and to the contralateral side. Quantitative RT-PCR analysis demonstrated expression of both M1- and M2-associated markers, which peaked at 5 days post-TBI.


CONCLUSIONS
The responses of macrophagic and microglial cells to histologically severe CCI in the female rat are maximal between days 3 and 7 postinjury. The response to injury is a mixture of M1 and M2 phenotypes.
Center for Neuroscience and Regenerative Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA. turtzolc@mail.nih.gov.
L Christine
LC
Turtzo
Jacob
J
Lescher
Lindsay
L
Janes
Dana D
DD
Dean
Matthew D
MD
Budde
Joseph A
JA
Frank
D000704:Analysis of Variance; D000818:Animals; D001921:Brain / Q000378:metabolism / Q000473:pathology*; D001930:Brain Injuries / Q000473:pathology* / Q000503:physiopathology; D002135:Calcium-Binding Proteins / Q000378:metabolism; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D005260:Female; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000502:physiology; D008264:Macrophages / Q000473:pathology*; D008279:Magnetic Resonance Imaging; D008840:Microfilament Proteins / Q000378:metabolism; D017628:Microglia / Q000473:pathology*; D008856:Microscopy, Fluorescence; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors
C100745:Aif1 protein, rat; D002135:Calcium-Binding Proteins; D016207:Cytokines; D008840:Microfilament Proteins