J. Lee1, K. Sail1, T. Costantini1, B. Eliceiri1, R. Coimbra1, V. Bansal1 1University Of California – San Diego,Department Of Surgery, Division Of Trauma, Surgical Critical Care And Burns,San Diego, CA, USA
Introduction: The invasion of leukocytes following traumatic brain injury (TBI) may have significant consequences. Excessive leukocytes are not beneficial and instead may cause an outsized inflammatory response initiating secondary neuronal injury and exacerbating long-term cognitive dysfunction. Therefore, modulating leukocyte migration may lead to novel therapy. Previously, we have shown that Esophageal Cancer Related Gene 4 (Ecrg4) is a novel leukocyte chemoattractant in glioblastoma. We hypothesize that Ecrg4 is a potent mediator of TBI induced leukocyte recruitment.
Methods: A controlled cortical impact model was used to create moderate TBI in 12 wk old wild type (WT) or Ecrg4 knock-out (KO) mice. Three days post-TBI, cells were isolated and flow cytometry analysis was used gating for microglia (CD45lowCD11b+ cells), monocytes (CD45highCD11b+ cells), and lymphocytes (CD45highCD11b- cells). Brain sections were immunostained with Iba-1 and CD11b to assess recruitment and activation of microglia and migrating leukocytes. Brain homogenates were collected following TBI and cytokine levels (IL-12p70, TNF, IFN-γ , MCP-1, IL-10, and IL-6) were measured by multiplex enzyme-linked immunosorbent assay (ELISA).
Results: TBI resulted in a rapid invasion of activated microglia and inflammatory monocytes, peaking at post-TBI Day 3 and persisting at Day 7 (19 ±30 vs. 1018 ± 694; p<0.05). Invading cells expressed surface markers for activation (MHCII+ and CD86+). Ecrg4 KO mice demonstrated reduced infiltration of microglia (101 ±694 vs. 299 ±143; p<0.05) and inflammatory monocytes (314 ±155 vs. 163 ±118; p<0.05) compared with Ecrg4 WT after TBI. Activated microglia (471 ±326 vs. 125 ±63; p<0.05) were also reduced in Ecrg4 KO mice when compared to Ecrg4 WT mice. TBI induced an increase in pro-inflammatory mediators (TNF, MCP-1, and IL-6), peaking at six hours post-TBI. TNF levels in the brain were significantly decreased in Ecrg4 KO mice compared with Ecrg4 WT mice (31± 12 vs. 4±2pg/ml; p<0.05) following TBI.
Conclusion: Genetically deficient Ecrg4 mice had altered leukocyte invasion and microglial activation following TBI, confirming Ecrg4’s role as a novel chemoattractant. Future studies will assess whether Ecrg4-mediated leukocyte invasion alters post-TBI histopathology and neurocognitive recovery.