Early monitoring and quantitative evaluation of macrophage infiltration after experimental traumatic brain injury: A magnetic resonance imaging and flow cytometric analysis

- Macrophages were labeled in vivo via intravenous injection of USPIO in TBI mice.
- Maximum macrophage infiltration occurred between 66 and 72 h post injuries in MRI study.
- A 7-fold increase in infiltrating macrophages occurred after 72 h post injury.
- PB staining and F4/80 staining were well supported with quantitative T2* data.

The inflammatory response following traumatic brain injury (TBI) is regulated by phagocytic cells. These cells comprising resident microglia and infiltrating macrophages play a pivotal role in the interface between early detrimental and delayed beneficial effects of inflammation. The aim of the present study was to monitor the early effect of monocyte/phagocytic accumulation and further to explore its kinetics in TBI mice. Localized macrophage population was monitored using ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle enhanced in vivo serial magnetic resonance imaging (MRI). Flow cytometry based gating study was performed to discriminate between resident microglia (Ly6G− CD11b+ CD45low) and infiltrating macrophages (Ly6G− CD11b+ CD45high) at the injury site. The T2* relaxation analysis revealed that maximum macrophage infiltration occurs between 66 and 72 h post injury (42-48 h post administration of USPIO) at the site of inflammation. This imaging data was well supported by iron oxide specific Prussian blue staining and macrophage specific F4/80 immunohistochemistry (IHC) analysis. Quantitative real-time PCR analysis found significant expression of monocyte chemoattractant protein-1 (MCP-1) at 72 h post injury. Also, we found that flow cytometric analysis demonstrated a 7-fold increase in infiltrating macrophages around 72 h post injuries as compared to control. The MR imaging in combination with flow cytometric analysis enabled the dynamic measurement of macrophage infiltration at the injury site. This study may help in setting an optimal time window to intervene and prevent damage due to inflammation and to increase the therapeutic efficacy.

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