Introduction: Recent studies have demonstrated that lymphocytes play a key role in ischemic brain injury. However, there is still a lack of viable approaches to non-invasively track infiltrating lymphocytes and reveal their key spatiotemporal events in the inflamed central nervous system (CNS). In the present study, we describe an in vivo imaging approach for sequential monitoring of brain-infiltrating CD4+ T cells in experimental ischemic stroke.
Methods: CD4+ T cells were obtained from splenocytes of C57BL/6 mice. After in vitro incubation with SPIO-Molday ION Rhodamine-B (MIRB) for 24 h, CD4+Rh-B+ T cells were sorted and purified with FACS followed by subsequent passive transfer into Rag2-/- recipient mice (lack of T and B cells). After sham or 60 min of MCAO procedures with designated time of reperfusion, MIRB-labeled cells were sequentially visualized using 7T-MRI and Xenogen imaging. In separate groups of Rag2-/- mice that received MIRB-labeled CD4+ T cells prior to sham or MCAO surgeries, brain, liver and spleen sections were obtained from these animals after MRI scans and immunostained with CD4 specific antibody, and the images were captured by a fluorescence microscopy.
Results: We show that MRI or Xenogen imaging combined with labeling of MIRB can be used to monitor the dynamics of CD4+ T cells in a passive transfer model (Figure 1). MIRB-labeled CD4+ T cells can be longitudinally visualized in the mouse brain and peripheral organs such as the spleen and liver after cerebral ischemia. Immunostaining of tissue sections showed similar kinetics of MIRB-labeled CD4+ T cells when compared with in vivo observations.
Conclusion: Our results demonstrated the use of MIRB coupled with in vivo imaging as a valid method to track CD4+ T cells in ischemic brain injury. This approach will facilitate future investigations to identify the dynamics and key spatiotemporal events for brain-infiltrating lymphocytes in CNS inflammatory diseases.