Tracking labeled stem cells in TBI model by cellular MRI

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Abstract

The pathology of traumatic brain injury in experimental models includes acute inflammatory reaction, blood brain barrier disruption, hemorrhage, demyelination, axonal transection and chronically with axonal neuronal loss and gliosis. Stem cell (SC) therapy is a potential treatment either as replacement therapy or via paracrine effect with release of growth factors and anti-inflammatory cytokines for TBI injury. Experimental studies in rodent models of TBI have been limited and usually a single dose of cells is administered within 24 to 72 hours after experimental injury. The optimal timing and dose of cell delivery to maximize functional recovery and transplantation survival during the acute inflammatory and edematous phase of damage is unknown. We evaluated the natural history of control cortical impact (CCI) to induce TBI in the rat brain by serial MRI. CCI was performed in left motor cortex (Bregma +1 lateral 2 mm) with impact time 50 msec at depth of 2mm and velocity 5 meters/seconds in female Wistar rats. MRI was performed at 7 Tesla. T2w and T2*w images with image resolution of 117x117x500m (days 2,9,30 and 58) and behavior (days 1,14, 28 and 56) were obtained at various time points after CCI. We observed that the appearance and volume of CCI-induced lesions at days 2, 9, and 30 was highly variable despite identical CCI injury settings. There was little correlation between the percent change of CCI side to contralateral side cortical volumes on days 2 to subsequent exams on 9, and 30 (mean percent change CCI cortical/contralateral cortex volume: Day 2=2611%; Day 9 = -214%; Day 30 = -1014%). Hemorrhagic conversion within the CCI lesion occurred in 45% of rats between days 2 and 9 post CCI. MRI and pathology demonstrate significant variation in cortical loss, gliosis, axonal loss and cyst formation using identical impact parameters and techniques. Although the variation in CCI lesions may be attributed to differences in technique, the divergence of similar lesions between days 2 and 30 demonstrates the inherent biological variability of the CCI rat model. The possibility of hemorrhagic evolution between days 2 and 9 also raised concerns about previous interpretation of using magnetically labeled cells to track their delivery to CCI lesions without obtaining a pre-stem cell infusion MRI, as hemorrhage appears identical on MRI and Prussian blue staining. Based on this study, MRI lesion or cortical volumes could be used as an outcome measure for novel therapies of experimental TBI, however the ability to detect 10 to 20% difference in cortical volume with alpha 0.05 and power =0.8 as a result of treatment, would require between 22-64 subjects would be needed to see significant differences in a randomized parallel group trial design. There was no correlation of lesion volume or percent cortical differences and behavioral measures at 30 days. We have developed an image analysis method for fusing MRI diffusion tensor imaging (DTI) findings to immunohistological stains. In models, increased Fractional Anisotropy (FA) has been argued to be associated with axonal reorganization and re-growth following stroke or TBI, but the histological evidence for such changes appeared tenuous. Histological sections from rats at 2 months post CCI were stained for GFAP (astrocytes), SMI31 & SMI32 (neurofilaments), MAP2 (microtubules), and MBP (myelin). The histological-based Fourier analysis captured the microscopic anisotropy and orientation as measured by DTI. In the CCI injured white matter (WM), FA decreased and was consistent with axonal and myelin injury, although astrocytes also had high anisotropy. In gray matter, FA increased and this was consistent with coherent astrogliosis and to a lesser extent, dendrite organization. These results demonstrate the microscopic cellular and subcellular elements that contribute to anisotropy in normal brain (axons and myelin) are not necessarily the only contributors to anisotropy in the injured brain. Astrocyte remodeling and migration also appears consistent with the DTI-measured anisotropy. This analytical method should help provide a quantitative and specific relationship between the microscopic anisotropy and DTI-derived anisotropy.