MRI evaluation of axonal reorganization after bone marrow stromal cell treatment of traumatic brain injury

Abstract

We treated traumatic brain injury (TBI) with human bone marrow stromal cells (hMSCs) and evaluated the effect of treatment on white matter reorganization using MRI. We subjected male Wistar rats (n = 17) to controlled cortical impact and either withheld treatment (controls; n = 9) or inserted collagen scaffolds containing hMSCs (n = 8). Six weeks later, the rats were sacrificed and MRI revealed selective migration of grafted neural progenitor cells towards the white matter reorganized boundary of the TBI-induced lesion. Histology confirmed that the white matter had been reorganized, associated with increased fractional anisotropy (FA; p < 0.01) in the recovery regions relative to the injured core region in both treated and control groups. Treatment with hMSCs increased FA in the recovery regions, lowered T(2) in the core region, decreased lesion volume and improved functional recovery relative to untreated controls. Immunoreactive staining showed axonal projections emanating from neurons and extruding from the corpus callosum into the ipsilateral cortex at the boundary of the lesion. Fiber tracking (FT) maps derived from diffusion tensor imaging confirmed the immunohistological data and provided information on axonal rewiring. The apparent kurtosis coefficient (AKC) detected additional axonal remodeling regions with crossing axons, confirmed by immunohistological staining, compared with FA. Our data demonstrate that AKC, FA, FT and T(2) can be used to evaluate treatment-induced white matter recovery, which may facilitate restorative therapy in patients with TBI.

Figure 1

Labeled cell migration and distribution after bone marrow stromal cell (MSC) treatment of traumatic brain injury (TBI). (A, B) Three-dimensional MRI (A) and T₂ (B) maps from 2 days (5 days before labeled MSC injection) to 6 weeks after TBI. (C–E) Prussian blue-stained sections (D and E from the boxes in C) obtained from the same rat 6 weeks after TBI, showing clusters of blue cells around the boundary of the lesion and the corpus callosum (D, E, blue cells, red arrowheads), as demonstrated by three-dimensional MRI (A, 6 weeks, red arrowheads).

Figure 2

Identification of labeled human bone marrow stromal cells (hMSCs). Immunofluorescent staining shows human mitochondrial cells (A, red, arrows) positive for Prussian blue (PB) (B, arrows). (C) Merging of (A) and (B). Nuclei were stained with 4′,6-diamidino-2-phenylindole (dapi) (blue). Bar, 20 μm.

Figure 3

(A, B) Quantitative characterization of MRI parameters in traumatic brain injury (TBI)-damaged tissue with and without human bone marrow stromal cell (hMSC) treatment. The graphs show the evolution of changes in fractional anisotropy (FA) and T₂ with and without scaffold + hMSC treatment of TBI. Significant differences were detected in FA (p <0.05 at 6 weeks in the recovery region) and T₂ (p < 0.01 at 2 and 3 weeks and p <0.05 at 4 and 6 weeks in the core region) between treated and nontreated groups. ^*p <0.05 and ^**p <0.01 comparing treated and nontreated groups.

Figure 4

Functional recovery after bone marrow stromal cell (MSC) treatment of traumatic brain injury (TBI). The graphs show the functional improvement as detected with the modified neurological severity score (mNSS, A) and modified Morris water maze test (B). ^*p <0.05 and ^**p <0.01 comparing treated and nontreated groups.

Figure 5

MRI detection of white matter remodeling after bone marrow stromal cell (MSC) treatment of traumatic brain injury (TBI). Evolution of in vivo trace apparent diffusion coefficient (ADC) and fractional anisotropy (FA) maps (A), corresponding ex vivo FA, radial (λ_⊥) and axial (λ_||) diffusivity, apparent kurtosis coefficient (AKC), fiber tracking, Gaussian and q-ball fiber orientation maps (B), and Bielshowsky and Luxol fast blue immunoreactive staining images (C–G) measured in the fixed animal brain. (D–G) High-magnification images from the areas shown in the box in (C) as indicated in the top right-hand corners of (D)–(G).