Ex vivo diffusion tensor imaging and neuropathological correlation in a murine model of hypoxia-ischemia-induced thrombotic stroke

Abstract

Diffusion tensor imaging (DTI) is a powerful method to visualize white matter, but its use in patients with acute stroke remains limited because of the lack of corresponding histologic information. In this study, we addressed this issue using a hypoxia-ischemia (HI)-induced thrombotic model of stroke in adult mice. At 6, 15, and 24 hours after injury, animals were divided into three groups for (1) in vivo T2- and diffusion-weighted magnetic resonance imaging, followed by histochemistry, (2) ex vivo DTI and electron microscopy, and (3) additional biochemical or immunochemical assays. The temporal changes of diffusion anisotropy and histopathology were compared in the fimbria, internal capsule, and external capsule. We found that HI caused a rapid reduction of axial and radial diffusivities in all three axonal bundles. A large decrease in fractional anisotropy, but not in axial diffusivity per se, was associated with structural breakdown of axons. Furthermore, the decrease in radial diffusivity correlated with swelling of myelin sheaths and compression of the axoplasma. The gray matter of the hippocampus also exhibited a high level of diffusion anisotropy, and its reduction signified dendritic degeneration. Taken together, these results suggest that cross-evaluation of multiple DTI parameters may provide a fuller picture of axonal and dendritic injury in acute ischemic stroke.

Figures

Figure 1

Evaluation of HI-induced brain damage by in vivo T2- and diffusion-weighted MRI. (A to C, E to G, and I to K) T2-weighted MRI of the brains at 6 hours (panels A to C), 15 hours (panels E to G), or 24 hours (panels I to K) after unilateral HI challenge (indicated by asterisks (*)). The representative images shown for each time point in the coronal, sagittal, and horizontal views of the same brain are reconstructed from 3D in vivo T2-weighted images. The corresponding planes for sagittal (S) and horizontal (H) images are indicated. The increase of T2 signals and expansion of the affected area from 6 to 24 hours after HI, which spared the cerebellum (CB) receiving blood supply from vertebrate arteries must be noted. The hyperintensity of T2 signals in the external capsule (ec), alveus/stratum oriens (alv/Or), and the molecular layer of the dentate gyrus (MoDG) at 24 hours after HI must also be noted. (D, H, and L) Apparent diffusion coefficient (ADC) maps at 6 hours (panel D), 15 hours (panel H), or 24 hours (panel L) after HI. (M, N, and O) Quantification of ADC values in the striatum (panel M), hippocampus (panel N), and cerebral cortex (panel O) on the HI-injured (black columns) and the contralateral hemispheres (white columns) at indicated times (n=4 for each time points). Mean and variance values are shown. P-value is determined by the paired t-test. Ctx, cerebral cortex; HI, hypoxia–ischemia; Hip, hippocampus; MRI, magnetic resonance imaging; Se, the septal area; St, striatum; Th, thalamus; 3D, three dimensional.

Figure 2

Assessment of HI-induced WM injury by ex vivo diffusion tensor imaging (DTI). (A) Representative images of transverse/horizontal (the first column) and coronal views (from the rostral to caudal in three columns) of directionally encoded color (DEC) map of the brains at 6, 15, or 24 hours after HI. The directions of color-encoded water diffusion are indicated in the x–y–z axes. The loss of contrast on these maps in the anterior commissure (ac), the external capsule (ec), the internal capsule (ic), and the fimbria/fornix (fm) on the HI-challenged (R) side of the brain at 15 and 24 hours recovery must be noted. (B) The temporal changes of three commonly used DTI parameters (FA, fractional anisotropy; λII, axial diffusivity; λ⊥, radial diffusivity) in the fimbria/fornix, internal capsule, and external capsule. The changes are presented as the ratio of each metric to its counterpart on the contralateral hemisphere at 6, 15, and 24 hours after HI (n=3 for each time point). Mean and s.e. are shown *P<0.05 compared with the individual DTI parameters at 6 hours by ANOVA. The decrease in λII in all three axon tracts must be noted, while a steep decline of FA was only observed in the external capsule. ANOVA, analysis of variance; HI, hypoxia–ischemia; MoDG, molecular layer of the dentate gyrus; opt, optic tract; rad, stratum radiatum; WM, white matter.

Figure 3

Histologic correlates of HI-induced changes in fractional anisotropy. (A to D and G to H) Sections taken from Thy1-YFP mice at 15 hours after HI (n=5) to detect axonal integrity in the external capsule (ec), internal capsule (ic), and the fimbria/fornix (fm). The selective axonal degeneration in the external capsule, but not in the internal capsule or the fimbria/fornix, on the HI-challenged side of the brain must be noted (panels B, D, and H). (E, F, I, and J) Electron microscopy study confirmed extensive axonal injury in the external capsule (panel F), but not in the internal capsule (panel J), on the HI-challenged side of the brain. The decrease in axonal bundles and increase in the empty space in the HI-injured external capsule must be noted (panel F). Scale bars: 200 μm in panels A and B; 50 μm in panels C, D, G, and H; 5 μm in panels E and F; and 1 μm in panels I and J. HI, hypoxia–ischemia.

Figure 4

Histologic correlates of HI-induced changes in radial diffusivity. (A, D) HI causes marked reduction of myelin basic protein (MBP) staining in the cerebral cortex (Ctx) and hippocampus at 15 hours of recovery (n=6). (B to F) Representative electron micrographs of the external capsule at 15 hours after HI on the contralateral (panels B and C) and the HI-injured side of the brain (panels E and F). The separation of myelin sheaths (M), increased empty space (red asterisks in panel F), and large vesicles within a myelinated axon (black asterisks in panel F) must be noted. The vesicles often contain myelin on the surface (arrows in panel F) and compress onto the axoplasma (colored in pink in panel C and F). Scale bars: 125 μm in panels A and D; 1 μm in panels B to F. DG, dentate gyrus; fm, fimbria/fornix; HI, hypoxia–ischemia.

Figure 5

Cerebral HI induces oxidative stress and oligodendrocyte injury. (A, B) Cerebral HI induces superoxide production along blood vessels in the ipsilateral brain at 1 hour of recovery, as revealed by dihydroethidium (DHE) staining. (C) HI also induces lipid peroxidation at 6 hours recovery, indicated by an increased amount of malondialdehyde (MDA) in cellular extracts of the cerebral cortex. Mean and s.d. are shown (n=4 for each). *P<0.05 compared with the other groups. (D, G) HI induces a high content of ferrous iron [Fe(II)] in oligodendrocytes in the corpus callosum (CC, arrows) (n=4). (E, H) TUNEL and Olig2 double labeling showed reduction of Olig2 immunoreactivity and the presence of many apoptotic nuclei immediately next to Olig2+ cells (arrows) in the CC on the HI-injured side of the brain at 15 hours recovery (n=3). (F, I) EM detected many pyknotic nuclei (L) of oligodendrocytes in the HI side of the CC at 15 hours. Scale bars: 100 μm in panels A and B; 5 μm in panels E and H; and 1 μm in panels F and I. Con, the contralateral side; EM, electron microscopy; HI, hypoxia–ischemia; St, striatum; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; UN, unchallenged animals.

Figure 6

DTI detects HI-induced dendritic degeneration in hippocampal neurons. (A) Comparison of the Nissl stain, ADC, FA, and DEC maps of the hippocampus on the same plane to determine the locations of stratum radiatum (rad) and molecular layer of the dentate gyrus (MoDG) on the DEC map. (B, C) Sections of the hippocampus taken from HI-injured Thy1-YFP mice at 15 hours recovery showed a marked reduction of apical dendrites in the stratum radiatum (st. rad) on the ipsilateral hemisphere (panel C). (D) T2-weighted MRI of an animal killed at 6 hours after HI, whose brain was examined by electron microscopy (EM), as shown in E to J. (Panels D to I) Electron micrographs of the indicated hippocampal subfields on the contralateral (panels E to G) and the HI-challenged hemispheres (panels H to J) at 6 hours after HI. The pervasive cytoskeletal dissolution inside dendrites (Den, colored in pink), swelling of the mitochondria (mit), and numerous intraaxonal vesicles (asterisks (*)) that compress onto the axoplasma (colored in green) must be noted. In contrast, there were few, if any, incidences of nuclear pyknosis inside the pyramidal neuron layer at the same time point, suggesting that dendritic and axonal histopathology precedes the apoptosis of hippocampal neurons after cerebral HI. Scale bars: 50 μm in panels B and C; 5 μm in panels E to I. ADC, apparent diffusion coefficient; DEC, directionally encoded color; DG, dentate gyrus; DTI, diffusion tensor imaging; ec, external capsule; FA, fractional anisotropy; HI, hypoxia–ischemia; Hip, hippocampus; MRI, magnetic resonance imaging.