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. 2013 Sep 18;8(9):e74631.
doi: 10.1371/journal.pone.0074631. eCollection 2013.

The ins and outs of the BCCAo model for chronic hypoperfusion: a multimodal and longitudinal MRI approach

Guadalupe Soria  1 , Raúl Tudela, Ana Márquez-Martín, Lluïsa Camón, Dafnis Batalle, Emma Muñoz-Moreno, Elisenda Eixarch, Josep Puig, Salvador Pedraza, Elisabet Vila, Alberto Prats-Galino, Anna M Planas
Affiliations

The ins and outs of the BCCAo model for chronic hypoperfusion: a multimodal and longitudinal MRI approach

Guadalupe Soria et al. PLoS One. .

Abstract

Cerebral hypoperfusion induced by bilateral common carotid artery occlusion (BCCAo) in rodents has been proposed as an experimental model of white matter damage and vascular dementia. However, the histopathological and behavioral alterations reported in this model are variable and a full characterization of the dynamic alterations is not available. Here we implemented a longitudinal multimodal magnetic resonance imaging (MRI) design, including time-of-flight angiography, high resolution T1-weighted images, T2 relaxometry mapping, diffusion tensor imaging, and cerebral blood flow measurements up to 12 weeks after BCCAo or sham-operation in Wistar rats. Changes in MRI were related to behavioral performance in executive function tasks and histopathological alterations in the same animals. MRI frequently (70%) showed various degrees of acute ischemic lesions, ranging from very small to large subcortical infarctions. Independently, delayed MRI changes were also apparent. The patterns of MRI alterations were related to either ischemic necrosis or gliosis. Progressive microstructural changes revealed by diffusion tensor imaging in white matter were confirmed by observation of myelinated fiber degeneration, including severe optic tract degeneration. The latter interfered with the visually cued learning paradigms used to test executive functions. Independently of brain damage, BCCAo induced progressive arteriogenesis in the vertebrobasilar tree, a process that was associated with blood flow recovery after 12 weeks. The structural alterations found in the basilar artery were compatible with compensatory adaptive changes driven by shear stress. In summary, BCCAo in rats induces specific signatures in multimodal MRI that are compatible with various types of histological lesion and with marked adaptive arteriogenesis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Longitudinal multimodal MRI.
Representative coronal section of two BCCAO animals (A, B) are shown at different time points. BCCAO induced variable degrees of focal acute ischemic lesions (see right striatum at 24 h in A and B) and delayed damage (see left striatum from day 10). For both animals (A, B), images in first row are T2-weighted (T2W), second row are T2 relaxometry map (T2R) and third row are 3D MDEFT T1-weighted (MDEFT) images. The box highlights MDEFT alterations at 12 weeks in the same animal shown in Figure 3 for histological damage. Color scale represents T2 relaxometry time (ms) for images shown in second row of panels A and S (T2R).
Figure 2
Figure 2. Statistical maps showing Diffusion Tensor Imaging (DTI) indexes.
Regions showing statistically significant differences of fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD) and radial diffusivity (RD), between sham and BCCAo animals 7 weeks after surgery. Background images are the corresponding averaged DTI index calculated for the sham group (left: coronal, middle: sagittal, and right: axial slices). Red areas (sham > BCCAo) have a significance of P <0.05 (Mann-Whitney U test). Blue areas (sham < BCCAo) have a significance of P <0.05 (Mann-Whitney U test). The slices show representative anatomical structures (see Results section for details).
Figure 3
Figure 3. Histological evaluation of brain lesions 12 weeks after surgery.
A-D) Macroscopic images of coronal brain sections (at the level of Bregma +0.2) of a BCCAo rat showing bilateral alterations in the striatum. This animal corresponds to the same rat shown in the MRI image in Figure 1A. Sections show tissue structure (hematoxylin-eosin, HE) (A), myelinated fiber tracts (B), and astroglial (GFAP) (C) and microglial (Iba1) (D) reactions. Corresponding magnifications of the above images are shown in the middle row (C-L) for the areas illustrated with rectangles in the image (A). Alterations (arrowheads) are apparent in both hemispheres compared to the tissue of a sham-operated rat (M-P). Arrows in M-P point to healthy structures and cells. The features of the alterations in the left and right hemisphere of this BCCAo rat differ, with more prominent changes in the right striatum, thus illustrating the variability in the extent and severity of the damage after BCCAo. Q) Luxol staining of a brain section (Bregma -2.3) indicating with a rectangle the genus region of the corpus callosum shown in higher magnification in (R) and (S) for a sham-operated and BCCAo rat, respectively. BCCAo induces rarefaction with pallor of the fiber staining and vacuolization (arrowheads). T) Quantification of fiber density shows a significant reduction in the genus of the corpus callosum 12 weeks after BCCAo. Data are represented as mean ± SEM. ** P< 0.01 (Unpaired Student’s t-test). Scale bar a-d, q = 0.5 cm; e-p, r, s = 50 µm.
Figure 4
Figure 4. Signs of optic tract degeneration 12 weeks after surgery.
A) Macroscopic images of the optic tract (arrows) show reduce fiber volume in BCCAo rats compared to sham-operated rats. B) Histological evaluation of fiber density assed with Luxol staining of the optic tract (at the level of Bregma -2.3) and immunostaining to show microglia reactivity (Iba-1). C) Quantification of fiber tract density and number of Iba-1+ cells per field (objective x40) show significant increases in BCCAo rats (n=5) versus sham rats (n=3). Data are represented as mean ± SEM. ** P< 0.01 (Unpaired Student’s t-test). D) Reversal learning executive function as expressed by trials performed correctly to acquire each reversal test, white and black bars represent sham (n=4) and BCCAo animals (n=7) respectively. C) Set-shifting executive function as expressed by correct and incorrect responses during the 6 testing weeks in sham (n=4) and BCCAo animals (n=7). Asterisks indicate significant differences between correct and incorrect responses (Bonferroni test). ** P< 0.01.
Figure 5
Figure 5. Time course changes in vertebro-basilar artery length and tortuosity following BCCAo in rats.
A) Segmentation of the vertebro-basilar artery for representative sham and BCCAo rats for all the time points evaluated. The ratio between the hypothetical minimum length and the true length was expressed as the arterial tortuosity (bottom right picture) (B, C) Measurements of length and tortuosity of the vertebro-basilar arteries (see Methods). The minimal distance from the base of the basilar artery to the final points evaluated is schematically drawn (in green) on the bottom right image of panel a. Vertebro-basilar artery length (B) and tortuosity (C) progressively increased after BCCAo from day 10. A measure of the effect size was determined by calculating the eta squared (η2), which for the treatment effect over the length was 0.077 and 0.242 over the tortuosity, suggesting 7% of the effect observed in length and 24% of the effect observed in the tortuosity accounts for BCCA occlusion. White and black circles represent mean±SEM for sham (n=4) and BCCAo (n=6) groups, respectively. * P< 0.05, ** P< 0.01.
Figure 6
Figure 6. Total arterial area progressively increases after BCCAo.
A) Maximal intensity projection (MIP) of the brain basal segment and arteries from representative sham (n = 4) and BCCAO (n = 6) rats at all the time points evaluated. B) Ratio between the brain surface and the vascular surface of the previous MIP calculated at all the time points evaluated. Data are represented as mean ± SEM. Black stars indicate significant differences versus the pre-occlusion data (Fisher’s LSD test), and white stars significant differences versus the sham-operated animals at corresponding time points. ★ P< 0.05; ★★ P< 0.01.
Figure 7
Figure 7. Structural evaluation of the basilar artery (BA), 12 weeks after BCCAo.
A) Representative coronal sections of the BA from a sham and a BCCAo rat. Red shows collagen immunostaining and blue shows the cell nuclei. B) Cross-sectional area and C) total number of smooth muscle cells (SMC)/ring calculated for the BA of sham (n = 5) and BCCAo (n = 6) group 12 weeks after surgery. Data are represented as mean ± SEM. Asterisks indicate significant differences between the 2 groups. ★★ P< 0.01, ★★★ P< 0.001 (Unpaired Student’s t-test).
Figure 8
Figure 8. Dynamic susceptibility contrast imaging for evaluation of cerebral perfusion.
A) Scheme of the gadodiamide (Gd) bolus track and the evaluated perfusion-related parameters. The maximum (max) is the peak of signal intensity. Time to peak (TTP) is the period of time from gadodiamide injection to the peak of signal intensity. The area under the curve (AUC) provides an estimation of relative cerebral blood volume (relCBV). B) Relative cerebral blood flow (relCBF); C) maximum of signal intensity; D) time to peak; E) relative CBV (relCBV); F) full width at high maximum (FWHM) as an estimation of the mean transit time in several brain regions. White bars represent sham animals (n = 3) and black bars BCCAO animals (n = 7), 12 weeks after surgery. Black stars indicate significant differences between groups (Bonferroni test). ★ P< 0.05 CP: caudate-putamen; pCx: medial prefrontal cortex; rCx: retrosplenial cortex.
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This work was supported by Instituto de Salud Carlos III, Subdirección General de Evaluación y Fomento de la Investigación (PS09/00527) and Ministerio de Economía y Competitividad ERANET-NEURON (PRI-PIMNEU-2011-1340) and the FP7/2007-2013 project ARISE (grant agreement number 201024). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.