Permanent bilateral common carotid arteries occlusion (2VO) is a commonly used model of chronic cerebral hypoperfusion for inducing vascular dementia[1]. It is known that 2VO results in white matter (WM) damages that are observable to diffusion tensor imaging[2], and gray matter (GM) abnormalities such as neuronal loss in the hippocampus and frontal cortex[1,3]. However, no previous study has used volumetric MRI to assess whether 2VO-induced chronic hypoperfusion is associated with regional brain atrophy, a feature that is not only of diagnostic value for clinical dementia[4], but also useful for assessing the efficacy of prevention/treatment measures[5]. It has been shown previously that cognitive rehabilitation in the 2VO model normalized hippocampal CA1 soma size, and significantly attenuated cognitive deficits[6]. In this study, we assessed regional brain atrophy in a rat model of 2VO with voxel-based morphometry (VBM), and tested the hypothesis that 2VO-induced regional brain atrophy can be alleviated by environmental enrichment (EE), an intervention that is well-known to be effective in treating neurological/psychiatric diseases[7].

Adult male Wistar rats, weighing180-220 g, were anesthetized by i.p. injection of 5% chloral hydrate (6 ml/kg). For the animals assigned to the 2VO group (n=24), bilateral common carotid arteries were exposed and double-ligated with 4-0 silk sutures. After the surgeries, a half of the animals were raised in EE cages (i.e., Group 2VOE) for 60 days for imaging, with 6 animals per cage; the other half was raised in standard rat cages (i.e., Group 2VOS) with 4 animals per cage. The EE cage (80 cm×60 cm×45 cm) contained a platform with different levels, toys of different shapes and colors, two wheels and several tunnels with shading. All objects in the EE cages were replaced every week. Eight animals received sham operations (i.e., Group SHAM), and were raised in cohort of 4 in two standard cages.

All animals were imaged on a 7 T/20 cm Bruker Biospec scanner. A volume coil was used for RF transmission, and a quadrature surface coil for signal detection. For each rat, T2-weighted images were acquired from 52 contiguous coronal slices with a RARE pulse sequence, RARE factor 4, TR 5800 ms, TEeff 40 ms, FOV 3 cm × 3 cm, matrix size 256 × 256, slice thickness 0.6 mm and 4 averages. VBM was used to analyze changes of GM/WM volumes in the 2VO rats with and without EE. With a set of custom-built tissue probability maps having a spatial resolution of 125 μm × 125 μm × 125 μm, the DARTEL algorithm in SPM8 was used for image segmentation and co-registration. For each animal, the modulated GM/WM maps were smoothed with a 0.3-mm FWHM Gaussian kernel, and compared with voxel-wise two-sample t-tests, with the whole brain volume as the covariate. Statistical significance level was set to p < 0.006, uncorrected, and cluster size = 10.

Figure 1 shows the VBM results. Compared to the SHAM group, the 2VOS group had significant GM atrophy in the superficial layers of primary (M1) and secondary (M2) motor cortex, primary sensory cortex (S1), dorsal cingulate cortex (Cg1), dorsal striatum (Dstr), and dentate gyrus (DG) of the hippocampus at 60 days after surgeries. EE rehabilitation significantly alleviated GM atrophy in these regions. Compared to the SHAM group, the 2VOE group had scattered GM volumes increases, involving mainly the medial prefrontal cortex and amygdala. The GM volume measured by VBM has been shown to be quantitatively related to spine density[8]. Decreases of GM volume in the 2VOS group is consistent with previous histology results showing decreased spine density in the frontal cortex and hippocampus after 2VO[9]. EE, as a powerful modulator of brain structural plasticity, has been found to increase spine density in the somatosensory cortex of mice that received infant-start enrichment [10], and preserve hippocampal dendritic spine density in a rat model of cerebral hypoxia-ischemia[11]. A VBM study has also reported increased GM volume in the hippocampus, sensorimotor cortex and striatum of adult mice treated with enriched environment for 3 weeks[12]. The observation that 2-month of EE intervention alleviates 2VO-induced GM atrophy is in line with these previous results. In conclusion, high resolution anatomical imaging combined with VBM is potentially a useful tool for assessing structural plasticity induced by chronic cerebral hypoperfusion, and testing the efficacy of novel prevention/treatment/intervention measures for vascular dementia.