Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Variations in post-perfusion immersion fixation and storage alter MRI measurements of mouse brain morphometry

Ex vivo magnetic resonance imaging (MRI) requires chemical fixation to preserve tissue during storage or extended imaging sessions. Although it is commonly understood that fixation may alter tissue volume and shape, the potential confounding effects of fixation and storage on morphometric analyses have not been well characterized. With increasing use of ex vivo MRI for mouse brain phenotying and opportunities for inter-study comparisons, we sought to characterize how changes in fixation and/or storage times affected tissue volume, and how this might impact phenotyping results. Mouse brain samples that had been perfusion fixed, within the skull as per our standard protocol, were immersed in formaldehyde-based fixative for 1 to 5days before being stored in saline or water. Throughout fixation and storage, samples were repeatedly scanned using magnetic resonance imaging, and analyzed for volume expansion or shrinkage. We found that most of the brain continued to shrink post fixation, with the rate of shrinkage dependent on the solution in which the samples were submerged. Maximum changes in volume of 3.5% per day and 3% per month were detected during fixation and storage (in PBS), respectively. Most notably, changes were non-uniform, with some structures shrinking slower, or even expanding, when compared to other structures in the brain. Our results highlight that caution is necessary when interpreting results from experiments with inconsistent fixation and storage protocols, so as not to mistake these changes for phenotypic differences.The Mouse Imaging Centre acknowledges funding from the Canada Foundation for Innovation and the Ontario Innovation Trust for providing facilities along with The Hospital for Sick Children. Operating funds from the Canadian Institutes of Health Research (CIHR), the Ontario Institute for Cancer Research (OICR) and the Natural Sciences and Engineering Research Council of Canada (NSERC) are also acknowledged

A method for 3D immunostaining and optical imaging of the mouse brain demonstrated in neural progenitor cells.

It is important to understand changes in cell distribution that occur as a part of disease progression. This is typically achieved using standard sectioning and immunostaining, however, many structures and cell distribution patterns are not readily appreciated in two-dimensions, including the distribution of neural stem and progenitor cells in the mouse forebrain. Three-dimensional immunostaining in the mouse brain has been hampered by poor penetration. For this reason, we have developed a method that allows for entire hemispheres of the mouse brain to be stained using commercially available antibodies. Brains stained for glial fibrillary acidic protein, doublecortin and nestin were imaged in three-dimensions using optical projection tomography and serial two-photon tomography. This staining method is simple, using a combination of heat, time and specimen preparation procedures readily available, so that it can be easily implemented without the need for specialized equipment, making it accessible to most laboratories

3D OPT imaging of DCX and nestin stained mouse brain.

<p>3D optical imaging with OPT was used to image a single hemisphere stained with DCX (red) and nestin (blue) simultaneously. Optical slices through the tissue are shown to represent the 3D nature of the sample (A). The slice orientation is shown on a cartoon. A 3D representation shows the distribution of DCX- and nestin-stained cells along the surface of the lateral ventricle (B).</p

3D serial two-photon tomography imaging of DCX staining in the mouse brain.

<p>Serial two-photon tomography was used to image a single hemisphere stained with DCX. Five individual slices are shown, with the slice location shown in the cartoon at lower right. For each slice, the grayscale image shows autofluorescence for anatomical reference and the color overlay shows the DCX staining. Neuronal processes can be distinguished (DCX in grayscale insets i, ii; arrowhead, inset i). The staining pattern is comparable to traditional 2D sections (insets with red color, iv and v), although with lower contrast to background signal.</p

3D serial two-photon tomography imaging of nestin staining in the mouse brain.

<p>Serial two-photon tomography was used to image a single brain hemisphere stained with nestin. The slice locations are shown in the cartoon. The staining pattern (grayscale inset) is comparable to the 2D histology section (color inset).</p

3D serial two-photon tomography imaging of GFAP staining in the mouse brain.

<p>Serial two-photon tomography was used to image a single hemisphere stained with GFAP. Optical slices through the brain show the staining pattern, which is widespread throughout the brain. The slice location is shown in the cartoon. The grayscale inset shows the typical astrocyte morphology, which is comparable to the 2D histology section (colour inset).</p

3D OPT imaging of GFAP staining in the mouse brain.

<p>3D optical imaging with OPT was used to image a GFAP-stained brain hemisphere. Optical slices of the brain are shown and GFAP-positive cells are seen throughout the brain (A). A cartoon shows the location of the slices. A 3D representation shows the distribution of GFAP staining along the corpus callosum (B).</p

Comparison of serial two-photon tomography in GFAP-stained brains and traditional immunohistochemistry in sections.

<p>Slices from a 3D serial two-photon tomography data set (A, C) are compared with traditional sections (B, D). Expanded images are shown for comparable regions (E, G, I and F, H, J). Rostral sections (A, B and E, F) show comparable results. Staining in the hippocampus is lighter in the 3D stained section (G) than in the traditional section (H). At the periphery, the 3D stain produces more intense GFAP signal with a higher background (I) than does the traditional section (J). Examples of lightly-stained cells are highlighted with arrowheads in (G) and (J). The arrow in (I) highlights an example of the characteristic astrocyte morphology. Maps of cell density are shown in (K, M). Rostral cell densities (K, L) are similar between the two methods. In the caudal regions (M, N), the density in the dentate gyrus (white arrowheads) is reduced in the 3D stain while it is appears increased in some regions of the cortex (asterisks). Cell density maps (K–N) are shown with black and white corresponding to <30 cells/mm² and >1200 cells/mm² respectively.</p

3D OPT imaging of DCX staining in the mouse brain.

<p>3D OPT of a DCX-stained single hemisphere are shown as optical slices. DCX staining is visible surrounding the lateral ventricle (A; i–iii) and in the subgranular zone of the dentate gyrus (A; iv). A cartoon shows the location of the slices. The chains of DCX migrating neuroblasts along the lateral ventricle can be visualized on the ventricle surface in 3D (B). In (C), a maximum intensity projection of a full brain shows cells migrating along the RMS to the olfactory bulbs (shaded green).</p