Quantitative Analysis of CSF Spaces

The delineated CSF space was separated manually from the final CSF mask in ITK-SNAP into seven compartments (Appendix 1—figure 3B– ‘Filtration and labeling’), for further statistical comparison: lateral ventricles; third ventricle; fourth ventricle; basilar artery; basal perivascular space at the skull base surrounding the Circle of Willis; parietal perivascular spaces and cisterns (ventrally from the position of posterior cerebral artery, via space neighboring the transverse sinuses and dorsally to the junction of the superior sagittal sinus and transverse sinuses); remaining perivascular space within the olfactory area, surrounding anterior cerebral and frontopolaris arteries, middle cerebral arteries branches, and posterior cisterns including pontine and cisterna magna. For supplementary comparison, the segmented lateral, third and fourth ventricular spaces were considered jointly as the ventricular space, and the basilar, basal and the remaining anterior/posterior CSF spaces were considered jointly as the whole perivascular space. Number of voxels was counted, and the volume of each segment was calculated by multiplying the voxels count by the voxel dimension from the original 3D-CISS image, for subsequent statistical comparison.
To compensate for the brain capsule volume differences and provide a reliable measure of the brain’s CSF space volume between animals, a ratio of the CSF to the brain volume (intracranial volume) was calculated for each delineated CSF segment as: $Rati{o}_{CSFspace}=\frac{CS{F}_{compartmentvolume}}{Brai{n}_{volume}-CS{F}_{wholesegmentedvolume}}$
The ratios obtained for each of the CSF compartments, as well as the segmented brain volumes were compared between KO and WT animals using nonparametric Mann-Whitney U-test.