Three-dimensional quantification using contrast-enhanced micro-CT reveals regional heterogeneity of microhemorrhagic burden after focal cerebral ischemia in mice

Abstract Hemorrhagic transformation (HT) after ischemic stroke reflects spatially heterogeneous neurovascular pathology, but the regional distribution of individual hemorrhagic lesions and their relationship to ischemic injury severity remain incompletely defined. Conventional histological and imaging approaches do not readily combine non-destructive preservation of whole-brain three-dimensional anatomical continuity with object-based quantification of individual hemorrhagic lesions alongside infarction and edema in the same experimental brain. Here, we applied ex vivo phosphotungstic acid-enhanced micro-computed tomography (micro-CT) to 121 mouse brains collected 1, 3, 7, and 14 days after transient middle cerebral artery occlusion. Whole-brain three-dimensional analyses were used to quantify infarction, hemispheric volume changes, HT, and individual microhemorrhagic lesions. Micro-CT findings were validated against immunohistological markers and a genetic edema model, and associations with sensorimotor outcomes were assessed. CT-defined hypodense lesions corresponded histologically to regions of neuronal loss surrounded by glial scar, and micro-CT detected aggravated edema in Ndrg2 knockout mice. Hyperdense regions corresponded to Ter119-positive erythrocyte clusters, supporting their hemorrhagic nature, and HT incidence increased with longer ischemia duration. Mice with HT exhibited greater edema and poorer performance on rotarod and grid-walk tests. Object-based three-dimensional analysis of individual microhemorrhagic lesions characterized a region-specific pattern, with declining cortical and persistent striatal microhemorrhagic burden during the first week after ischemia-reperfusion. At day 7, microhemorrhagic burden was greater in the caudate putamen than in the cortex, and caudate putamen microhemorrhage number correlated positively with both infarct volume and neurological deficit score, whereas no significant correlations were observed in the cortex. These findings demonstrate spatial heterogeneity of post-ischemic microhemorrhagic burden, and highlight contrast-enhanced micro-CT as a quantitative platform for integrated, spatially resolved 3D assessment of infarction, edema, and hemorrhagic lesions after cerebral ischemia.