In Vivo Wide-Field Mapping of Microvascular Dynamics in Orthotopic GL261 Gliomas by Super-Resolution Ultrasound Imaging.

PURPOSE: The development and progression of gliomas are intimately linked to abnormal tumor angiogenesis. Different grades of gliomas exhibit distinct vascular distribution and structural characteristics. Therefore, analyzing vascular architecture and hemodynamics can provide crucial insights into tumor growth and invasion. METHODS: Ultrasound localization microscopy (ULM) was applied to image the cerebrovascular changes in an orthotopic glioma mouse model at two different stages of tumor progression (14- and 21-days post-implantation). A total of twenty mice with intracranial glioma were imaged on the 14th day (n = 9) and on the 21st day (n = 11) after implantation. Pathological staining, vascular endothelial immunofluorescence examinations, and fluorescence dye perfusion were conducted to compare the ULM imaging results of the microvascular network morphology. RESULTS: By separating slow and fast blood flow signals, the ULM images revealed distinct vascular changes that visually correlated with H&E-derived pathological features. The vascular densities in glioma regions showed a significant increase compared to the contralateral side: 30.4% on day 14 (39.91% ± 4.09% vs. 30.60% ± 3.17%, p < 0.001) and 56.5% on day 21 (42.54% ± 3.78% vs. 27.17% ± 4.56%, p < 0.001) post-glioma implantation. The vascular densities obtained from ULM images correlated strongly and significantly with pathological and vascular perfusion results (CD31 and DiI perfusion). ULM-derived multiparametric analysis further revealed significantly reduced average vessel length in glioma regions at both Day 14 (123.72 ± 28.06 μm vs. 192.90 ± 27.75 μm, p < 0.001) and Day 21 (87.07 ± 22.92 μm vs. 233.79 ± 38.94 μm, p < 0.001), decreased blood flow velocity at Day 21 (3.70 ± 0.66 mm/s vs. 4.64 ± 0.82 mm/s, p < 0.001), and increased flow-orientation heterogeneity at both Day 14 (0.838 ± 0.082 vs. 0.595 ± 0.174, p < 0.001) and Day 21 (0.812 ± 0.086 vs. 0.625 ± 0.083, p < 0.001). CONCLUSION: ULM imaging modality enables in vivo simultaneous structural-functional imaging of glioma microvasculature at micron-level resolution, capturing microcirculatory alterations across glioma regions. Its feasibility and reliability in visualizing vascular abnormalities were validated against ex vivo pathological and vascular perfusion, providing preclinical evidence for research and clinical applications in glioma therapy assessment and aggressiveness monitoring.