Background: The blood–brain barrier (BBB) tightly regulates the movement of molecules between the bloodstream and the brain, ensuring a stable environment for neuronal function. With aging, structural and functional changes in the cerebrovascular system contribute to increased BBB permeability, which is considered an early hallmark of neurovascular dysfunction and cognitive decline. Despite this, the specific vascular segments driving age-related leakage remain poorly defined. Identifying which vessels become permeable earliest or most prominently is crucial for enhancing detection and understanding the mechanisms of BBB breakdown in aging. Methods: A cranial window was implanted over the somatosensory barrel cortex in male and female young (3–5 months) and old (18–20 months) C57BL/6J mice (n = 5/group). In vivo two-photon imaging of retro-orbitally injected 40 kDa fluorescein 5-isothiocyanate (FITC)–dextran and 150 kDa tetramethylrhodamine (TRITC)–dextran, was used to assess age- and time-dependent differences in vascular permeability of penetrating arterioles (PAs), capillaries (CAPs), and ascending venules (AVs). Post-acquisition image processing and quantification were performed using ImageJ and IMARIS software. Mean intensity in the extravascular region was used as the surrogate measure of BBB permeability. Results: We analyzed PAs, CAPs, and AVs in young and old mice at three time points (40, 80, and 120 minutes) following the injection of high (150 kDa) and low (40 kDa) molecular weight fluorescent dextrans. Extravascular mean intensity distributions across structures and time revealed that AVs exhibited significantly increased BBB permeability in aged mice. Mean intensity values in PAs showed no age-related differences, consistent with previous literature. CAPs did not show significant permeability changes across time points; however, they displayed a subtle age-related trend toward increased permeability. The relatively low permeability observed in CAPs may reflect their more restricted perivascular space. Although BBB permeability in AVs was significantly elevated in aged mice, no differences were detected across the 40-, 80-, and 120-minute post-injection time points within the aged group. This pattern suggests that AV-associated BBB leakage may be assessed as early as 40 minutes post-injection, with 40 kDa FITC–dextran in aged mice. Conclusions: Our results suggest aging significantly increases BBB permeability in ascending venules, with no detectable differences across the measured time points. These results indicate that ascending venules may serve as a sensitive and potentially major site of BBB dysfunction in aging. Funding support: NIH grants: R01NS136041(PKC), R56AG075988 (DWB), R01NS114286 (RM), and R01AG074489 (RM). This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Hernandez et al. (Fri,) studied this question.