Repeated moderate hypothermia leads to sustained glymphatic dysfunction and loss of vascular AQP4 polarization

Cerebrospinal fluid (CSF) enters the brain along perivascular spaces in a manner that depends on aquaporin-4 (AQP4) polarization, facilitating brain clearance and is called the glymphatic system. While physiological factors such as cardiovascular activity and respiration are known to influence glymphatic function, the effects of hypothermia remain poorly understood. In this study, we used quantitative optical and light-sheet microscopy to track fluorescent CSF tracers in mice under ketamine–xylazine (KX) anesthesia at normothermia (37 °C), mild hypothermia (33 °C), and moderate hypothermia (30 °C). Acute mild hypothermia did not affect CSF influx, indicating tolerance to modest temperature fluctuations in KX anesthetized mice, whereas acute moderate hypothermia markedly impaired glymphatic transport by reducing tracer penetration and CSF outflow. Notably, repeated exposure to moderate hypothermia over four consecutive days induced a sustained suppression of glymphatic influx, which persisted even when assessed under normothermic conditions. This persistent dysfunction strongly correlated with the loss of perivascular AQP4 polarization. These findings demonstrate that repeated hypothermia exerts cumulative effects on glymphatic function, which may compromise brain waste clearance and contribute to cognitive impairment in vulnerable populations with impaired thermoregulatory capacity.