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

## TL;DR

Repeated moderate hypothermia in mice causes long-term brain fluid clearance issues by disrupting AQP4 polarization, which could lead to cognitive problems.

## Contribution

This study reveals that repeated moderate hypothermia causes sustained glymphatic dysfunction and loss of AQP4 polarization in mice.

## Key findings

- Acute mild hypothermia does not impair glymphatic function in anesthetized mice.
- Moderate hypothermia acutely impairs glymphatic transport by reducing tracer penetration and CSF outflow.
- Repeated moderate hypothermia leads to sustained glymphatic dysfunction and loss of AQP4 polarization.

## Abstract

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.

The online version contains supplementary material available at 10.1186/s12987-026-00770-0.