Melatonin Enhances Aquaporin 4 and Alpha‐Syntrophin Interaction by Inhibiting Cyclin‐Dependent Kinase 5 Activity to Preserve Glymphatic Function in Neonatal Hypoxic‐Ischemic Encephalopathy

ABSTRACT The glymphatic system is a critical waste clearance system in the brain, playing an essential role in maintaining homeostasis within the central nervous system. Aquaporin 4 (AQP4), an indispensable component of the glymphatic system, is vital for ensuring the proper function of this system. Melatonin has been proven to be protective in treating hypoxic‐ischemic encephalopathy (HIE). The aim of this study was to examine if alterations occur in the glymphatic system function in the brain of HIE model rats, and to determine whether melatonin can enhance the function of the glymphatic system by regulating AQP4, along with elucidating the mechanisms underlying melatonin's effects on AQP4. 10‐day‐old rat pups were subjected to hypoxic‐ischemic (HI) injury; melatonin and roscovitine (an inhibitor of cyclin‐dependent kinase 5) were injected intraperitoneally at 10 min following HI induction. At 24 h post‐HI, intracisternal tracer infusion, neurobehavioral tests, immunofluorescence staining, western blot analysis, Evans blue (EB) permeability assay, brain water content test, ELISA detection, and co‐immunoprecipitation tests were performed. At 28 days post‐HI, neurobehavioral tests, intracisternal EB infusion, Nissl staining, and cerebral blood flow (CBF) evaluations were performed. The results showed that melatonin improved neurological function, restored glymphatic function, maintained blood–brain barrier integrity, alleviated brain edema, increased CBF, and reduced brain atrophy; both melatonin and roscovitine inhibited cyclin‐dependent kinase 5 (CDK5) activity, enhanced the interaction between AQP4 and alpha‐syntrophin (α‐Syn), and maintained AQP4 polarity. In conclusion, the current study suggests that melatonin may enhance the interaction between AQP4 and α‐Syn by inhibiting CDK5 activity after HI to maintain glymphatic function.