Lentivirus-mediated inhibition of AQP4 accelerates motor function recovery associated with NGF in spinal cord contusion rats

Abstract

Aquaporin-4 (AQP4) is a water channel protein in spinal cord and plays a critical role in the pathophysiological process of spinal cord injury (SCI). However, little is known about the molecular mechanism of AQP4 involved in SCI. The present study was performed to investigate the possible molecules regulated by AQP4 after SCI by use of lentivirus-mediated RNA interference (RNAi). First, the motor function was evaluated by Basso, Beattie, Bresnahan (BBB) scale and the expression of AQP4 was measured by western blot, immunohistochemical staining and immunofluorescence in rats after contusion spinal cord injury (cSCI). After cSCI, the rats exhibited a gradual motor function recovery from 3dpo to 28dpo. And the expression and localization of AQP4 changed with different post-injury stages. At 3d after SCI, AQP4 located mainly in vascular endothelial cells (VECs) in the anterior horn of spinal cord, which was similar to the sham rats. At 7d and 28d after SCI, AQP4 was expressed both in VECs and the position of neuron membranes. The protein level of AQP4 increased significantly at 12h, 1d and 3d after SCI, and decreased slightly at 7d after SCI. Then lentivirus-mediated AQP4 RNA interference (AQP4-RNAi) was constructed and used to inhibit AQP4 expression in cSCI rats. The results from real-time quantitative polymerase chain reactions (RT-qPCR) and immunohistochemical staining suggested that the expression of nerve growth factor (NGF) was up-regulated by lentivirus-mediated AQP4 inhibition in cSCI rats, while the motor function recovery was accelerated. The results suggested that the acceleration of motor function recovery by AQP4 inhibition was associated with NGF up-regulation. This is the first report on the relationship between AQP4 and NGF in SCI, which may shed light on illustrating the role of AQP4 in SCI. These findings may also provide strategies of the clinical treatment for SCI in the future.

Keywords: Aquaporin-4; Lentivirus-mediated RNA interference; Nerve growth factor; Spinal cord injury.