Fasudil reduces GFAP expression after hypoxic injury

Fasudil (HA-1077), a specific Rho kinase II (ROCKII) inhibitor, is in clinical trials for recovery from spinal cord injury (SCI). The primary role of Fasudil is in axonal regeneration, as it inhibits ROCKII, the key signaling molecule involved in collapse of axon growth cone. Astrogliosis, due to the activation of astrocytes is an indicator of CNS injury. In early stages of injury, GFAP expression increases, helping to restore the integrity of the CNS. An increase in GFAP expression is also a marker of astrogliosis. Thus, reducing GFAP and hence astrogliosis at later stages of SCI is important for neuroregeneration and functional recovery. CoCl2 was used to induce hypoxic injury in astrocytic cell lines A172 (24 h) and in spinal cord dorsal column white matter (8 h). Several different techniques were used to study the changes in GFAP expression such as real-time PCR, western blotting and immunofluorescence staining with confocal microscopy. Hypoxia increased the expression of GFAP in A172 cells and in the spinal cord dorsal column after CoCl2 (100 μM) treatment for 24 h and 8 h, respectively. We observed 11 folds increase in protein expression in A172 cells (24 h) and 4.5 folds in spinal cord dorsal column (8 h). The RNA expression was increased 3 folds in A172 cells after 24 h of treatment and 4 folds in spinal cord dorsal column after 8 h of treatment with 100 μM CoCl2. Treatment with fasudil (20 μM) significantly reduces the expression of GFAP in A172 cells and in spinal cord dorsal column. Fasudil also decreased activation of NF-κB in A172 cells after hypoxic injury. In the present study, we observed that fasudil reduces the expression of GFAP (consequently, astrogliosis) after hypoxic injury to A172 cells and spinal cord dorsal column. Our studies demonstrate that fasudil also plays a role in GFAP expression by reducing NF-κB activation at the injury site which could further help in axonal regeneration.