Please cite this article in press as: V. Kesherwani, et al., Fasudil reduces GFAP expression after hypoxic injury, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.053 ARTICLE IN PRESS G Model NSL 30619 1–6 Neuroscience Letters xxx (2014) xxx–xxx Contents lists available at ScienceDirect Neuroscience Letters jo ur nal ho me page: www.elsevier.com/locate/neulet Fasudil reduces GFAP expression after hypoxic injury Varun Kesherwani a , Shikha Tarang b , Robert Barnes c , Sandeep K. Agrawal a,∗ Q1 a Division of Neurosurgery, Department of Surgery, University of Nebraska Medical Center, Omaha, NE 68198-7690, USA b Department of Oral Biology, Creighton University School of Dentistry, Omaha, NE 68178, USA c Northwestern University, Evanston, IL 60201, USA h i g h l i g h t s • CoCl 2 induced GFAP expression in astrocytes and spinal cord dorsal column. • Fasudil reduced CoCl2 induced GFAP expression in astrocytes and spinal cord dorsal column. • Fasudil can be used to reduce astrogliosis after CNS injury. a r t i c l e i n f o Article history: Received 17 February 2014 Received in revised form 23 May 2014 Accepted 27 May 2014 Available online xxx Keywords: Spinal cord Hypoxia Fasudil GFAP Astrogliosis a b s t r a c t 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 astro- cytes 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 recov- ery. CoCl 2 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 expres- sion 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 CoCl 2 (100 M) 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 M CoCl 2. Treatment with fasudil (20 M) significantly reduces the expression of GFAP in A172 cells and in spinal cord dorsal column. Fasudil also decreased activation of NF-B 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-B activation at the injury site which could further help in axonal regeneration. © 2014 Published by Elsevier Ireland Ltd. 1. Introduction Every year more than 10,000 new cases of spinal cord injury (SCI) are reported in United States [1]. Although not fatal in most of the cases, SCI significantly affects the life of survivor. In majority of the cases recovery is poor and patients are unable to perform day-to-day life activities. Management of patients after SCI is very costly with high socio-economic impact [2]. ∗ Corresponding author. Tel.: +1 402 559 4567. E-mail address: agrawals@unmc.edu (S.K. Agrawal). SCI has two phases, primary and secondary. Primary phase is physical damage of spinal cord tissue, while secondary injury is due to inflammation and oxidative stress which leads to cell death at the injury site and the surrounding tissues [3]. Much of the efforts in the last decade focused on reducing the secondary injury using anti- inflammatory and antioxidant molecules. Later, it was realized that reducing secondary injury will not significantly help in recovery unless there are approaches to reverse the primary injury to the spinal cord [4]. Fasudil (HA-1077) [(1-(5-isoquinolinesulfonyl)- homopiperazine, monohydrochloride], is a specific inhibitor of ROCK. While ROCKI is expressed in non-neuronal tissues, http://dx.doi.org/10.1016/j.neulet.2014.05.053 0304-3940/© 2014 Published by Elsevier Ireland Ltd. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46