Role of c-Jun N-Terminal Kinase in Early Brain Injury After Subarachnoid Hemorrhage Hiroshi Yatsushige, 1 Robert P. Ostrowski, 1 Tamiji Tsubokawa, 1 Austin Colohan, 2 and John H. Zhang 1,2,3 * 1 Department of Physiology, Loma Linda University, Loma Linda, California 2 Department of Neurosurgery, Loma Linda University, Loma Linda, California 3 Department of Anesthesiology, Loma Linda University, Loma Linda, California The c-Jun N-terminal kinase (JNK) is induced by cere- bral ischemia and injurious blood components acutely after subarachnoid hemorrhage (SAH). We hypothe- sized that inhibition of JNK will prevent damage to the neurovascular unit in the early brain injury period after SAH. Ninety-nine male SD rats (300–350 g) were ran- domly assigned to sham, SAH, and SAH treated with JNK inhibitor groups. SAH was induced by endovascu- lar perforation. The JNK inhibitor SP600125 was admin- istered intraperitoneally at 1 hr before and 6 hr after SAH. At 24 hr after SAH, we observed increased phos- phorylation of JNK and c-Jun. Signs of neurovascular damage were observed in the hemorrhagic brains; these included the increases of aquaporin (AQP)-1 expression and brain water content as well as enhanced matrix metalloproteinase (MMP)-9 activity, vascular collagen IV loss, increased VEGF tissue level, and Evans blue ex- travasation. The appearances of cleaved caspase-3 expression, TUNEL-positive cells, and apoptotic mor- phology in cerebral tissues were associated with neuro- logical deficit after SAH. JNK inhibition prevented c-Jun phosphorylation and suppressed AQP1, MMP-9, VEGF, and caspase-3 activation, with concomitant diminution of neuronal injury, blood–brain barrier preservation, reduced brain swelling, and improved neurological defi- cit in rats after SAH. This study demonstrates a multi- tude of beneficial effects of JNK inhibition, including protection of the neurovascular unit in early brain injury after SAH. V V C 2007 Wiley-Liss, Inc. Key words: SAH; JNK; VEGF; neurovascular protection; SP600125 The key mediators of multiple signaling pathways in early brain injury after subarachnoid hemorrhage (SAH) are reasonable targets for novel therapeutic inter- vention in SAH, presumably in support of surgical treat- ment. The role of JNK in SAH-induced early neurovas- cular injury was first suggested when it was found to be activated at 24 hr after experimental SAH in the cerebral arteries (Kusaka et al., 2004). JNK phosphorylates c-Jun, which up-regulates apoptotic cascades by inducing expression of the proapoptotic member of Bcl-2 family Hrk/DP5, Bim, and Fas (Yuan and Yankner, 2000; Kuan et al., 2003). In addition, c-Jun mRNA has been found to be up-regulated acutely after SAH in the rat cerebral cortex and hippocampus (Harada et al., 1997; Kawamura et al., 2000). So far, the complex roles of JNK for acute brain in- jury have been investigated in ischemic models of stroke. In transient focal cerebral ischemia, inhibition of JNK prevented caspase-9 and -3 activation; it also reduced apoptotic cell death and infarct volume (Borsello et al., 2003; Gao et al., 2005). Cerebral ischemia, induced by a rise in intracranial pressure (ICP), acute arterial spasm, and generalized activation of coagulation cascades, is a major component of acute SAH pathophysiology. How- ever, only its combination with injurious blood-derived factors produces severe brain injury (Solomon et al., 1985). JNK responds to ischemic insult, but it can also respond to SAH-induced inflammation (Yatsushige et al., 2005). Vascular MAP kinases have also been found acti- vated by blood components (Zubkov et al., 2000). JNK may have a distinctively powerful effect on apoptosis induced after SAH because of its combined response to ischemia, blood stimulation, and consecutive inflamma- tion. Therefore, the involvement of JNK in the SAH- induced brain injury may be very different from the ischemic pattern and closely related to vascular injury. Blockade of Src kinase, a VEGF downstream, re- sulted in diminished vascular JNK activation and reduced Contract grant sponsor: NIH; Contract grant number: NS43338 (to J.H.Z.); Contract grant number: NS45694 (to J.H.Z.); Contract grant number: NS53407 (to J.H.Z.); Contract grant sponsor: National Hospital Organization (Japan; to H.Y.). *Correspondence to: John H. Zhang, MD, PhD, Department of Neurosurgery, Loma Linda University Medical Center, 11234 Anderson Street, Room 2562B, Loma Linda, CA 92354. E-mail: johnzhang3910@yahoo.com Received 15 November 2006; Revised 29 December 2006; Accepted 16 January 2007 Published online 4 April 2007 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.21281 Journal of Neuroscience Research 85:1436–1448 (2007) ' 2007 Wiley-Liss, Inc.