Induction of ezrin–radixin–moesin molecules after cryogenic traumatic brain injury of the mouse cortex Younghye Moon b , Joo Yeon Kim a , So Yoen Choi a , Kyungjin Kim b , Hyun Kim a and Woong Sun a Traumatic brain injury promotes rapid induction of microglial cells and infiltration of peripheral macrophages to the injury sites. Such inflammatory responses are mediated by the activation and migration of immune cells, which are influenced by the actin cytoskeleton remodeling. In this study, we observed that the phosphorylation and expressions of ezrin–radixin–moesin (ERM) proteins, which are linkers for cell surface with actin cytoskeleton, are induced in the activated microglia/macrophages, whereas ERM molecules are only marginally expressed in quiescent microglia in the normal brain. These results suggest that ERM activation in the injury penumbra is implicated in the inflammatory immune responses after traumatic brain injury. NeuroReport 22:304–308  c 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins. NeuroReport 2011, 22:304–308 Keywords: ezrin–radixin–moesin, macrophage, microglia, traumatic brain injury a Department of Anatomy and Division of Brain Korea 21 Biomedical Science, Korea University College of Medicine and b Brain and Neuroendocrine Laboratory, School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, Korea Correspondence to Professor Woong Sun, PhD, Korea University College of Medicine, 126-1, Anam-Dong, Sungbuk-Gu, Seoul 136 705, Korea Tel: + 82 2 920 6404; fax: + 82 2 929 5696; e-mail: woongsun@korea.ac.kr Received 31 December 2010 accepted 21 February 2011 Introduction Traumatic brain injury causes immediate death of neurons and glial cells, a physical impact itself, and subsequently complex series of physiological responses including demyelination, inflammatory responses, and glial scar formation [1,2]. These secondary responses may impede axonal regeneration, and thus a better understanding of injury-related responses is important in the development of effective therapeutic strategy to traumatic brain injury. Ezrin–radixin–moesin (ERM) family molecules are essential in the remodeling of tissues because they not only link membrane integral proteins with cortical actin cytoskeleton, but also participate in the signal-transduction pathways [3]. Modification of ERM activity thus triggers a wide variety of actin-mediated cellular events such as cell–cell interaction, cell shape change, cell migration, apoptosis, and membrane trafficking [4]. Owing to the fact that brain damage markedly disturbs normal cell–cell interac- tions and evokes major tissue remodeling, ERM mole- cules may contribute to the brain responses to injury. In this study, we found that the ERM gene expres- sion is markedly induced in the penumbra regions of injury. Further characterization of the cell types exhibit- ing ERM induction showed that they are mainly microglia/ macrophages in the penumbra expressed ERM, which suggests that ERM activation is implicated in the inflam- matory immune responses after traumatic brain injury. Materials and methods Animals and cryogenic traumatic brain injury Two-month-old C57BL/6 male mice were used (Orient Bio, Seongnam, Korea). Cryogenic traumatic brain injury was performed as described earlier [5]. Under deep anesthesia, a metal probe (5 mm in diameter) chilled by liquid nitrogen was placed on the right side of cranium for 1 min. The cut skin was sutured and the animals were allowed to move freely in their home cage until they were killed. All experiments were performed in accordance with the regulations and approval of the Animal Care and Use Committee of the Korea University. In-situ hybridization The brains were frozen in liquid nitrogen-chilled isopentane. Frozen sections (12 mm) were fixed with 4% paraformaldehyde, treated with 0.25% acetic anhydride in 0.1 M triethanolamine buffer (pH 8.0), dehydrated, and dried in air. The riboprobes were produced from the plasmids containing complementary DNA, corresponding to sequence containing 659–1174 nucleotides of ezrin (GenBank accession number: NM_009510), 1230–1661 nucleotides of radixin (GenBank accession number: NM_ 009041), or 486–840 nucleotides of moesin (GenBank accession number: NM_010833). The antisense probes were transcribed in vitro in the presence of a-[ 35 S] uridine triphosphate and the sections were hybridized overnight with the labeled probes at 631C. On the next day, the sections were washed for 5 min in 4 sodium saline citrate (SSC), treated with ribonuclease A (20 mg/ml) for 30 min at 371C, washed for 1 h in 2 SSC, 1h in 1 SSC, and 1 h in 0.5 SSC at room temperature, incub- ated in 0.1 SSC for 30 min, rinsed in graded series of ethanol containing 1 mM dithiothreitol, and dried. Radio- active signals were visualized by exposure to b-max film 304 Clinical neuroscience and neuropathology 0959-4965  c 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins DOI: 10.1097/WNR.0b013e3283460265 Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.