Ž . Molecular Brain Research 71 1999 304–312 www.elsevier.comrlocaterbres Research report Molecular cloning and expression of the rat monocyte chemotactic protein-3 gene: a possible role in stroke Xinkang Wang a, ) , Xiang Li a , Shoshanit Yaish-Ohad a , Henry M. Sarau b , Frank C. Barone a , Giora Z. Feuerstein a,1 a Department of CardioÕascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA b Department of Pulmonary Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA Accepted 8 June 1999 Abstract Ž . Using the suppression subtractive hybridization SSH strategy for differential gene cloning, we identified the induced expression of a Ž . rat homologue to murine and human monocyte chemotactic protein-3 MCP-3 in ischemic brain. The 2.4-kilobase rat MCP-3 gene features high homology in gene structure and sequence to murine MCP-3. The temporal expression of MCP-3 mRNA was examined in Ž . brain tissue rendered ischemia by permanent or temporary occlusion of the middle cerebral artery MCAO . A marked increase in MCP-3 Ž . mRNA was observed 12 h post-ischemia, with 49-fold and 17-fold increase n s4, p -0.01 over control in the permanent or temporary MCAO, respectively. Significant induction of MCP-3 in the ischemic cortex was sustained up to 5 days after ischemic injury. The profile of MCP-3 mRNA induction paralleled leukocyte infiltration and accumulation that occur after focal stroke, suggesting a role for MCP-3 in recruiting these inflammatory cells into the ischemic tissue. Molecular cloning of rat MCP-3 should provide a valuable tool, as demonstrated in the present work, for the investigation of MCP-3 expression and function in rat disease models. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Brain; Chemokine; Inflammation; Ischemia 1. Introduction Differential gene expression plays a critical role in normal development and in disease processes of the central nervous system. Focal brain ischemia occurs frequently in humans and usually results in significant brain damage and neurological deficits. In an effort to further understand the Ž. molecular mechanism s associated with brain injury fol- lowing focal stroke, we have applied the suppression sub- Ž . wx tractive hybridization SSH method 8 to identify genes that are specifically regulated in focal stroke. As illustrated in this work, a gene that encodes a rat homologue to AbbreÕiations: MCAO, Occlusion of the middle cerebral artery; ICAM, Intercellular adhesion molecule; IL, Interleukin; MCP, Monocyte chemotactic protein; SSH, Suppression subtractive hybridization ) Corresponding author. Department of Cardiovascular Sciences, Dupont Pharmaceuticals, Experimental Station, E400r3420B, Wilming- ton, DE 19880-0400, USA. Fax: q1-302-695-8210; E-mail: xinkang.wang@dupontpharma.com 1 Current address: Department of Cardiovascular Sciences, Dupont Pharmaceuticals, Wilmington, DE 19880-0400, USA. Ž . human and murine monocyte chemotactic protein MCP -3 was discovered in the ischemic cortex using the SSH approach. Chemokines are low molecular weight, secreted pro- teins that chemoattract and activate specific subpopulations of leukocytes and play an important role in regulating leukocyte trafficking. MCP-3 is a member of the CC or b-chemokine subfamily. Similar to other CC chemokines such as MCP-1 and macrophage inflammatory protein Ž . MIP , MCP-3 demonstrates chemotactic preference for w x monocytes, lymphocytes, eosinophils and basophils 24 . MCP-3 interacts with two primary CC chemokine recep- w x tors, i.e., CCR2 and CCR3 22 . A rat model of cerebral ischemia has been well-char- acterized and involves a robust leukocyte infiltration and w x accumulation 5,9,12 . Significant polymorphonuclear cell infiltration and accumulation occurs between 1 and 48 h, which is followed by a sustained mononuclear cell infiltra- w x tion at 2–15 days after ischemic injury 5,14 . The inflam- matory reaction in the ischemic brain tissue is believed to be mediated by de novo expression of endothelial adhesion 0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0169-328X 99 00203-X