European Journal of Neuroscience, Vol. 10, pp. 680–688, 1998 © European Neuroscience Association Focal ischaemia of the rat brain elicits an unusual inflammatory response: early appearance of CD8+ macrophages/microglia Sebastian Jander, Michael Schroeter, Donatella D’Urso, Clemens Gillen, Otto W. Witte and Guido Stoll Department of Neurology and Center for Biological and Medical Research, Heinrich-Heine-University, PO Box 10 10 07, D-40001 Du ¨ sseldorf, Germany Keywords: CD4, experimental autoimmune encephalomyelitis, Wallerian degeneration. Abstract Cerebral ischaemia leads to profound glial activation and leukocyte infiltration into the infarct area. In this study, we provide evidence for a dual macrophage response in focal ischaemic lesions of the rat brain. We show that a considerable proportion of macrophages in the ischaemic lesions express the CD8αβ heterodimer to date only described on CD8+ T cells. As known from other lesion paradigms, CD4+ macrophages were also present. Interestingly, CD8- and CD4-expressing macrophages formed two non-overlapping subpopulations. CD8+ macrophages reached their maximum during the first week with pronounced downregulation thereafter whereas CD4+ cells persisted at high levels into the second week. In contrast to cerebral ischaemia, macrophages in the spleen and in Wallerian degeneration after optic nerve axotomy expressed CD4, but not CD8. In experimental autoimmune encephalomyelitis, CD8 was mainly associated with T cells and very weakly detectable on some ramified cells resembling activated microglia. In conclusion, we show that cerebral ischaemia triggers an unusual inflammatory response characterized by the appearance of CD8+/CD4– macrophages that might exert specific functions in the pathogenesis of ischaemic brain damage. Introduction Both resident microglia and infiltrating blood-derived macrophages contribute to the population of activated macrophages that appear in response to different kinds of central nervous system (CNS) injury (Perry & Gordon, 1988; Streit et al., 1988; Perry et al., 1993; Schroeter et al., 1997). Activated CNS macrophages can exert cytotoxic effector functions (Banati et al., 1993; Giulian et al., 1993). In neurological disorders like stroke and spinal cord injury, these cells may therefore exacerbate the tissue damage induced by the primary ischaemic or traumatic insult (Giulian & Robertson, 1990; Lees, 1993; Dusart & Schwab, 1994; Gehrmann et al., 1995). On the other hand, activated macrophages secrete growth promoting substances and remove inhibit- ory tissue debris (Lindholm et al., 1987; Schwab & Caroni, 1988; David et al., 1990). Thereby, they are equally important in repair and regeneration processes after CNS injury. Irrespective of the underlying disease mechanism, activated CNS macrophages uniformly upregulate immunomolecules—most promin- ently major histocompatibility complex (MHC) class II and CD4 antigens—on their surface (Matsumoto et al., 1986; Perry & Gordon, 1987; Stoll et al., 1989; Streit et al., 1989; Matsushima et al., 1994). To date, no phenotypic markers that could differentiate functionally specialized macrophage subpopulations in the injured CNS have been described. We have previously shown that the macrophage response to focal cerebral ischaemia in the rat is accompanied by the appearance Correspondence: Sebastian Jander, as above. Received 20 March 1997, revised 18 August 1997, accepted 6 October 1997 of substantial numbers of CD8+ cells (Schroeter et al., 1994; Jander et al., 1995). These cells do not express markers for T cells or natural killer (NK) cells, the only two established CD8+ cell populations in the rat (Lawetzky et al., 1990; Torres-Nagel et al., 1992). In the present study, we used double labelling immunofluorescence in combination with confocal microscopy and mRNA analysis by reverse transcriptase–polymerase chain reaction (RT–PCR) to study the pre- cise cellular localization and time course of CD8 expression in ischaemic lesions induced by either middle cerebral artery occlusion (MCAO) or photothrombosis of cortical microvessels (Watson et al., 1985). We provide evidence that CD8 after ischaemia is expressed on a hitherto unrecognized subpopulation of activated CNS macro- phages that may play a specific functional role in the pathogenesis of ischaemic brain damage. Materials and methods Animals Focal cerebral infarcts were induced in adult male Wistar and female Lewis rats by permanent MCAO or photothrombosis of cortical microvessels as described in detail elsewhere (Schroeter et al., 1994; Jander et al., 1995). Animals were killed 24 h later and on days 3, 6, 10 and 14 after ischaemia. Experimental autoimmune encephalomyelitis