Mice deficient in fractalkine are less susceptible to cerebral ischemia-reperfusion injury
Introduction
Cerebral ischemia is associated with activation and migration of inflammatory cells such as leukocytes and microglia. Inflammatory processes in the brain have been associated with increased expression of different classes of adhesion molecules and chemokines, which mediate leukocyte trafficking and microglial activation (Springer, 1995). Inactivation of adhesion molecules ICAM-1 and Mac-1 in an experimental rodent stroke model resulted in a significant reduction in ischemic damage, indicating the importance of the adhesion cascade Soriano et al., 1996, Soriano et al., 1999. Distinct microglial activation also occurs immediately after transient focal ischemia and peaks between 16 and 24 h after middle cerebral artery occlusion (Mabuchi et al., 2000). Chemokines mediate both leukocyte migration and microglial activation and are extensively expressed after cerebral ischemia (Wang et al., 1995). However, studies examining the direct role of chemokines after ischemic insults are lacking.
Fractalkine (FKN), also known as neurotactin, is one of many chemokines that are cell-type-selective proteins that facilitate directional migration of leukocytes and microglial activation.(Pan et al., 1997) Recent investigations have shown that FKN may facilitate inflammatory processes in the central nervous system (Schwaeble et al., 1998). To test the hypothesis that a deficiency in FKN leads to a reduction in infarct size following stroke, we generated FKN-deficient mice and measured the extent of histopathological damage after transient cerebral ischemia-reperfusion.
Section snippets
Construction of targeting vector and the generation of FKN deficient mice
A mouse FKN genomic clone was isolated from a mouse (129Sv/J) genomic library using FKN cDNA as a probe. 5′ and 3′ FKN flanking regions were subcloned and sequenced. A HindIII fragment containing the first exon of FKN, which only encoded the signal peptide, and a SalI/NcoI fragment containing the 3′ UTR were inserted into a targeting vector (Fig. 1a). The resulting targeting vector has approximately 3 and 4.3 kb of FKN homology, 5′ and 3′ of the Neo cassette, and is designed to delete the
Generation of mice deficient in FKN
We generated FKN-deficient mice by targeted gene disruption of the fractalkine gene by insertion of a neomycin cassette (Fig. 1a). The mice were genotyped by Southern analysis of EcoRI digested tails from F2 progeny mice (Fig. 1b). The FKN gene disruption was confirmed by Northern blot analysis (Fig. 1c). Heterozygous and homozygous mice developed normally and were fertile. The absence of FKN expression in the FKN-deficient mice was also confirmed by both immunohistochemistry and in situ
Discussion
In the setting of cerebral ischemia and reperfusion, FKN deficiency provides some degree of neuroprotection. However, the mechanism of this phenomenon remains elusive. Two potential pathways include an impairment of leukocyte recruitment by separation from selectin- and integrin-mediated pathways and/or microglial activation Fong et al., 1998, Imai et al., 1997. Given the short time course (24 h) of our experimental model, the former might be operational. A recent analysis of fractalkine
Acknowledgments
We thank K. McDonald and A.J. Goodearl for affinity-purified polyclonal antibodies to FKN and G. Kingsbury for polyclonal antibodies to CX3CR1.
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