(Discussion) Chemokine receptor-like 2 is involved in ischemic brain injury

Discussion

In this study, we show that the expression of CCRL2, a chemokine receptor, is up-regulated in mouse brain slices under ischemic conditions. The deletion of CCRL2 not only reduced the ischemia-induced cell death in brain slices but attenuated tMCAO-induced brain injury and neurological deficits in mice. Therefore, CCRL2 is involved in brain injury in the mouse stroke model.

Previous studies have shown that CCRL2 mRNA is expressed in mouse brain and particularly in glial cells under pathological conditions (Zuurman et al., 2003). In a transient MCAO model (30 min MCAO + reperfusion), a significant up-regulation of CCRL2 mRNA in the mouse brain was observed after a 24 h- or 72 h-reperfusion (Brait et al., 2011). In our previous study, we used microarray analysis to show that CCRL2 mRNA was upregulated in brain slices treated with ischemia solution (Yao et al., 2009). The current work has further confirmed that CCRL2 mRNA and protein were increased by 14 h of ischemia. Our data also show an increase in cleaved caspase 3 which is suggestive of apoptotic cell death and increased PI uptake which indicates both apoptotic and necrotic cell death at a later time point. The early increase in CCRL2 expression may indicate that CCRL2 initiates an inflammatory cascade that induces both apoptosis and necrosis. Indeed, chemokine receptors in the C-C chemokine receptor family, such as CCR1, CCR2 and CCR5, have been implicated in CNS disorders (Offner et al., 2006, Reichel et al., 2006, Dimitrijevic et al., 2007). Although we have not analyzed the function of CCRL2 in the current study, it is tempting to believe that the CCRL2 could be a new target that contributes to CNS physiology and pathology.

Our results show that mice lacking CCRL2 had major decreases in infarct volume and showed significant improvement in neurological outcomes in tMCAO+24 h-reperfusion group. The mechanism underlying the protective effect of CCRL2 deletion is not immediately clear. Available data and our observations suggest that there is no abnormal phenotype found in the CCRL2 KO mice (Zabel et al., 2008). Arterial blood gases and pressure were not significantly different between CCRL2 KO and WT before or after MCAO. This supports the idea that the differences in the response to MCAO with CCRL2 deletion are not the result of differences in respiratory or cardiovascular function prior to ischemia. However, there was a non-significant trend for lower arterial PCO2 in CCRL2 KO mice before MCAO (Table 1), suggesting hyperventilation. Further experiments are necessary to determine if CCRL2 normally plays a role in control of breathing by recruiting proinflammatory signals to respiratory centers in the brain since inflammatory signals have been recognized recently as important modulators of cardiovascular and respiratory control (Powell and Kou, 2011, Waki et al., 2011).

The potential alteration of cerebral vascular structure in the CCRL2 KO mice could be a factor that needs further investigation since gene knockout in mice could change the cerebral vascular anatomy as documented previously (Yang et al., 2007). As such, it is possible that the neurovascular territory of the middle cerebral artery in CCRL2 KO mice could have been changed as compared to WT controls and which, as a result, could have led to reduced infarct sizes. Nevertheless, we believe it is less likely a major factor contributing to the reduction of brain injury in this study since the attenuation of brain injury only occurred in the tMCAO+24 h-reperfusion group but not in the 24 h-pMCAO group. This suggests that an increase in CCRL2 expression may have a detrimental effect on brain injury at cellular and molecular levels.

Our IS model was developed to mimic penumbral cell death in ischemic stroke (Yao et al., 2007). The significant reduction of IS-induced cell death in the in vitro CCRL2 KO brain slices indicates that a profound protection of penumbral cells should occur in the in vivo penumbral area. Along the same line, the larger the penumbral region that exists, the more protection will be demonstrated in CCRL2 KO mice subjected to MCAO. Indeed, after the deletion of CCRL2 gene, our in vivo tMCAO mice showed more pronounced brain protection than pMCAO mice which may be indicative of the prior having a larger penumbral region than the latter. This is in agreement with the well known fact that pMCAO produces a more severe and rapid brain infarction with a smaller and more short-lived penumbra than is seen with tMCAO, in which, infarcts are surrounded by a large ischemic penumbra(Lo et al., 2003). Thus, the neuroprotective effect of CCRL2 deletion found in the in vitro brain slices and in vivo stroke model may suggest that CCRL2 plays a role in ischemia-induced cell death.

Taken together, our study suggests that CCRL2 is an important component of ischemia/reperfusion brain injury. As a C-C chemokine receptor, CCRL2 may not only mediate proinflammatory processes in the lesion area but also participate in the direct modulation of cell death following ischemia. Therefore, CCRL2 is a potential therapeutic target for stroke treatment.

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