Exacerbation of ischemic brain injury in hypercholesterolemic mice is associated with pronounced changes in peripheral and cerebral immune responses
Introduction
Inflammation is involved in stroke-induced brain damage (Iadecola and Anrather, 2011). However, difficulties remain with regard to the translation of inflammation-targeting therapeutic approaches from pre-clinical to clinical studies (Endres et al., 2008, Enlimomab Acute Stroke Trial Investigators, 2001, Fisher et al., 2009), which might be due to a neglect of co-morbidities in clinical trial designs.
Stroke induces inflammatory responses in the ischemic brain. Glial and endothelial cell activation lead to immune cell infiltration, exacerbating ischemic tissue damage (Dirnagl et al., 1999). Nevertheless, stroke also provokes peripheral immune responses, which influence secondary lesion growth and thus modulate long-term outcome (Macrez et al., 2011). These systemic immune reactions involve a rapid activation of the immune system with an increased cytokine and chemokine production in the blood and spleen (Emsley et al., 2003, Offner et al., 2006, Smith et al., 2004), followed by secondary immunosuppression and increased susceptibility to bacterial infections (Dirnagl et al., 2007, Meisel et al., 2005, Prass et al., 2003).
There is emerging evidence that inflammatory factors outside the brain markedly influence stroke susceptibility and outcome. Systemic inflammation has been shown to exacerbate brain damage in experimental models of cerebral ischemia (Langdon et al., 2010, McColl et al., 2009). As such, peripherally delivered IL-1β is known to increase tissue damage via mechanisms involving modulation of tight junction proteins and priming of peripheral innate immune cells (McColl et al., 2008). The clinical relevance of the interaction between peripheral inflammation and brain tissue injury is emphasized by epidemiological and clinical studies, demonstrating a close correlation between increased plasma IL-6 levels and stroke severity (Smith et al., 2004).
Nevertheless, the majority of pre-clinical studies do not consider co-morbidities such as hypercholesterolemia, which are frequently shown in stroke patients. Hypercholesterolemia does not only promote atherosclerotic plaque development but also induces local inflammation within the vessel wall of peripheral arteries, which is associated with broad systemic immune changes, affecting almost all immune cell subtypes of the innate and adaptive immune system (Drechsler et al., 2010, Smith et al., 2010, Wu et al., 2009).
A few pre-clinical studies emphasize the role of the immune system in the combined setting of hyperlipidemia and stroke, but are hampered due to focusing on single molecules and immune cell subsets (Kim et al., 2008). Until now, there is only limited information about the modulation of peripheral immune responses which cannot be appropriately attributed to effects either of hypercholesterolemia or brain ischemia due to insufficient controls. Finally, most studies do not discriminate how hypercholesterolemia is induced. Thus, dietary and genetic interventions for induction of hypercholesterolemia are often combined without assessing individual effects of each of these interventions.
In a comprehensive approach we investigated ischemia-induced peripheral immune responses and their concomitant reactions in the ischemic brain with special emphasis on immune cell infiltrates, endothelial cell activation and brain tissue injury. Wildtype and ApoE−/− mice fed with a regular or cholesterol-rich food were used in order to mimic different severities of hypercholesterolemia.
Section snippets
Mice
Experiments were performed in accordance to National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals with local government approval. Male wildtype and ApoE−/− mice which were generated on the same C57BL/6 genetic background were either fed with a normal chow or a cholesterol rich chow, so called Western diet (TD.88137 Adjusted Calories Diet, Harlan Laboratories) for 6 weeks and submitted to left-sided middle cerebral artery occlusion (MCAO) or sham surgery as
ApoE−/− mice fed with Western diet reveal increased stroke-induced tissue damage
Induction of 20 min of MCAO resulted in reproducible infarcts of the left-sided striatum in wildtype mice as revealed by cresyl violet staining. Significantly increased infarct volumes were noticed on day 3 in ApoE−/− mice fed with a high cholesterol diet (Fig. 1A). Accordingly, two-way ANOVA showed a significant interaction effect for ApoE−/− × diet (F1,23 = 7.753; p < 0.05). We and others have recently shown that these mice exhibit the highest cholesterol levels compared with the other experimental
Discussion
In the present study, we demonstrate that hyperlipidemia as well as stroke induces marked changes in peripheral immune responses, which are differentially regulated in a combined setting. The systemic immunomodulation is accompanied by an increased endogenous stroke-induced endothelial cell and microglia activation, resulting in enhanced leukocyte, particularly granulocyte, accumulation in ischemic brains and increased tissue injury.
Hypercholesterolemia can be induced by genetic intervention,
Conflict of interest
We declare no conflict of interest.
Acknowledgments
We thank Britta Kaltwasser and Beate Karow for technical assistance and Janine Gronewold for discussion of statistical analysis. This work was supported by grants of the German Research Foundation (HE3173/2-1 and HE3173/3-1 to D.M.H.), the Dr. Werner Jackstätt Foundation (Az. S 134-10.071 to J.H.) and Mercator Research Center Ruhr (An-2011-0081 to J.H.).
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