Multiplex immunoassay characterization and species comparison of inflammation in acute and non-acute ischemic infarcts in human and mouse brain tissue

Abstract

This study provides a parallel characterization of the cytokine and chemokine response to stroke in the human and mouse brain at different stages of infarct resolution. The study goal was to address the hypothesis that chronic inflammation may contribute to stroke-related dementia. We used C57BL/6 and BALB/c mice to control for strain related differences in the mouse immune response. Our data indicate that in both mouse strains, and humans, there is increased granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), interleukin-12 p70 (IL-12p70), interferon gamma-induced protein-10 (IP-10), keratinocyte chemoattractant/interleukin-8 (KC/IL-8), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), macrophage inflammatory protein-1β (MIP-1β), regulated on activation, normal T cell expressed and secreted (RANTES), and Tumor necrosis factor-α (TNF-α) in the infarct core during the acute time period. Nevertheless, correlation and two-way ANOVA analyses reveal that despite this substantial overlap between species, there are still significant differences, particularly in the regulation of granulocyte colony-stimulating factor (G-CSF), which is increased in mice but not in humans. In the weeks after stroke, during the stage of liquefactive necrosis, there is significant resolution of the inflammatory response to stroke within the infarct. However, CD68+ macrophages remain present, and levels of IL-6 and MCP-1 remain chronically elevated in infarcts from both mice and humans. Furthermore, there is a chronic T cell response within the infarct in both species. This response is differentially polarized towards a T helper 1 (Th1) response in C57BL/6 mice, and a T helper 2 (Th2) response in BALB/c mice, suggesting that the chronic inflammatory response to stroke may follow a different trajectory in different patients. To control for the fact that the average age of the patients used in this study was 80 years, they were of both sexes, and many had suffered from multiple strokes, we also present findings that reveal how the chronic inflammatory response to stroke is impacted by age, sex, and multiple strokes in mice. Our data indicate that the chronic cytokine and chemokine response to stroke is not substantially altered in 18-month old compared to 3-month old C57BL/6 mice, although T cell infiltration is attenuated. We found a significant correlation in the chronic cytokine response to stroke in males and females. However, the chronic cytokine response to stroke was mildly exacerbated by a recurrent stroke in both C57BL/6 and BALB/c mice.

Glial scars are permeable to the neurotoxic environment of chronic stroke infarcts

“…Distal middle cerebral artery occlusion (DMCAO) plus hypoxia (DH stroke) was performed on C57BL/6 mice, as previously described (Doyle et al, 2012; Nguyen et al, 2016). Mice were anesthetized by isoflurane inhalation, and an incision was made to expose the temporalis muscle.…”

“…Hypoxia is necessary with this model because C57BL/6 mice that undergo DMCAO without hypoxia have significantly smaller infarcts (Doyle et al, 2012). BALB/c mice underwent DMCAO without hypoxia, as previously described (Doyle et al, 2012; Nguyen et al, 2016). DMCAO by itself generates an infarct in BALB/c mice that is equivalent in size and location to the infarct generated by DH stroke in C57BL/6 mice (Doyle et al, 2012).…”

“…Free floating sections were then immunostained using standard techniques (Doyle et al, 2015; Nguyen et al, 2016). The following primary antibodies were used: immunoglobulin (IgG; Vector Laboratories, Cat.…”

“…It persists for many months following stroke and other brain injuries, is largely uncharacterized in terms of its constituents and toxicity to surrounding brain regions, and the exact time frame of its resolution is unknown. The eventual outcome of liquefactive necrosis is cystic encephalomalacia, the term given to the end stage of infarct resolution when all that remains is an area of astrogliosis and a cerebrospinal fluid (CSF) filled cavity (Nguyen et al, 2016). …”

“…Although the stage of liquefactive necrosis is largely uncharacterized, it is known that in both mice and humans, infarcts at the stage of liquefactive necrosis are sites of chronic inflammation (Doyle et al, 2015; Nguyen et al, 2016). In C57BL/6 mice we recently demonstrated with our colleagues that this inflammation includes a chronic B-lymphocyte response in the infarct and an increased concentration of antibodies in the surrounding brain tissue for months after the injury (Doyle et al, 2015).…”