CD69 Plays a Beneficial Role in Ischemic Stroke by Dampening Endothelial Activation.

RATIONALE: CD69 is an immunomodulatory molecule induced during lymphocyte activation. Following stroke, T-lymphocytes upregulate CD69 but its function is unknown. OBJECTIVE: We investigated whether CD69 was involved in brain damage following an ischemic stroke. METHODS AND RESULTS: We used adult male mice on the C57BL/6 or BALB/c backgrounds, including wild-type mice and CD69-/- mice, and CD69+/+ and CD69-/- lymphocyte-deficient Rag2-/- mice, and generated chimeric mice. We induced ischemia by transient or permanent middle cerebral artery occlusion. We measured infarct volume, assessed neurological function, and studied CD69 expression, as well as platelet function, fibrin(ogen) deposition, and VWF (von Willebrand factor) expression in brain vessels and VWF content and activity in plasma, and performed the tail-vein bleeding test and the carotid artery ferric chloride-induced thrombosis model. We also performed primary glial cell cultures and sorted brain CD45-CD11b-CD31+ endothelial cells for mRNA expression studies. We blocked VWF by intravenous administration of anti-VWF antibodies. CD69-/- mice showed larger infarct volumes and worse neurological deficits than the wild-type mice after ischemia. This worsening effect was not attributable to lymphocytes or other hematopoietic cells. CD69 deficiency lowered the time to thrombosis in the carotid artery despite platelet function not being affected. Ischemia upregulated Cd69 mRNA expression in brain endothelial cells. CD69-deficiency increased fibrin(ogen) accumulation in the ischemic tissue, and plasma VWF content and activity, and VWF expression in brain vessels. Blocking VWF reduced infarct volume and reverted the detrimental effect of CD69-/- deficiency. CONCLUSIONS: CD69 deficiency promotes a prothrombotic phenotype characterized by increased VWF and worse brain damage after ischemic stroke. The results suggest that CD69 acts as a downregulator of endothelial activation.

The IMPROVE Guidelines (Ischaemia Models: Procedural Refinements Of in Vivo Experiments).

Most in vivo models of ischaemic stroke target the middle cerebral artery and a spectrum of stroke severities, from mild to substantial, can be achieved. This review describes opportunities to improve the in vivo modelling of ischaemic stroke and animal welfare. It provides a number of recommendations to minimise the level of severity in the most common rodent models of middle cerebral artery occlusion, while sustaining or improving the scientific outcomes. The recommendations cover basic requirements pre-surgery, selecting the most appropriate anaesthetic and analgesic regimen, as well as intraoperative and post-operative care. The aim is to provide support for researchers and animal care staff to refine their procedures and practices, and implement small incremental changes to improve the welfare of the animals used and to answer the scientific question under investigation. All recommendations are recapitulated in a summary poster (see supplementary information).

Uric Acid Is Protective After Cerebral Ischemia/Reperfusion in Hyperglycemic Mice.

Hyperglycemia at stroke onset is associated with poor long-term clinical outcome in numerous studies. Hyperglycemia induces intracellular acidosis, lipid peroxidation, and peroxynitrite production resulting in the generation of oxidative and nitrosative stress in the ischemic tissue. Here, we studied the effects of acute hyperglycemia on in vivo intercellular adhesion molecule-1 (ICAM-1) expression, neutrophil recruitment, and brain damage after ischemia/reperfusion in mice and tested whether the natural antioxidant uric acid was protective. Hyperglycemia was induced by i.p. administration of dextrose 45 min before transient occlusion of the middle cerebral artery. Magnetic resonance imaging (MRI) was performed at 24 h to measure lesion volume. A group of normoglycemic and hyperglycemic mice received an i.v. injection of micron-sized particles of iron oxide (MPIOs), conjugated with either anti-ICAM-1 antibody or control IgG, followed by T2*w MRI. Neutrophil infiltration was studied by immunofluorescence and flow cytometry. A group of hyperglycemic mice received an i.v. infusion of uric acid (16 mg/kg) or the vehicle starting after 45 min of reperfusion. ICAM-1-targeted MPIOs induced significantly larger MRI contrast-enhancing effects in the ischemic brain of hyperglycemic mice, which also showed more infiltrating neutrophils and larger lesions than normoglycemic mice. Uric acid reduced infarct volume in hyperglycemic mice but it did not prevent vascular ICAM-1 upregulation and did not significantly reduce the number of neutrophils in the ischemic brain tissue. In conclusion, hyperglycemia enhances stroke-induced vascular ICAM-1 and neutrophil infiltration and exacerbates the brain lesion. Uric acid reduces the lesion size after ischemia/reperfusion in hyperglycemic mice.

Immature monocytes recruited to the ischemic mouse brain differentiate into macrophages with features of alternative activation.

Acute stroke induces a local inflammatory reaction causing leukocyte infiltration. Circulating monocytes are recruited to the ischemic brain and become tissue macrophages morphologically indistinguishable from reactive microglia. However, monocytes are a heterogeneous population of cells with different functions. Herein, we investigated the infiltration and fate of the monocyte subsets in a mouse model of focal brain ischemia by permanent occlusion of the distal portion of the middle cerebral artery. We separated two main subtypes of CD11b(hi) monocytes according to their expression of the surface markers Ly6C and CD43. Using adoptive transfer of reporter monocytes and monocyte depletion, we identified the pro-inflammatory Ly6C(hi)CD43(lo)CCR2(+) subset as the predominant monocytes recruited to the ischemic tissue. Monocytes were seen in the leptomeninges from where they entered the cortex along the penetrating arterioles. Four days post-ischemia, they had invaded the infarcted core, where they were often located adjacent to blood vessels. At this time, Iba-1(-) and Iba-1(+) cells in the ischemic tissue incorporated BrdU, but BrdU incorporation was rare in the reporter monocytes. The monocyte phenotype progressively changed by down-regulating Ly6C, up-regulating F4/80, expressing low or intermediate levels of Iba-1, and developing macrophage morphology. Moreover, monocytes progressively acquired the expression of typical markers of alternatively activated macrophages, like arginase-1 and YM-1. Collectively, the results show that stroke mobilized immature pro-inflammatory Ly6C(hi)CD43(lo) monocytes that acutely infiltrated the ischemic tissue reaching the core of the lesion. Monocytes differentiated to macrophages with features of alternative activation suggesting possible roles in tissue repair during the sub-acute phase of stroke.

Neutrophil recruitment to the brain in mouse and human ischemic stroke.

Neutrophils are rapidly recruited in response to local tissue infection or inflammation. Stroke triggers a strong inflammatory reaction but the relevance of neutrophils in the ischemic brain is not fully understood, particularly in the absence of reperfusion. We investigated brain neutrophil recruitment in two murine models of permanent ischemia induced by either cauterization of the distal portion of the middle cerebral artery (c-MCAo) or intraluminal MCA occlusion (il-MCAo), and three fatal cases of human ischemic stroke. Flow cytometry analyses revealed progressive neutrophil recruitment after c-MCAo, lesser neutrophil recruitment following il-MCAo, and absence of neutrophils after sham operation. Confocal microscopy identified neutrophils in the leptomeninges from 6 h after the occlusion, in the cortical basal lamina and cortical Virchow-Robin spaces from 15 h, and also in the cortical brain parenchyma at 24 h. Neutrophils showed signs of activation including histone-3 citrullination, chromatin decondensation, and extracellular projection of DNA and histones suggestive of extracellular trap formation. Perivascular neutrophils were identified within the entire cortical infarction following c-MCAo. After il-MCAo, neutrophils prevailed in the margins but not the center of the cortical infarct, and were intraluminal and less abundant in the striatum. The lack of collaterals to the striatum and a collapsed pial anastomotic network due to brain edema in large hemispheric infarctions could impair neutrophil trafficking in this model. Neutrophil extravasation at the leptomeninges was also detected in the human tissue. We concluded that neutrophils extravasate from the leptomeningeal vessels and can eventually reach the brain in experimental animal models and humans with prolonged arterial occlusion.

Infarct volume prediction using apparent diffusion coefficient maps during middle cerebral artery occlusion and soon after reperfusion in the rat.

Middle cerebral artery occlusion (MCAO) in rodents causes brain infarctions of variable sizes that depend on multiple factors, particularly in models of ischemia/reperfusion. This is a major problem for infarct volume comparisons between different experimental groups since unavoidable variability can induce biases in the results and imposes the use of large number of subjects. MRI can help to minimize these difficulties by ensuring that the severity of ischemia is comparable between groups. Furthermore, several studies showed that infarct volumes can be predicted with MRI data obtained soon after ischemia onset. However, such predictive studies require multiparametric MRI acquisitions that cannot be routinely performed, and data processing using complex algorithms that are often not available. The aim here was to provide a simplified method for infarct volume prediction using apparent diffusion coefficient (ADC) data in a model of transient MCAO in rats. ADC images were obtained before, during MCAO and after 60 min of reperfusion. Probability histograms were generated using ADC data obtained either during MCAO, after reperfusion, or both combined. The results were compared to real infarct volumes, i.e.T2 maps obtained at day 7. Assessment of the performance of the estimations showed better results combining ADC data obtained during occlusion and at reperfusion. Therefore, ADC data alone can provide sufficient information for a reasonable prediction of infarct volume if the MRI information is obtained both during the occlusion and soon after reperfusion. This approach can be used to check whether drug administration after MRI acquisition can change infarct volume prediction.

IL-10 deficiency exacerbates the brain inflammatory response to permanent ischemia without preventing resolution of the lesion.

Stroke induces inflammation that can aggravate brain damage. This work examines whether interleukin-10 (IL-10) deficiency exacerbates inflammation and worsens the outcome of permanent middle cerebral artery occlusion (pMCAO). Expression of IL-10 and IL-10 receptor (IL-10R) increased after ischemia. From day 4, reactive astrocytes showed strong IL-10R immunoreactivity. Interleukin-10 knockout (IL-10 KO) mice kept in conventional housing showed more mortality after pMCAO than the wild type (WT). This effect was associated with the presence of signs of colitis in the IL-10 KO mice, suggesting that ongoing systemic inflammation was a confounding factor. In a pathogen-free environment, IL-10 deficiency slightly increased infarct volume and neurologic deficits. Induction of proinflammatory molecules in the IL-10 KO brain was similar to that in the WT 6 hours after ischemia, but was higher at day 4, while differences decreased at day 7. Deficiency of IL-10 promoted the presence of more mature phagocytic cells in the ischemic tissue, and enhanced the expression of M2 markers and the T-cell inhibitory molecule CTLA-4. These findings agree with a role of IL-10 in attenuating local inflammatory reactions, but do not support an essential function of IL-10 in lesion resolution. Upregulation of alternative immunosuppressive molecules after brain ischemia can compensate, at least in part, the absence of IL-10.