Barry W. McColl

Systemic inflammatory stimulus potentiates the acute phase and CXC chemokine responses to experimental stroke and exacerbates brain damage via interleukin-1- and neutrophil-dependent mechanisms.

Systemic inflammatory stimuli, such as infection, increase the risk of stroke and are associated with poorer clinical outcome. The mechanisms underlying the impact of systemic inflammatory stimuli on stroke are not well defined. We investigated the impact of systemic inflammation on experimental stroke and potential mechanisms involved. Focal cerebral ischemia was induced by intraluminal filament occlusion of the middle cerebral artery (MCAo). Brain damage and neurological deficit 24 h after MCAo were exacerbated by systemic lipopolysaccharide (LPS) administration. This exacerbation was critically dependent on interleukin (IL)-1, because coadministration of IL-1 receptor antagonist abolished the effect of LPS on brain damage. Systemic administration of IL-1 increased ischemic damage to a similar extent as LPS and also exacerbated brain edema. IL-1 markedly potentiated circulating levels of the acute phase proteins, serum amyloid A and IL-6, and the neutrophil-selective CXC chemokines, KC and macrophage inflammatory protein-2. Neutrophil mobilization and cortical neutrophil infiltration were aggravated by IL-1 before changes in ischemic damage. Neutropenia abolished the damaging effects of systemic IL-1. These data show for the first time that an acute systemic inflammatory stimulus is detrimental to outcome after experimental stroke and highlight IL-1 as a critical mediator in this paradigm. Our data suggest IL-1-induced potentiation of neutrophil mobilization via CXC chemokine induction is a putative mechanism underlying this effect. Our results may help to explain the poorer outcome in stroke patients presenting with infection and may have implications for neurodegenerative diseases involving neurovascular alterations, such as Alzheimers disease, in which systemic inflammation can modulate disease progression.

Systemic Inflammation Alters the Kinetics of Cerebrovascular Tight Junction Disruption after Experimental Stroke in Mice

Systemic inflammatory events, such as infection, increase the risk of stroke and are associated with worse outcome, but the mediators of this clinically important effect are unknown. Our aim here was to elucidate mechanisms contributing to the detrimental effects of systemic inflammation on mild ischemic brain injury in mice. Systemic inflammation was induced in mice by peripheral interleukin-1β (IL-1β) challenge and focal cerebral ischemia by transient middle cerebral artery occlusion (MCAo). Systemic inflammation caused an alteration in the kinetics of blood–brain barrier (BBB) disruption through conversion of a transient to a sustained disruption of the tight junction protein, claudin-5, and also markedly exacerbated disruption to the cerebrovascular basal lamina protein, collagen-IV. These alterations were associated with a systemic inflammation-induced increase in neurovascular gelatinolytic activity that was mediated by a fivefold increase in neutrophil-derived matrix metalloproteinase-9 (MMP-9) in the brains of IL-1β-challenged mice after MCAo. Specific inhibition of MMP-9 abrogated the effects of systemic inflammation on the sustained but not the acute disruption of claudin-5, which was associated with phosphorylation of cerebrovascular myosin light chain. MMP-9 inhibition also attenuated the deleterious impact of systemic inflammation on brain damage, edema, neurological deficit, and incidence of hemorrhagic transformation. These data indicate that a transformation from transient to sustained BBB disruption caused by enhanced neutrophil-derived neurovascular MMP-9 activity is a critical mechanism underlying the exacerbation of ischemic brain injury by systemic inflammation. These mechanisms may contribute to the poor clinical outcome in stroke patients presenting with antecedent infection.

Proliferating resident microglia after focal cerebral ischaemia in mice.

Cerebral ischaemia usually results in the rapid death of neurons within the immediate territory of the affected artery. Neuronal loss is accompanied by a sequence of events, including brain oedema, blood-brain barrier (BBB) breakdown, and neuroinflammation, all of which contribute to further neuronal death. Although the role of macrophages and mononuclear phagocytes in the expansion of ischaemic injury has been widely studied, the relative contribution of these cells, either of exogenous or intrinsic central nervous system (CNS) origin is still not entirely clear. The purpose of this study, therefore, was to use different durations of transient middle cerebral artery occlusion (tMCAo) in the mouse to investigate fully post-occlusion BBB permeability and cellular changes in the brain during the 72 h post-MCAo period. This was achieved using in vivo magnetic resonance imaging (MRI) and cell labelling techniques. Our results show that BBB breakdown and formation of the primary ischaemic damage after tMCAo is not associated with significant infiltration of neutrophils, although more are observed with longer periods of MCAo. In addition, we observe very few infiltrating exogenous macrophages over a 72 h period after 30 or 60 mins of occlusion, instead a profound increase in proliferating resident microglia cells was observed. Interestingly, the more severe injury associated with 60 mins of MCAo leads to a markedly reduced proliferation of resident microglial cells, suggesting that these cells may play a protective function, possibly through phagocytosis of infiltrating neutrophils. These data further support possible beneficial actions of microglial cells in the injured brain.

Microglial brain region−dependent diversity and selective regional sensitivities to aging

Microglia have critical roles in neural development, homeostasis and neuroinflammation and are increasingly implicated in age-related neurological dysfunction. Neurodegeneration often occurs in disease-specific, spatially restricted patterns, the origins of which are unknown. We performed to our knowledge the first genome-wide analysis of microglia from discrete brain regions across the adult lifespan of the mouse, and found that microglia have distinct region-dependent transcriptional identities and age in a regionally variable manner. In the young adult brain, differences in bioenergetic and immunoregulatory pathways were the major sources of heterogeneity and suggested that cerebellar and hippocampal microglia exist in a more immune-vigilant state. Immune function correlated with regional transcriptional patterns. Augmentation of the distinct cerebellar immunophenotype and a contrasting loss in distinction of the hippocampal phenotype among forebrain regions were key features during aging. Microglial diversity may enable regionally localized homeostatic functions but could also underlie region-specific sensitivities to microglial dysregulation and involvement in age-related neurodegeneration.

Systemic infection, inflammation and acute ischemic stroke

Extensive evidence implicates inflammation in multiple phases of stroke etiology and pathology. In particular, there is growing awareness that inflammatory events outside the brain have an important impact on stroke susceptibility and outcome. Numerous conditions, including infection and chronic non-infectious diseases, that are established risk factors for stroke are associated with an elevated systemic inflammatory profile. Recent clinical and pre-clinical studies support the concept that the systemic inflammatory status prior to and at the time of stroke is a key determinant of acute outcome and long-term prognosis. Here, we provide an overview of the impact of systemic inflammation on stroke susceptibility and outcome. We discuss potential mechanisms underlying the impact on ischemic brain injury and highlight the implications for stroke prevention, therapy and modeling.

Brain inflammation is induced by co-morbidities and risk factors for stroke

Highlights ► Risk factors for stroke include atherosclerosis, obesity, diabetes and hypertension. ► Stroke risk factors are associated with peripheral inflammation. ► Corpulent rats and atherogenic mice show increased inflammation in the brain. ► Pilot data show that patients at risk of stroke may also develop brain inflammation. ► Chronic peripheral inflammation can drive inflammatory changes in the brain.

Platelet interleukin-1alpha drives cerebrovascular inflammation.

White blood cell infiltration across an activated brain endothelium contributes to neurologic disease, including cerebral ischemia and multiple sclerosis. Identifying mechanisms of cerebrovascular activation is therefore critical to our understanding of brain disease. Platelet accumulation in microvessels of ischemic mouse brain was associated with endothelial activation in vivo. Mouse platelets expressed interleukin-1alpha (IL-1alpha), but not IL-1beta, induced endothelial cell adhesion molecule expression (ICAM-1 and VCAM-1), and enhanced the release of CXC chemokine CXCL1 when incubated with primary cultures of brain endothelial cells from wild-type or IL-1alpha/beta-deficient mice. A neutralizing antibody to IL-1alpha (but not IL-1beta) or application of IL-1 receptor antagonist inhibited platelet-induced endothelial activation by more than 90%. Platelets from IL-1alpha/beta-deficient mice did not induce expression of adhesion molecules in cerebrovascular endothelial cells and did not promote CXCL1 release in vitro. Conditioned medium from activated platelets induced an IL-1alpha-dependent activation of mouse brain endothelial cells and supported the transendothelial migration of neutrophils in vitro. Thus, we have identified platelets as a key source of IL-1alpha and propose that platelet activation of brain endothelium via IL-1alpha is a critical step for the entry of white blood cells, major contributors to inflammation-mediated injury in the brain.

A rapid and transient peripheral inflammatory response precedes brain inflammation after experimental stroke

Increasing evidence suggests that peripheral inflammatory responses to stroke and other brain injuries have an important role in determining neurological outcome. The mediators of this response and the temporal relationships between peripheral and central inflammatory alterations are poorly understood. In this study, we show that experimental stroke in mice induces a peripheral inflammatory response that peaks 4 h after stroke, and precedes the peak in brain inflammation 24 h after stroke. This peripheral response is dominated by the induction of the chemokine CXCL-1 and the proinflammatory cytokine interleukin-6 and could serve as an accessible target for therapy and as a source of biomarkers predictive of prognosis.

Extension of cerebral hypoperfusion and ischaemic pathology beyond MCA territory after intraluminal filament occlusion in C57Bl/6J mice

Rodent models of focal cerebral ischaemia are critical for understanding pathophysiological concepts in human stroke. The availability of genetically modified mice has prompted the adaptation of the intraluminal filament occlusion model of focal ischaemia for use in mice. In the present study, we investigated the effects of increasing duration of intraluminal occlusion on the extent and distribution of ischaemic pathology and local cerebral blood flow (LCBF) in C57Bl/6J mice, the most common background mouse strain. Volumetric assessment of ischaemic damage was performed after 15, 30 or 60 min occlusion followed by 24 h reperfusion. LCBF was measured after 15 and 60 min occlusion using quantitative 14C-iodoantipyrine autoradiography. The extent and distribution of ischaemic damage was highly sensitive to increasing occlusion duration. Recruitment of tissue outside MCA territory produced a steep increase in the volume of damage with increasing occlusion duration: 15 min (9+/-2 mm3); 30 min (56+/-6 mm3); 60 min (69+/-2 mm3). Significant increases in the severity of cerebral hypoperfusion were observed after 60 min compared to 15 min occlusion within and outside MCA territory, e.g. caudate nucleus (9+/-6 ml per 100 g per min at 60 min vs. 33 ml per 100 g per min at 15 min) and hippocampus (16+/-14 ml per 100 g per min at 60 min vs. 61+/-16 ml per 100 g per min at 15 min). MABP remained stable for 25 min after occlusion onset and declined thereafter. The integrity of the circle of Willis was examined by carbon black perfusion of the vasculature. A complete circle of Willis was present in only one of 10 mice. These results demonstrate that intraluminal filament occlusion in C57Bl/6J mice leads to an occlusion duration-dependent increase in severity of cerebral hypoperfusion and extension of ischaemic pathology beyond MCA territory.

Neutrophil Cerebrovascular Transmigration Triggers Rapid Neurotoxicity through Release of Proteases Associated with Decondensed DNA

Cerebrovascular inflammation contributes to diverse CNS disorders through mechanisms that are incompletely understood. The recruitment of neutrophils to the brain can contribute to neurotoxicity, particularly during acute brain injuries, such as cerebral ischemia, trauma, and seizures. However, the regulatory and effector mechanisms that underlie neutrophil-mediated neurotoxicity are poorly understood. In this study, we show that mouse neutrophils are not inherently toxic to neurons but that transendothelial migration across IL-1–stimulated brain endothelium triggers neutrophils to acquire a neurotoxic phenotype that causes the rapid death of cultured neurons. Neurotoxicity was induced by the addition of transmigrated neutrophils or conditioned medium, taken from transmigrated neutrophils, to neurons and was partially mediated by excitotoxic mechanisms and soluble proteins. Transmigrated neutrophils also released decondensed DNA associated with proteases, which are known as neutrophil extracellular traps. The blockade of histone–DNA complexes attenuated transmigrated neutrophil-induced neuronal death, whereas the inhibition of key neutrophil proteases in the presence of transmigrated neutrophils rescued neuronal viability. We also show that neutrophil recruitment in the brain is IL-1 dependent, and release of proteases and decondensed DNA from recruited neutrophils in the brain occurs in several in vivo experimental models of neuroinflammation. These data reveal new regulatory and effector mechanisms of neutrophil-mediated neurotoxicity (i.e., the release of proteases and decondensed DNA triggered by phenotypic transformation during cerebrovascular transmigration). Such mechanisms have important implications for neuroinflammatory disorders, notably in the development of antileukocyte therapies.