The innate immune response in the brain is initiated by pathogen-associated molecular patterns (PAMPS) or danger-associated molecular patterns (DAMPS) produced in response to central nervous system (CNS) infection or injury. These molecules activate members of the Toll-like receptor (TLR) family, of which TLR4 is the receptor for bacterial lipopolysaccharide (LPS). Although neurons have been reported to express TLR4, the function of TLR4 activation in neurons remains unknown. TLR4 mRNA expression in primary mouse glial and neuronal cultures was assessed by RT-PCR. Mouse mixed glial, neuronal or endothelial cell cultures were treated with LPS in the absence or the presence of a TLR4 specific antagonist (VIPER) or a specific JNK inhibitor (SP600125). Expression of inflammatory mediators was assayed by cytometric bead array (CBA) and ELISA. Activation of extracellular-signal regulated kinase 1/2 (ERK1/2), p38, c-Jun-N-terminal kinase (JNK) and c-Jun was assessed by Western blot. The effect of conditioned media of untreated- versus LPS-treated glial or neuronal cultures on endothelial activation was assessed by neutrophil transmigration assay, and immunocytochemistry and ELISA were used to measure expression of intercellular cell adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1). LPS induces strong release of the chemokines RANTES and CXCL1 (KC), tumor necrosis factor-α (TNFα) and IL-6 in primary mouse neuronal cultures. In contrast, LPS induced release of IL-1α, IL-1β and granulocyte-colony stimulating factor (G-CSF) in mixed glial, but not in neuronal cultures. LPS-induced neuronal KC expression and release were completely blocked by VIPER. In glial cultures, LPS induced activation of ERK1/2, p38 and JNK. In contrast, in neuronal cultures, LPS activated JNK but not ERK1/2 or p38, and the specific JNK inhibitor SP600125 significantly blocked LPS-induced KC expression and release. Finally, conditioned medium of LPS-treated neuronal cultures induced strong expression of ICAM-1 and VCAM-1 on endothelial cells, and induced infiltration of neutrophils across the endothelial monolayer, which was inhibited by VIPER. These data demonstrate for the first time that neurons can play a role as key sensors of infection to initiate CNS inflammation.

Acute-phase proteins (APPs) are key effectors of the immune response and are routinely used as biomarkers in cerebrovascular diseases, but their role during brain inflammation remains largely unknown. Elevated circulating levels of the acute-phase protein pentraxin-3 (PTX3) are associated with worse outcome in stroke patients. Here we show that PTX3 is expressed in neurons and glia in response to cerebral ischemia, and that the proinflammatory cytokine interleukin-1 (IL-1) is a key driver of PTX3 expression in the brain after experimental stroke. Gene deletion of PTX3 had no significant effects on acute ischemic brain injury. In contrast, the absence of PTX3 strongly compromised blood–brain barrier integrity and resolution of brain edema during recovery after ischemic injury. Compromised resolution of brain edema in PTX3-deficient mice was associated with impaired glial scar formation and alterations in scar-associated extracellular matrix production. Our results suggest that PTX3 expression induced by proinflammatory signals after ischemic brain injury is a critical effector of edema resolution and glial scar formation. This highlights the potential role for inflammatory molecules in brain recovery after injury and identifies APPs, in particular PTX3, as important targets in ischemic stroke and possibly other brain inflammatory disorders.

Obesity increases the risk for ischaemic stroke and is associated with worse outcome clinically and experimentally. Most experimental studies have used genetic models of obesity. Here, a more clinically relevant model, diet-induced obesity, was used to study the impact of obesity over time on the outcome and inflammatory response after stroke. Male C57BL/6 mice were maintained on a high-fat (60 % fat) or control (12 % fat) diet for 2, 3, 4 and 6 months when experimental stroke was induced by transient occlusion of the middle cerebral artery (MCAo) for either 20 (6-month diet) or 30 min (2-, 3-, 4- and 6-month diet). Ischaemic damage, blood–brain barrier (BBB) integrity, neutrophil number and chemokine expression in the brain were assessed at 24 h. Plasma chemokine levels (at 4 and 24 h) and neutrophil number in the liver (at 24 h) were measured. Physiological parameters (body weight and blood glucose) were measured in naïve control- and high-fat-fed mice at all time points and blood pressure at 3 and 6 months. Blood cell counts were also assessed in naïve 6-month control- and high-fat-fed mice. Mice fed a high-fat diet for 6 months had greater body weight, blood glucose and white and red blood cell count but no change in systolic blood pressure. After 4 and 6 months of high-fat feeding, and in the latter group with a 30-min (but not 20-min) occlusion of the MCA, obese mice had greater ischaemic brain damage. An increase in blood–brain barrier permeability, chemokine expression (CXCL-1 and CCL3), neutrophil number and microglia/macrophage cells was observed in the brains of 6-month high-fat-fed mice after 30-min MCAo. In response to stroke, chemokine (CXCL-1) expression in the plasma and liver was significantly different in obese mice (6-month high-fat fed), and a greater number of neutrophils were detected in the liver of control but not obese mice. The detrimental effects of diet-induced obesity on stroke were therefore dependent on the severity of obesity and length of ischaemic challenge. The altered inflammatory response in obese mice may play a key role in its negative impact on stroke.

Stroke represents a global challenge and is a leading cause of permanent disability worldwide. Despite much effort, translation of research findings to clinical benefit has not yet been successful. Failure of neuroprotection trials is considered, in part, due to the low quality of preclinical studies, low level of reproducibility across different laboratories and that stroke co-morbidities have not been fully considered in experimental models. More rigorous testing of new drug candidates in different experimental models of stroke and initiation of preclinical cross-laboratory studies have been suggested as ways to improve translation. However, to our knowledge, no drugs currently in clinical stroke trials have been investigated in preclinical cross-laboratory studies. The cytokine interleukin 1 is a key mediator of neuronal injury, and the naturally occurring interleukin 1 receptor antagonist has been reported as beneficial in experimental studies of stroke. In the present paper, we report on a preclinical cross-laboratory stroke trial designed to investigate the efficacy of interleukin 1 receptor antagonist in different research laboratories across Europe. Our results strongly support the therapeutic potential of interleukin 1 receptor antagonist in experimental stroke and provide further evidence that interleukin 1 receptor antagonist should be evaluated in more extensive clinical stroke trials.

The rate of ischemic brain injury varies with the brain region, requiring only hours in striatum but days in hippocampus. Such maturation implies the existence of endogenous neuroprotective mechanisms. Adenosine is an endogenous neuroprotectant regulated by adenosine kinase (ADK). To investigate, whether adenosine might play a role in protecting the hippocampus after focal ischemia, we subjected transgenic mice, which overexpress ADK in hippocampal neurons (Adk-tg mice) to transient middle cerebral artery occlusion (MCAO). Although the hippocampus of wild-type (wt) mice was consistently spared from injury after 60 mins of MCAO, hippocampal injury became evident in Adk-tg mice after only 15 mins of MCAO. To determine, whether downregulation of hippocampal ADK might qualify as candidate mechanism mediating endogenous neuroprotection, we evaluated ADK expression in wt mice after several periods of reperfusion after 15 or 60 mins of MCAO. After 60 mins of MCAO, hippocampal ADK was significantly reduced in both hemispheres after 1, 3, and 24 h of reperfusion. Reduction of ADK-immunoreactivity corresponded to a 2.2-fold increase in hippocampal adenosine at 3 h of reperfusion. Remarkably, a significant reduction of ADK immunoreactivity was also found in the ipsilateral (stroked) hippocampus after 15 mins of MCAO and 3 h of reperfusion. Thus, transient downregulation of hippocampal ADK after stroke might be a protective mechanism during maturation hippocampal cell loss.

Background and Purpose—: The debate over the fact that experimental drugs proposed for the treatment of stroke fail in the translation to the clinical situation has attracted considerable attention in the literature. In this context, we present a retrospective pooled analysis of a large data set from preclinical studies, to examine the effects of early versus late administration of intravenous recombinant tissue-type plasminogen activator. Methods—: We collected data from 26 individual studies from 9 international centers (13 researchers; 716 animals) that compared recombinant tissue-type plasminogen activator with controls, in a unique mouse model of thromboembolic stroke induced by an in situ injection of thrombin into the middle cerebral artery. Studies were classified into early (<3 hours) versus late (≥3 hours) drug administration. Final infarct volumes, assessed by histology or magnetic resonance imaging, were compared in each study, and the absolute differences were pooled in a random-effect meta-analysis. The influence of time of administration was tested. Results—: When compared with saline controls, early recombinant tissue-type plasminogen activator administration was associated with a significant benefit (absolute difference, −6.63 mm ³ ; 95% confidence interval, −9.08 to −4.17; I ² =76%), whereas late recombinant tissue-type plasminogen activator treatment showed a deleterious effect (+5.06 mm ³ ; 95% confidence interval, +2.78 to +7.34; I ² =42%; P _int <0.00001). Results remained unchanged after subgroup analyses. Conclusions—: Our results provide the basis needed for the design of future preclinical studies on recanalization therapies using this model of thromboembolic stroke in mice. The power analysis reveals that a multicenter trial would require 123 animals per group instead of 40 for a single-center trial.