Efficacy of Alteplase in a Mouse Model of Acute Ischemic Stroke: A Retrospective Pooled Analysis.

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%; Pint<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.

Prolonged diet-induced obesity in mice modifies the inflammatory response and leads to worse outcome after stroke.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Neuronal Toll-like receptor 4 signaling induces brain endothelial activation and neutrophil transmigration in vitro.

BACKGROUND: 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. METHODS: 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). RESULTS: 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-1ß 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. CONCLUSION: These data demonstrate for the first time that neurons can play a role as key sensors of infection to initiate CNS inflammation.

Changes in NMDA Receptor Function in Rapid Ischemic Tolerance: A Potential Role for Tri-Heteromeric NMDA Receptors.

In this study, we characterize biophysical changes in NMDA receptor function in response to brief non-injurious ischemic stress (ischemic preconditioning). Electrophysiological studies show NMDA receptor function is reduced following preconditioning in cultured rat cortical neurons. This functional change is not due to changes in the reversal potential of the receptor, but an increase in desensitization. We performed concentration-response analysis of NMDA-evoked currents, and demonstrate that preconditioned neurons show a reduced potency of NMDA to evoke currents, an increase in Mg2+ sensitivity, but no change in glycine sensitivity. Antagonists studies show a reduced inhibition of GluN2B antagonists that have an allosteric mode of action (ifenprodil and R-25-6981), but competitive antagonists at the GluR2A and 2B receptor (NVP-AMM077 and conantokin-G) appear to have similar potency to block currents. Biochemical studies show a reduction in membrane surface GluN2B subunits, and an increased co-immunoprecipitation of GluN2A with GluN2B subunits, suggestive of tri-heteromeric receptor formation. Finally, we show that blocking actin remodeling with jasplakinolide, a mechanism of rapid ischemic tolerance, prevents NMDA receptor functional changes and co-immunoprecipitation of GluN2A and 2B subunits. Together, this study shows that alterations in NMDA receptor function following preconditioning ischemia are associated with neuroprotection in rapid ischemic tolerance.

Inhibition of acid sensing ion channel currents by lidocaine in cultured mouse cortical neurons.

BACKGROUND: Lidocaine is a local anesthetic that has multiple pharmacological effects including antiarrhythmia, antinociception, and neuroprotection. Acid sensing ion channels (ASICs) are proton-gated cation channels that belong to the epithelial sodium channel/degenerin superfamily. Activation of ASICs by protons results in sodium and calcium influx. ASICs have been implicated in various physiological processes including learning/memory, nociception, and in acidosis-mediated neuron injury. In this study, we examined the effect of lidocaine on ASICs in cultured mouse cortical neurons. METHODS: ASIC currents were activated and recorded using a whole-cell patch-clamp technique in cultured mouse cortical neurons. The effects of lidocaine at different concentrations were examined. To determine whether the inhibition of lidocaine on ASIC currents is subunit specific, we examined the effect of lidocaine on homomeric ASIC1a and ASIC2a currents expressed in Chinese hamster ovary cells. RESULTS: Lidocaine significantly inhibits the ASIC currents in mouse cortical neurons. The inhibition was reversible and dose dependent. A detectable effect was noticed at a concentration of 0.3 mM lidocaine. At 30 mM, ASIC current was inhibited by approximately 90%. Analysis of the complete dose-response relationship yielded a half-maximal inhibitory concentration of 11.79 ± 1.74 mM and a Hill coefficient of 2.7 ± 0.5 (n = 10). The effect is rapid and does not depend on pH. In Chinese hamster ovary cells expressing different ASIC subunits, lidocaine inhibits the ASIC1a current without affecting the ASIC2a current. CONCLUSION: ASIC currents are significantly inhibited by lidocaine. Our finding reveals a new pharmacological effect of lidocaine in neurons.

Proliferating progenitor cells: a required cellular element for induction of ischemic tolerance in the brain.

Permanent cerebral blood flow reduction results in brain injury (stroke), whereas transient ischemic stress results in preconditioning, which can ameliorate the extent of irreversible brain injury from subsequent ischemia-the phenomena of ischemic tolerance. Neurogenesis in the brain occurs after both ischemic injury and the brief ischemia resulting in preconditioning. As neurogenesis is regarded as having an intrinsic neuroprotective role in the brain, we investigated the possible role of these endogenous progenitor cells in the induction of ischemic tolerance. Methylazoxymethanol acetate (MAM) was injected in wild-type mice to attenuate precursor cell proliferation and ganciclovir was used to diminish newly generated cells in GFAP/HSV-TK mice. Both MAM and ganciclovir significantly attenuated ischemia-induced progenitor cell proliferation in the subventricular zone, dentate gyrus, penumbra, and corpus callosum as quantified by 5-bromo-2'-deoxyuridine- or Ki-67-positive cells. Attenuation of ischemia-induced progenitor cell proliferation in the brain blocked the induction of ischemic tolerance. Further the number of TUNEL (TdT-mediated dUTP nick end labeling)-positive cells was considerably increased in MAM-treated animals, whereas MAM did not cause cell death in sham-operated controls. The results of this study suggest a role for endogenous progenitors in the protective effect of ischemic tolerance.

Downregulation of hippocampal adenosine kinase after focal ischemia as potential endogenous neuroprotective mechanism.

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.

Effects of interferon-beta on oligodendroglial cells.

The effect of interferon-beta (IFN-beta) for the treatment of multiple sclerosis (MS) is thought to be mediated by the modulation of immune cells. In addition, it has been shown that glial cells may be influenced by IFN-beta and a role during remyelination has been suggested. However, the mechanism is not yet clear and there are conflicting data. We have therefore systematically investigated proliferation, differentiation, toxicity, and cytoprotection of IFN-beta on oligodendroglia, both as a direct effect and mediated indirectly via other glial cells. Differentiation of oligodendrocyte progenitor cells (OPC) was significantly (p<0.01) inhibited by IFN-beta only when cultured in the presence with astrocytes and microglia. Proliferation was not changed, neither was IFN-beta toxic. There was no cytoprotective effect of IFN-beta on oligodendroglia injury induced by H2O2, NO, complement, or glutamate. Similarly, there was no cytoprotective effect mediated via treatment of astrocytes with IFN-beta. These data demonstrate that IFN-beta is neither toxic nor cytoprotective for oligodendrocytes. In summary, the only effect of IFN-beta was the inhibition of differentiation of OPC mediated indirectly via other glial cells. In vivo experiments will show how this effect may influence remyelination.

Oligodendrocyte precursor cells express a functional chemokine receptor CCR3: implications for myelination.

Myelination in the central nervous system requires an accurate interplay between oligodendrocyte precursor cells (OPC) and axons. By as yet not fully understood mechanisms, OPC proliferate, migrate to the axon to be myelinated and finally differentiate into mature oligodendrocytes. The recent finding that OPC express CXC chemokine receptors led us to the investigation of the expression and functional importance of CC chemokine receptors. Using RT-PCR, we show that primary OPC from neonatal rats express CCR3, while CCR1, CCR2, CCR4, CCR5, and CCR7 are not expressed. Immunofluorescence staining of OPC could further demonstrate protein expression of CCR3. A rise of intracellular Ca2+ upon stimulation with the appropriate ligand CCL11 showed that this receptor is functional. Moreover, CCL11 led to a concentration specific increase in proliferation, inhibition of migration, and augmentation of differentiation in primary OPC. Thus, CCR3 may influence the process of myelination. This is of general importance for both developmental tissue patterning and for repair processes in demyelinating diseases like multiple sclerosis.

Modulation of rat oligodendrocyte precursor cells by the chemokine CXCL12.

Migration, proliferation, and differentiation of oligodendrocyte precursor cells are essential for the assembly of myelin in the central nervous system. Knowledge on the regulation of these precursor cells is therefore of great importance for the understanding of developmental myelination and remyelination in demyelinating diseases. Here, we show that primary rat oligodendrocyte precursor cells express the chemokine receptor CXCR4. Stimulation with the ligand CXCL12 (SDF-1 alpha) leads to intracellular Ca elevation. Furthermore, 10 ng/ml CXCL12 augmented differentiation of precursors into mature oligodendrocytes. Migration toward growth factor conditioned medium was inhibited by CXCL12, while proliferation was only slightly modulated. The effect of CXCL12 on both migration and differentiation was blocked using a G protein antagonist. These data suggest a role for CXCL12 and oligodendroglial CXCR4 receptors during developmental myelination and repair in demyelinating diseases of the central nervous system.

A cross-laboratory preclinical study on the effectiveness of interleukin-1 receptor antagonist in 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 acute-phase protein PTX3 is an essential mediator of glial scar formation and resolution of brain edema after ischemic injury.

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.