Carles Justicia

Expression and activation of matrix metalloproteinase-2 and -9 in rat brain after transient focal cerebral ischemia

Matrix metalloproteinases (MMPs) degrade the extracellular matrix and carry out key functions during development and after injury. By means of zymography, Western blot and immunohistochemistry, we studied MMP-2 (gelatinase A) and MMP-9 (gelatinase B) in rat brain after focal cerebral ischemia. The control rat brain showed constitutive MMP-2 and, to a lesser extent, MMP-9, which were mainly present as prozymogens. MMP-2 protein was located in the cell body of neurons, glia, and endothelium, whereas MMP-9 was associated to neurons and myelinated fibre tracts. Ischemia greatly increased MMP activation in two temporal waves, in the first one, MMP-9 protein was induced from 4 h to 4 days, and also a small and short-lasting increase in MMP-2 was detected at 4 h. The second wave showed a massive increase in MMP-2 protein expression and activation by day 4, which was compatible with abundant MMP-2 in reactive microglia/macrophages. Our results are compatible with progressive induction of MMP-9 proform, likely in neurons, shortly after ischemia. For MMP-2, the results suggest a discrete production immediately after reperfusion, while a very enhanced expression and activation of MMP-2 attributable to microglia/macrophages occurs on day 4, and it might contribute to the phagocytic action of these reactive cells.

Neutrophil infiltration increases matrix metalloproteinase-9 in the ischemic brain after occlusion/reperfusion of the middle cerebral artery in rats.

Matrix metalloproteinase-9 (MMP-9) activity increases in the brain during the first day after focal ischemia and might be involved in the pathogenesis of tissue damage. We previously showed MMP-9 in the extracellular space of brain parenchyma along with neutrophil recruitment after ischemia. In the present study, we tested whether neutrophils were a direct source of enhanced MMP-9 in the ischemic brain. Neutrophil infiltration was prevented either by injecting an antibody against ICAM-1, which abrogates neutrophil adhesion to the endothelial vessel wall, or by inducing neutropenia. One-hour intraluminal middle cerebral artery occlusion with reperfusion was induced, and studies were performed at 24 hours. Circulating neutrophils expressed 95-kDa MMP-9 and dimers, and infiltrated neutrophils stained positive for MMP-9. The expression of MMP-9 (mainly 95-kDa proform and dimers and, to a lesser extent, 88-kDa form) increased in brain after ischemia/reperfusion. Treatments preventing neutrophil infiltration failed to preclude the ischemia-induced increase in 88-kDa MMP-9 form and gelatinase activity in neurons and blood vessels. However, these treatments prevented the major increase in 95-kDa MMP-9 form and dimers. We conclude that neutrophil infiltration highly contributes to enhanced MMP-9 in the ischemic brain by releasing MMP-9 proform, which might participate in the tissular inflammatory reaction.

Activation of the JAK/STAT pathway following transient focal cerebral ischemia: Signaling through Jak1 and Stat3 in astrocytes

JAK/STAT is one of the pathways bearing signals from the cell membrane to the nucleus in response to extracellular growth factors and cytokines. In the present study, we examined the cellular distribution of Jak1 and Stat3, and activation of the JAK/STAT pathway following transient focal cerebral ischemia in the rat. Jak1 was mainly seen in white matter astrocytes and in certain neurons. Notably, large pyramidal neurons of cortical layer V showed the highest neuronal Jak1 expression within cerebral cortex and, in addition, expressed Stat3 indicating that the JAK/STAT pathway is involved in signaling in the corticofugal projection system. Shortly following ischemia, Jak1 immunoreactive astrocytes located in the ipsilateral neighbouring white matter and ischemic cortex and striatum showed nuclear translocation of Stat3. These features were maintained in large reactive astrocytes that surrounded the infarct from 3 to 7 days. At these later times, the abundant reactive microglia/macrophages were strongly immunoreactive to Stat3 and, to a lesser extent, Jak1. Two main protein complexes showing DNA binding activity at the sis‐inducible element site were found under basal conditions, followed by changes in this pattern following ischemia concomitant with neuronal cell loss and activation of glia. This study showed basal cerebral activity of JAK/STAT signaling pathway, involving Jak1 and Stat3 proteins, and selective activation following ischemia. It is suggested that the kinase activity of Jak1 mediates nuclear translocation of Stat3 in astrocytes, and that this signaling pathway is involved in the astroglial response to focal cerebral ischemia. GLIA 30:253–270, 2000.

Activation of nuclear factor-κB in the rat brain after transient focal ischemia

Abstract Nuclear factor-kappaB (NF-κB) becomes activated under inflammatory conditions and triggers induction of gene expression. Here, activation of NF-κB was studied after transient middle cerebral artery occlusion in the rat. Expression of p65 and p50, protein subunits of NF-κB, was examined by Western blotting, and immunohistochemistry for p65 was carried out. Double-labelling with specific markers for astroglia and microglia was used for cell type identification. Neurons located within and surrounding the ischemic core were identified during the first 24 h post-ischemia by using an antibody against 72-kDa heat shock protein. NF-κB binding activity was evaluated at different times post-ischemia with electrophoretic mobility gel shift assays. The results showed constitutive expression of p65 and p50, and NF-κB binding activity. Basal p65 was seen in certain neurons and resting astrocytes. Constitutive NF-κB binding activity was attributable to one main protein complex possibly formed in neurons and astrocytes, although two minor complexes were also detected. At 1 day post-ischemia selective induction of p65 was seen in neurons located in a penumbra-like area. At this time, however, no disturbances of basal NF-κB binding activity were found. Western blotting showed delayed induction of p65 several days after ischemia, whereas no changes were detected for p50. From 4 days post-ischemia, a substantial increase in the amount of p65 was detected due to induction in reactive astrocytes and microglia/macrophages. This was correlated with a robust enhancement of NF-κB binding activity with formation of three major specific complexes binding DNA. It is proposed that the highly inducible NF-κB complexes resulted from induction of p65 and activation of NF-κB in post-ischemic reactive glia.

Genetically-Defined Deficiency of Mannose-Binding Lectin Is Associated with Protection after Experimental Stroke in Mice and Outcome in Human Stroke

Background The complement system is a major effector of innate immunity that has been involved in stroke brain damage. Complement activation occurs through the classical, alternative and lectin pathways. The latter is initiated by mannose-binding lectin (MBL) and MBL-associated serine proteases (MASPs). Here we investigated whether the lectin pathway contributes to stroke outcome in mice and humans. Methodology/Principal Findings Focal cerebral ischemia/reperfusion in MBL-null mice induced smaller infarctions, better functional outcome, and diminished C3 deposition and neutrophil infiltration than in wild-type mice. Accordingly, reconstitution of MBL-null mice with recombinant human MBL (rhMBL) enhanced brain damage. In order to investigate the clinical relevance of these experimental observations, a study of MBL2 and MASP-2 gene polymorphism rendering the lectin pathway dysfunctional was performed in 135 stroke patients. In logistic regression adjusted for age, gender and initial stroke severity, unfavourable outcome at 3 months was associated with MBL-sufficient genotype (OR 10.85, p = 0.008) and circulating MBL levels (OR 1.29, p = 0.04). Individuals carrying MBL-low genotypes (17.8%) had lower C3, C4, and CRP levels, and the proinflammatory cytokine profile was attenuated versus MBL-sufficient genotypes. Conclusions/Significance In conclusion, genetically defined MBL-deficiency is associated with a better outcome after acute stroke in mice and humans.

Induction of Stat3, a Signal Transducer and Transcription Factor, in Reactive Microglia following Transient Focal Cerebral Ischaemia

Stat3, a member of the family of cytoplasmic signal transducers and activators of transcription, was found in the rat brain in vivo under physiological conditions and was stimulated following transient focal cerebral ischaemia. A transient episode of middle cerebral artery occlusion induced a strong microglial response in the areas undergoing neural cell death from 4 days after middle cerebral artery occlusion. This was accompanied by increased expression of Stat3 in the ipsilateral cortex and striatum, as revealed by Western blotting of tissue extracts. immunohistochemistry showed strong induction of Stat3 in reactive microglial cells 4, 7 and 15 days after cerebral ischaemia. Stat3 was seen in the microglia cytoplasm, but in many microglial cells immunoreactivity was also distributed within the nucleus. These results suggest that Stat3 mediates signal transduction and activates transcription in reactive microglia in vivo following brain ischaemia.

Epidermal growth factor receptor in proliferating reactive glia following transient focal ischemia in the rat brain

Severe transient focal cerebral ischemia causes brain infarction with a strong glial reaction. We have studied whether postischemic reactive glial cells express epidermal growth factor receptor (EGFR) following middle cerebral artery occlusion in the rat. We have also looked for signs of proliferating activity, as EGFR is known to be involved in cell growth and proliferation in certain non‐neural cells. EGFR was studied using three different antibodies which were found to stain for a tyrosine‐phosphorylated protein (p170) corresponding to the membrane‐anchored EGFR. Neurons of the control brain were strongly immunoreactive to EGFR, but a decrease of EGFR‐immunoreactivity was seen in the ipsilateral brain side from 24 h postischemia due to neuronal loss. However, the presence of abundant glial cells strongly immunoreactive to EGFR became apparent in this area from 4 days postischemia onward. The use of microglial (lectin or OX‐42) and astroglial (GFAP) markers showed that these postischemic EGFR‐stained cells were reactive microglia/macrophages and astroglia. The subcellular localization of EGFR in reactive microglia/macrophages was compatible with the network of the Golgi apparatus, as revealed with an antibody against a peripheral membrane‐bound protein of the Golgi. The presence of abundant proliferating cells in the ischemic brain was detected from 4 days postischemia with an antibody against proliferating cell nuclear antigen. Proliferating reactive microglia/macrophages were abundant within the infarcted brain side, whereas proliferating astrocytes were found mainly in the immediate periphery of the infarct limiting the necrotic area from the undamaged tissue. These proliferating cells were immunoreactive to EGFR. The results show the presence of EGFR in postischemic reactive glial cells and suggest that EGFR‐dependent pathways mediate signal transduction in reactive glia following transient focal cerebral ischemia. GLIA 23:120–129, 1998.

Caspase-dependent and caspase-independent signalling of apoptosis in the penumbra following middle cerebral artery occlusion in the adult rat

Transient focal ischaemia by middle cerebral artery occlusion (MCAO) may produce cell death, but the mechanisms leading to cell death differ in the infarct core and in the penumbra, the immediate zone surrounding the infarct core. In the present study, transient focal ischaemia to adult rats was produced by intraluminal occlusion of the middle cerebral artery for 1 h followed by 0 h (n = 6), 1 h (n = 10), 4 h (n = 8), 6 h (n = 2) and 12 h (n = 3) of reperfusion. The present model of ischaemia causes a large cortico‐striatal infarct extending through the mediolateral cortex and dorsolateral striatum at 12 h. The expression and subcellular distribution of several proteins involved in apoptosis have been examined in the penumbra and in the infarct core by using combined methods of immunohistochemistry, cell subfractionation and Western blotting. Transient focal ischaemia by MCAO results in activation of complex signal pathways for cell death in the penumbra. Increased expression of Bcl‐2 and Bax, but not of Bcl‐x, occurs in the penumbra at the time when Bax translocates from the cytosol to the mitochondria, cytochrome c is released to the cytoplasm and active caspase‐3 is expressed. Bax translocation, cytochrome c release and active caspase‐3 are observed at 4 h, but not at 1 h, following reperfusion, and together indicate activation of the caspase‐dependent pathway of apoptosis in the penumbra. In contrast, reduced Bax expression but not Bax translocation and cytochrome c release occurs in the infarct core, thus suggesting apoptosis signals restricted to the penumbra. In addition, increased expression of an apoptosis‐inducing factor in the cytoplasm and nuclei of selected cells shows, for the first time, activation of the caspase‐independent mitochondrial pathway in the penumbra following transient focal ischaemia and reperfusion.

Anti-VCAM-1 antibodies did not protect against ischemic damage either in rats or in mice

Cerebral ischemia triggers an inflammatory process involving the infiltration of leukocytes to the parenchyma. Circulating leukocytes adhere to the vascular wall through adhesion molecules. Here we quantified the in vivo expression of vascular cell adhesion molecule-1 (VCAM-1) in the brain, heart and lungs from 6 to 48 h after transient middle cerebral artery (MCA) occlusion in rats, by intravenous injection of a tracer radiolabeled anti-VCAM-1 antibody. The vascular localization of VCAM-1 was verified by immunohistochemistry after in vivo injection of the antibody. Vascular cell adhesion molecule-1 was strongly induced (4-fold at 24 h) in the microvasculature of the ischemic area, and, to a lesser extent, in the contralateral hemisphere and in a remote organ, the heart, but not in the lungs, indicating that the inflammatory process propagates beyond the injured brain. We injected intravenously either blocking doses of anti-VCAM-1 antibodies or control antibodies after MCA occlusion in rats and mice. We evaluated the neurological score in rats, and infarct volume at 2 days in rats and at 4 days in mice. Anti-VCAM-1 did not protect against ischemic damage either in rats or in mice. Vascular cell adhesion molecule-1 blockade significantly decreased the number of ED1 (labeling monocytes/macrophages/reactive microglia)-positive cells in the ischemic rat brain. However, it did not reduce the numbers of infiltrating neutrophils and lymphocytes, and total leukocytes (CD45 positive), which showed a trend to increase. The results show vascular upregulation of VCAM-1 after transient focal ischemia, but no benefits of blocking VCAM-1, suggesting that this is not a therapeutical strategy for stroke treatment.

Cell tracking using magnetic resonance imaging

Cell tracking by in vivo magnetic resonance imaging (MRI) requires strategies of labelling the cells with MRI contrast agents. The principal routes to achieve efficient cell labelling for neurological applications are discussed with methodological advantages and caveats. Beyond temporo‐spatial localization of labelled cells, the investigation of functional cell status is of great interest to allow studies of functional cell dynamics. The two major approaches to reach this goal, use of responsive contrast agents and generation of transgenic cell lines, are discussed.