Soluble endoglin reduces thrombus formation and platelet aggregation via interaction with αIIbß3 integrin.

BACKGROUND: The circulating form of human endoglin (sEng) is a cleavage product of membrane-bound endoglin present on endothelial cells. Because sEng encompasses an RGD motif involved in integrin binding, we hypothesized that sEng would be able to bind integrin αIIbß3, thereby compromising platelet binding to fibrinogen and thrombus stability. METHODS: In vitro human platelet aggregation, thrombus retraction, and secretion-competition assays were performed in the presence of sEng. Surface plasmon resonance (SPR) binding and computational (docking) analyses were carried out to evaluate protein-protein interactions. A transgenic mouse overexpressing human sEng (hsEng+) was used to measure bleeding/rebleeding, prothrombin time (PT), blood stream, and embolus formation after FeCl3-induced injury of the carotid artery. RESULTS: Under flow conditions, supplementation of human whole blood with sEng led to a smaller thrombus size. sEng inhibited platelet aggregation and thrombus retraction, interfering with fibrinogen binding, but did not affect platelet activation. SPR binding studies demonstrated that the specific interaction between αIIbß3 and sEng and molecular modeling showed a good fitting between αIIbß3 and sEng structures involving the endoglin RGD motif, suggesting the possible formation of a highly stable αIIbß3/sEng. hsEng+ mice showed increased bleeding time and number of rebleedings compared to wild-type mice. No differences in PT were denoted between genotypes. After FeCl3 injury, the number of released emboli in hsEng+ mice was higher and the occlusion was slower compared to controls. CONCLUSIONS: Our results demonstrate that sEng interferes with thrombus formation and stabilization, likely via its binding to platelet αIIbß3, suggesting its involvement in primary hemostasis control.

From positron emission tomography to cell analysis of the 18-kDa Translocator Protein in mild traumatic brain injury.

Traumatic brain injury (TBI) leads to a deleterious neuroinflammation, originating from microglial activation. Monitoring microglial activation is an indispensable step to develop therapeutic strategies for TBI. In this study, we evaluated the use of the 18-kDa translocator protein (TSPO) in positron emission tomography (PET) and cellular analysis to monitor microglial activation in a mild TBI mouse model. TBI was induced on male Swiss mice. PET imaging analysis with [18F]FEPPA, a TSPO radiotracer, was performed at 1, 3 and 7 days post-TBI and flow cytometry analysis on brain at 1 and 3 days post-TBI. PET analysis showed no difference in TSPO expression between non-operated, sham-operated and TBI mice. Flow cytometry analysis demonstrated an increase in TSPO expression in ipsilateral brain 3 days post-TBI, especially in microglia, macrophages, lymphocytes and neutrophils. Moreover, microglia represent only 58.3% of TSPO+ cells in the brain. Our results raise the question of the use of TSPO radiotracer to monitor microglial activation after TBI. More broadly, flow cytometry results point the lack of specificity of TSPO for microglia and imply that microglia contribute to the overall increase in TSPO in the brain after TBI, but is not its only contributor.

Antioxidant Activity and Toxicity Study of Cerium Oxide Nanoparticles Stabilized with Innovative Functional Copolymers.

Oxidative stress, which is one of the main harmful mechanisms of pathologies including ischemic stroke, contributes to both neurons and endothelial cell damages, leading to vascular lesions. Although many antioxidants are tested in preclinical studies, no treatment is currently available for stroke patients. Since cerium oxide nanoparticles (CNPs) exhibit remarkable antioxidant capacities, the objective is to develop an innovative coating to enhance CNPs biocompatibility without disrupting their antioxidant capacities or enhance their toxicity. This study reports the synthesis and characterization of functional polymers and their impact on the enzyme-like catalytic activity of CNPs. To study the toxicity and the antioxidant properties of CNPs for stroke and particularly endothelial damages, in vitro studies are conducted on a cerebral endothelial cell line (bEnd.3). Despite their internalization in bEnd.3 cells, coated CNPs are devoid of cytotoxicity. Microscopy studies report an intracellular localization of CNPs, more precisely in endosomes. All CNPs reduces glutamate-induced intracellular production of reactive oxygen species (ROS) in endothelial cells but one CNP significantly reduces both the production of mitochondrial superoxide anion and DNA oxidation. In vivo studies report a lack of toxicity in mice. This study therefore describes and identifies biocompatible CNPs with interesting antioxidant properties for ischemic stroke and related pathologies.

Behavioral tests that reveal long-term deficits after permanent focal cerebral ischemia in mouse.

Efforts are still needed regarding the research of therapeutics for ischemic stroke. While in experimental studies the protective effect of pharmacological agents is often highlighted by a reduction of the lesion size evaluated in the short term (days), in clinical studies a functional recovery of patients suffering from stroke is expected on the long-term (months and years). Long-term functional preclinical studies are highly recommended to evaluate potential neuroprotective agents for stroke, rather than an assessment of the infarction size at a short time point. The present study thus aimed to select among various behavioral tests those able to highlight long-term deficits (3 months) after cerebral ischemia in mice. Permanent focal cerebral ischemia was carried out in male Swiss mice by intraluminal occlusion of the left middle cerebral artery (MCA). Fourteen behavioral tests were assessed from 7 days to 90 days after ischemia (locomotor activity, neurological score, exit circle test, grip and string tests, chimney test, adhesive removal test, pole test, beam-walking tests, elevated plus maze, marble burying test, forced swimming test, novel object recognition test). The present study clearly identified a battery of behavioral tests able to highlight deficits up to 3 months in our mouse model of permanent MCA occlusion (locomotor activity, neurological score, adhesive removal test, pole test, beam-walking tests, elevated plus maze, marble burying test, forced swimming test and novel object recognition test). This battery of behavioral tests highlighting long-term deficits is useful to study future neuroprotective strategies for stroke treatment.

Improved Reperfusion and Vasculoprotection by the Poly(ADP-Ribose)Polymerase Inhibitor PJ34 After Stroke and Thrombolysis in Mice.

Benefits from thrombolysis with recombinant tissue plasminogen activator (rt-PA) after ischemic stroke remain limited due to a narrow therapeutic window, low reperfusion rates, and increased risk of hemorrhagic transformations (HT). Experimental data showed that rt-PA enhances the post-ischemic activation of poly(ADP-ribose)polymerase (PARP) which in turn contributes to blood-brain barrier injury. The aim of the present study was to evaluate whether PJ34, a potent PARP inhibitor, improves poor reperfusion induced by delayed rt-PA administration, exerts vasculoprotective effects, and finally increases the therapeutic window of rt-PA. Stroke was induced by thrombin injection (0.75 UI in 1 µl) in the left middle cerebral artery (MCA) of male Swiss mice. Administration of rt-PA (0.9 mg kg-1) or saline was delayed for 4 h after ischemia onset. Saline or PJ34 (3 mg kg-1) was given intraperitoneally twice, just after thrombin injection and 3 h later, or once, 3 h after ischemia onset. Reperfusion was evaluated by laser Doppler, vascular inflammation by immunohistochemistry of vascular cell adhesion molecule-1 (VCAM-1) expression, and vasospasm by morphometric measurement of the MCA. Edema, cortical lesion, and sensorimotor deficit were evaluated. Treatment with PJ34 improved rt-PA-induced reperfusion and promoted vascular protection including reduction in vascular inflammation (decrease in VCAM-1 expression), HT, and MCA vasospasm. Additionally, the combined treatment significantly reduced brain edema, cortical lesion, and sensorimotor deficit. In conclusion, the combination of the PARP inhibitor PJ34 with rt-PA after cerebral ischemia may be of particular interest in order to improve thrombolysis with an extended therapeutic window.

Recombinant tissue plasminogen activator enhances microparticle release from mouse brain-derived endothelial cells through plasmin.

Thrombolysis with recombinant tissue plasminogen activator (rt-PA) is currently the only approved pharmacological strategy for acute ischemic stroke. However, rt-PA exhibits vascular toxicity mainly due to endothelial damage. To investigate the mechanisms underlying rt-PA-induced endothelial alterations, we assessed the role of rt-PA in the generation of endothelial microparticles (EMPs), emerging biological markers and effectors of endothelial dysfunction. The mouse brain-derived endothelial cell line bEnd.3 was used. Cells were treated with rt-PA at 20, 40 or 80µg/ml for 15 or 24h, and EMPs were quantified in the culture media using Annexin-V staining coupled with flow cytometry. Rt-PA enhanced EMP release from bEnd.3 cells with a maximal increase at the 40µg/ml dose for 24h (+78% compared to controls). Using tranexamic acid and aprotinin we demonstrated that plasmin is responsible for rt-PA-induced EMP release. The p38 MAPK inhibitor SB203580 and the poly(ADP-ribose)polymerase (PARP) inhibitor PJ34 also reduced rt-PA-induced EMP production, suggesting that p38 MAPK and PARP are downstream intracellular effectors of rt-PA/plasmin. Rt-PA also altered through plasmin the morphology and the confluence of bEnd.3 cells. By contrast, these changes did not implicate p38 MAPK and PARP. This study demonstrates that rt-PA induces the production of microparticles by cerebral endothelial cells, through plasmin, p38 MAPK and PARP pathways. Determining the phenotype of these EMPs to clarify their role on the endothelium in ischemic conditions could thus be of particular interest.

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.

Another "string to the bow" of PJ34, a potent poly(ADP-Ribose)polymerase inhibitor: an antiplatelet effect through P2Y12 antagonism?

BACKGROUND: Neuro- and vasoprotective effects of poly(ADP-ribose)polymerase (PARP) inhibition have been largely documented in models of cerebral ischemia, particularly with the potent PARP inhibitor PJ34. Furthermore, after ischemic stroke, physicians are faced with incomplete tissue reperfusion and reocclusion, in which platelet activation/aggregation plays a key role. Data suggest that certain PARP inhibitors could act as antiplatelet agents. In that context, the present in vitro study investigated on human blood the potential antiplatelet effect of PJ34 and two structurally different PARP inhibitors, DPQ and INO-1001. METHODS AND RESULTS: ADP concentrations were chosen to induce a biphasic aggregation curve resulting from the successive activation of both its receptors P2Y(1) and P2Y(12). In these experimental conditions, PJ34 inhibited the second phase of aggregation; this effect was reduced by incremental ADP concentrations. In addition, in line with a P2Y(12) pathway inhibitory effect, PJ34 inhibited the dephosphorylation of the vasodilator stimulated phosphoprotein (VASP) in a concentration-dependent manner. Besides, PJ34 had no effect on platelet aggregation induced by collagen or PAR1 activating peptide, used at concentrations inducing a strong activation independent on secreted ADP. By contrast, DPQ and INO-1001 were devoid of any effect whatever the platelet agonist used. CONCLUSIONS: We showed that, in addition to its already demonstrated beneficial effects in in vivo models of cerebral ischemia, the potent PARP inhibitor PJ34 exerts in vitro an antiplatelet effect. Moreover, this is the first study to report that PJ34 could act via a competitive P2Y(12) antagonism. Thus, this antiplatelet effect could improve post-stroke reperfusion and/or prevent reocclusion, which reinforces the interest of this drug for stroke treatment.

Prevention of rt-PA induced blood-brain barrier component degradation by the poly(ADP-ribose)polymerase inhibitor PJ34 after ischemic stroke in mice.

Recombinant tissue plasminogen activator (rt-PA) is the only pharmacological treatment approved for thrombolysis in patients suffering from ischemic stroke, but its administration aggravates the risk of hemorrhagic transformations. Experimental data demonstrated that rt-PA increases the activity of poly(ADP-ribose)polymerase (PARP). The aim of the present study was to investigate whether PJ34, a potent (PARP) inhibitor, protects the blood-brain barrier components from rt-PA toxicity. In our mouse model of cerebral ischemia, administration of rt-PA (10 mg/kg, i.v.) 6h after ischemia aggravated the post-ischemic degradation of ZO-1, claudin-5 and VE-cadherin, increased the hemorrhagic transformations (assessed by brain hemoglobin content and magnetic resonance imaging). Furthermore, rt-PA also aggravated ischemia-induced functional deficits. Combining PJ34 with rt-PA preserved the expression of ZO-1, claudin-5 and VE-cadherin, reduced the hemorrhagic transformations and improved the sensorimotor performances. In vitro studies also demonstrated that PJ34 crosses the blood-brain barrier and may thus exert its protective effect by acting on endothelial and/or parenchymal cells. Thus, co-treatment with a PARP inhibitor seems to be a promising strategy to reduce rt-PA-induced vascular toxicity after stroke.

Protective effects of angiopoietin-like 4 on cerebrovascular and functional damages in ischaemic stroke.

AIMS: Given the impact of vascular injuries and oedema on brain damage caused during stroke, vascular protection represents a major medical need. We hypothesized that angiopoietin-like 4 (ANGPTL4), a regulator of endothelial barrier integrity, might exert a protective effect during ischaemic stroke. METHODS AND RESULTS: Using a murine transient ischaemic stroke model, treatment with recombinant ANGPTL4 led to significantly decreased infarct size and improved behaviour. Quantitative characteristics of the vascular network (density and branchpoints) were preserved in ANGPTL4-treated mice. Integrity of tight and adherens junctions was also quantified and ANGPTL4-treated mice displayed increased VE-cadherin and claudin-5-positive areas. Brain oedema was thus significantly decreased in ANGPTL4-treated mice. In accordance, vascular damage and infarct severity were increased in angptl4-deficient mice thus providing genetic evidence that ANGPTL4 preserves brain tissue from ischaemia-induced alterations. Altogether, these data show that ANGPTL4 protects not only the global vascular network, but also interendothelial junctions and controls both deleterious inflammatory response and oedema. Mechanistically, ANGPTL4 counteracted VEGF signalling and thereby diminished Src-signalling downstream from VEGFR2. This led to decreased VEGFR2-VE-cadherin complex disruption, increased stability of junctions and thus increased endothelial cell barrier integrity of the cerebral microcirculation. In addition, ANGPTL4 prevented neuronal loss in the ischaemic area. CONCLUSION: These results, therefore, show ANGPTL4 counteracts the loss of vascular integrity in ischaemic stroke, by restricting Src kinase signalling downstream from VEGFR2. ANGPTL4 treatment thus reduces oedema, infarct size, neuronal loss, and improves mice behaviour. These results suggest that ANGPTL4 constitutes a relevant target for vasculoprotection and cerebral protection during stroke.

MFGE8 inhibits inflammasome-induced IL-1ß production and limits postischemic cerebral injury.

Milk fat globule-EGF 8 (MFGE8) plays important, nonredundant roles in several biological processes, including apoptotic cell clearance, angiogenesis, and adaptive immunity. Several recent studies have reported a potential role for MFGE8 in regulation of the innate immune response; however, the precise mechanisms underlying this role are poorly understood. Here, we show that MFGE8 is an endogenous inhibitor of inflammasome-induced IL-1ß production. MFGE8 inhibited necrotic cell-induced and ATP-dependent IL-1ß production by macrophages through mediation of integrin ß(3) and P2X7 receptor interactions in primed cells. Itgb3 deficiency in macrophages abrogated the inhibitory effect of MFGE8 on ATP-induced IL-1ß production. In a setting of postischemic cerebral injury in mice, MFGE8 deficiency was associated with enhanced IL-1ß production and larger infarct size; the latter was abolished after treatment with IL-1 receptor antagonist. MFGE8 supplementation significantly dampened caspase-1 activation and IL-1ß production and reduced infarct size in wild-type mice, but did not limit cerebral necrosis in Il1b-, Itgb3-, or P2rx7-deficient animals. In conclusion, we demonstrated that MFGE8 regulates innate immunity through inhibition of inflammasome-induced IL-1ß production.

Combined therapy with PJ34, a poly(ADP-ribose)polymerase inhibitor, reduces tissue plasminogen activator-induced hemorrhagic transformations in cerebral ischemia in mice.

Recombinant tissue-type plasminogen activator (rt-PA) is presently the only pharmacological treatment approved for thrombolysis in patients suffering from ischemic stroke. Although reperfusion of ischemic tissue is essential, the use of rt-PA is limited due to its narrow therapeutic window and risk of hemorrhagic transformations. Recent studies have shown that rt-PA amplifies the post-ischemic activation of the nuclear enzyme poly(ADP-ribose)polymerase (PARP). This enzyme has been shown to contribute to both the breakdown of the blood brain barrier and spontaneous hemorrhagic transformations after ischemia. We therefore examined the capacity of PJ34 (N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-2-(N,N-dimethylamino) acetamide hydrochloride), a potent inhibitor of PARP, to reduce the hemorrhagic transformations that occur after rt-PA in mice with permanent focal cerebral ischemia. Ischemia was produced by intraluminal occlusion of the left middle cerebral artery and treated with vehicle, rt-PA (10 mg/kg, i.v., 6 h after occlusion) or rt-PA plus PJ34 (3, 6 or 12 mg/kg, i.p., at ischemia onset and 4 h later). Hemorrhagic transformations, neurological examination, and infarct volumes were evaluated 48 h after the onset of ischemia. Delayed administration of rt-PA resulted in increased hemorrhagic transformations and aggravated the neurological deficit. Giving PJ34 (3 mg/kg) markedly reduced the hemorrhagic transformations, an effect not owing to a modification of matrix metalloprotease activity. Furthermore, PJ34 improved the neurological functions of rt-PA-treated ischemic mice. To conclude, the PARP inhibitor PJ34 makes rt-PA safer in experimental ischemic stroke.

Experimental modeling of recombinant tissue plasminogen activator effects after ischemic stroke.

Recombinant tissue plasminogen activator (rt-PA) is currently the only approved drug for ischemic stroke treatment, with a dose of 0.9 mg/kg. Since the fibrinolytic activity of rt-PA has been reported in vitro to be 10-fold less potent in rodent than in human, in most in vivo experimental models of cerebral ischemia rt-PA is used at 10 mg/kg. The purpose of this study was to compare the effects of the "human" (0.9 mg/kg) and "rodent" (10 mg/kg) doses of rt-PA given at an early or a delayed time point in a mouse model of cerebral ischemia. Cerebral ischemia was induced by thrombin injection into the left middle cerebral artery of mice. Rt-PA (0.9 or 10 mg/kg) was intravenously administered 30 min or 4 h after the onset of ischemia. The degree of reperfusion after rt-PA was followed for 90 min after its injection. The neurological deficit, infarct volumes, edema and hemorrhagic transformations (HT) were assessed at 24 h. Reperfusion was complete after early administration of rt-PA at 10 mg/kg but partial with rt-PA at 0.9 mg/kg. Both doses given at 4 h induced partial reperfusion. Early administration of both doses of rt-PA reduced the neurological deficit, lesion volume and brain edema, without modifying post-ischemic HT. Injected at 4 h, rt-PA at 0.9 and 10 mg/kg lost its beneficial effects and worsened HT. In conclusion, in the mouse thrombin stroke model, the "human" dose of rt-PA exhibits effects close to those observed in clinic.

Induction of P-glycoprotein and Bcrp at the rat blood-brain barrier following a subchronic morphine treatment is mediated through NMDA/COX-2 activation.

Subchronic morphine treatment induces P-glycoprotein (P-gp) up-regulation at the blood-brain barrier. This study investigates the rate and extent to which P-gp and breast cancer-resistance protein (Bcrp) increase at the rat blood-brain barrier following subchronic morphine treatment. Rats were given increasing doses of morphine (10-40 mg/kg) or saline i.p. twice daily for 5 days. The brain cortex large vessels and microvessels were then mechanical isolated 6, 9, 12, 24, and 36 h after the last injection. The gene and protein expression of P-gp and Bcrp in morphine-treated and control rats were compared by qRT-PCR and western blotting. The levels of Mdr1a and Bcrp mRNAs were not significantly modified 6 h post morphine, but the Mdr1a mRNA increased 1.4-fold and Bcrp mRNA 2.4-fold at 24 h. P-gp and Bcrp protein expression in brain microvessels was unchanged 6 h post morphine and increased 1.5-fold at 24 h. This effect was more pronounced in large vessels than in microvessels. However, extracellular morphine concentrations of 0.01-10 µM did not modify the expressions of the MDR1 and BCRP genes in hCMEC/D3 human endothelial brain cells in vitro. MK-801 (NMDA antagonist) and meloxicam (cyclo-oxygenase-2 inhibitor) given after morphine treatment completely blocked P-gp and Bcrp up-regulation. Interestingly, misoprostol and iloprost, two well-known agonists of prostaglandin E2 receptors induced both MDR1 and BCRP mRNA levels in hCMEC/D3. Thus, morphine does not directly stimulate P-gp and Bcrp expression by the brain endothelium, but glutamate released during morphine withdrawal may do so by activating the NMDA/cyclo-oxygenase-2 cascade.

Progesterone receptors: a key for neuroprotection in experimental stroke.

Progesterone receptors (PR) are expressed throughout the brain. However, their functional significance remains understudied. Here we report a novel role of PR as crucial mediators of neuroprotection using a model of transient middle cerebral artery occlusion and PR knockout mice. Six hours after ischemia, we observed a rapid increase in progesterone and 5α-dihydroprogesterone, the endogenous PR ligands, a process that may be a part of the natural neuroprotective mechanisms. PR deficiency, and even haploinsufficiency, increases the susceptibility of the brain to stroke damage. Within a time window of 24 h, PR-dependent signaling of endogenous brain progesterone limits the extent of tissue damage and the impairment of motor functions. Longer-term improvement requires additional treatment with exogenous progesterone and is also PR dependent. The potent and selective PR agonist Nestorone is also effective. In contrast to progesterone, levels of the neurosteroid allopregnanolone, which modulates γ-aminobutyric acid type A receptors, did not increase after stroke, but its administration protected both wild-type and PR-deficient mice against ischemic damage. These results show that 1) PR are linked to signaling pathways that influence susceptibility to stroke, and 2) PR are direct key targets for both endogenous neuroprotection and for therapeutic strategies after stroke, and they suggest a novel indication for synthetic progestins already validated for contraception. Although allopregnanolone may not be an endogenous neuroprotective agent, its administration protects the brain against ischemic damage by signaling mechanisms not involving PR. Collectively, our data clarify the relative roles of PR and allopregnanolone in neuroprotection after stroke.

Neuroblast survival depends on mature vascular network formation after mouse stroke: role of endothelial and smooth muscle progenitor cell co-administration.

Pro-angiogenic cell-based therapies constitute an interesting and attractive approach to enhancing post-stroke neurogenesis and decreasing neurological deficit. However, most new stroke-induced neurons die during the first few weeks after ischemia, thus impairing total recovery. Although the neovascularization process involves different cell types and various growth factors, most cell therapy protocols are based on the biological effects of single-cell-type populations or on the administration of heterogeneous populations of progenitors, namely human cord blood-derived CD34(+) cells, with scarce vascular progenitor cells. Tight cooperation between endothelial cells and smooth muscle cells/pericytes is critical for the development of functional neovessels. We hypothesized that neuroblast survival in stroke brain depends on mature vascular network formation. In this study, we injected a combination of endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SMPCs), isolated from human umbilical cord blood, into a murine model of permanent focal ischemia induced by middle cerebral artery occlusion. The co-administration of SMPCs and EPCs induced enhanced angiogenesis and vascular remodeling in the peri-infarct and infarct areas, where vessels exhibited a more mature phenotype. This activation of vessel growth resulted in the maintenance of neurogenesis and neuroblast migration to the peri-ischemic cortex. Our data suggest that a mature vascular network is essential for neuroblast survival after cerebral ischemia, and that co-administration of EPCs and SMPCs may constitute a novel therapeutic strategy for improving the treatment of stroke.

Simvastatin in traumatic brain injury: effect on brain edema mechanisms.

OBJECTIVES: Traumatic brain injury causes deleterious brain edema, leading to high mortality and morbidity. Brain edema exacerbates neurologic deficits and may be attributable to the breakdown of endothelial cell junction protein, leukocyte infiltration, and matrix metalloproteinase activation. These all contribute to loss of blood-brain barrier integrity. The pleiotropic effects of statins, hydroxymethylglutaryl-coenzyme A reductase inhibitors, may inhibit posttraumatic brain edema. We therefore investigated the effect of acute simvastatin on neurologic deficits, cerebral edema, and its origins. DESIGN: Randomized laboratory animal study. SETTINGS: University-affiliated research laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Rats were subjected to lateral fluid percussion traumatic brain injury. Our preliminary dose-effect study indicated that 37.5 mg/kg simvastatin, administered orally 1 hr and 6 hrs after traumatic brain injury, has the greatest anti-edematous effect. This dose was used to study its effects on brain edema and on its mechanisms. MEASUREMENTS AND MAIN RESULTS: We first assessed the effects of simvastatin 24 hrs after traumatic brain injury on brain edema, brain claudin-5 expression, and the vascular endothelial-cadherin (pTyr731)/total vascular endothelial-cadherin ratio, matrix metalloproteinase-9 activity, intercellular adhesion molecule-1 expression, and polymorphonuclear neutrophil infiltration. We also evaluated blood-brain barrier permeability by measuring Evans blue and fluorescein sodium salt extravasation into the cerebral parenchyma. We then investigated whether simvastatin reduces neurologic deficits, edema, and blood-brain barrier permeability earlier than 24 hrs; these effects were evaluated 6 hrs after traumatic brain injury. The anti-edematous effect of simvastatin 24 hrs after traumatic brain injury was associated with increased claudin-5 and decreased intercellular adhesion molecule-1, polymorphonuclear neutrophil infiltration, and blood-brain barrier permeability, with no effect on matrix metalloproteinase-9 activity or vascular endothelial-cadherin phosphorylation. Earlier, 6-hrs after traumatic brain injury, simvastatin reduced neurologic deficits, cerebral edema, and blood-brain barrier permeability. CONCLUSIONS: Simvastatin could be a new therapy for reducing posttraumatic edema by preventing damage to tight junctions and neutrophil infiltration into the parenchyma, thus preserving blood-brain barrier integrity.

trans-Resveratrol downregulates Txnip overexpression occurring during liver ischemia-reperfusion.

Txnip (thioredoxin-interacting protein) is a protein with multifunctional roles in cellular responses and stress-related diseases. Txnip is involved in intracellular redox regulation and has been recently described as a possible link between redox state and metabolism. trans-Resveratrol (T-res) is a natural phytoalexin with antiproliferative, antiapoptotic and antioxidative effects. However, to date there have been no reports of the implication of Txnip in a model of liver acute stress such as ischemia-reperfusion (I/R) and no work has looked for a T-res effect on Txnip. Here we studied the effects of a post-ischemic treatment of T-res on the liver thioredoxin (Trx)/Txnip system and investigated whether the T-res effects were dependent on *NO production. In this work, liver I/R induced hepatic Txnip expression and T-res inhibited I/R Txnip expression. This decrease in Txnip expression by T-res was associated with an increase in liver Trx redox activity and a decrease in hepatic I/R-induced Trx-1 expression with no effect on Trx-2, on plasma Trx redox activity or on liver and plasma Trx reductase activity, independently of *NO production. In conclusion, these results show that in our model, not only did T-res protect Trx redox activity by diminishing the Txnip protein expression; it also reduced secretion of Trx1. This is the first report of a major implication of the Trx1/Txnip system in hepatic I/R injuries. It also affirms the importance of the antioxidant effect of T-res on the Trx1/Txnip system.

Long-term histological and behavioural characterisation of a collagenase-induced model of intracerebral haemorrhage in rats.

Although intracerebral haemorrhage (ICH) entails the highest rates of mortality and disability of all stroke subtypes, efficient neuroprotective therapy is still needed. As functional recovery is a major endpoint in clinical trials, preclinical studies must demonstrate the potential of drugs to improve the sensorimotor and cognitive function of animals. In addition, behavioural studies should be performed on the long-term in order to truly mimic clinical needs. The aim of our study was to characterise a model of intracerebral haemorrhage using both histology and long-term behaviour. ICH was induced in rats by an intrastriatal injection of collagenase. Histology was performed 24h, 7 days and 2 months after ICH. Among a set of sensorimotor tests, we discriminate those able to reveal long-term deficits (up to 2 months) after cerebral haemorrhage. Our five behavioural tests (a neurological score, an adhesive removal test, two beam-walking tests and ipsilateral circling induced by dexamphetamine) proved to be effective in revealing sensorimotor deficits up to 35 days or more after cerebral haemorrhage. In conclusion, these behavioural tests appear of particular interest to screen protective agents that may exhibit benefits in patients who suffer ICH.

Reduction of hemorrhagic transformation by PJ34, a poly(ADP-ribose)polymerase inhibitor, after permanent focal cerebral ischemia in mice.

Hemorrhagic transformation is an aggravating event that occurs in 15 to 43% of patients suffering from ischemic stroke. This phenomenon due to blood-brain barrier breakdown appears to be mediated in part by matrix metalloproteinases (MMPs) among which MMP-2 and MMP-9 could be particularly involved. Recent experimental studies demonstrated that post-ischemic MMP-9 overexpression is regulated by poly(ADP-ribose)polymerase (PARP). In this context, our study aimed to evaluate the effect of PJ34 (N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-2-(N,N-dimethylamino)acetamide), a potent PARP inhibitor, on MMP-2 and MMP-9 levels and on hemorrhagic transformations in a model of permanent focal cerebral ischemia in mice. PJ34 (6.25-12.5 mg/kg, i.p.) was given at the time of ischemia onset and 4 h later. Hemorrhagic transformations, divided into microscopic and macroscopic hemorrhages, were counted 48 h after ischemia on 12 coronal brain slices. Microscopic and macroscopic hemorrhages were respectively reduced by 38% and 69% with 6.25 mg/kg PJ34. The anti-hemorrhagic effect of PJ34 was associated with a 57% decrease in MMP-9 overexpression assessed by gelatin zymography. No increase in MMP-2 activity was observed after ischemia in our model. The vascular protection achieved by PJ34 was associated with a reduction in the motor deficit (P<0.05) and in infarct volume (-31%, P<0.01). In conclusion, our study demonstrates for the first time that PJ34 reduces hemorrhagic transformations after cerebral ischemia. Thus this PARP inhibitor exhibits both anti-hemorrhagic and neuroprotective effects that may be of valuable interest for the treatment of stroke.