Acute Microbleeds and Microinfarcts Within the Perihematomal Area After Intracerebral Hemorrhage.

BACKGROUND: To further our understanding of the pathophysiology of spontaneous intracerebral hemorrhage (ICH) and related injury, we provided a postmortem neuropathological examination of acute microvascular lesions (microbleeds and microinfarcts) within the perihematomal area. METHODS: We included all consecutive cases (2005-2019) from the Lille University Hospital brain bank of ICH patients who died within the first month. Paraffin-embedded tissue sections from the perihematomal area were processed for several stainings and immunolabelings to investigate the presence of acute microbleeds and microinfarcts in the perihematomal area and to characterize surrounding neuronal and systemic inflammatory reaction (macrophages and neutrophils). RESULTS: We included 14 ICH cases (median age, 78 years; 10 females). Acute microbleeds were observed in the perihematomal area in 12/14 patients (86%, ranging from 1 through >10) and microinfarcts in 5/14 (36%, ranging from 1 through 4). Microbleeds were observed whatever the delay from ICH onset to death was, while most cases with acute microinfarcts were observed between day 3 and day 7 (n=3/5). Both lesions were characterized by an abundant accumulation of systemic inflammatory cells and necrotic areas. CONCLUSIONS: Acute microbleeds and microinfarcts might contribute to the propagation of secondary brain tissue damages after ICH. Our examinations also question the potential role of massive systemic inflammatory cells recruitment in the genesis of these microvascular injuries.

Intracerebral Hemorrhage-Induced Cognitive Impairment in Rats Is Associated With Brain Atrophy, Hypometabolism, and Network Dysconnectivity.

The mechanisms underlying intracerebral hemorrhage (ICH)-related cognitive impairment (CI) remain unclear. Long-term structural and functional changes were investigated in the brains of healthy male and female Wistar rats after experimental ICH. Following double injection of autologous blood, rats underwent short-term (onset, 3 and 7 days) and long-term (3 and 6 months) radiological assessment and behavioral tests exploring spontaneous locomotion, anxiety-like behavior and working memory, spatial recognition memory and visual recognition memory. Volumetric and metabolic changes in brain areas were examined by 7Tesla-MRI and [18F] FDG-PET, respectively. Brain connectomic disorders and maladaptive processes were seeked through brain metabolic connectivity analysis and atrophy-related network analysis. From an initial hematoma mean volume of 23.35 ± 9.50 mm3, we found early spontaneous locomotor recovery and significant spontaneous blood resorption (≈ 40% of the initial lesion) from days 0 to 7. After 3 and 6 months, ICH rats exhibited CI in several domains as compared to the sham group (working memory: 58.1 ± 1.2 vs. 70.7 ± 1.2%, p < 0.001; spatial recognition memory: 48.7 ± 1.9 vs. 64 ± 1.8%, p < 0.001 and visual recognition memory: 0.14 ± 0.05 vs. 0.33 ± 0.04, p = 0.013, in female only). Rats that experienced ICH had remote and concomitant cerebral atrophy and hypometabolism of ipsilateral striatum, thalamus, limbic system and cortical areas (temporal and parietal lobes). Interestingly, both structural and metabolic deterioration was found in the limbic system connected to the affected site, but remotely from the initial insult. On the other hand, increased activity and functional connectivity occurred in the contralateral hemisphere. These connectomics results showed that both maladaptative and compensation processes coexist in the rat brain following ICH, even at young age and in a disease-free setting. These radiological findings deepen our understanding of ICH-related CI and may serve as biomarkers in the view of future therapeutic intervention.

Brain Peri-Hematomal Area, a Strategic Interface for Blood Clearance: A Human Neuropathological and Transcriptomic Study.

BACKGROUND: Enhancing the blood clearance process is a promising therapeutic strategy for intracerebral hemorrhage (ICH). We aimed to investigate the kinetic of this process after ICH in human brain tissue through the monocyte-macrophage scavenger receptor (CD163)/HO-1 (hemoxygenase-1) pathway. METHODS: We led a cross-sectional post-mortem study including 22 consecutive ICH cases (2005-2019) from the Lille Neurobank. Cases were grouped according to the time of death: ≤72 hours, 4 to 7 days, 8 to 15 days, 16 to 90 days, and >90 days after ICH onset. Paraffin-embedded tissue was extracted from 4 strategic areas, including hematoma core and peri-hematomal area to perform histological investigations. Additionally, we extracted RNA from the peri-hematomal area of 6 cases to perform transcriptomic analysis. RESULTS: We included 19 ICH cases (median age: 79 [71-89] years; median delay ICH-death: 13 [5-41] days). The peri-hematomal area concentrated most of reactive microglia, CD163/HO-1 and iron deposits as compared with other brain areas. We found a surge in the blood clearance process from day 8 to day 15 after ICH onset. Transcriptomic analysis showed that HO-1 was the most upregulated gene (2.81±0.39, adjusted P=1.11×10-10) and CD163 the sixth (1.49±0.29, adjusted P=1.68×10-5). We also identified several upregulated genes that exert a beneficial role in terminating inflammation and enhancing tissue repair. CONCLUSIONS: We provide histological and transcriptomic-based evidence in humans for the key role of peri-hematomal area in endogenous blood clearance process through the CD163/HO-1 pathway, especially from day 8 after ICH and favored by an anti-inflammatory environment. Our findings contribute to identify innovative therapeutic strategies for ICH.

In vitro differentiation modifies the neurotoxic response of SH-SY5Y cells.

The SH-SY5Y cell line is commonly used for the assessment of neurotoxicity in drug discovery. These neuroblastoma-derived cells can be differentiated into neurons using many methods. The present study has compared 24 of these differentiation methods on SH-SY5Y cells. After morphologic selection of the three most differentiating media (retinoic acid in 10% fetal bovine serum (FBS), staurosporine in 1% FBS medium, and cyclic adenosine monophosphate (cAMP) in B21-supplemented neurobasal medium), cells were analyzed for pan-neuronal and specific neuronal protein expression by fluorescent automated imaging. The response of SH-SY5Y to a set of compounds of known toxicity was examined in these culture conditions performed in 2D, and also in a 3D hyaluronic acid-based hydroscaffold™ which mimics the extracellular matrix. The extent of neuronal markers expression and the sensitivity to neurotoxic compounds varied according to the differentiation medium. The cAMP B21-supplemented neurobasal medium led to the higher neuronal differentiation, and the higher sensitivity to neurotoxic compounds. The culture in 3D modified the neurotoxic response, through a lower sensitivity of cells compared to the 2D culture. The in vitro differentiation environment influences the neurotoxic response of SH-SY5Y cells and thus should be considered carefully in research as well as in drug discovery.

Neutrophil extracellular traps (NETs) infiltrate haematoma and surrounding brain tissue after intracerebral haemorrhage: A post-mortem study.

AIMS: Because of their prothrombotic and neuroinflammatory effects, neutrophils and neutrophil extracellular traps (NETs) represent interesting therapeutic targets for spontaneous intracerebral haemorrhage (sICH). We investigated the presence, spatial and temporal distribution of NETs in a human sICH post-mortem study. METHODS: From 2005 to 2019, all sICH patients who came to autopsy within the first month after stroke were included and grouped according to the timing of death: 72 h, 4-7 days, 8-15 days and >15 days after ICH onset. Paraffin-embedded tissue was extracted from four strategic areas: haematoma, peri-haematomal area, ipsilateral surrounding brain tissue and a control contralateral area. Myeloperoxidase and histone H3 citrulline were immunolabelled to detect neutrophils and NETs respectively. RESULTS: Neutrophils were present in the brains of the 14 cases (4 men, median age: 78 years) and NETs were found in 7/14 cases. Both neutrophils and NETs were detected within the haematoma but also in the surrounding tissue. The appearance of neutrophils and NETs was time-dependent, following a two-wave pattern: during the first 72 h and between 8 and 15 days after ICH onset. Qualitative examination showed that neutrophils and NETs were mainly located around dense fibrin fibres within the haematoma. CONCLUSIONS: These observations provide evidence for NETs infiltration in the brain of patients who die from sICH. NETs might interact with early haemostasis within the haematoma core, and with the surrounding neuroinflammatory response. These findings open research perspectives for NETs in the treatment of sICH injuries.

Age and diet modify acute microhemorrhage outcome in the mouse brain.

Cerebral microhemorrhages (CMHs) are considered as asymptomatic lesions, but might impair cognition in non-demented elderly individuals. The aging process includes poor vascular health, enhanced at midlife by metabolic disturbances upon high-fat diet (HFD). The onset of CMHs could thus have more serious consequences in midlife subjects with metabolic disturbances. This hypothesis was tested through the induction of multiple CMHs, using cyclodextrin nanoparticles injection, in mice at midlife (14 month old) or at a younger stage (5 month old) after 12 months or 3 months of normal diet or HFD (40% of animal fat) respectively. When induced at 14 months of age, CMHs were not larger but were more numerous (+25%) in mice on HFD compared with mice on normal diet. They slowed down the locomotor activity significantly but caused neither a change in the working memory nor a difference in the visual recognition memory decline. When induced at 5 months of age, CMHs provoked slighter locomotor and cognitive symptoms, regardless the diet. No spontaneous progression of CMHs toward larger hemorrhages was observed after onset when HFD was prolonged up to midlife. Consistently, no precipitated cognitive decline was observed. Middle-age plus time of metabolic disturbances represent enhanced risk factors for CMH outcome.

Visceral adiposity links cerebrovascular dysfunction to cognitive impairment in middle-aged mice.

Midlife cognitive decline is now recognized as a factor of poor prognosis for late-life dementia. Although an epidemiological link has been suggested with high fat diet (HFD)-induced metabolic disorders, the effect of a long period of HFD on midlife cerebrovascular and cognitive functions remains unproven. A cohort of 216 young mice was fed with HFD up to middle age (12 months), and kinetically characterized for metabolic status, including weight, blood lipid profile, hepatic fat accumulation, glucose intolerance, and visceral adiposity. Metabolic disorders were evidenced from 3 months of HFD. Visual recognition memory and flexibility were significantly altered and associated to a visceral adiposity whereas spatial reference memory and working memory did not. Concomitantly, a progressive dysfunction of the vascular endothelium-dependent relaxation was detected in both middle cerebral artery and parenchymal arterioles, with consequences on the regulation of cerebral blood flow, but without any modification of the basal brain tissue MRI perfusion signal. Our data collection empowered us to stratify the mice according to their heterogeneous response to diet, and to propose a statistical prediction model for cognitive impairment, combining visceral adiposity and cerebral vasomotion in a diagnostic perspective of early neurological deficits.

Safety of oral anticoagulants on experimental brain microbleeding and cognition.

This study aims at determining the ability of clinical-based doses of four oral anticoagulants to transform the onset of a cerebral microhemorrhages (CMH) burden into a symptomatic intracerebral hemorrhage (ICH) in the healthy brain, and precipitate cognitive impairment. Wild-type mice were anticoagulated for 10 days using apixaban, rivaroxaban or dabigatran as direct oral anticoagulants (DOACs), or warfarin as vitamin K-antagonist. Meanwhile, a burden of ∼20 CMHs was induced in the Sylvian territory by intra-carotid injection of cyclodextrin nanoparticles. At bleeding onset, only warfarin provoked deadly hematoma, and dramatically increased mortality (+45%). All the DOACs enhanced CMH burden through a greater number of intermediate-sized microhemorrhages (+80% to +180%). Although silent at onset, both baseline- and anticoagulant-enhanced CMH burdens increased mortality (+11% to +58%) along the following year without statistical difference among groups, and despite cessation of anticoagulation and absence of CMH progression or transformation into ICH. All survivor mice exhibited reduction in visual recognition memory from 9 months. In the healthy brain, DOACs preserve the onset of microhemorrhages from transformation into ICH, and do not precipitate cognitive impairment despite enhancement of CMH burden. High CMH burdens should however be considered for early detection and preventive memory care apart from anticoagulation decisions.

Cerebral microbleeds: Beyond the macroscope.

While being increasingly recognized in clinical routine, brain microbleeds remain a puzzling finding for physicians. These small dot-like lesions are thought to be old perivascular collections of hemosiderin deposits. They can be found in different neurological settings such as cerebrovascular or neurodegenerative diseases. While their microscopic size would suggest considering these lesions as anecdotal, they are now regarded as biomarkers of severity of an underlying cerebrovascular disease. Their natural history and the interactions with surrounding brain cells remain unknown. However, their presence may impact therapeutic decisions. Deciphering the biological mechanisms leading to, or following microbleeds would enable us to address a key question: do microbleeds arise and impact the surrounding parenchyma like a miniature version of intracerebral hemorrhages or do they represent a different kind of injury? We hereby discuss, based on both clinical and experimental literature, the gap between the definition of microbleeds coming from neuroimaging and the pathophysiological hypotheses raised from histopathological and experimental data. Our analysis supports the need for a convergent effort from clinicians and basic scientists to go beyond the current "macro" view and disclose the cellular and molecular insights of these cerebral hemorrhagic microlesions.

Neither nefopam nor acetaminophen can be used as postoperative analgesics in a rat model of ischemic stroke.

Analgesics such as opioid agonists are usually not given during the postoperative phase of experimental stroke because they are susceptible to interfere with the evaluation of neuroprotective therapies. Here, we investigate the potential of acetaminophen and nefopam, two nonopioid analgesic drugs, to exert an analgesic effect without inducing neuroprotection in a murine model of ischemic stroke. We demonstrate that acetaminophen (200 mg/kg, PO) induces a significant decrease in the infarct volume, particularly in the cortex (VEHICLE: 200.1 mm3 vs. ACETAMINOPHEN: 140.9 mm3 , P < 0.05), while nefopam (2, 20 or 40 mg/kg, IM), administered at the end of middle cerebral artery occlusion (MCAO), do not influence the infarct size (VEHICLE: 268.6 mm3 vs. NEFOPAM 2: 248.8 mm3 , NEFOPAM 20: 250.6 mm3 and NEFOPAM 40: 215.9 mm3 , P > 0.05). Moreover, we find that nefopam administration (20 mg/kg, IM) in the acute postoperative phase do not change the level of neuroprotection induced by MK801 (3 mg/kg, IV), a well-known neuroprotectant (VEHICLE: 268.6 mm3 vs. MK801: 194.4 mm3 and vs. MK801 + NEFOPAM 20: 195.2 mm3 ). On the other hand, although nefopam induces analgesia in healthy animals, it is not the case when administered during MCAO (behavior scores at 5 min: HEALTHY: 2.1 vs. HEALTHY + NEFOPAM 20: 0.6, P < 0.5; IR: 0.40 vs. IR + NEFOPAM 20: 0.67, P > 0.05). Our data suggest that neither acetaminophen nor nefopam can be used as analgesic agents to meet the needs of limiting rodent pain and distress during experimental stroke surgery.

PPAR-Alpha Agonist Used at the Acute Phase of Experimental Ischemic Stroke Reduces Occurrence of Thrombolysis-Induced Hemorrhage in Rats.

The impact of fenofibrate, a peroxisome proliferator-activated receptor-alpha (PPAR-α) agonist, on the risk of thrombolysis-induced hemorrhage during the acute phase of stroke in a rat model of stroke was studied. One-hour middle cerebral artery occlusion followed by thrombolysis with tissue plasminogen activator was made in rats receiving either fenofibrate or vehicle for 72 h after stroke. Evaluation of infarct, hemorrhage, middle cerebral artery vasoreactivity, and immunochemistry (CD11b for microglial activation, myeloperoxidase, and ICAM-1 for neutrophil infiltration) was performed. The PPAR-alpha agonist significantly reduced the risk of hemorrhage after thrombolysis in parallel with a decrease in the infarct volume and in the stroke-induced vascular endothelial dysfunction. These effects are concomitant with a reduction in microglial activation and neutrophil infiltration in infarct area. Our results strengthen the idea that using drugs such as fenofibrate, with pleiotropic properties due to PPAR-alpha agonism, may be of value to reduce thrombolysis-induced hemorrhage during acute stroke.

Protein kinase C restricts transport of carnitine by amino acid transporter ATB(0,+) apically localized in the blood-brain barrier.

Carnitine (3-hydroxy-4-trimethylammoniobutyrate) is necessary for transfer of fatty acids through the inner mitochondrial membrane. Carnitine, not synthesized in the brain, is delivered there through the strongly polarized blood-brain barrier (BBB). Expression and presence of two carnitine transporters - organic cation/carnitine transporter (OCTN2) and amino acid transporter B(0,+) (ATB(0,+)) have been demonstrated previously in an in vitro model of the BBB. Due to potential protein kinase C (PKC) phosphorylation sites within ATB(0,+) sequence, the present study verified effects of this kinase on transporter function and localization in the BBB. ATB(0,+) can be regulated by estrogen receptor α and up-regulated in vitro, therefore its presence in vivo was verified with the transmission electron microscopy. The analyses of brain slices demonstrated ATB(0,+) luminal localization in brain capillaries, confirmed by biotinylation experiments in an in vitro model of the BBB. Brain capillary endothelial cells were shown to control carnitine gradient. ATB(0,+) was phosphorylated by PKC, what correlated with inhibition of carnitine transport. PKC activation did not change the amount of ATB(0,+) present in the apical membrane of brain endothelial cells, but resulted in transporter exclusion from raft microdomains. ATB(0,+) inactivation by a lateral movement in plasma membrane after transporter phosphorylation has been postulated.

Stroke-induced brain parenchymal injury drives blood-brain barrier early leakage kinetics: a combined in vivo/in vitro study.

The disappointing clinical outcomes of neuroprotectants challenge the relevance of preclinical stroke models and data in defining early cerebrovascular events as potential therapeutic targets. The kinetics of blood-brain barrier (BBB) leakage after reperfusion and the link with parenchymal lesion remain debated. By using in vivo and in vitro approaches, we conducted a kinetic analysis of BBB dysfunction during early reperfusion. After 60 minutes of middle cerebral artery occlusion followed by reperfusion times up to 24 hours in mice, a non-invasive magnetic resonance imaging method, through an original sequence of diffusion-weighted imaging, determined brain water mobility in microvascular compartments (D*) apart from parenchymal compartments (apparent diffusion coefficient). An increase in D* found at 4 hours post reperfusion concurred with the onset of both Evans blue/Dextran extravasations and in vitro BBB opening under oxygen-glucose deprivation and reoxygenation (R). The BBB leakage coincided with an emerging cell death in brain tissue as well as in activated glial cells in vitro. The co-culture of BBB endothelial and glial cells evidenced a recovery of endothelium tightness when glial cells were absent or non-injured during R. Preserving the ischemic brain parenchymal cells within 4 hours of reperfusion may improve therapeutic strategies for cerebrovascular protection against stroke.

Transient oxygen-glucose deprivation sensitizes brain capillary endothelial cells to rtPA at 4h of reoxygenation.

Thrombolysis treatment of acute ischemic stroke is limited by the pro-edematous and hemorrhagic effects exerted by reperfusion, which disrupts the blood-brain barrier (BBB) capillary endothelium in the infarct core. Most studies of the ischemic BBB overlook the complexity of the penumbral area, where the affected brain cells are still viable following deprivation. Our present objective was to examine in vitro the kinetic impact of reoxygenation on the integrity of ischemic BBB cells after oxygen-glucose deprivation. Through the use of a co-culture of brain capillary endothelial cells and glial cells, we first showed that the transendothelial permeability increase induced by deprivation can occur with both preserved cell viability and interendothelial tight junction network. The subtle and heterogeneous alteration of the tight junctions was observable only through electron microscopy. A complete permeability recovery was then found after reoxygenation, when Vimentin and Actin networks were reordered. However, still sparse ultrastructural alterations of tight junctions suggested an acquired vulnerability. Endothelial cells were then exposed to recombinant tissue-type plasminogen activator (rtPA) to define a temporal profile for the toxic effect of this thrombolytic on transendothelial permeability. Interestingly, the reoxygenated BBB broke down with aggravated tight junction disruption when exposed to rtPA only at 4h after reoxygenation. Moreover, this breakdown was enhanced by 50% when ischemic glial cells were present during the first hours of reoxygenation. Our results suggest that post-stroke reoxygenation enables retrieval of the barrier function of brain capillary endothelium when in a non-necrotic environment, but may sensitize it to rtPA at the 4-hour time point, when both endothelial breakdown mechanisms and glial secretions could be identified and targeted in a therapeutical perspective.

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.

Diesel exhaust particles impair endothelial progenitor cells, compromise endothelial integrity, reduce neoangiogenesis, and increase atherogenesis in mice.

The mechanisms of the harmful cardiovascular effects of small particulate matter are incompletely understood. Endothelial progenitor cells (EPCs) predict outcome of patients with vascular disease. The aim of our study was to examine the effects of diesel exhaust particles (DEP) on EPC and on the associated vascular damage in mice. C57Bl/6 mice were exposed to DEP. 2 µg DEP/day was applicated intranasally for 3 weeks. Exposure to DEP reduced DiLDL/lectin positive EPC to 58.4 ± 5.6% (p < 0.005). Migratory capacity was reduced to 65.8 ± 3.9% (p < 0.0001). In ApoE(-/-) mice, DEP application reduced the number of EPC to 75.6 ± 6.4% (p < 0.005) and EPC migration to 58.5 ± 6.8% (p < 0.005). Neoangiogenesis was reduced to 39.5 ± 14.6% (p < 0.005). Atherogenesis was profoundly increased by DEP treatment (157.7 ± 18.1% vs. controls, p < 0.05). In cultured human EPC, DEP (0.1-100 µg/mL) reduced migratory capacity to 25 ± 2.6% (p < 0.001). The number of colony-forming units was reduced to 8.8 ± 0.9% (p < 0.001) and production of reactive oxygen species was elevated by DEP treatment (p < 0.001). Furthermore, DEP treatment increased apoptosis of EPC (to 266 ± 62% of control, p < 0.05). In a blood-brain barrier model, DEP treatment impaired endothelial cell integrity during oxygen-glucose deprivation (p < 0.001). Diesel exhaust particles impair endothelial progenitor cell number and function in vivo and in vitro. The reduction in EPC was associated with impaired neoangiogenesis and a marked increase in atherosclerotic lesion formation.

High t-PA release by neonate brain microvascular endothelial cells under glutamate exposure affects neuronal fate.

Glutamate excitotoxicity is a consolidated hypothesis in neonatal brain injuries and tissue plasminogen activator (t-PA) participates in the processes through proteolytic and receptor mediated effects. In brain microvascular endothelial cell (nBMEC) cultures from neonates, t-PA content and release upon glutamate are higher than in adult (aBMECs) cultures. Owing to the variety of t-PA substrates and receptor targets, the study was aimed at determining the putative roles of endothelial t-PA in the neonatal brain parenchyma under glutamate challenge. Basal t-PA release was 4.4 fold higher in nBMECs vs aBMECs and glutamate was 20 fold more potent to allow Evans blue vascular permeability in neonate microvessels indicating that, under noxious glutamate (50 µM) exposure, high amounts of endothelial t-PA stores may be mobilized and may access the nervous parenchyma. Culture media from nBMECS or aBMECs challenged by excitotoxic glutamate were applied to neuron cultures at DIV 11. While media from adult cells did not evoke more LDH release in neuronal cultures that under glutamate alone, media from nBMECs enhanced 2.2 fold LDH release. This effect was not observed with media from t-PA(-/-) nBMECs and was inhibited by hr-PAI-1. In Cortical slices from 10 day-old mice, hrt-PA associated with glutamate evoked neuronal necrosis in deeper (more mature) layers, an effect reversed by NMDA receptor GluN1 amino-terminal domain antibody capable of inhibiting t-PA potentiation of the receptor. In superficial layers (less mature), hrt-PA alone inhibited apoptosis, an effect reversed by the EGF receptor antagonist AG1478. Applied to immature neurons in culture (DIV5), media from nBMEC rescued 85.1% of neurons from cell death induced by serum deprivation. In cortical slices, the anti-apoptotic effect of t-PA fitted with age dependent localization of less mature neurons. These data suggest that in the immature brain, propensity of vessels to release high amounts of t-PA may not only impact vascular integrity but may also influence neuronal fate, via regulation of apoptosis in immature cells and, as in adult by potentiating glutamate toxicity in mature neurons. The data point out putative implication of microvessels in glutamate neurotoxicity in the development, and justify research towards vessel oriented neuroprotection strategies in neonates.

[Dysfunction of the blood-brain barrier during ischaemia: a therapeutic concern]. / La barrière hémato-encéphalique lors de l'ischémie cérébrale : une cible thérapeutique.

Since it was discovered and its brain-protective role characterized, the blood-brain barrier (BBB), through the permeability-restricting action of the brain capillary endothelial cells, has been representing a hurdle for 95% of new medical compounds targeting the central nervous system. Recently, a BBB dysfunction is being found in an increasing number of pathologies such as brain ischaemic stroke, whose only therapy consists in a pharmacological thrombolysis limited to a small percentage of the admitted patients, because of the toxical effects of thrombolytics. And since the clinical failure of promising neuroprotectants, numerous studies of brain ischaemia were carried out, with physiopathological or pharmacological approaches refocused on the BBB, whose structural complexity is now expanded to perivascular cells, all forming a functional unit named the neurovascular unit (NVU). Nevertheless, in spite of the numerous molecular mechanisms identified, the process of BBB dysfunction in the ischaemia/reperfusion cascade remains insufficiently established to explain the pleiotropic action exerted by new pharmacological compounds, possibly protecting the entire NVU and representing potential treatments.

Endothelial cells and astrocytes: a concerto en duo in ischemic pathophysiology.

The neurovascular/gliovascular unit has recently gained increased attention in cerebral ischemic research, especially regarding the cellular and molecular changes that occur in astrocytes and endothelial cells. In this paper we summarize the recent knowledge of these changes in association with edema formation, interactions with the basal lamina, and blood-brain barrier dysfunctions. We also review the involvement of astrocytes and endothelial cells with recombinant tissue plasminogen activator, which is the only FDA-approved thrombolytic drug after stroke. However, it has a narrow therapeutic time window and serious clinical side effects. Lastly, we provide alternative therapeutic targets for future ischemia drug developments such as peroxisome proliferator- activated receptors and inhibitors of the c-Jun N-terminal kinase pathway. Targeting the neurovascular unit to protect the blood-brain barrier instead of a classical neuron-centric approach in the development of neuroprotective drugs may result in improved clinical outcomes after stroke.

HMGB-1 promotes fibrinolysis and reduces neurotoxicity mediated by tissue plasminogen activator.

Owing to its ability to generate the clot-dissolving protease plasmin, tissue plasminogen activator (tPA) is the only approved drug for the acute treatment of ischemic stroke. However, tPA also promotes hemorrhagic transformation and excitotoxic events. High mobility group box-1 protein (HMGB-1) is a non-histone transcription factor and a pro-inflammatory cytokine, which has also been shown to bind to both tPA and plasminogen. We thus investigated the cellular and molecular effects through which HMGB-1 could influence the vascular and parenchymal effects of tPA during ischemia. We demonstrate that HMGB-1 not only increases clot lysis by tPA, but also reduces the passage of vascular tPA across the blood-brain barrier, as well as tPA-driven leakage of the blood-brain barrier. In addition, HMGB-1 prevents the pro-neurotoxic effect of tPA, by blocking its interaction with N-methyl-D-aspartate (NMDA) receptors and the attendant potentiation of NMDA-induced neuronal Ca²âº influx. In conclusion, we show in vitro that HMGB-1 can promote the beneficial effects of tPA while counteracting its deleterious properties. We suggest that derivatives of HMGB-1, devoid of pro-inflammatory properties, could be used as adjunctive therapies to improve the overall benefit of tPA-mediated thrombolysis following stroke.