Welcome to the ESOC 2021 Virtual Conference Calendar

Background And Aims

Blood-brain barrier (BBB) damage constitutes the main cause of death following ischaemic stroke associated with neuroinflammation. Outgrowth endothelial cells (OECs) play a pivotal role in the maintenance of endothelial integrity through various paracrine factors that they release. This study investigated whether OEC-derived conditioned medium (OEC-CM) may be protective against inflammatory mediator, TNF-a.

Methods

An in vitro model of human blood-brain barrier was established by co-culture of human astrocytes, pericytes and human brain microvascular endothelial cells (HBMECs) alone or mixed with OECs before exposing to TNF-α (10 ng/mL, 6 h) in the presence or absence of OEC-CM.

Results

The data generated show that OEC-CM potentiate the regenerative potential of both HBMECs and OECs, as ascertained by substantial increases in their wound reparative and proliferative capacity. Significant increases in transendothelial electrical resistance and concomitant decreases in sodium fluorescein flux across obtained by OEC-CM further confirm the BBB-protective impact of OEC-CM against TNF-α. Suppression of NADPH oxidase activity, superoxide anion production, endostatin expression, stress fibres formation as well as HBMEC and OEC apoptosis appear to account for the barrier-restorative effects of OEC-CM in conditions associated with excessive availability of TNF-α. Elevated capacities of HBMEC and OEC to adhere extracellular matrix and form tubules on matrigel also contribute the beneficial effects of OEC-CM.

Conclusions

OEC-CM protects BBB integrity and function against TNF-α and may be a new therapeutic option in suppressing oedema following ischaemic stroke.

Trial Registration Number

Not applicable

Background And Aims

Mechanical thrombectomy (MT) has opened the window to brain delivery of therapeutic agents, which could be further improved by nanomedicine. Our aim is to administer endothelial progenitor cells secretome in magnetized poly(D-L-lactic-co-glycolic acid) (PLGA) nanocarriers through endovascular delivery (EVD) to improve brain targeting with a safe and sustained release after stroke.

Methods

Secretome-loaded nanocarriers (270nm) were functionalized with superparamagnetic iron-oxide nanoparticles (SPIONs) and fluorescent tags for magnetic guidance (MG) and magnetic resonance/fluorescent molecular imaging (MRI/FMI), respectively.

Biodistribution was assessed by MRI/FMI in naïve/ischemic mice (n=110) from 30min to 7 days after receiving intravenous/intraarterial nanocarriers, with 30min-3h MG by different magnets (M1-permanent/M2-focused, 8mm/6mm) implanted on the ipsilateral brain. The safety of acute EVD through the ICA (75µl/min) after intraluminal MCAO (n=69) was assessed at 48h-4 weeks by brain infarct/hemorrhage evaluation, motor/neurological tests and markers of systemic toxicity.

The EVD/MG of the nanocarriers was also assessed by FMI in a humanized 3D-printed supraaortic vascular model at 1h after catheter-guided MCA infusion (580µl/min) with a focused magnet (40mm) placed on a 3D-printed temporal bone.

Results

The intraarterial route enhanced brain delivery (2.53±0.95-fold/5.65±0.96-fold increase in vivo/ex vivo, respectively, vs. intravenous) with specific cortical targeting improvement by MG, showing no adverse effects in acute brain damage nor short/long-term mortality/toxicity. MG towards the target MCA was also achieved in a humanized model.

Conclusions

The challenging brain delivery of therapeutic nanomaterials could be efficiently and safely overcome with a controlled EVD further improved with magnetic targeting, which could be considered in the context of MT delivering multiple treatments for stroke.

Trial Registration Number

Not applicable

Background And Aims

The utilisation of outgrowth endothelial cells (OECs) as a regenerative therapy in stroke medicine requires an extensive ex vivo expansion to generate sufficient cell numbers. The current study investigated the impact of such expansion on cerebral barrier-forming capacity of OECs.

Methods

OECs were cultured to point of replicative senescence by repetitive passaging. Cerebral barrier-forming capacity of young (P7) and senescent (P14) OECs were assessed in vitro using a laboratory model of human blood-brain barrier (BBB) established by co-culture of astrocytes, pericytes and brain microvascular endothelial cells.

Results

In contrast to P7, P14 OECs exhibit a largely flattened and enlarged cell shape coupled with increases in actin stress fibre formation and impairments in tubulogenesis. They also exhibit decreases in proliferative, migratory and wound-reparative capacity alongside increases in β-galactosidase and γ-H2AX staining, a marker of DNA damage, suggesting replicative senescence. The senescent OECs possess higher NADPH oxidase (Nox) activity and superoxide anion level, lower total anti-oxidant capacity, and fail to form functional BBB. Intriguingly, co-culture of senescent OECs with young cerebral endothelial cells (CECs) adversely affect the barrier-forming capacity of CECs, proving the detrimental effect of senescent OECs on resident ECs. Targeting Nox activity by a specific inhibitor (VAS2870, 5μM) or anti-oxidant vitamin C (0.5μM) in late passage cells (P12) delayed senescence, enhanced regenerative potential and restored the capacity of OECs to form BBB.

Conclusions

Long-term in vitro expansion of OECs leads to replicative senescence and triggers cellular dysfunction. Suppression of Nox activity by using vitamin C or VAS2870 delay senescence and augments cerebral barrier-restorative capacity of OECs.

Trial Registration Number

Not applicable

Background And Aims

Ischemic stroke is a primary cause of morbidity and mortality worldwide. Beyond the standard thrombolytic therapies, there is still no effective treatment for stroke disease. Drug repositioning has become a promising alternative approach to identify new uses of existing drugs outside its original scope. Besides, combinational treatments are also emerging as powerful strategies to simultaneously target more than one disease-response mechanism underlying complex pathologies.

Methods

Here, we used a systems biology-based approach based on artificial intelligence and pattern recognition tools to generate in silico mathematical models that mimic the ischemic stroke pathology. Through the screening of these artificial models with multiple two-by-two combinations of FDA-approved drugs, we identified a novel combinational neuroprotective treatment formed by ceruletide and alpha-1 antitrypsin. The safety, synergistic neuroprotective effects and their compatibility with tPA were then evaluated in pre-clinical stroke models in mice.

Results

The administration of the drug combination resulted safe and effective in attenuating infarct volume and neurological deficits 24h after transient cerebral ischemia (fMCAO) (p=0.022, n=10/group). Individual drugs, however, did not reduce the ischemic lesion. Moreover, incompatibilities of the drug combination with tPA were discarded both in an in vitro model of tPA-induced clot lysis and in mice submitted to the thromboembolic stroke model (n=10/group). Finally, we identified that the underlying synergistic action of ceruletide and alpha-1 antitrypsin is mainly mediated by the modulation of EGFR and ANGPT-1 levels in circulation.

Conclusions

In conclusion, we have identified a promising combinational treatment with neuroprotective effects for ischemic stroke disease.

Background And Aims

We tested the neuroprotective effects of a novel prodrug, NTS-104, in a clinically relevant rat embolic model of middle cerebral artery occlusion (MCAo). NTS-104 is a highly druggable novel neuroactive steroid whose active moiety crosses the blood brain barrier (BBB) and has unique receptor pharmacology, including agonism of the progesterone receptor and antagonism of the mineralocorticoid and androgen receptors.

Methods

Stroke was induced by placing a fibrin-rich clot at the origin of the MCA. Rats (n=15/group) randomly received either vehicle or NTS-104 at 1, 3, 10, or 30 mg/kg intramuscularly at 4, 10, 24, 48, 72 and 96 hours after the onset of MCAo. Sensorimotor endpoints including modified neurological severity score (mNSS, prespecified primary endpoint), adhesive tape removal, and foot faults were blindly assessed at pre-treatment, 1, 7, 14, 21 and 28 days after injury. Independently held blinding codes were revealed after completing all assessments. Group means were compared using ANOVA followed by a post-hoc Dunnett’s test (α=0.05).

Results

NTS-104 produced a robust and dose-related improvement from baseline in the mNSS at 28 days after injury relative to vehicle (change from baseline of -3.7±0.3). At doses of 10 and 30 mg/kg, we measured differences in mNSS relative to baseline of -5.7±0.4 (mean±SEM; p=0.0003) and -6.2±0.4 points (p<0.0001). Doses of 1 and 3mg/kg showed no significant differences in mNSS vs. vehicle treated animals. Similar dose-related responses were observed in adhesive tape and foot fault assessments (see figure).

Conclusions

NTS-104 robustly protects against MCAo-induced neurologic deficits in a dose-dependent manner.

Background And Aims

Novel therapeutic paradigms remain a high unmet need in intracerebral hemorrhage (ICH). Current low clinical translation of preclinical efficacy highlights the need for ICH-models that better recapitulate ICH-pathogenesis. Therefore, the aim of our study was to test whether antibody-targeted inhibition of the dual endothelin-1/signal peptide receptor (DEspR), expressed on apoptotic-resistant, activated neutrophil subset associated with ARDS-mortality, would improve median overall survival (mOS) in a model of spontaneous ICH: the Dahl Salt-sensitive hypertensive, ICH-prone rat model that presents with acute-onset and progressive neurological deficits, 200-mmHg-SBP, and histopathology/MRI-documented ICH.

Methods

We tested single iv-dose anti-DEspR 10a3[mIgG1] monoclonal antibody (mAb) therapy in acute ICH after video-recording of non-transient neurological deficits (study-1); and as 4-weekly iv-doses given at pre-stroke stage (study-2). In study-3: we tested humanized anti-DEspR hIgG4^S228P mAb, hu6g8, similar to study-1 design. Response was defined by video-recording of improved neurological status, and by duration to 2^nd stroke as survival endpoint.

Results

Immunofluorescence-staining of rat ICH-brains obtained at stroke onset documented DEspR+ neutrophils peri-bleed areas. Anti-DEspR[10a3]-treatment increased mOS in study-1 [females: 7-treated; 10-controls: 5-isotype, 5-vehicle], Kaplan-Meier Curve (KMC) survival-analysis: Mantel-Cox test p<0.0001, and delayed ICH-onset in study-2: [females: 9-treated; 7-vehicle] KMC stroke-onset analysis p<0.0001. In study 3: hu6g8 increased mOS [females/males: 11-treated; 10-nonTx] KMC survival-analysis p<0.0001. Post-mortem brain-MRI documented supra/infra-tentorial ICH and PHE and confirmed ICH-death.

Conclusions

Altogether, data show that targeted-inhibition of DEspR+ neutrophil subset is a nodal therapeutic paradigm with promising efficacy and safety potential to improve outcomes in the acute ICH-stage, and/or to preempt ICH-pathogenesis.

Background And Aims

Brain glucose metabolism is altered after stroke, and its level of disturbance depends on stroke severity. We applied a novel MR spectroscopy-based technique, deuterium metabolic imaging (DMI), in a mouse stroke model to distinguish different degrees of brain injury based on metabolic activity.

Methods

12–14 weeks old male C57BL/6 mice underwent 60-min unilateral middle cerebral artery occlusion (tMCAO) (n=14). Healthy animals served as controls (n=3). T2-weighted MRI followed by DMI combined with intravenous infusion of deuterium-labeled glucose were executed in a preclinical 9.4 T MR system at 48 h or 11 days after tMCAO. Deuterated metabolite concentrations were quantified from the MR spectroscopy signals.

Results

T2-weighted MRI showed that mice had different lesion sizes that could be classified as either small subcortical or large cortical and subcortical lesions. Oxidative glucose metabolism, characterized by active formation of (deuterated) glutamate or glutamine (Glx), was preserved in the subcortical lesion of animals with a small lesion at 48h, but not in those with a large lesion. Increased formation of deuterated lactate, representing active anaerobic glycolysis, was observed in the subcortical area of large lesions at 48h. Active formation of Glx and lactate was diminished after 11 days.

Conclusions

DMI, a clinically applicable non-invasive imaging method, can reveal distinct metabolic profiles in post-ischemic tissue, which depend on the level of stroke severity. Mapping of the brain’s metabolic status with DMI may aid in detection or monitoring of (potentially) viable tissue after stroke.

Trial Registration Number

Not applicable

Background And Aims

Approximately 11% of patients with cerebral venous thrombosis (CVT) develop late seizures (>7 days after diagnosis). Although highly relevant for treatment decisions and patient education, it is unclear how to estimate risk of late seizures for individual patients. Therefore, our aim was to develop a model for prediction of late seizures within 1 and 3 years after CVT.

Methods

We used data of the International CVT Consortium to develop a Cox proportional hazards model. Predictors were chosen based on literature review and usability in clinical practice. Optimism was adjusted for using a Ridge penalty. Internal validation was assessed in terms of discrimination (C-statistic; 1000 bootstrap samples) and goodness-of-fit (calibration plots).

Results

Analysis of data of 808 patients (mean age 43±16, 72% female) with 90 first late seizures led to the following model: intracranial hemorrhage HR 1.55 (95% CI 1.16-2.07), acute symptomatic seizure(s) without status epilepticus HR 1.59 (95% CI 1.18-2.14), status epilepticus in the acute phase HR 1.97 (95% CI 1.16-3.33), subdural hematoma HR 1.25 (95% CI 0.58-2.66), decompressive hemicraniectomy HR 2.44 (95% CI 1.45-4.10), and age HR 1.00 (95% CI 0.58-2.66). Internal validation showed good predictive performance (C-statistic 0.74 (95% CI 0.68-0.80) and adequate calibration.

Conclusions

The newly developed prediction model is a promising tool to predict the individual risk of late seizures within 1 and 3 years after CVT diagnosis. We will use the model to develop an easy-to-use tool to calculate individual patient risk of late seizures, and thus identify patients at high risk of developing late seizures.

Trial Registration Number

Not applicable