Endogenous tPA levels: A biomarker for discriminating hemorrhagic stroke from ischemic stroke and stroke mimics.

OBJECTIVE: Stroke is the leading cause of death and disability. Timely differentiation between ischemic stroke, hemorrhagic stroke, and stroke mimics is critical for tailored treatment and triage. To accelerate the identification of stroke's subtype, we propose to use the levels of circulating tPA as a biomarker. METHODS: Biostroke is an observational study performed at the Caen Hospital. We quantified tPA levels in 110 patients with ischemic strokes, 30 patients with hemorrhagic strokes, and 67 stroke mimic patients upon their arrival at the emergency. Two logistic regression models were formulated: one with parameters measurable in an ambulance (Model A) and one with parameters measurable at the hospital (Model H). These models were both tested with or without plasma tPA measurements. Our initial assessment involved evaluating the effectiveness of both models in distinguishing between hemorrhagic strokes, ischemic strokes, and stroke mimics within our study cohort. RESULTS: Plasmatic tPA levels exhibit significant distinctions between hemorrhagic, ischemic, and mimic stroke patients (1.8; 2.5; 2.4 ng/mL, respectively). The inclusion of tPA in model A significantly enhances the classification accuracy of hemorrhagic patients only, increasing identification from 0.67 (95% CI, 0.59 to 0.75) to 0.78 (95% CI, 0.7 to 0.85) (p = 0.0098). Similarly, in model H, classification accuracy of hemorrhagic patients significantly increased with the addition of tPA, rising from 0.75 (95% CI, 0.67 to 0.83) without tPA to 0.86 (95% CI, 0.81 to 0.91) with tPA (p = 0.024). INTERPRETATIONS: Our findings underscore the valuable role of tPA levels in distinguishing between stroke subtypes.

DNA-sensing inflammasomes cause recurrent atherosclerotic stroke.

The risk of early recurrent events after stroke remains high despite currently established secondary prevention strategies1. Risk is particularly high in patients with atherosclerosis, with more than 10% of patients experiencing early recurrent events1,2. However, despite the enormous medical burden of this clinical phenomenon, the underlying mechanisms leading to increased vascular risk and recurrent stroke are largely unknown. Here, using a novel mouse model of stroke-induced recurrent ischaemia, we show that stroke leads to activation of the AIM2 inflammasome in vulnerable atherosclerotic plaques via an increase of circulating cell-free DNA. Enhanced plaque inflammation post-stroke results in plaque destabilization and atherothrombosis, finally leading to arterioarterial embolism and recurrent stroke within days after the index stroke. We confirm key steps of plaque destabilization also after experimental myocardial infarction and in carotid artery plaque samples from patients with acute stroke. Rapid neutrophil NETosis was identified as the main source of cell-free DNA after stroke and NET-DNA as the causative agent leading to AIM2 inflammasome activation. Neutralization of cell-free DNA by DNase treatment or inhibition of inflammasome activation reduced the rate of stroke recurrence after experimental stroke. Our findings present an explanation for the high recurrence rate after incident ischaemic events in patients with atherosclerosis. The detailed mechanisms uncovered here provide clinically uncharted therapeutic targets for which we show high efficacy to prevent recurrent events. Targeting DNA-mediated inflammasome activation after remote tissue injury represents a promising avenue for further clinical development in the prevention of early recurrent events.

Calibration trial of an innovative medical device (NEVVA© ) for the evaluation of pain in non-communicating patients in the intensive care unit.

Background: Pain management is an essential and complex issue for non-communicative patients undergoing sedation in the intensive care unit (ICU). The Behavioral Pain Scale (BPS), although not perfect for assessing behavioral pain, is the gold standard based partly on clinical facial expression. NEVVA© , an automatic pain assessment tool based on facial expressions in critically ill patients, is a much-needed innovative medical device. Methods: In this prospective pilot study, we recorded the facial expressions of critically ill patients in the medical ICU of Caen University Hospital using the iPhone and Smart Motion Tracking System (SMTS) software with the Facial Action Coding System (FACS) to measure human facial expressions metrically during sedation weaning. Analyses were recorded continuously, and BPS scores were collected hourly over two 8 h periods per day for 3 consecutive days. For this first stage, calibration of the innovative NEVVA© medical device algorithm was obtained by comparison with the reference pain scale (BPS). Results: Thirty participants were enrolled between March and July 2022. To assess the acute severity of illness, the Sequential Organ Failure Assessment (SOFA) and the Simplified Acute Physiology Score (SAPS II) were recorded on ICU admission and were 9 and 47, respectively. All participants had deep sedation, assessed by a Richmond Agitation and Sedation scale (RASS) score of less than or equal to -4 at the time of inclusion. One thousand and six BPS recordings were obtained, and 130 recordings were retained for final calibration: 108 BPS recordings corresponding to the absence of pain and 22 BPS recordings corresponding to the presence of pain. Due to the small size of the dataset, a leave-one-subject-out cross-validation (LOSO-CV) strategy was performed, and the training results obtained the receiver operating characteristic (ROC) curve with an area under the curve (AUC) of 0.792. This model has a sensitivity of 81.8% and a specificity of 72.2%. Conclusion: This pilot study calibrated the NEVVA© medical device and showed the feasibility of continuous facial expression analysis for pain monitoring in ICU patients. The next step will be to correlate this device with the BPS scale.

Central nervous system-associated macrophages modulate the immune response following stroke in aged mice.

Age is a major nonmodifiable risk factor for ischemic stroke. Central nervous system-associated macrophages (CAMs) are resident immune cells located along the brain vasculature at the interface between the blood circulation and the parenchyma. By using a clinically relevant thromboembolic stroke model in young and aged male mice and corresponding human tissue samples, we show that during aging, CAMs acquire a central role in orchestrating immune cell trafficking after stroke through the specific modulation of adhesion molecules by endothelial cells. The absence of CAMs provokes increased leukocyte infiltration (neutrophils and CD4+ and CD8+ T lymphocytes) and neurological dysfunction after stroke exclusively in aged mice. Major histocompatibility complex class II, overexpressed by CAMs during aging, plays a significant role in the modulation of immune responses to stroke. We demonstrate that during aging, CAMs become central coordinators of the neuroimmune response that ensure a long-term fine-tuning of the immune responses triggered by stroke.

MRI-based microthrombi detection in stroke with polydopamine iron oxide.

In acute ischemic stroke, even when successful recanalization is obtained, downstream microcirculation may still be obstructed by microvascular thrombosis, which is associated with compromised brain reperfusion and cognitive decline. Identifying these microthrombi through non-invasive methods remains challenging. We developed the PHySIOMIC (Polydopamine Hybridized Self-assembled Iron Oxide Mussel Inspired Clusters), a MRI-based contrast agent that unmasks these microthrombi. In a mouse model of thromboembolic ischemic stroke, our findings demonstrate that the PHySIOMIC generate a distinct hypointense signal on T2*-weighted MRI in the presence of microthrombi, that correlates with the lesion areas observed 24 hours post-stroke. Our microfluidic studies reveal the role of fibrinogen in the protein corona for the thrombosis targeting properties. Finally, we observe the biodegradation and biocompatibility of these particles. This work demonstrates that the PHySIOMIC particles offer an innovative and valuable tool for non-invasive in vivo diagnosis and monitoring of microthrombi, using MRI during ischemic stroke.

Effects of general anaesthesia in early childhood on local and global visual processing: a post hoc analysis of the APEX cohort study.

BACKGROUND: Preclinical studies suggest that early exposure to anaesthesia alters the visual system in mice and non-human primates. We investigated whether exposure to general anaesthesia leads to visual attention processing changes in children, which could potentially impact essential life skills, including learning. METHODS: This was a post hoc analysis of data from the APprentissages EXécutifs et cerveau chez les enfants d'âge scolaire (APEX) cohort study. A total of 24 healthy 9-10-yr-old children who were or were not exposed to general anaesthesia (for surgery) by a mean age of 3.8 (2.6) yr performed a visual attention task to evaluate ability to process either local details or general global visual information. Whether children were distracted by visual interference during global and local information processing was also assessed. RESULTS: Participants included in the analyses (n=12 participants exposed to general anaesthesia and n=12 controls) successfully completed (>90% of correct answers) the trial tasks. Children from both groups were equally distracted by visual interference. However, children who had been exposed to general anaesthesia were more attracted to global visual information than were control children (P=0.03). CONCLUSIONS: These findings suggest lasting effects of early-life exposure to general anaesthesia on visuospatial abilities. Further investigations of the mechanisms by which general anaesthesia could have delayed effects on how children perceive their visual environment are needed.

Intestinal MAdCAM-1 imaging as biomarker for prognostic in murine models of multiple sclerosis.

INTRODUCTION: Multiple sclerosis (MS) is an autoimmune disease of the central nervous system. Recent evidence suggests that lymphocyte trafficking in the intestines could play a key role in its etiology. Nevertheless, it is not clear how intestinal tissue is involved in the disease onset nor its evolution. In the present study, we aimed to evaluate intestinal inflammation dynamic throughout the disease course and its potential impact on disease progression. METHODS: We used tissue immunophenotyping (immunohistofluorescence and flow cytometry) and a recently described molecular magnetic resonance imaging (MRI) method targeting mucosal addressin cell adhesion molecule-1 (MAdCAM-1) to assess intestinal inflammation in vivo in two distinct animal models of MS (Experimental Autoimmune Encephalomyelitis - EAE) at several time points of disease progression. RESULTS: We report a positive correlation between disease severity and MAdCAM-1 MRI signal in two EAE models. Moreover, high MAdCAM-1 MRI signal during the asymptomatic phase is associated with a delayed disease onset in progressive EAE and to a lower risk of conversion to a secondary-progressive form in relapsing-remitting EAE. During disease evolution, in line with a bi-directional immune communication between the gut and the central nervous system, we observed a decrease in T-CD4+ and B lymphocytes in the ileum concomitantly with their increase in the spinal cord. CONCLUSION: Altogether, these data unveil a crosstalk between intestinal and central inflammation in EAE and support the use of molecular MRI of intestinal MAdCAM-1 as a new biomarker for prognostic in MS patients.

Pharmacological inhibition of mTORC1 reduces neural death and damage volume after MCAO by modulating microglial reactivity.

Ischemic stroke is a sudden and acute disease characterized by neuronal death, increment of reactive gliosis (reactive microglia and astrocytes), and a severe inflammatory process. Neuroinflammation is an early event after cerebral ischemia, with microglia playing a leading role. Reactive microglia involve functional and morphological changes that drive a wide variety of phenotypes. In this context, deciphering the molecular mechanisms underlying such reactive microglial is essential to devise strategies to protect neurons and maintain certain brain functions affected by early neuroinflammation after ischemia. Here, we studied the role of mammalian target of rapamycin (mTOR) activity in the microglial response using a murine model of cerebral ischemia in the acute phase. We also determined the therapeutic relevance of the pharmacological administration of rapamycin, a mTOR inhibitor, before and after ischemic injury. Our data show that rapamycin, administered before or after brain ischemia induction, reduced the volume of brain damage and neuronal loss by attenuating the microglial response. Therefore, our findings indicate that the pharmacological inhibition of mTORC1 in the acute phase of ischemia may provide an alternative strategy to reduce neuronal damage through attenuation of the associated neuroinflammation.

The Janus face of endogenous neuronal tPA: promoting self-protection and worsening the death of neighboring neurons.

Recombinant tissue-type plasminogen activator (r-tPA/Actilyse) stands as the prevailing pharmacological solution for treating ischemic stroke patients, of whom because their endogenous circulating tPA alone is not sufficient to rescue reperfusion and to promote favorable outcome. Beyond the tPA contributed by circulating endothelial cells and hepatocytes, neurons also express tPA, sparking debates regarding its impact on neuronal fate ranging from pro-survival to neurotoxic properties. In order to investigate the role of neuronal tPA during brain injuries, we developed models leading to its conditional deletion in neurons, employing AAV9-pPlat-GFP and AAV9-pPlat-Cre-GFP along with tPA floxed mice. These models were subjected to N-methyl-D-aspartate (NMDA)-induced excitotoxicity or thromboembolic ischemic stroke in mice. Initially, we established that our AAV9 constructs selectively transduce neurons, bypassing other brain cell types. Subsequently, we demonstrated that tPA-expressing neurons exhibit greater resistance against NMDA-induced excitotoxicity compared to tPA negative neurons. The targeted removal of tPA in neurons heightened the susceptibility of these neurons to cell death and prevented a paracrine neurotoxic effect on tPA non-expressing neurons. Under ischemic conditions, the self-neuroprotective influence of tPA encompassed both excitatory (GFP+/Tbr1+) and inhibitory (GFP+/GABA+) neurons. Our data indicate that endogenous neuronal tPA is a protective or deleterious factor against neuronal death in an excitotoxic/ischemic context, depending on whether it acts as an autocrine or a paracrine mediator.

CNS-associated macrophages contribute to intracerebral aneurysm pathophysiology.

Intracerebral aneurysms (IAs) are pathological dilatations of cerebral arteries whose rupture leads to subarachnoid hemorrhage, a significant cause of disability and death. Inflammation is recognized as a critical contributor to the formation, growth, and rupture of IAs; however, its precise actors have not yet been fully elucidated. Here, we report CNS-associated macrophages (CAMs), also known as border-associated macrophages, as one of the key players in IA pathogenesis, acting as critical mediators of inflammatory processes related to IA ruptures. Using a new mouse model of middle cerebral artery (MCA) aneurysms we show that CAMs accumulate in the IA walls. This finding was confirmed in a human MCA aneurysm obtained after surgical clipping, together with other pathological characteristics found in the experimental model including morphological changes and inflammatory cell infiltration. In addition, in vivo longitudinal molecular MRI studies revealed vascular inflammation strongly associated with the aneurysm area, i.e., high expression of VCAM-1 and P-selectin adhesion molecules, which precedes and predicts the bleeding extent in the case of IA rupture. Specific CAM depletion by intracerebroventricular injection of clodronate liposomes prior to IA induction reduced IA formation and rupture rate. Moreover, the absence of CAMs ameliorated the outcome severity of IA ruptures resulting in smaller hemorrhages, accompanied by reduced neutrophil infiltration. Our data shed light on the unexplored role of CAMs as main actors orchestrating the progression of IAs towards a rupture-prone state.

Modulation of Abundance and Location of High-Mobility Group Box 1 in Human Microglia and Macrophages under Oxygen-Glucose Deprivation.

While stroke represents one of the main causes of death worldwide, available effective drug treatment options remain limited to classic thrombolysis with recombinant tissue plasminogen activator (rtPA) for arterial-clot occlusion. Following stroke, multiple pathways become engaged in producing a vicious proinflammatory cycle through the release of damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB1) and heat shock protein 70 kDa (HSP72). HMGB1, in particular, can activate proinflammatory cytokine production when acetylated (AcHMGB1), a form that prefers cytosolic localization and extracellular release. This study aimed at determining how HMGB1 and HSP72 are modulated and affected following treatment with the anti-inflammatory compound resveratrol and novel platelet membrane-derived nanocarriers loaded with rtPA (CSM@rtPA) recently developed by our group for ischemic artery recanalization. Under ischemic conditions of oxygen-glucose deprivation (OGD), nuclear abundance of HMGB1 and AcHMGB1 in microglia and macrophages decreased, whereas treatment with CSM@rtPA did not alter nuclear or cytosolic abundance. Resveratrol treatment markedly increased the cytosolic abundance of HSP72 in microglia. Using proximity ligation assays, we determined that HSP72 interacted with HMGB1 and with acetylated HMGB1. The interaction was differentially affected under the OGD conditions. Resveratrol treatment under the OGD further decreased HSP72-HMGB1 interactions, whereas, in contrast, treatment increased HSP72-AcHMGB1 interactions in microglia. This study points out a salient molecular interaction suited for a two-pronged nanotherapeutic intervention in stroke: enhancement of rtPA's thrombolytic activity and modulation of cytosolic interactions between HMGB1 and HSP72 by resveratrol.

Associations Between Repetitive Negative Thinking and Objective and Subjective Sleep Health in Cognitively Healthy Older Adults.

Objective: Poor sleep and high levels of repetitive negative thinking (RNT), including future-directed (ie, worry) and past-directed (ie, brooding) negative thoughts, have been associated with markers of dementia risk. The relationship between RNT and sleep health in older adults is unknown. This study aimed to investigate this association and its specificities including multiple dimensions of objective and subjective sleep. Methods: This study used a cross sectional quantitative design with baseline data from 127 cognitively healthy older adults (mean age 69.4 ± 3.8 years; 63% female) who took part in the Age-Well clinical trial, France. RNT (ie, worry and brooding) levels were measured using the Penn State Worry Questionnaire and the Rumination Response Scale (brooding subscale). Polysomnography was used to assess sleep objectively, and the Pittsburgh Sleep Quality Index and the St. Mary's Hospital Sleep Questionnaire were used to measure sleep subjectively. In primary analyses the associations between RNT and sleep (ie, objective sleep duration, fragmentation and efficiency and subjective sleep disturbance) were assessed via adjusted regressions. Results: Higher levels of RNT were associated with poorer objective sleep efficiency (worry: ß=-0.32, p<0.001; brooding: ß=-0.26, p=0.002), but not objective sleep duration, fragmentation, or subjective sleep disturbance. Additional analyses, however, revealed differences in levels of worry between those with short, compared with typical and long objective sleep durations (p < 0.05). Conclusion: In cognitively healthy older adults, RNT was associated with sleep characteristics that have been implicated in increased dementia risk. It will take additional research to ascertain the causal link between RNT and sleep characteristics and how they ultimately relate to the risk of developing dementia.

N-Acetylcysteine to Reduce Mortality for Patients Requiring Cardiac Catheterization or Cardiac Surgery: A Systematic Review and Meta-analysis.

ABSTRACT: Multimers of von Willebrand factor play a critical role in various processes inducing morbidity and mortality in cardiovascular-risk patients. With the ability to reduce von Willebrand factor multimers, N-acetylcysteine (NAC) could reduce mortality in patients undergoing coronary catheterization or cardiac surgery. However, its impact in perioperative period has never been studied so far in regard of its potential cardiovascular benefits. Then, 4 databases were searched for randomized controlled trials that compared in-hospital mortality between an experimental group, with NAC, and a control group without NAC, in patients undergoing coronary catheterization or cardiac surgery. The primary efficacy outcome was in-hospital mortality. Secondary outcomes were the occurrence of thrombotic events, major cardiovascular events, myocardial infarction, and contrast-induced nephropathy. The safety outcome was occurrence of hemorrhagic events. Nineteen studies totaling 3718 patients were included. Pooled analysis demonstrated a reduction of in-hospital mortality associated with NAC: odds ratio, 0.60; 95% confidence interval, 0.39-0.92; P = 0.02. The occurrence of secondary outcomes was not significantly reduced with NAC except for contrast-induced nephropathy. No difference was reported for hemorrhagic events. Subgroup analyses revealed a life-saving effect of NAC in a dose-dependent manner with reduction of in-hospital mortality for the NAC high-dose group, but not for the NAC standard-dose (<3500-mg) group. In conclusion, without being able to conclude on the nature of the mechanism involved, our review suggests a benefit of NAC in cardiovascular-risk patients in perioperative period in terms of mortality and supports prospective confirmatory studies.

Pharmacological preclinical comparison of tenecteplase and alteplase for the treatment of acute stroke.

Alteplase (rtPA) remains the standard thrombolytic drug for acute ischemic stroke. However, new rtPA-derived molecules, such as tenecteplase (TNK), with prolonged half-lives following a single bolus administration, have been developed. Although TNK is currently under clinical evaluation, the limited preclinical data highlight the need for additional studies to elucidate its benefits. The toxicities of rtPA and TNK were evaluated in endothelial cells, astrocytes, and neuronal cells. In addition, their in vivo efficacy was independently assessed at two research centers using an ischemic thromboembolic mouse model. Both therapies were tested via early (20 and 30 min) and late administration (4 and 4.5 h) after stroke. rtPA, but not TNK, caused cell death only in neuronal cultures. Mice were less sensitive to thrombolytic therapies than humans, requiring doses 10-fold higher than the established clinical dose. A single bolus dose of 2.5 mg/kg TNK led to an infarct reduction similar to perfusion with 10 mg/kg of rtPA. Early administration of TNK decreased the hemorrhagic transformations compared to that by the early administration of rtPA; however, this result was not obtained following late administration. These two independent preclinical studies support the use of TNK as a promising reperfusion alternative to rtPA.

The niche matters: origin, function and fate of CNS-associated macrophages during health and disease.

There are several cellular and acellular structural barriers associated with the brain interfaces, which include the dura, the leptomeninges, the perivascular space and the choroid plexus epithelium. Each structure is enriched by distinct myeloid populations, which mainly originate from erythromyeloid precursors (EMP) in the embryonic yolk sac and seed the CNS during embryogenesis. However, depending on the precise microanatomical environment, resident myeloid cells differ in their marker profile, turnover and the extent to which they can be replenished by blood-derived cells. While some EMP-derived cells seed the parenchyma to become microglia, others engraft the meninges and become CNS-associated macrophages (CAMs), also referred to as border-associated macrophages (BAMs), e.g., leptomeningeal macrophages (MnMΦ). Recent data revealed that MnMΦ migrate into perivascular spaces postnatally where they differentiate into perivascular macrophages (PvMΦ). Under homeostatic conditions in pathogen-free mice, there is virtually no contribution of bone marrow-derived cells to MnMΦ and PvMΦ, but rather to macrophages of the choroid plexus and dura. In neuropathological conditions in which the blood-brain barrier is compromised, however, an influx of bone marrow-derived cells into the CNS can occur, potentially contributing to the pool of CNS myeloid cells. Simultaneously, resident CAMs may also proliferate and undergo transcriptional and proteomic changes, thereby, contributing to the disease outcome. Thus, both resident and infiltrating myeloid cells together act within their microenvironmental niche, but both populations play crucial roles in the overall disease course. Here, we summarize the current understanding of the sources and fates of resident CAMs in health and disease, and the role of the microenvironment in influencing their maintenance and function.

Tissue-plasminogen activator effects on the phenotype of splenic myeloid cells in acute inflammation.

Tissue-plasminogen activator (tPA) is a serine protease well known for its fibrinolytic function. Recent studies indicate that tPA could also modulate inflammation via plasmin generation and/or by receptor mediated signalling in vitro. However, the contribution of tPA in inflammatory processes in vivo has not been fully addressed. Therefore, using tPA-deficient mice, we have analysed the effect of lipopolysaccharide (LPS) challenge on the phenotype of myeloid cells including neutrophils, macrophages and dendritic cells (DCs) in spleen. We found that LPS treatment upregulated the frequency of major histocompatibility class two (MHCII+) macrophages but also, paradoxically, induced a deep downregulation of MHCII molecule level on macrophages and on conventional dendritic cells 2 (cDC2). Expression level of the CD11b integrin, known as a tPA receptor, was upregulated by LPS on MHCII+ macrophages and cDC2, suggesting that tPA effects could be amplified during inflammation. In tPA-/- mice under inflammatory conditions, expression of costimulatory CD86 molecules on MHCII+ macrophages was decreased compared to WT mice, while in steady state the expression of MHCII molecules was higher on macrophages. Finally, we reported that tPA deficiency slightly modified the phenotype of DCs and T cells in acute inflammatory conditions. Overall, our findings indicate that in vivo, LPS injection had an unexpectedly bimodal effect on MHCII expression on macrophages and DCs that consequently might affect adaptive immunity. tPA could also participate in the regulation of the T cell response by modulating the levels of CD86 and MHCII molecules on macrophages.

Leptomeningeal collaterals regulate reperfusion in ischemic stroke and rescue the brain from futile recanalization.

Recanalization is the mainstay of ischemic stroke treatment. However, even with timely clot removal, many stroke patients recover poorly. Leptomeningeal collaterals (LMCs) are pial anastomotic vessels with yet-unknown functions. We applied laser speckle imaging, ultrafast ultrasound, and two-photon microscopy in a thrombin-based mouse model of stroke and fibrinolytic treatment to show that LMCs maintain cerebral autoregulation and allow for gradual reperfusion, resulting in small infarcts. In mice with poor LMCs, distal arterial segments collapse, and deleterious hyperemia causes hemorrhage and mortality after recanalization. In silico analyses confirm the relevance of LMCs for preserving perfusion in the ischemic region. Accordingly, in stroke patients with poor collaterals undergoing thrombectomy, rapid reperfusion resulted in hemorrhagic transformation and unfavorable recovery. Thus, we identify LMCs as key components regulating reperfusion and preventing futile recanalization after stroke. Future therapeutic interventions should aim to enhance collateral function, allowing for beneficial reperfusion after stroke.

Improving stroke outcomes in hyperglycemic mice by modulating tPA/NMDAR signaling to reduce inflammation and hemorrhages.

ABSTRACT: The pharmacological intervention for ischemic stroke hinges on intravenous administration of the recombinant tissue-type plasminogen activator (rtPA, Alteplase/Actilyse) either as a standalone treatment or in conjunction with thrombectomy. However, despite its clinical significance, broader use of rtPA is constrained because of the risk of hemorrhagic transformations (HTs). Furthermore, the presence of diabetes or chronic hyperglycemia is associated with an elevated risk of HT subsequent to thrombolysis. This detrimental impact of tPA on the neurovascular unit in patients with hyperglycemia has been ascribed to its capacity to induce endothelial N-methyl-D-aspartate receptor (NMDAR) signaling, contributing to compromised blood-brain barrier integrity and neuroinflammatory processes. In a mouse model of thromboembolic stroke with chronic hyperglycemia, we assessed the effectiveness of rtPA and N-acetylcysteine (NAC) as thrombolytic agents. We also tested the effect of blocking tPA/NMDAR signaling using a monoclonal antibody, Glunomab. Magnetic resonance imaging, speckle contrast imaging, flow cytometry, and behavioral tasks were used to evaluate stroke outcomes. In hyperglycemic animals, treatment with rtPA resulted in lower recanalization rates and increased HTs. Conversely, NAC treatment reduced lesion sizes while mitigating HTs. After a single administration, either in standalone or combined with rtPA-induced thrombolysis, Glunomab reduced brain lesion volumes, HTs, and neuroinflammation after stroke, translating into improved neurological outcomes. Additionally, we demonstrated the therapeutic efficacy of Glunomab in combination with NAC or as a standalone strategy in chronic hyperglycemic animals. Counteracting tPA-dependent endothelial NMDAR signaling limits ischemic damages induced by both endogenous and exogenous tPA, including HTs and inflammatory processes after ischemic stroke in hyperglycemic animals.

Thrombolytic therapy based on lyophilized platelet-derived nanocarriers for ischemic stroke.

BACKGROUND: Intravenous administration of fibrinolytic drugs, such as recombinant tissue plasminogen activator (rtPA) is the standard treatment of acute thrombotic diseases. However, current fibrinolytics exhibit limited clinical efficacy because of their short plasma half-lives and risk of hemorrhagic transformations. Platelet membrane-based nanocarriers have received increasing attention for ischemic stroke therapies, as they have natural thrombus-targeting activity, can prolong half-life of the fibrinolytic therapy, and reduce side effects. In this study we have gone further in developing platelet-derived nanocarriers (defined as cellsomes) to encapsulate and protect rtPA from degradation. Following lyophilization and characterization, their formulation properties, biocompatibility, therapeutic effect, and risk of hemorrhages were later investigated in a thromboembolic model of stroke in mice. RESULTS: Cellsomes of 200 nm size and loaded with rtPA were generated from membrane fragments of human platelets. The lyophilization process did not influence the nanocarrier size distribution, morphology, and colloidal stability conferring particle preservation and long-term storage. Encapsulated rtPA in cellsomes and administered as a single bolus showed to be as effective as a continuous clinical perfusion of free rtPA at equal concentration, without increasing the risk of hemorrhagic transformations or provoking an inflammatory response. CONCLUSIONS: This study provides evidence for the safe and effective use of lyophilized biomimetic platelet-derived nanomedicine for precise thrombolytic treatment of acute ischemic stroke. In addition, this new nanoformulation could simplify the clinical use of rtPA as a single bolus, being easier and less time-consuming in an emergency setting than a treatment perfusion, particularly in stroke patients. We have successfully addressed one of the main barriers to drug application and commercialization, the long-term storage of nanomedicines, overcoming the potential chemical and physical instabilities of nanomedicines when stored in an aqueous buffer.