MMP-9 release into collateral blood vessels before endovascular thrombectomy to assess the risk of major intracerebral haemorrhages and poor outcome for acute ischaemic stroke: a proof-of-concept study.

BACKGROUND: Matrix metalloproteinases (MMPs) are implied in blood-brain barrier degradation and haemorrhagic transformation following ischaemic stroke, but their local relevance in the hyperacute disease phase is unknown. We aimed to examine ultra-early MMP-9 and MMP-2 release into collateral blood vessels, and to assess its prognostic value before therapeutic recanalisation by endovascular thrombectomy (EVT). METHODS: We report a cross-sectional proof-of-concept study including patients undergoing EVT for large-vessel ischaemic stroke at the University Hospital Würzburg, Germany. We obtained liquid biopsies from the collateral circulation before recanalisation, and systemic control samples. Laboratory workup included quantification of MMP-9 and MMP-2 plasma concentrations by cytometric bead array, immunohistochemical analyses of cellular MMP-9 and MMP-2 expression, and detection of proteolytic activity by gelatine zymography. The clinical impact of MMP concentrations was assessed by stratification according to intracranial haemorrhagic lesions on postinterventional computed tomography (Heidelberg Bleeding Classification, HBC) and early functional outcome (modified Rankin Scale, mRS). We used multivariable logistic regression, receiver-operating-characteristic (ROC) curves, and fixed-level estimates of test accuracy measures to study the prognostic value of MMP-9 concentrations. FINDINGS: Between August 3, 2018, and September 16, 2021, 264 matched samples from 132 patients (86 [65.2%] women, 46 [34.8%] men, aged 40-94 years) were obtained. Median (interquartile range, IQR) MMP-9 (279.7 [IQR 126.4-569.6] vs 441 [IQR 223.4-731.5] ng/ml, p < 0.0001) but not MMP-2 concentrations were increased within collateral blood vessels. The median MMP-9 expression level of invading neutrophils was elevated (fluorescence intensity, arbitrary unit: 2276 [IQR 1007-5086] vs 3078 [IQR 1108-7963], p = 0.0018). Gelatine zymography experiments indicated the locally confined proteolytic activity of MMP-9 but not of MMP-2. Pretherapeutic MMP-9 release into stroke-affected brain regions predicted the degree of intracerebral haemorrhages and clinical stroke severity after recanalisation, and independently increased the odds of space-occupying parenchymal haematomas (HBC1c-3a) by 1.54 times, and the odds of severe disability or death (mRS ≥5 at hospital discharge) by 2.33 times per 1000 ng/ml increase. Excessive concentrations of MMP-9 indicated impending parenchymal haematomas and severe disability or death with high specificity. INTERPRETATION: Measurement of MMP-9 within collateral blood vessels is feasible and identifies patients with stroke at risk of major intracerebral haemorrhages and poor outcome before therapeutic recanalisation by EVT, thereby providing evidence of the concept validity of ultra-early local stroke biomarkers. FUNDING: This work was funded by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) and the Interdisciplinary Centre for Clinical Research (IZKF) at the University of Würzburg.

Dimethyl Fumarate Attenuates Lymphocyte Infiltration and Reduces Infarct Size in Experimental Stroke.

Ischemic stroke is associated with exacerbated tissue damage caused by the activation of immune cells and the initiation of other inflammatory processes. Dimethyl fumarate (DMF) is known to modulate the immune response, activate antioxidative pathways, and improve the blood-brain barrier (BBB) after stroke. However, the specific impact of DMF on immune cells after cerebral ischemia remains unclear. In our study, male mice underwent transient middle cerebral artery occlusion (tMCAO) for 30 min and received oral DMF (15 mg/kg) or a vehicle immediately after tMCAO, followed by twice-daily administrations for 7 days. Infarct volume was assessed on T2-weighted magnetic resonance images on days 1 and 7 after tMCAO. Brain-infiltrating immune cells (lymphocytes, monocytes) and microglia were quantified using fluorescence-activated cell sorting. DMF treatment significantly reduced infarct volumes and brain edema. On day 1 after tMCAO, DMF-treated mice showed reduced lymphocyte infiltration compared to controls, which was not observed on day 7. Monocyte and microglial cell counts did not differ between groups on either day. In the acute phase of stroke, DMF administration attenuated lymphocyte infiltration, probably due to its stabilizing effect on the BBB. This highlights the potential of DMF as a therapeutic candidate for mitigating immune cell-driven damage in stroke.

Regulation and Release of Vasoactive Endoglin by Brain Endothelium in Response to Hypoxia/Reoxygenation in Stroke.

In large vessel occlusion stroke, recanalization to restore cerebral perfusion is essential but not necessarily sufficient for a favorable outcome. Paradoxically, in some patients, reperfusion carries the risk of increased tissue damage and cerebral hemorrhage. Experimental and clinical data suggest that endothelial cells, representing the interface for detrimental platelet and leukocyte responses, likely play a crucial role in the phenomenon referred to as ischemia/reperfusion (I/R)-injury, but the mechanisms are unknown. We aimed to determine the role of endoglin in cerebral I/R-injury; endoglin is a membrane-bound protein abundantly expressed by endothelial cells that has previously been shown to be involved in the maintenance of vascular homeostasis. We investigated the expression of membranous endoglin (using Western blotting and RT-PCR) and the generation of soluble endoglin (using an enzyme-linked immunosorbent assay of cell culture supernatants) after hypoxia and subsequent reoxygenation in human non-immortalized brain endothelial cells. To validate these in vitro data, we additionally examined endoglin expression in an intraluminal monofilament model of permanent and transient middle cerebral artery occlusion in mice. Subsequently, the effects of recombinant human soluble endoglin were assessed by label-free impedance-based measurement of endothelial monolayer integrity (using the xCELLigence DP system) and immunocytochemistry. Endoglin expression is highly inducible by hypoxia in human brain endothelial monolayers in vitro, and subsequent reoxygenation induced its shedding. These findings were corroborated in mice during MCAO; an upregulation of endoglin was displayed in the infarcted hemispheres under occlusion, whereas endoglin expression was significantly diminished after transient MCAO, which is indicative of shedding. Of note is the finding that soluble endoglin induced an inflammatory phenotype in endothelial monolayers. The treatment of HBMEC with endoglin resulted in a decrease in transendothelial resistance and the downregulation of VE-cadherin. Our data establish a novel mechanism in which hypoxia triggers the initial endothelial upregulation of endoglin and subsequent reoxygenation triggers its release as a vasoactive mediator that, when rinsed into adjacent vascular beds after recanalization, can contribute to cerebral reperfusion injury.

NLRP3 Inhibition Reduces rt-PA Induced Endothelial Dysfunction under Ischemic Conditions.

Thrombolysis with recombinant tissue plasminogen activator (rt-PA) is a mainstay of acute ischemic stroke treatment but is associated with bleeding complications, especially after prolonged large vessel occlusion. Recently, inhibition of the NLRP3 inflammasome led to preserved blood-brain barrier (BBB) integrity in experimental stroke in vivo. To further address the potential of NLRP3 inflammasome inhibition as adjunct stroke treatment we used immortalized brain derived endothelial cells (bEnd5) as an in vitro model of the BBB. We treated bEnd5 with rt-PA in combination with the NLRP3 specific inhibitor MCC950 or vehicle under normoxic as well as ischemic (OGD) conditions. We found that rt-PA exerted a cytotoxic effect on bEnd5 cells under OGD confirming that rt-PA is harmful to the BBB. This detrimental effect could be significantly reduced by MCC950 treatment. Moreover, under ischemic conditions, the Cell Index-a sensible indicator for a patent BBB-and the protein expression of Zonula occludens 1 stabilized after MCC950 treatment. At the same time, the extent of endothelial cell death and NLRP3 expression decreased. In conclusion, NLRP3 inhibition can protect the BBB from rt-PA-induced damage and thereby potentially increase the narrow time window for safe thrombolysis in stroke.

Defining cerebral leukocyte populations in local ischemic blood samples from patients with hyperacute stroke.

In acute stroke, neuroinflammation can nowadays be analyzed by local cerebral aspiration of pial-ischemic blood during mechanical thrombectomy. Recently, Shaw et al. reported on differences in leukocyte subpopulations within the occluded cerebrovascular compartment. In their study, a main proportion of granulocytes was lost during isolation. By immediate analysis, we found a reproducible increase in absolute local granulocytes without variations in absolute lymphocyte and monocyte numbers. Flow-cytometric phenotyping confirmed a high proportion of granulocytes and a local shift towards CD4+ T cells. Thus, immediate analysis appears to be critical to observe distinct local responses of leukocytes to acute ischemic stroke.

Infarct growth precedes cerebral thrombosis following experimental stroke in mice.

Ischemic stroke is among the leading causes of disability and death worldwide. In acute ischemic stroke, successful recanalization of occluded vessels is the primary therapeutic aim, but even if it is achieved, not all patients benefit. Although blockade of platelet aggregation did not prevent infarct progression, cerebral thrombosis as cause of secondary infarct growth has remained a matter of debate. As cerebral thrombi are frequently observed after experimental stroke, a thrombus-induced impairment of the brain microcirculation is considered to contribute to tissue damage. Here, we combine the model of transient middle cerebral artery occlusion (tMCAO) with light sheet fluorescence microscopy and immunohistochemistry of brain slices to investigate the kinetics of thrombus formation and infarct progression. Our data reveal that tissue damage already peaks after 8 h of reperfusion following 60 min MCAO, while cerebral thrombi are only observed at later time points. Thus, cerebral thrombosis is not causative for secondary infarct growth during ischemic stroke.

Treatment of radiation-induced maculopathy with fluocinolone acetonide.

PURPOSE: Chronic macular oedema is a well-known presentation of radiation-induced maculopathy (RM) following external beam photon therapy, plaque radiotherapy and proton beam radiotherapy for choroidal tumours. Current therapies vary in respect of efficacy and clinical benefit. The potential of fluocinolone acetonide (FAc) slow-release implants is unknown. We hypothesised that local continuous delivery of low-dose corticosteroids might improve symptoms of RM. METHODS: Five-two male and three female-patients from 37 to 68 years presented with RM following 106Ru-plaque brachytherapy or stereotactic radiation therapy (STx) with photons using a hypofractionated schedule of 5 × 10 Gy. All were treated with triamcinolone injections in first line and proofed to be refractory to steroids. In addition, two patients had received Ozurdex® implants as a second-line treatment, though without any clinical benefit. FAc slow-release implants were injected, and patients were followed up to monitor clinical improvement. RESULTS: All patients responded to therapy by means of a decrease in macular oedema. In four of five (80%) patients, visual acuity improved, and one patient showed stable visual acuity. No toxic effects or complications were observed. CONCLUSION: Slow-release implants of FAc are a promising therapeutic potent steroid treatment option to benefit anatomical structures of the fovea and visual function. Slow-release implants with FAc reduce the frequency of intravitreal injections and the therapeutic burden.

Epigenetic heterogeneity of developmentally important genes in human sperm: implications for assisted reproduction outcome.

The molecular basis of male infertility is poorly understood, the majority of cases remaining unsolved. The association of aberrant sperm DNA methylation patterns and compromised semen parameters suggests that disturbances in male germline epigenetic reprogramming contribute to this problem. So far there are only few data on the epigenetic heterogeneity of sperm within a given sample and how to select the best sperm for successful infertility treatment. Limiting dilution bisulfite sequencing of small pools of sperm from fertile donors did not reveal significant differences in the occurrence of abnormal methylation imprints between sperm with and without morphological abnormalities. Intracytoplasmic morphologically selected sperm injection was not associated with an improved epigenetic quality, compared to standard intracytoplasmatic sperm injection. Deep bisulfite sequencing (DBS) of 2 imprinted and 2 pluripotency genes in sperm from men attending a fertility center showed that in both samples with normozoospermia and oligoasthenoteratozoospermia (OAT) the vast majority of sperm alleles was normally (de)methylated and the percentage of epimutations (allele methylation errors) was generally low (<1%). However, DBS allowed one to identify and quantify these rare epimutations with high accuracy. Sperm samples not leading to a pregnancy, in particular in the OAT group, had significantly more epimutations in the paternally methylated GTL2 gene than samples leading to a live birth. All 13 normozoospermic and 13 OAT samples leading to a child had <1% GTL2 epimutations, whereas one (7%) of 14 normozoospermic and 7 (50%) of 14 OAT samples without pregnancy displayed 1-14% GTL2 epimutations.