Cerebral Microbleeds in Murine Amyloid Angiopathy: Natural Course and Anticoagulant Effects.

BACKGROUND AND PURPOSE: Cerebral microbleeds (CMBs) predispose patients to intracerebral hemorrhage. Preclinical models to examine the effects of antithrombotic treatments on the development of clinically overt intracerebral hemorrhage are needed. We examined the natural course of CMB development and the effects of long-term anticoagulation with warfarin or dabigatran on cerebral micro- and macrohemorrhage in mice overexpressing the APP23 (amyloid precursor protein). METHODS: Repeated susceptibility-weighted magnetic resonance imaging was performed in APP23 mice at the age of 18 and 21 months, respectively. After establishing stable long-term anticoagulation effects of warfarin and dabigatran on number and total volume of CMBs, the outcome parameters were compared with nonanticoagulated control. RESULTS: CMBs were equally located in lobar and deep brain regions, and number and total volume of CMBs increased over time. Anticoagulation with either warfarin or dabigatran did not increase CMBs in APP23 significantly. Mice treated with warfarin numerically had a higher mortality (nonanticoagulated: 31%; dabigatran: 35% versus warfarin: 55%; P=0.21). In postmortem brains of prematurely dying animals warfarin caused significantly more frequently large intracerebral hemorrhage than control and dabigatran. CONCLUSIONS: Anticoagulation with warfarin or dabigatran for 3 to 4 months does not promote the formation of CMBs in aged APP23 mice. Nevertheless, warfarin but not dabigatran is associated with a higher risk of extensive intracerebral hemorrhage, suggesting that this model may allow preclinical safety evaluation of antithrombotic therapies.

Only very early oxygen therapy attenuates posthemorrhagic edema formation and blood-brain barrier disruption in murine intracerebral hemorrhage.

BACKGROUND AND PURPOSE: Perihematomal edema exacerbates the mass effect of hematoma and contributes to early neurological deterioration after intracerebral hemorrhage (ICH). Oxygen therapy has protective effects on the blood-brain barrier (BBB). We aimed to examine the effects of oxygen therapy on edema formation and BBB permeability after ICH. METHODS: ICH was induced in mice by injecting autologous blood (30 µL) or collagenase (0.03 U) intrastriatally. Development of posthemorrhagic edema formation and BBB disruption was characterized by wet-dry-weight assays and sodium- fluorescein fluorospectrometry 1d, 3d, and 7d after ICH induction. In subsequent experiments, mice received air, normobaric (NBO), or hyperbaric oxygen (HBO; 3ata) for 60 min starting either 30, 60, or 120 min after ICH induction. Expression of occludin, claudin-5, zonula occludens-1, matrix metalloproteinases (MMPs), and hypoxia-inducible factor-1α (HIF-1α) was measured by Western blot and zymography. RESULTS: Posthemorrhagic edema formation (water content: blood-injection model 80.6 ± 0.3 %; collagenase injection model 83.3 ± 0.7 %) and BBB disruption (interhemispheric ratio of extravasated sodium fluorescein: blood-injection model 1.75 ± 0.07; collagenase injection model 3.02 ± 0.15) peaked 3d after ICH. NBO and HBO initiated within 30 min of ICH induction attenuated edema formation and BBB disruption (interhemispheric ratio of fluorescein; blood-injection air 1.75 ± 0.12, NBO 1.52 ± 0.08, HBO 1.49 ± 0.09; collagenase: air 3.04 ± 0.23, NBO 2.25 ± 0.21, HBO 2.17 ± 0.23) 3d after ICH, whereas delayed oxygen therapy had no effect. Early oxygen therapies prevented occludin degradation, MMP-9 activation, and reduced HIF-1α expression. CONCLUSION: Very early oxygen therapy can attenuate edema formation and BBB disruption after ICH, but the brief therapeutic time window suggests that the translational potential is limited.

MRI assessment of the intra-carotid route for macrophage delivery after transient cerebral ischemia.

The broad aim underlying the present research was to investigate the distribution and homing of bone marrow-derived macrophages in a rodent model of transient middle cerebral artery occlusion using MRI and ultrasmall superparamagnetic iron oxide (USPIO) to magnetically label bone marrow-derived macrophages. The specific aim was to assess the intra-carotid infusion route for bone marrow-derived macrophage delivery at reperfusion. Fifteen Sprague-Dawley rats sustained 1 h of middle cerebral artery occlusion. USPIO-labeled bone marrow-derived macrophages were slowly injected for 5 min immediately after reperfusion in ischemic animals (n=7), 1 h after the end of surgery in sham animals (n=5) and very shortly after anesthesia in healthy animals (n=3). Multiparametric MRI was performed at day 0, just after cell administration, and repeated at day 1. Immunohistological analysis included Prussian blue for iron detection and rat endothelial cell antigen-1 for endothelium visualization. Intra-carotid cell delivery brought a large number of cells to the ipsilateral hemisphere of the brain, as seen on both MRI and immunohistology. However, it was associated with high mortality (50%). The study of sham animals demonstrated that intra-carotid cell delivery could induce ischemic lesions and may thus favor additional brain damage. The present study highlights severe drawbacks to the intra-carotid delivery of macrophages at the time of reperfusion in this rodent model of transient cerebral ischemia. Multiparametric MRI appears to be a method of choice to monitor longitudinally the effects of cell infusion, allowing the assessment of both cell fate with the help of magnetic labeling and of potential tissue damage.

Monitoring therapeutic effects in experimental stroke by serial USPIO-enhanced MRI.

OBJECTIVES: This study sought to evaluate whether the therapeutic effects of an anti-inflammatory drug such as minocycline could be monitored by serial ultrasmall superparamagnetic particles of iron oxide (USPIO)-enhanced MRI in experimental stroke. METHODS: Mice received a three-dose minocycline treatment (n = 12) or vehicle (n = 12) after permanent middle cerebral artery occlusion. USPIOs were administered 5 h post-surgery. MRI was performed before, 24 h and 48 h post-USPIO administration. MRI endpoints were the extent of signal abnormalities on R2 maps (=1/T2) and quantitative R2 changes over time (∆R2). Post-mortem brains were prepared either for immunohistology (n = 16) or for iron dosage (n = 8). RESULTS: As expected, treatment with minocycline significantly reduced infarct size, blood-brain barrier permeability and F4/80 immunostaining for microglia/macrophages. Areas of R2 maps > 35 ms(-1) also appeared significantly decreased in minocycline-treated mice (ANOVA for repeated measures, P = 0.017). There was a fair correlation between these areas and the amount of iron in the brain (R(2) = 0.69, P = 0.010), but no significant difference in ∆R2 was found between the two groups. CONCLUSIONS: This study showed that the extent of signal abnormalities on R2 maps can be used as a surrogate marker to detect minocycline effects in a murine experimental model of stroke.

Extent of white matter lesion is associated with early hemorrhagic transformation in acute ischemic stroke related to atrial fibrillation.

BACKGROUND: Hemorrhagic transformation (HT) after stroke, related to atrial fibrillation (AF), is a frequent complication, and it can be associated with a delay in the (re-)initiation of oral anticoagulation therapy. We investigated the effect of the presence and severity of white matter disease (WMD) on early HT after stroke related to AF. METHODS: A consecutive series of patients with recent (<4 weeks) ischemic stroke and AF, treated at the Hyper Acute Stroke Unit of the Imperial College London between 2010 and 2017, were enrolled. Patients with brain MRI performed 24-72 h from stroke onset and not yet started on anticoagulant treatment were included. WMD was graded using the Fazekas score. RESULTS: Among the 441 patients eligible for the analysis, 91 (20.6%) had any HT. Patients with and without HT showed similar clinical characteristics. Patients with HT had a larger diffusion-weighted imaging (DWI) infarct volume compared to patients without HT (p < .001) and significant difference in the distribution of the Fazekas score (p = .001). On multivariable analysis, HT was independently associated with increasing DWI infarct volume (odd ratio (OR), 1.03; 95% confidence interval (CI), 1.01-1.05; p < .001), higher Fazekas scores (OR, 1.94; 95% CI, 1.47-2.57; p < .001) and history of previous intracranial hemorrhage (OR, 4.80; 95% CI, 1.11-20.80; p = .036). CONCLUSIONS: Presence and severity of WMD is associated with increased risk of development of early HT in patients with stroke and AF. Further evidence is needed to provide reliable radiological predictors of the risk of HT in cardioembolic stroke.

Experimental ischaemic stroke induces transient cardiac atrophy and dysfunction.

BACKGROUND: Stroke can lead to cardiac dysfunction in patients, but the mechanisms underlying the interaction between the injured brain and the heart are poorly understood. The objective of the study is to investigate the effects of experimental murine stroke on cardiac function and molecular signalling in the heart. METHODS AND RESULTS: Mice were subjected to filament-induced left middle cerebral artery occlusion for 30 or 60 min or sham surgery and underwent repetitive micro-echocardiography. Left ventricular contractility was reduced early (24-72 h) but not late (2 months) after brain ischaemia. Cardiac dysfunction was accompanied by a release of high-sensitive cardiac troponin (hsTNT (ng/ml): d1: 7.0 ± 1.0 vs. 25.0 ± 3.2*; d3: 7.3 ± 1.1 vs. 52.2 ± 16.7*; d14: 5.7 ± 0.8 vs. 5.2 ± 0.3; sham vs. 60 min. MCAO; mean ± SEM; *p < 0.05); reduced heart weight (heart weight/tibia length ratio: d1: 6.9 ± 0.2 vs. 6.4 ± 0.1*; d3: 6.7 ± 0.2 vs. 5.8 ± 0.1*; d14: 6.7 ± 0.2 vs. 6.4 ± 03; sham vs. 60 min. MCAO; mean ± SEM; *p < 0.05); resulting from cardiomyocyte atrophy (cardiomyocyte size: d1: 12.8% ± 0.002**; d3: 13.5% ± 0.002**; 14d: 6.3% ± 0.003*; 60 min. MCAO vs. sham; mean ± SEM; **p < 0.01; *p < 0.05), accompanied by increased atrogin-1 and the E3 ubiquitin ligase murf-1. Net norepinephrine but not synthesis was increased, suggesting a reduced norepinephrine release or an increase of norepinephrine re-uptake, resulting in a functional denervation. Transcriptome analysis in cardiac tissue identified the transcription factor peroxisome proliferator-activated receptor gamma as a potential mediator of stroke-induced transcriptional dysregulation involved in cardiac atrophy. CONCLUSIONS: Stroke induces a complex molecular response in the heart muscle with immediate but transient cardiac atrophy and dysfunction.

Clot injection technique affects thrombolytic efficacy in a rat embolic stroke model: implications for translaboratory collaborations.

Current recommendations encourage the use of embolic stroke (ES) models and replication of results across laboratories in preclinical research. Since such endeavors employ different surgeons, we sought to ascertain the impact of injection technique on outcome and response to thrombolysis in an ES model. Embolic stroke was induced in Male Wistar Kyoto rats (n=166) by a fast or a slow clot injection (CI) technique. Saline or recombinant tissue plasminogen activator (rtPA) was given at 1 hour after stroke. Flow rate curves were assessed in 24 animals. Cerebral perfusion was assessed using laser Doppler flowmetry. Edema corrected infarct volume, hemispheric swelling, hemorrhagic transformation, and neurologic outcome were assessed at 24 hours after stroke. Clot burden was estimated in a subset of animals (n=40). Slow CI resulted in significantly smaller infarct volumes (P=0.024) and better neurologic outcomes (P=0.01) compared with fast CI at 24 hours. Unexpectedly, rtPA treatment attenuated infarct size in fast (P<0.001) but not in slow CI experiments (P=0.382), possibly related to reperfusion injury as indicated by greater hemorrhagic transformation (P<0.001) and hemispheric swelling (P<0.05). Outcome and response to thrombolysis after ES are operator dependent, which needs to be considered when comparing results obtained from different laboratories.

In vitro and in vivo models of cerebral ischemia show discrepancy in therapeutic effects of M2 macrophages.

THE INFLAMMATORY RESPONSE FOLLOWING ISCHEMIC STROKE IS DOMINATED BY INNATE IMMUNE CELLS: resident microglia and blood-derived macrophages. The ambivalent role of these cells in stroke outcome might be explained in part by the acquisition of distinct functional phenotypes: classically (M1) and alternatively activated (M2) macrophages. To shed light on the crosstalk between hypoxic neurons and macrophages, an in vitro model was set up in which bone marrow-derived macrophages were co-cultured with hippocampal slices subjected to oxygen and glucose deprivation. The results showed that macrophages provided potent protection against neuron cell loss through a paracrine mechanism, and that they expressed M2-type alternative polarization. These findings raised the possibility of using bone marrow-derived M2 macrophages in cellular therapy for stroke. Therefore, 2 million M2 macrophages (or vehicle) were intravenously administered during the subacute stage of ischemia (D4) in a model of transient middle cerebral artery occlusion. Functional neuroscores and magnetic resonance imaging endpoints (infarct volumes, blood-brain barrier integrity, phagocytic activity assessed by iron oxide uptake) were longitudinally monitored for 2 weeks. This cell-based treatment did not significantly improve any outcome measure compared with vehicle, suggesting that this strategy is not relevant to stroke therapy.