Translational Stroke Research

Although the association between patent foramen ovale and ischemic stroke is controversial, the evaluation for a right-to-left shunt remains part of the standard workup for cryptogenic stroke. Transthoracic and transesophageal echocardiogram (TTE and TEE) are the screening test and gold standard to evaluate for right-to-left shunt, respectively. Studies comparing TTE or TEE to transcranial Doppler (TCD) have shown that 15–25 % of patients test positive for right-to-left shunt on TCD but are negative on TTE or TEE. We sought to further explore this phenomenon in patients with recent ischemic stroke. Between 2011 and 2013, 109 ischemic stroke patients had both a TCD and TTE or TEE bubble study. We abstracted 12 comorbid medical conditions and stroke subtype according to the TOAST classification. The majority of TCD and TTE or TEE showed agreement on right-to-left shunt status (80/109, 73 %). Two percent (2/109) of patients were negative on TCD and positive on TTE or TEE, while 25 % (27/109) had a positive TCD and negative TTE or TEE (TCD+Echo−). The TCD+Echo− patients were more likely to have active malignancy and the delayed arrival of contrast bubbles than the remainder of the cohort (15 vs. 2 %, p = 0.032; 51 vs. 18 %, p = 0.001). Our results confirm previous reports that TCD is superior to echocardiography in the detection of right-to-left shunt. The TCD+Echo− patients were more likely to have active malignancy and findings suggestive of an extracardiac shunt. These results could lead to more comprehensive evaluation for occult malignancy or a pulmonary arteriovenous malformation, both potentially treatable etiologies of ischemic stroke.

Systemic lupus erythematosus (SLE) is an autoimmune disease associated with significant morbidity, including premature cardiovascular disease, and mortality. Platelets bearing complement protein C4d (P-C4d) were initially determined to be specific for diagnosis of SLE and were later found to be associated with acute ischemic stroke in non-SLE patients. P-C4d may identify a subset of SLE patients with a worse clinical prognosis. This study investigated the associations of P-C4d with all-cause mortality and vascular events in a lupus cohort. A cohort of 356 consecutive patients with SLE was followed from 2001 to 2009. Primary outcome was all-cause mortality. Secondary outcomes were vascular events (myocardial infarction, coronary artery bypass graft, percutaneous coronary transluminal angioplasty, ischemic stroke, venous thromboembolism, pulmonary embolism, or other thrombosis). P-C4d was measured at study baseline. Seventy SLE patients (19.7 %) had P-C4d. Mean follow-up was 4.7 years. All-cause mortality was 4 %. P-C4d was associated with all-cause mortality (hazard ratio 7.52, 95 % confidence interval (CI) 2.14–26.45, p = 0.002) after adjusting for age, ethnicity, sex, cancer, and anticoagulant use. Vascular event rate was 21.6 %. Patients with positive P-C4d were more likely to have had vascular events compared to those with negative P-C4d (35.7 vs. 18.2 %, p = 0.001). Specifically, P-C4d was associated with ischemic stroke (odds ratio 4.54, 95 % CI 1.63–12.69, p = 0.004) after adjusting for age, ethnicity, and antiphospholipid antibodies. Platelet-C4d is associated with all-cause mortality and stroke in SLE patients. P-C4d may be a prognostic biomarker as well as a pathogenic clue that links platelets, complement activation, and thrombosis.

Early brain injury (EBI) following  subarachnoid hemorrhage (SAH) is characterized by rapid development of neuron apoptosis and dysregulated inflammatory response. Microglia efferocytosis plays a critical role in the clearance of apoptotic cells, attenuation of inflammation, and minimizing brain injury in various pathological conditions. Here, using a mouse SAH model, we aim to investigate whether microglia efferocytosis is involved in post-SAH inflammation and to determine the underlying signaling pathway. We hypothesized that TAM receptors and their ligands regulate this process. To prove our hypothesis, the expression and cellular location of TAM (Tyro3, Axl, and Mertk) receptors and their ligands growth arrest-specific 6 (Gas6) and Protein S (ProS1) were examined by PCR, western blots, and fluorescence immunostaining. Thirty minutes after SAH, mice received an intraventricular injection of recombinant Gas6 (rGas6) or recombinant ProS1 (rPros1) and underwent evaluations of inflammatory mediator expression, neurological deficits, and blood–brain barrier integrity at 24 h. Microglia efferocytosis of apoptotic neurons was analyzed in vivo and in vitro. The potential mechanism was determined by inhibiting or knocking down TAM receptors and Rac1 by specific inhibitors or siRNA. SAH induced upregulation of Axl and its ligand Gas6. The administration of rGas6 but not rPros1 promoted microglia efferocytosis, alleviated inflammation, and ameliorated SAH-induced BBB breakdown and neurological deficits. The beneficial effects of rGas6 were arrogated by inhibiting or knocking down Axl and Rac1. We concluded that rGas6 attenuated the development of early brain injury in mice after SAH by facilitating microglia efferocytosis and preventing inflammatory response, which is partly dependent on activation of Axl and Rac1.

Endovascular treatment (EVT) has been proven to be the standard treatment for acute vertebrobasilar artery occlusion (VBAO). This study aimed to analyze the effects of international normalized ratio (INR) indicators on outcomes in patients with acute VBAO treated with EVT. Dynamic data on INR in patients with VBAO who received endovascular treatment (EVT) at 65 stroke centers in China were retrospectively enrolled. Outcome measures included the modified Rankin Scale (mRS) score at 90 days and 1 year and symptomatic intracranial hemorrhage (sICH). The associations between elevated INR (INR > 1.1), INR variability (time-weighted variance of INR changes), and various clinical outcomes were analyzed in all patients and subgroups stratified by oral anticoagulation (OAC) by mixed logistic regression analysis. A total of 1825 patients met the study criteria, of which 1384 had normal INR and 441 had elevated INR. Multivariate analysis showed that elevated INR was significantly associated with poor functional outcomes (mRS 4–6) at 90 days (odds ratio [OR] 1.36, 95% confidence interval [CI] 1.08–1.72) and 1 year (OR 1.32, 95% CI 1.05–1.66), but was not associated with an increased risk of sICH (OR 1.00, 95% CI 0.83–1.20). Similar associations exist between INR variability and poor functional outcomes at 90 days (OR 2.17, 95% CI 1.09–4.30), 1 year (OR 2.28, 95% CI 1.16–4.46), and sICH (OR 1.11, 95% CI 0.93–1.33). Subgroup analyses further revealed that elevated INR and INR variability remained associated with poor functional outcomes in patients not receiving oral anticoagulation (OAC) therapy, while no significant associations were observed in OAC-treated patients, regardless of whether they were on warfarin or direct oral anticoagulants. Elevated INR and INR variability in VBAO patients treated with EVT were associated with poor functional outcomes. The mechanism underlying the association between elevated INR and poor functional outcomes might be attributed to the fact that elevated INR indirectly reflects the burden of comorbidities, which could independently worsen outcomes. These findings underscore the importance of a comprehensive and dynamic evaluation of INR levels in the management of VBAO patients receiving EVT, providing valuable insights for optimizing patient outcomes.

Cerebral blood volume mapping can characterize hemodynamic changes within brain tissue, particularly after stroke. This study aims to quantify blood volume changes in the perihematomal parenchyma and pericavity parenchyma after minimally invasive intracerebral hemorrhage evacuation (MIS for ICH). Thirty-two patients underwent MIS for ICH with pre- and post-operative CT imaging and intraoperative perfusion imaging (DynaCT PBV Neuro, Artis Q, Siemens). The pre-operative and post-operative CT scans were segmented using ITK-SNAP software to calculate hematoma volumes and to delineate the pericavity tissue. Helical CT segmentations were registered to cone beam CT data using elastix software. Mean blood volumes were computed inside subvolumes by dilating the segmentations at increasing distances from the lesion. Pre-operative perihematomal blood volumes and post-operative pericavity blood volumes (PBV) were compared. In 27 patients with complete imaging, post-operative PBV significantly increased within the 6-mm pericavity region after MIS for ICH. The mean relative PBV increased by 21.6 and 9.1% at 3 mm and 6 mm, respectively (P = 0.001 and 0.016, respectively). At the 9-mm pericavity region, there was a 2.83% increase in mean relative PBV, though no longer statistically significant. PBV analysis demonstrated a significant increase in pericavity cerebral blood volume after minimally invasive ICH evacuation to a distance of 6 mm from the border of the lesion.

Choline acetyltransferase-positive (ChAT+) neurons within the subventricular zone (SVZ) have been shown to promote neurogenesis after stroke in mice by secreting acetylcholine (ACh); however, the mechanisms remain unclear. Receptors known to bind ACh include the nicotinic ACh receptors (nAChRs), which are present in the SVZ and have been shown to be important for cell proliferation, differentiation, and survival. In this study, we investigated the neurogenic role of the alpha-7 nAChR (α7 nAChR) in a mouse model of middle cerebral artery occlusion (MCAO) by using α7 nAChR inhibitor methyllycaconitine. Mice subjected to MCAO exhibited elevated expression of cytomembrane and nuclear fibroblast growth factor receptor 1 (FGFR1), as well as increased expression of PI3K, pAkt, doublecortin (DCX), polysialylated - neuronal cell adhesion molecule (PSA-NCAM), and mammalian achaete-scute homolog 1 (Mash1). MCAO mice also had more glial fibrillary acidic protein (GFAP)/5-bromo-2′-deoxyuridine (BrdU)-positive cells and DCX-positive cells in the SVZ than did the sham-operated group. Methyllycaconitine treatment increased cytomembrane FGFR1 expression and GFAP/BrdU-positive cells, upregulated the levels of phosphoinositide 3-kinase (PI3K) and phospho-Akt (pAkt), decreased nuclear FGFR1 expression, decreased the number of DCX-positive cells, and reduced the levels of DCX, PSA-NCAM, and Mash1 in the SVZ of MCAO mice compared with levels in vehicle-treated MCAO mice. MCAO mice treated with α7 nAChR agonist PNU-282987 exhibited the opposite effects. Our data show that α7 nAChR may decrease the proliferation of neural stem cells and promote differentiation of existing neural stem cells after stroke. These results identify a new mechanism of SVZ ChAT+ neuron-induced neurogenesis.

The use of genetic engineering to develop important neuropathological mouse models has made cerebrovascular imaging essential for the investigation of numerous brain disorders, especially cerebrovascular disorders, such as aneurysms, arteriovenous malformations, and ischemic and hemorrhagic stroke. New laboratory-based X-ray microimagers exist that provide easy access, reliable operation, and performance previously found only in synchrotron-based instruments. Here, we reported a novel approach using such a system to detect intracerebral hemorrhage and resultant cerebrovascular pathology. Adult male C57BL/6 mice (n = 12) underwent 30 μl autologous blood injection into the right basal ganglia region. After sacrificing the animals and vascular perfusion with Microfil® MV-122 Yellow to opacify vascular and microvascular structures, the brain was post-fixed and partially hydrated for 3D imaging with a MicroXCT-400® at 30 KeV and 2-μm resolution. Tomographic reconstruction of high-resolution microimages was accomplished with Amira® software. High-quality 3D images included cerebrocortical microvessels, the circle of Willis, the sagittal sinus, transverse sinus, and other arterial and venous systems. In the ipsilateral hemisphere, there clearly were early-stage vasodilatation and later-stage neovascularization. Very high-resolution, laboratory-based, X-ray micro-CT contrast imaging can accomplish sensitive quantifications of normal and pathological small cerebrovascular changes, especially in hemorrhagic stroke and subsequent hemorrhage-induced neovascularization.

The pathophysiology and treatment of post-stroke cognitive impairment (PSCI) are not clear. Stroke triggers an inflammatory response, which might affect synapse function and cognitive status. We performed a systematic review and meta-analysis to assess whether patients with PSCI have increased levels of inflammatory markers and whether anti-inflammatory interventions in animals decrease PSCI. We systematically searched PubMed, EMBASE, and PsychInfo for studies on stroke. For human studies, we determined the standardized mean difference (SMD) on the association between PSCI and markers of inflammation. For animal studies, we determined the SMD of post-stroke cognitive outcome after an anti-inflammatory intervention. Interventions were grouped based on proposed mechanism of action. In patients, the SMD of inflammatory markers for those with versus those without PSCI was 0.46 (95% CI 0.18; 0.76; I2 = 92%), and the correlation coefficient between level of inflammation and cognitive scores was − 0.25 (95% CI − 0.34; − 0.16; I2 = 75%). In animals, the SMD of cognition for those treated with versus those without anti-inflammatory interventions was 1.43 (95% CI 1.12; 1.74; I2 = 83%). The largest effect sizes in treated animals were for complement inhibition (SMD = 1.94 (95% CI 1.50; 2.37), I2 = 51%) and fingolimod (SMD = 2.1 (95% CI 0.75; 3.47), I2 = 81%). Inflammation is increased in stroke survivors with cognitive impairment and is negatively correlated with cognitive functioning. Anti-inflammatory interventions seem to improve cognitive functioning in animals. Complement inhibition and fingolimod are promising therapies on reducing PSCI.

Some researchers have previously shown that RNAi knockdown of arginyl-tRNA synthetase (ArgRS) before or after a hypoxic injury can rescue animals from death, based on the model organism, C. elegans. However, there has been no study on the application of arginyl-tRNA synthetase knockdown in treating mammalian ischemic stroke, and its potential mechanism and effect on ischemic brain damage are still unknown. Here, we focused on the Rars gene, which encodes an arginyl-tRNA synthetase, and examined the effects of Rars knockdown in a permanent middle cerebral artery occlusion model in rats. To achieve this aim, adult male Sprague-Dawley (SD) rats were given right cerebral cortex injections of short hairpin RNA (shRNA) adenovirus (AV) particles to knock down arginyl-tRNA synthetase, and a non-targeting control (NTC) vector or phosphate-buffered solution served as the controls. After 4 days, the rats were exposed to permanent middle cerebral artery occlusion (pMCAO). Then, the right cerebral cortex level of arginyl-tRNA synthetase was examined, and the effects of the Rars knockdown were evaluated by differences in infarction volume, oxidative stress, blood-brain barrier, mitochondrial function, and glucose metabolism at 1 day and 3 days after MCAO. The injection of shRNA adenovirus particles successfully suppressed the expression of arginyl-tRNA synthetase in the cerebral cortex. We observed an improvement in oxidative stress, mitochondrial function, and glucose utilization and a reduction in brain edema compared with the non-targeting control rats with suppressed expression of arginyl-tRNA synthetase mRNA in the ipsilateral ischemic cortex of the brain. Our findings indicate that knockdown of arginyl-tRNA synthetase in the cerebral cortex exerted neuroprotective effects, which were achieved not only by the improvement of oxidative stress and glucose utilization but also by the maintenance of mitochondrial morphological integrity and the preservation of mitochondrial function. Knockdown of ArgRS administration could be a promising approach to protect ischemic stroke.