Current Neurovascular Research - Volume 15, Issue 1, 2018

Background: Mutant transactive response DNA-binding protein (TDP-43) is closely correlated to the inherited form of amyotrophic lateral sclerosis (ALS). TDP-43 transgenic rats can reproduce the core phenotype of ALS and constitutive expression of TDP-43 caused postnatal death. Objective: The study aimed to understand whether neurologic deficiency caused by mutant TDP- 43 is dependent on its temporal expression. Method: Transgenic rats were established that express mutant human TDP-43 (M337V substitution) in neurons, then a Tet-off system was used to regulate its expression. Results: TDP-43 mutant transgenic rats developed significant weakness after the transgene was activated. Rats with expression of mutant TDP-43 at 30 days showed a more aggressive phenotype. More severe pathological changes in neurogenic atrophy were observed in these rats. Conclusion: Temporal expression of mutant TDP-43 in neurons promoted serious phenotype in rats. The dysfunction of TDP-43 had a profound impact on the development of motor neurons and skeletal muscles.

Background: The prognostic value of White Blood Cell (WBC) counts and C-reactive Protein (CRP) in clinical outcomes of Acute Ischemic Stroke (AIS) patients after Intravenous Thrombolysis (IVT) remains unknown. We investigated the association of WBC counts and CRP with 3-month functional outcomes and all-cause mortality in AIS patients. Methods: 447 AIS patients treated with IVT between May 2010 and May 2017 were enrolled. WBC counts and CRP were measured within 24 hours after IVT. The main outcomes included poor functional outcomes (modified Rankin score ≥3) at 3 months and 3-month all-cause mortality. Results: High WBC counts were associated with poor functional outcomes (adjusted OR (odds ratio) 4.48; 95% CI (confidence interval) 2.00-10.03; P-trend<0.001) and with all-cause mortality (adjusted HR (hazard ratio) 2.19; 95% CI 1.07-4.49; P-trend=0.018). In addition, high CRP levels were associated with poor functional outcomes (adjusted OR 4.95; 95% CI 1.39-17.65; Ptrend= 0.002). However, no significant association between high CRP levels and all-cause mortality was observed (adjusted HR 2.61; 95% CI 0.80-8.47; P-trend=0.138). Conclusion: Our analysis indicated that elevated WBC counts and CRP levels after IVT can independently predict poor outcome among AIS patients.

Background: We hypothesized that the inflammatory markers (IM) could be the independent predictors of Carotid Stenosis Progression (CSP) after Carotid Artery Stenting (CAS). Methods: Between 2010 and 2012, 122 patients undergoing cervicocranial revascularization in our hospital were prospectively recruited. Patients undergoing revascularizations other than CAS were excluded. Carotid duplex ultrasonography was performed before and at 1 week, 6 months (6M), 1 year, and 2 years after CAS. IM levels were recorded before CAS and were followed up immediately and 6M after CAS. The data was analyzed retrospectively. Patients were categorized into the Progression Group (PG) and Nonprogression Group (NPG) based on the presence or absence of CSP, including in-stent restenosis (ISR) and worsening contralateral carotid stenosis. Receiver operating characteristic and multivariate logistic regression analyses were conducted. Results: In Total, 77 patients were enrolled. The frequency of CSP was 24.7% (ISR: 14.3%; worsening contralateral carotid stenosis: 14.3%). Compared with the NPG, the PG had lower E-selectin levels before CAS [PG vs. NPG, 47.90 (42.80, 64.90) vs. 68.25 (52.08, 92.30); p = .01] and a nonreduced E-selectin levels at 6M after CAS [PG vs. NPG, 7.65 (-2.45, 25.75) vs. -16.10 (-33.45, 1.65); p = .002]. The E-selectin changes between 6M after and before CAS had highest predictive accuracy on CSP (area under the curve = 0.74, p = .002). The optimal cut-off level was a 2.95 ng/mL decrease and the adjusted odds ratio for CSP was 10.16 (p = .001). Conclusion: The E-selectin changes between 6M after and before CAS are independent predictors of CSP.

Background: C-X-C motif chemokine ligand 12 (CXCL12) may play an important role in the development of Intracranial Aneurysm (IA). Objective: The goal of this study was to explore the association between CXCL12 rs1746048 genotypes and circulating lipid concentrations along with the risk of IA. Methods: A total of 256 IA patients and 361 healthy volunteers were included in the case-control study. The genotypes of CXCL12 rs1746048 were detected by Melting Temperature shift (Tmshift) Polymerase Chain Reaction (PCR). Results: Significant higher levels were seen in Total Cholesterol (TC) (padjusted < 0.001), Highdensity Lipoprotein Cholesterol (HDL-C) (padjusted < 0.001), Low-density Lipoprotein Cholesterol (LDL-C) (padjusted < 0.001), Apolipoprotein A-I (ApoA-I) (padjusted = 0.040), and Apolipoprotein B (ApoB) (padjusted < 0.001) in IAs compared with controls. CXCL12 rs1746048 T allele frequency showed significant association with the risk of IA in the female group aged 65 or above (p = 0.019, Odds Ratio (OR) = 2.15, 95% confidence interval (95%CI) = 1.13 – 4.11, power = 64.8%). Moreover, CXCL12 rs1746048 was likely to be a risk variant of IA under the recessive model in females older than 65 years. (p = 0.030, OR = 3.77, 95%CI = 1.08 – 13.12, power = 81.8%). Additionally, we also found that the levels of LDL-C were significantly different among three genotypes (CC vs. CT vs. TT = 2.75±0.73 vs. 3.03±0.89 vs. 2.82±0.72, p = 0.035) in IA patients. Conclusion: Our results suggest that CXCL12 rs1746048 is significantly associated with IA risk in Han Chinese females aged 65 years and older. Additionally, the genotypes of CXCL12 rs1746048 may affect the LDL-C concentrations in IA patients.

Background: Cerebral collateral circulation is a network of arterial anastomotic channels capable of providing supplementary perfusion to brain regions in response to ischemic insults. Arterial stiffness could negatively affect collateral circulation development, by means of its effects on the structural intracerebral vasculature. Objective: The aim of our study is to investigate a possible link between arterial stiffness and presence of collateral circulation in patients with acute ischemic stroke. Methods: 113 patients (age: 74±12 years) with acute anterior ischemic stroke underwent neuroimaging examination and 24-hour blood pressure monitoring. Arterial Stiffness Index (ASI) and Pulse Pressure (PP) were assumed as surrogate measures of arterial stiffness. Collateral circulation was evaluated by means of the collateral grading system that was scored on a scale of 0–3. Results: According to TOAST classification, etiology of ischemic stroke was the following: Large-Artery Atherosclerosis (LAA)(n:41), Cardioembolism (CE)(n:60), Undetermined Etiology (UE)(n:12). Logistic regression analysis showed that good predictors of poor collaterals were ASI (OR 2.78 for 0.1, 95% CI:1.19–6.50, p=0.01) and PP (OR 1.81 for 10 mmHg, 95% CI:1.01–3.22, p=0.04) in stroke from LAA. Conclusion: Our results suggest that, in patients with ischemic stroke from LAA, arterial stiffness may contribute to the impairment of collateral circulation and, therefore, it could reduce the beneficial effects of acute treatments.

Objectives: To investigate the association of kidney function with the total burden of Cerebral Small Vessel Disease (CSVD) in primary Intracerebral Hemorrhage (pICH). Methods: Cerebral magnetic resonance imagings of consecutively enrolled pICH patients were reviewed to assess for lacunes, White Matter Hyperintensity (WMH), Cerebral Microbleeds (CMBs) and Enlarged Perivascular Spaces (EPVS). Minor refinements to the CSVD score, namely modified CSVD score 1 and 2, were made by incorporating different weightings of CSVD markers. Kidney function was assessed using the estimated Glomerular Filtration Rate (eGFR). We used ordinal regression analysis to assess the association of kidney function with the CSVD score and modified scores. Results: In the 108 patients included, the presence of lacunes, CMBs, MWH and basal ganglia EPVS>10 was 27.8%, 67.6%, 47.2% and 35.2%, respectively. In multivariable ordinal regression, a decreasing eGFR value was associated with an increased CSVD score [Odds Ratio (OR) 0.978, 95% Confidence Interval (CI) 0.962 to 0.995, P=0.013], modified CSVD score 1 (OR 0.973, 95% CI 0.957 to 0.990, P=0.002) and 2 (OR 0.969, 95% CI 0.953 to 0.986, P<0.001). The link between eGFR and the total burden of CSVD was significant in strictly deep pICH (P=0.011 for CSVD score; P=0.001 for modified score 1 and 2), but not strictly lobar pICH in subgroup analysis. Conclusions: Low eGFR is associated with a high burden of CSVD in patients with deep pICH, but not lobar pICH. Future studies are warranted to assess whether low eGFR is a potential therapeutic target for preventing the progress of CSVD burden for deep pICH.

Introduction: We previously demonstrated that microRNAs (miRNA) play an important role in Hypothermic Circulatory Arrest (DHCA)-associated neural injury. However, the specific role of miRNAs in the pathogenesis of DHCA-induced neuron death is still unclear. Material and Methods: Thus, in the present study, we investigated miR-29 expression and roles in neuronal HT-22 cells with Oxygen-glucose Deprivation/reoxygenation (OGD/R). In this study, the model of OGD/R was established using an airtight culture container and the anaeropack. Measurement of Reactive Oxygen Species (ROS) production and Mitochondrial Membrane Potential (MMP) was done using the probes of JC-1 and H2DCFDA. The microRNA (miRNA) profile in hippocampal neurons from rat model of DHCA was determined by miRNA deep sequencing. Results: We found that the expression of the miR-29 family (miR-29a/b/c) was significantly reduced in model of DHCA and OGD/R. Overexpression of the miR-29 family inhibited the OGD/R-induced elevation of ROS and reduction of MMP in HT-22 cells. In addition, administration of the miR-29 family suppressed proteins of Keap1, Bax and PUMA and increased Nrf2 expression. We further demonstrated that the miR-29 family targeted the PUMA by luciferase reporter assay and Western blot analysis. Conclusion: In conclusion, our data suggest that by targeting a pro-apoptotic BCL2 family member PUMA, the miR-29 family might emerge as a strategy for protection against DHCA-mediated neural cell injury.

Background: P-selectin is activated early after stroke, followed by a rapid decline. This time course can be used to generate important information on stroke onset. The latter is crucial for therapeutic decision-making of wake-up strokes (i.e. thrombolysis or not). Here, we evaluated the specific p-selectin inhibitor fucoidan labeled with gallium-68 (68Ga-Fucoidan) as an imaging biomarker for assessing p-selectin activation in acute ischemic stroke using Positron Emission Tomography (PET). Methods: 68Ga-Fucoidan was investigated in rats brain at 2-5 h (n=16), and additionally at 24-26 h (n=9) and 48 h (n=3) after induction of photothrombic stroke or in sham-operated animals (n=6). Correlation of cerebral 68Ga-Fucoidan uptake with p-selectin expression was determined by exposing freshly cut brain cryosections to autoradiography and immunostaining using specific antibodies against p-selectin. Results: PET scans showed an increased accumulation of 68Ga-Fucoidan in the histologically proven ischemic stroke, as compared to the corresponding contralateral hemisphere in all except one animal. The median ratio between the uptake in the ischemic lesion and the contralateral region was 1.95 (1.45-2.41) at 2-5 h, 1.38 (1.05-1.89) at 24-26 h, and 1.09 (0.81-1.38) at 48 h after stroke, compared to 1.22 (0.99-1.49) for sham-operated animals. In the ex vivo autoradiography, 68Ga-Fucoidan accumulation co-localized with p-selectin as assessed by immunostaining. Control animals and those scanned at 24-26 h and 48 h after stroke exhibited no elevated 68Ga-Fucoidan uptake in either hemisphere. Conclusion: PET imaging using 68Ga-Fucoidan represents a valuable tool for assessing p-selectin activation in vivo discriminating ischemic stroke early after stroke onset.

Objective: This study aims to explore the function of blood microRNA-15a (miR-15a) in the pathogenesis of acute cerebral ischemia (AIS). Methods: Blood samples were collected from healthy control and AIS patients within 72 h after onset. A model of ischemia in human umbilical vein endothelial cells (HUVECs) was established through oxygen and glucose deprivation (OGD). MiR-15a in patients and in cells was measured using real-time quantitative polymerase chain reaction (qPCR). The predicted target of miR-15a such as interleukin-6 (IL-6) and insulin-like growth factors-1 (IGF-1) in plasma was detected by enzyme-linked immunosorbent assay (ELISA). The relations between blood miR-15a and stroke severity, stroke etiology, infarct location, stroke prognosis, predicted targets were analyzed by Statistical Product and Service Solutions (SPSS) software respectively. Results: Higher miR-15a levels were found in AIS patients and ischemic cells within 72 h, compared to control (p < 0.05). Receiver Operating Characteristic (ROC) analysis showed that blood miR-15a predicted stroke onset with 98.67% specificity. Blood miR-15a had a negative correlation with National Institutes of Health Stroke Scale (NIHSS) scores (r = -0.3695, p < 0.01). The AIS patients with increased miR-15a levels had a better prognosis. MiR-15a was up-regulated in anterior circulation infarction and small-artery atherosclerosis stroke. Plasma levels of IL-6 and IGF-1 were associated with blood miR-15a (r = -0.6051, 0.3231, p < 0.05, respectively). Conclusion: Blood miR-15a associates with IL-6, IGF-1 and acute cerebral ischemia. It could serve as a potential diagnostic biomarker and therapeutic target for stroke.

Background: Delayed cerebral vasospasm (dCVS) following aneurysmal subarachnoid hemorrhage (aSAH) is (next to possible aneurysm rebleeding, cortical spreading depression and early brain injury) one of the main factors contributing to poor overall patient outcome. Since decades, intensive research has been ongoing with the goal of improving our understanding of the pathophysiological principles underlying dCVS. Endothelin-1 (ET-1) and prostaglandin F2 alpha (PGF2a) seem to play a major role during dCVS. The synthesis of ET-1 is enhanced after subarachnoid hemorrhage (SAH) to mediate a long-lasting vasoconstriction, and PGF2a contributes to cerebral inflammation and vasoconstriction. Under physiological conditions, levosimendan (LS) demonstrates an antagonistic effect on PGF2a-induced cerebral vasoconstriction. Thus, the intention of the present study was to analyze potentially beneficial interactions in a pathophysiological situation. Methods: A modified double hemorrhage model was used. Functional interactions between the calcium-sensitizing action of LS and the vasoconstrictive properties of PGF2a were investigated. Results: After pre-incubation with LS, followed by application of PGF2a, a significant decrease in maximum contraction (Emax) for sham-operated animals was found (Emax 28% with LS, Emax 56% without LS). Using the same setting after SAH, the vessel segments did not reach a statistically significant contraction (but similar like the sham-operated vessels), neither for Emax nor pD2 (-log10EC50) nor EC50 (i.e., the concentration at which half of the maximal effect occurs). LS series in sham animals were performed by pre-incubation with PGF2a. The resultant Emax showed a statistically strong significance concerning a higher vasorelaxation compared with a solvent control group. Vessel segment relaxation was significantly stronger in the same experimental setup after SAH. Conclusion: Under physiological and pathophysiological circumstances, LS reduced and dosedependently reversed PGF2a-induced vasoconstriction. These results can be applied to further developing methods to antagonize dCVS after aSAH.

Background: With the global increase in lifespan expectancy, neurodegenerative disorders continue to affect an ever-increasing number of individuals throughout the world. New treatment strategies for neurodegenerative diseases are desperately required given the lack of current treatment modalities. Methods: Here, we examine novel strategies for neurodegenerative disorders that include circadian clock genes, non-coding Ribonucleic Acids (RNAs), and the mammalian forkhead transcription factors of the O class (FoxOs). Results: Circadian clock genes, non-coding RNAs, and FoxOs offer exciting prospects to potentially limit or remove the significant disability and death associated with neurodegenerative disorders. Each of these pathways has an intimate relationship with the programmed death pathways of autophagy and apoptosis and share a common link to the silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae) (SIRT1) and the mechanistic target of rapamycin (mTOR). Circadian clock genes are necessary to modulate autophagy, limit cognitive loss, and prevent neuronal injury. Non-coding RNAs can control neuronal stem cell development and neuronal differentiation and offer protection against vascular disease such as atherosclerosis. FoxOs provide exciting prospects to block neuronal apoptotic death and to activate pathways of autophagy to remove toxic accumulations in neurons that can lead to neurodegenerative disorders. Conclusion: Continued work with circadian clock genes, non-coding RNAs, and FoxOs can offer new prospects and hope for the development of vital strategies for the treatment of neurodegenerative diseases. These innovative investigative avenues have the potential to significantly limit disability and death from these devastating disorders.