Ketogenic diet alleviates cognitive dysfunction and neuroinflammation in APP/PS1 mice via the Nrf2/HO-1 and NF-κB signaling pathways.

Alzheimer's disease is a progressive neurological disorder characterized by cognitive decline and chronic inflammation within the brain. The ketogenic diet, a widely recognized therapeutic intervention for refractory epilepsy, has recently been proposed as a potential treatment for a variety of neurological diseases, including Alzheimer's disease. However, the efficacy of ketogenic diet in treating Alzheimer's disease and the underlying mechanism remains unclear. The current investigation aimed to explore the effect of ketogenic diet on cognitive function and the underlying biological mechanisms in a mouse model of Alzheimer's disease. Male amyloid precursor protein/presenilin 1 (APP/PS1) mice were randomly assigned to either a ketogenic diet or control diet group, and received their respective diets for a duration of 3 months. The findings show that ketogenic diet administration enhanced cognitive function, attenuated amyloid plaque formation and proinflammatory cytokine levels in APP/PS1 mice, and augmented the nuclear factor-erythroid 2-p45 derived factor 2/heme oxygenase-1 signaling pathway while suppressing the nuclear factor-kappa B pathway. Collectively, these data suggest that ketogenic diet may have a therapeutic potential in treating Alzheimer's disease by ameliorating the neurotoxicity associated with Aß-induced inflammation. This study highlights the urgent need for further research into the use of ketogenic diet as a potential therapy for Alzheimer's disease.

The Association of Nocturnal Blood Pressure Patterns and Other Influencing Factors With Lacunes and Enlarged Perivascular Spaces in Hypertensive Patients.

Purpose: Nocturnal blood pressure dipping patterns have been associated with an increased risk of Cerebral Small Vessel Disease (CSVD), which has not been well-studied. This study is aimed to explore the association of dipping patterns and other factors with lacunes and enlarged perivascular spaces (EPVS) in patients with hypertension. Methods: We enrolled a total of 1,322 patients with essential hypertension in this study. Magnetic resonance imaging (MRI) scans and 24-h ambulatory blood pressure (BP) monitoring were completed. Nocturnal BP decline was calculated, and then dipping patterns were classified. Patients were classified into four groups according to the performance of lacunes and EPVS in the MRI scan for statistical analysis. Results: (1) Nocturnal BP decline showed independent negative correlation with both lacunes and EPVS while mean systolic BP (mSBP) level showed an independent positive correlation with them (P < 0.05). (2) The frequency of reverse-dippers in the control group was significantly lower than that in other groups; the frequency of non-dippers in the lacunes group and EPVS group was significantly lower than that in the control group; the frequency of extreme-dippers in the EPVS group was significantly higher than that in the mixed (lacunes with EPVS) group (P < 0.05). Conclusions: Both mSBP and dipping patterns might play an important role in developing lacunes and EPVS in patients with hypertension.

Higher Burden of Cerebral Small Vascular Disease Predicts Major Adverse Cardiac and Cerebrovascular Events and Is Related to Abnormal Blood Pressure Variability Pattern in Hypertension Patients.

Background and Objectives: The study aims to test the hypotheses that a higher burden of cerebral small vascular disease (CSVD) predicts major adverse cardiac and cerebrovascular events (MACCE) in patients with hypertension (HTN) and that abnormal blood pressure variability (BPV) pattern aggravates total CSVD burden. Methods: We retrospectively reviewed patients with HTN prospectively selected between February 2015 and February 2019 from three participating centers. Patients were included if they had HTN for over 1 year and had at least one MRI feature of CSVD. Independent predictors were found using multivariate logistic regression. Results: Among the 908 patients who finally enrolled in the study, the number of CSVD markers (OR = 1.940; 95% CI = 1.393-2.703; P < 0.001) independently predicted MACCE with acceptable predictive value (C-statistic = 0.730; 95% CI = 0.669-0.791; P < 0.001). An abnormal BPV pattern was identified as an independent risk factor for increased CSVD burden. Among them, reverse-dipper subtype demonstrated the most significant relationship (OR = 1.725; 95% CI = 1.129-2.633; P = 0.012). Conclusion: Total CSVD burden predicts an increased risk of composite MACCE independently. An abnormal BPV pattern is associated with a higher burden of CSVD.

Longitudinal white matter hyperintensity changes and cognitive decline in patients with minor stroke.

BACKGROUND AND OBJECTIVE: As reported, both minor stroke and white matter hyperintensities (WMHs) are associated with an increased risk of cognitive impairment and dementia. The underlying factors for dynamic changes in WMH volume and cognitive performances in patients with minor stroke remain poorly understood. A 2-year longitudinal study was designed to investigate the factors associated with the changes in white matter hyperintensity (WMH) volume on brain MRI and cognitive decline in patients with minor stroke. METHODS: A group of eligible patients with minor ischemic stroke was recruited in a row. At the initial and 2-year follow-up visits, all the participants underwent routine examinations, multimodal MRI, and cognitive assessment. Using a lesion prediction algorithm tool, we were able to automate the measurement of the change in WMH volume. During the 2-year follow-up, cognitive function was evaluated using Telephone Interview for Cognitive Status-Modified (TICS-m). Participants' demographic, clinical, and therapeutic data were collected and statistically analyzed. Regression analyses were used to test the relationships between risk factors and changes in WMH volume and cognitive decline. RESULTS: Finally, we followed up with 225/261 participants for 2 years, with a mean age of 65.67 ± 10.73 years (65.6% men). WMH volume was observed to be increased in 113 patients, decreased in 74 patients, and remained stable in 58 patients. Patients with WMH progression were more often had a history of hypertension (p = 0.006) and a higher CSVD burden both at baseline and follow-up visit (p < 0.05). Longitudinally, the proportion of patients taking antihypertension medications on a regular basis in the regression group was higher than in the stable group (p = 0.01). When compared to the stable group, the presence of lacunes (OR 9.931, 95% CI 1.597-61.77, p = 0.014) was a stronger predictor of progression in WMH volume. 87 subjects (38.6%) displayed incident cognitive impairment. The progression of WMH volume was a significant risk factor for cognitive decline (p < 0.001). CONCLUSIONS: The longitudinal change of WMH is dynamic. The regressive WMH volume was associated with the use of antihypertensive medications on a regular basis. The presence of lacunes at the initial visit of the study was a stronger predictor of WMH progression. The progression of WMH volume could be useful in predicting cognitive decline in patients with minor stroke.

Leukoaraiosis is associated with clinical symptom severity, poor neurological function prognosis and stroke recurrence in mild intracerebral hemorrhage: a prospective multi-center cohort study.

Leukoaraiosis (LA) results from ischemic injury in small cerebral vessels, which may be attributable to decreased vascular density, reduced cerebrovascular angiogenesis, decreased cerebral blood flow, or microcirculatory dysfunction in the brain. In this study, we enrolled 357 patients with mild intracerebral hemorrhage (ICH) from five hospitals in China and analyzed the relationships between LA and clinical symptom severity at admission, neurological function prognosis at 3 months, and 1-year stroke recurrence. Patients were divided into groups based on Fazekas scale scores: no LA (n = 83), mild LA (n = 64), moderate LA (n = 98) and severe LA (n = 112). More severe LA, larger hematoma volume, and higher blood glucose level at admission were associated with more severe neurological deficit. More severe LA, older age and larger hematoma volume were associated with worse neurological function prognosis at 3 months. In addition, moderate-to-severe LA, admission glucose and symptom-free cerebral infarction were associated with 1-year stroke recurrence. These findings suggest that LA severity may be a potential marker of individual ICH vulnerability, which can be characterized by poor tolerance to intracerebral attack or poor recovery ability after ICH. Evaluating LA severity in patients with mild ICH may help neurologists to optimize treatment protocols. This study was approved by the Ethics Committee of Ruijin Hospital Affiliated to Shanghai Jiao Tong University (approval No. 12) on March 10, 2011.

The Predictive Values of Different Small Vessel Disease Scores on Clinical Outcomes in Mild ICH Patients.

AIM: To explore the predictive values of different small vessel disease (SVD) scores on functional recoveries and the clinical cerebrovascular events in mild intracerebral hemorrhage (ICH). METHODS: In this study, we enrolled conscious and mild ICH patients without surgery and further divided them into the cerebral amyloid angiopathy (CAA)-ICH group and hypertension (HTN)-ICH group. The severity of individual SVD markers, including lacunes, cerebral microbleeds (CMBs), enlarged perivascular spaces (EPVS), white matter hyperintensity (WMH), and cortical superficial siderosis (cSS), was evaluated. The original SVD score, modified SVD score, refined SVD score, and CAA-SVD score and the total number of SVD markers were further calculated. Functional recoveries were evaluated using the modified Rankin scale. Recurrences of stroke were defined as readmission to the hospital with a definite diagnosis of stroke. RESULTS: A total of 163 ICH patients (60 CAA-ICH and 103 HTN-ICH) were included in the study. The CAA-SVD score (OR=3.429; 95% confidence interval (CI)=1.518-7.748) had the best predictive effect on functional dependence in the CAA-ICH group, among which cSS severities probably played a vital role (OR=4.665; 95% CI=1.388-15.679). The total number of SVD markers [hazard ratio (HR)=3.765; 95% CI=1.467-9.663] can better identify stroke recurrences in CAA-ICH. In HTN-ICH, while the total number of SVD markers (HR=2.136; 95% CI=1.218-3.745) also demonstrated association with recurrent stroke, this effect seems to be related with the influence of lacunes (HR=5.064; 95% CI=1.697-15.116). CONCLUSIONS: The CAA-SVD score and the total number of SVD markers might identify mild CAA-ICH patients with poor prognosis. However, it would be better to focus on lacunes rather than on the overall burden of SVD to predict recurrent strokes in HTN-ICH.

M2 microglial small extracellular vesicles reduce glial scar formation via the miR-124/STAT3 pathway after ischemic stroke in mice.

Rationale: Glial scars present a major obstacle for neuronal regeneration after stroke. Thus, approaches to promote their degradation and inhibit their formation are beneficial for stroke recovery. The interaction of microglia and astrocytes is known to be involved in glial scar formation after stroke; however, how microglia affect glial scar formation remains unclear. Methods: Mice were treated daily with M2 microglial small extracellular vesicles through tail intravenous injections from day 1 to day 7 after middle cerebral artery occlusion. Glial scar, infarct volume, neurological score were detected after ischemia. microRNA and related protein were examined in peri-infarct areas of the brain following ischemia. Results: M2 microglial small extracellular vesicles reduced glial scar formation and promoted recovery after stroke and were enriched in miR-124. Furthermore, M2 microglial small extracellular vesicle treatment decreased the expression of the astrocyte proliferation gene signal transducer and activator of transcription 3, one of the targets of miR-124, and glial fibrillary acidic protein and inhibited astrocyte proliferation both in vitro and in vivo. It also decreased Notch 1 expression and increased Sox2 expression in astrocytes, which suggested that astrocytes had transformed into neuronal progenitor cells. Finally, miR-124 knockdown in M2 microglial small extracellular vesicles blocked their effects on glial scars and stroke recovery. Conclusions: Our results showed, for the first time, that microglia regulate glial scar formation via small extracellular vesicles, indicating that M2 microglial small extracellular vesicles could represent a new therapeutic approach for stroke.

The Single Nucleotide Polymorphisms of Chromosome 9p21 and CD147 Were Relevant with the Carotid Plaque Risk in Acute Cerebral Infarction Patients Among Chinese Han Population.

This study focused on the relevance between the carotid plaque formation and the single nucleotide polymorphisms of chromosome 9p21 and CD147 in acute non-cardiogenic cerebral infarction. A total of 937 eligible patients were enrolled and categorized into carotid plaque group or non-carotid plaque group. The baseline data was analyzed, and the SNPs of chromosome 9p21 and CD147 were detected. After analyzing the results of clinic data and SNPs, we found that age, total cholesterol, low density lipoprotein, systolic blood pressure, fasting serum glucose, and NIHSS score are associated with plaque formation. Meanwhile, rs10757274, rs4977574, and rs4919862 existed statistical differences between two groups. We also analyzed linkage disequilibrium, haplotype, and inheritance models of these three SNPs, and drew the ROC curve to assess diagnostic efficiency. The results showed that mutations of SNP rs10757274 and rs4977574 in chromosome 9p21 together with SNP rs4919862 located in gene CD147 were highly relevant with the carotid plague formation in acute cerebral ischemia patients. Compared with single SNP genotype mutation, combined allele mutations on rs10757274 or rs4977574 in chromosome 9p21 with rs4919862 in CD147 resulted in much higher risks of patients, which might be associated with acute cerebral infarction happening.

P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis.

INTRODUCTION: Clearance of damaged cells and debris is beneficial for the functional recovery after ischemic brain injury. However, the specific phagocytic receptor that mediates microglial phagocytosis after ischemic stroke is unknown. AIM: To investigate whether P2Y6 receptor-mediated microglial phagocytosis is beneficial for the debris clearance and functional recovery after ischemic stroke. RESULTS: The expression of the P2Y6 receptor in microglia increased within 3 days after transient middle cerebral artery occlusion. Inhibition of microglial phagocytosis by the selective inhibitor MRS2578 enlarged the brain atrophy and edema volume after ischemic stroke, subsequently aggravated neurological function as measured by modified neurological severity scores and Grid walking test. MRS2578 treatment had no effect on the expression of IL-1α, IL-1ß, IL-6, IL-10, TNF-α, TGF-ß, and MPO after ischemic stroke. Finally, we found that the expression of myosin light chain kinase decreased after microglial phagocytosis inhibition in the ischemic mouse brain, which suggested that myosin light chain kinase was involved in P2Y6 receptor-mediated phagocytosis. CONCLUSION: Our results indicate that P2Y6 receptor-mediated microglial phagocytosis plays a beneficial role during the acute stage of ischemic stroke, which can be a therapeutic target for ischemic stroke.

Abnormal blood pressure circadian rhythms are relevant to cerebral infarction and Leukoaraiosis in hypertensive patients.

BACKGROUND: To investigate the relationships between blood pressure (BP) circadian rhythms and acute cerebral infarction (ACI), silent cerebral infarction (SCI) and the severity of leukoaraiosis in hypertensive patients. METHODS: A retrospective case-control study was performed among hypertensive patients with 24-h ambulatory blood pressure monitoring (ABPM) and cranial magnetic resonance imaging (MRI). RESULTS: A total of 1267 patients were enrolled. Lower nocturnal blood pressure (BP) decreases were observed in ACI patients than in controls (3.3% vs 8.2%, P<0.001). Reverse-dipper pattern (RD) and non-dipper pattern (ND) were found to be independent risk factors for ACI. In ACI patients, both RD and ND BP circadian rhythms were revealed to be independent risk factors for moderate-severe leukoaraiosis. In addition, in SCI patients, RD (OR = 1.7, 95% CI, 0.9-3.0; P = 0.047) or extreme-dipper pattern (ED) (OR = 2.9, 95% CI, 1.2-7.0; P = 0.015) were found to be independent risk factors for moderate-severe leukoaraiosis. Moreover, the greater the severity of leukoaraiosis was, the higher the ratio of abnormal BP circadian rhythms. CONCLUSION: RD and ND BP circadian rhythms might not only be relevant to the onset of ACI but also correlate with the severity of leukoaraiosis. Thus, when modulating BP with antihypertensive drugs, the BP circadian rhythms, and not merely the BP level, should warrant more attention.

DL-3n-Butylphthalide Improves Blood-Brain Barrier Integrity in Rat After Middle Cerebral Artery Occlusion.

Objective: DL-3n-butylphthalide (NBP) has beneficial effects in different stages of ischemic stroke. Our previous studies have demonstrated that NBP promoted angiogenesis in the perifocal region of the ischemic brain. However, the molecular mechanism of NBP for blood-brain barrier protection in acute ischemic stroke was unclear. Here, we explored the neuroprotective effects of NBP on blood-brain barrier integrity in the acute phase of ischemic stroke in a rat model. Methods: Adult male Sprague-Dawley rats (n = 82) underwent 2 h of transient middle cerebral artery occlusion and received 90 mg/kg of NBP for 3 days. Brain edema, infarct volume, surface blood flow, and neurological severity score were evaluated. Blood-brain barrier integrity was evaluated by Evans blue leakage and changes in tight junction proteins. We further examined AQP4 and eNOS expression, MMP-9 enzyme activity, and possible signaling pathways for the role of NBP after ischemic stroke. Results: NBP treatment significantly increased eNOS expression and surface blood flow in the brain, reduced brain edema and infarct volume, and improved neurological severity score compared to the control group (p < 0.05). Furthermore, NBP attenuated Evans blue and IgG leakage and increased tight junction protein expression compared to the control after 1 and 3 days of ischemic stroke (p < 0.05). Finally, NBP decreased AQP4 expression, MMP-9 enzyme activity, and increased MAPK expression during acute ischemic stroke. Conclusion: NBP protected blood-brain barrier integrity and attenuated brain injury in the acute phase of ischemic stroke by decreasing AQP4 expression and MMP-9 enzyme activity. The MAPK signaling pathway may be associated in this process.

Rapamycin Increases Collateral Circulation in Rodent Brain after Focal Ischemia as detected by Multiple Modality Dynamic Imaging.

Rationale: Brain collaterals contribute to improving ischemic stroke outcomes. However, dynamic and timely investigations of collateral blood flow and collateral restoration in whole brains of living animals have rarely been reported. Methods: Using multiple modalities of imaging, including synchrotron radiation angiography, laser speckle imaging, and micro-CT imaging, we dynamically explored collateral circulation throughout the whole brain in the rodent middle cerebral artery occlusion model. Results: We demonstrated that compared to control animals, 4 neocollaterals gradually formed between the intra- and extra-arteries in the skull base of model animals after occlusion (p<0.05). Two main collaterals were critical to the supply of blood from the posterior to the middle cerebral artery territory in the deep brain (p<0.05). Abundant small vessel and capillary anastomoses were detected on the surface of the cortex between the posterior and middle cerebral artery and between the anterior and middle cerebral artery (p<0.05). Collateral perfusion occurred immediately (≈15 min) and was maintained for up to 14 days after occlusion. Further study revealed that administration of rapamycin at 15 min after MCAO dilated the existing collateral vessels and promoted collateral perfusion. Principal conclusions: Our results provide evidence of collateral functional perfusion in the skull base, deep brain, and surface of the cortex. Rapamycin was capable of enlarging the diameter of collaterals, potentially extending the time window for ischemic stroke therapy.

Lysosome exocytosis is involved in astrocyte ATP release after oxidative stress induced by H2O2.

BACKGROUND AND PURPOSE: Studies demonstrated that oxidative damage decreased intracellular ATP level in astrocytes. However, the pathway mediated ATP level decrease is obscure. Our previous study found intracellular ATP could be released via lysosome exocytosis in astrocytes. Here, we explored whether lysosome exocytosis was involved in ATP release during oxidative stress induced by H2O2 in astrocytes. METHODS: Astrocytes were isolated from the cortex of neonatal rats. Intracellular lysosomes and calcium signals were stained in astrocytes before and after H2O2 stimulation. ATP molecules location and ATP level were detected by immunostaining and bioluminescence method, respectively. Extracellular ß-Hexosaminidase and LDH were examined by colorimetric method. RESULTS: We found that ATP located in lysosome of astrocytes. H2O2 stimulation resulted in the decrease of lysosomes staining and the increase of extracellular ATP, compared to the control (p < 0.05). At the same time, intracellular Fluo4 signals and ß-Hexosaminidase level were also increased (p < 0.05). Extracellular LDH level did not show an increase, suggesting that there is no cell membrane damage after H2O2 stimulation. Glycyl-phenylalanine 2-naphthylamide blocked lysosome exocytosis and inhibited ATP release in astrocytes after H2O2-treatment (p < 0.05). CONCLUSION: Our results indicated that H2O2 induced ATP release from intracellular to extracellular via lysosome exocytosis. The increase of intracellular Ca2+ was necessary for lysosome release under oxidative stress induced by H2O2.

Increased Inflammatory Response in Old Mice is Associated with More Severe Neuronal Injury at the Acute Stage of Ischemic Stroke.

Stroke occurs mostly in patients with advanced age. Elderly patients have a less favorable prognosis compared with young adult patients. To understand the underlying mechanisms, we tested our hypothesis that an increased inflammatory response to acute ischemic injury in old stroke mice leads to more severe brain damage and behavioral dysfunction. An ischemic stroke model was created in 2- and 12-month-old C57BL/6 mice through permanent occlusion of the left distal middle cerebral artery (dMCAO). Infarct/atrophy volumes were quantified by staining the brain sections with Cresyl Violet. Sensorimotor function was assessed using the corner test and adhesive removal test. Quantification of CD68+ cells in the peri-infarct region was performed at 1, 3 and 14 days after dMCAO. Interleukin-6 (IL-6), interleukin-1 ß (IL-1ß) and vascular endothelial growth factor (VEGF) levels in the ischemic brain tissue were measured using ELISA. Western blot was used to determine the expression levels of tight junction proteins, claudin-5 and zonula occludens (ZO)-1. Blood-brain barrier permeability was measured by Evans blue (EB) extravasation. Gelatinase B (MMP-9, type IV collagenase) was measured by gel zymography. Compared to 2-month-old mice, 12-month-old mice had more severe behavioral deficits at both the acute and chronic stages of stroke. Compared with the 2-month-old mice, 12-month-old mice had larger infarct/atrophy volumes at 1 and 14 days after dMCAO, higher levels of IL-6 and IL-1ß, higher MMP9 activity, and lower levels of claudin-5 and ZO-1 at 1 and 3 days after dMCAO. 12-month-old mice also had more CD68+ cells in the peri-infarct region at 1, 3 and 14 days after dMCAO and more EB leakage at 3 days after dMCAO. A higher inflammatory response at the acute stage of ischemic stroke in old mice is associated with more severe neuronal injury and long-term behavioral dysfunction.

Total MRI burden of cerebral vessel disease correlates with the progression in patients with acute single small subcortical strokes.

BACKGROUND: The patients of single small subcortical strokes (SS) commonly have neurological worsening with risk factors, and mechanisms remain unclear. Asymptomatic lacunes, white matter lesions, cerebral microbleeds, and enlarged perivascular spaces are MRI markers of cerebral small vessel disease (cSVD). Previous studies mostly explored the association between the neurological deterioration and presence of above markers separately. The relationship between progressive single small SS and the simultaneous presence of multiple markers of cSVD has not been fully identified. We aimed to investigate whether total burden of cSVD detected with MRI was associated with progressive small SS in this study. METHODS: Patients with single small SS (2.0 cm in diameter) were prospectively recruited during January 2016 and May 2018. Progression was defined as worsening by ≥1 point in National Institutes Health Stroke Scale (NIHSS) motor score within 72 hr from onset. The presence and burden of cSVD were determined by brain MRI, producing a score between 0 and 4. Besides, the patients' characteristics, clinical data, medical treatments during hospitalization stay were collected and statistically analyzed. Associations with progression were tested with forward stepwise regression analyses. RESULTS: Fifty-seven (35.6%) patients underwent progression. No significant difference was observed in the distribution of any single vascular risk factor and its related laboratory data among these patients. After adjustment for age, sex, NIHSS score at admission, and time from stroke to MRI in separate models, severe WMHs (OR = 4.892; 95% CI = 2.011-11.904, p = 0.016), moderate- and high-grade basal ganglia EPVS (OR = 2.970; 95% CI = 1.861-6.121, p = 0.009), and total cSVD score (OR = 3.359; 95% CI = 2.016-5.599, p = 0.010) were associated with progression. CONCLUSION: This study demonstrated that total MRI cSVD burden was independently associated with progression after single small subcortical strokes.

Thalidomide Reduces Hemorrhage of Brain Arteriovenous Malformations in a Mouse Model.

BACKGROUND AND PURPOSE: Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. Current treatments for bAVM are all associated with considerable risks. There is no safe method to prevent bAVM hemorrhage. Thalidomide reduces nose bleeding in patients with hereditary hemorrhagic telangiectasia, an inherited disorder characterized by vascular malformations. In this study, we tested whether thalidomide and its less toxic analog, lenalidomide, reduce bAVM hemorrhage using a mouse model. METHODS: bAVMs were induced through induction of brain focal activin-like kinase 1 (Alk1, an AVM causative gene) gene deletion and angiogenesis in adult Alk1-floxed mice. Thalidomide was injected intraperitoneally twice per week for 6 weeks, starting either 2 or 8 weeks after AVM induction. Lenalidomide was injected intraperitoneally daily starting 8 weeks after AVM induction for 6 weeks. Brain samples were collected at the end of the treatments for morphology, mRNA, and protein analyses. The influence of Alk1 downregulation on PDGFB (platelet-derived growth factor B) expression was also studied on cultured human brain microvascular endothelial cells. The effect of PDGFB in mural cell recruitment in bAVM was explored by injection of a PDGFB overexpressing lentiviral vector to the mouse brain. RESULTS: Thalidomide or lenalidomide treatment reduced the number of dysplastic vessels and hemorrhage and increased mural cell (vascular smooth muscle cells and pericytes) coverage in the bAVM lesion. Thalidomide reduced the burden of CD68+ cells and the expression of inflammatory cytokines in the bAVM lesions. PDGFB expression was reduced in ALK1-knockdown human brain microvascular endothelial cells and in mouse bAVM lesion. Thalidomide increased Pdgfb expression in bAVM lesion. Overexpression of PDGFB mimicked the effect of thalidomide. CONCLUSIONS: Thalidomide and lenalidomide improve mural cell coverage of bAVM vessels and reduce bAVM hemorrhage, which is likely through upregulation of Pdgfb expression.

Arterial stiffness and stroke: de-stiffening strategy, a therapeutic target for stroke.

Stroke is the second leading cause of mortality and morbidity worldwide. Early intervention is of great importance in reducing disease burden. Since the conventional risk factors cannot fully account for the pathogenesis of stroke, it is extremely important to detect useful biomarkers of the vascular disorder for appropriate intervention. Arterial stiffness, a newly recognised reliable feature of arterial structure and function, is demonstrated to be associated with stroke onset and serve as an independent predictor of stroke incidence and poststroke functional outcomes. In this review article, different measurements of arterial stiffness, especially pressure wave velocity, were discussed. We explained the association between arterial stiffness and stroke occurrence by discussing the secondary haemodynamic changes. We reviewed clinical data that support the prediction role of arterial stiffness on stroke. Despite the lack of long-term randomised double-blind controlled therapeutic trials, it is high potential to reduce stroke prevalence through a significant reduction of arterial stiffness (which is called de-stiffening therapy). Pharmacological interventions or lifestyle modification that can influence blood pressure, arterial function or structure in either the short or long term are promising de-stiffening therapies. Here, we summarised different de-stiffening strategies including antihypertension drugs, antihyperlipidaemic agents, chemicals that target arterial remodelling and exercise training. Large and well-designed clinical trials on de-stiffening strategy are needed to testify the prevention effect for stroke. Novel techniques such as modern microscopic imaging and reliable animal models would facilitate the mechanistic analyses in pathophysiology, pharmacology and therapeutics.

Soluble FLT1 Gene Therapy Alleviates Brain Arteriovenous Malformation Severity.

BACKGROUND AND PURPOSE: Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. Current therapies are associated with high morbidities. Excessive vascular endothelial growth factor has been implicated in bAVM pathophysiology. Because soluble FLT1 binds to vascular endothelial growth factor with high affinity, we tested intravenous delivery of an adeno-associated viral vector serotype-9 expressing soluble FLT1 (AAV9-sFLT1) to alleviate the bAVM phenotype. METHODS: Two mouse models were used. In model 1, bAVM was induced in R26CreER;Eng2f/2f mice through global Eng gene deletion and brain focal angiogenic stimulation; AAV2-sFLT02 (an AAV expressing a shorter form of sFLT1) was injected into the brain at the time of model induction, and AAV9-sFLT1, intravenously injected 8 weeks after. In model 2, SM22αCre;Eng2f/2f mice had a 90% occurrence of spontaneous bAVM at 5 weeks of age and 50% mortality at 6 weeks; AAV9-sFLT1 was intravenously delivered into 4- to 5-week-old mice. Tissue samples were collected 4 weeks after AAV9-sFLT1 delivery. RESULTS: AAV2-sFLT02 inhibited bAVM formation, and AAV9-sFLT1 reduced abnormal vessels in model 1 (GFP versus sFLT1: 3.66±1.58/200 vessels versus 1.98±1.29, P<0.05). AAV9-sFLT1 reduced the occurrence of bAVM (GFP versus sFLT1: 100% versus 36%) and mortality (GFP versus sFLT1: 57% [12/22 mice] versus 24% [4/19 mice], P<0.05) in model 2. Kidney and liver function did not change significantly. Minor liver inflammation was found in 56% of AAV9-sFLT1-treated model 1 mice. CONCLUSIONS: By applying a regulated mechanism to restrict sFLT1 expression to bAVM, AAV9-sFLT1 can potentially be developed into a safer therapy to reduce the bAVM severity.

Thromboxane A2 receptor antagonist SQ29548 reduces ischemic stroke-induced microglia/macrophages activation and enrichment, and ameliorates brain injury.

Thromboxane A2 receptor (TXA2R) activation is thought to be involved in thrombosis/hemostasis and inflammation responses. We have previously shown that TXA2R antagonist SQ29548 attenuates BV2 microglia activation by suppression of ERK pathway, but its effect is not tested in vivo. The present study aims to explore the role of TXA2R on microglia/macrophages activation after ischemia/reperfusion brain injury in mice. Adult male ICR mice underwent 90-min transient middle cerebral artery occlusion (tMCAO). Immediately and 24 h after reperfusion, SQ29548 was administered twice to the ipsilateral ventricle (10 µl, 2.6 µmol/ml, per dose). Cerebral infarction volume, inflammatory cytokines release and microglia/macrophages activation were measured using the cresyl violet method, quantitative polymerase chain reaction (qPCR), and immunofluorescence double staining, respectively. Expression of TXA2R was significantly increased in the ipsilateral brain tissue after ischemia/reperfusion, which was also found to co-localize with activated microglia/macrophages in the infarct area. Administration of SQ29548 inhibited microglia/macrophages activation and enrichment, including both M1 and M2 phenotypes, and attenuated ischemia-induced IL-1ß, IL-6, and TNF-α up-regulation and iNOS release. TXA2R antagonist SQ29548 inhibited ischemia-induced inflammatory response and furthermore reduced microglia/macrophages activation and ischemic/reperfusion brain injury.

Integrin ß8 Deletion Enhances Vascular Dysplasia and Hemorrhage in the Brain of Adult Alk1 Heterozygous Mice.

Brain arteriovenous malformation (bAVM), characterized by tangled dysplastic vessels, is an important cause of intracranial hemorrhage in young adults, and its pathogenesis and progression are not fully understood. Patients with haploinsufficiency of transforming growth factor-ß (TGF-ß) receptors, activin receptor-like kinase 1 (ALK1) or endoglin (ENG) have a higher incidence of bAVM than the general population. However, bAVM does not develop effectively in mice with the same haploinsufficiency. The expression of integrin ß8 subunit (ITGB8), another member in the TGF-ß superfamily, is reduced in sporadic human bAVM. Brain angiogenic stimulation results at the capillary level of vascular malformation in adult Alk1 haploinsufficient (Alk1 +/- ) mice. We hypothesized that deletion of Itgb8 enhances bAVM development in adult Alk1 +/- mice. An adenoviral vector expressing Cre recombinase (Ad-Cre) was co-injected with an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) into the brain of Alk1 +/-;Itgb8-floxed mice to induce focal Itgb8 gene deletion and angiogenesis. We showed that compared with Alk +/- mice (4.75 ± 1.38/mm2), the Alk1 +/-;Itgb8-deficient mice had more dysplastic vessels in the angiogenic foci (7.14 ± 0.68/mm2, P = 0.003). More severe hemorrhage was associated with dysplastic vessels in the brain of Itgb8-deleted Alk1 +/- , as evidenced by larger Prussian blue-positive areas (1278 ± 373 pixels/mm2 vs. Alk1 +/-  : 320 ± 104 pixels/mm2; P = 0.028). These data indicate that both Itgb8 and Alk1 are important in maintaining normal cerebral angiogenesis in response to VEGF. Itgb8 deficiency enhances the formation of dysplastic vessels and hemorrhage in Alk1 +/- mice.