Protocol to measure apoptosis-associated speck-like protein containing a CARD specks in human cerebrospinal fluid via imaging flow cytometry.

Apoptosis-associated speck-like protein containing a c-terminal caspase activation and recruitment domain (ASC) specks are elevated in the cerebrospinal fluid (CSF) of Alzheimer's disease and related dementias (AD/ADRDs) patients. Here, we present a flow cytometry protocol to quantify ASC specks. We describe steps for fluorescently labeling ASC specks using antibody technology, visualizing with imaging flow cytometry, and gating based on physical characteristics. CSF ASC specks levels positively correlate with phosphorylated tau (Thr181) and negatively correlate with amyloid ß ratio (42/40), thus serving as a neuroinflammatory biomarker for diagnosing AD/ADRDs. For complete details on the use and execution of this protocol, please refer to Jiang et al.1.

Progressive Disruption of Sphingosine-1-Phosphate Receptor 1 Correlates with Blood-Brain Barrier Leakage in A Rat Model of Chronic Hypoxic Hypoperfusion.

Endothelial dysfunction and blood-brain barrier (BBB) leakage have been suggested as a fundamental role in the development of cerebral small vessel disease (SVD) pathology. However, the molecular and cellular mechanisms that link cerebral hypoxic hypoperfusion and BBB disruption remain elusive. Sphingosine-1-phosphate (S1P) regulates the BBB integrity by binding to its receptor isoform 1 (S1PR1) on endothelial cells. This study tested the hypothesis that hypoxic hypoperfusion triggers capillary endothelial S1PR1 disruption, which compromises BBB integrity and leads to SVD-related neuropathological changes, using a chronic hypoxic hypoperfusion model with BBB dysfunction. Spontaneously hypertensive rat stroke-prone underwent unilateral carotid artery occlusion (UCAO) followed by a Japanese permissive diet (JPD) for up to 9 weeks. Selective S1PR1 agonist SEW2871 was used to activate S1PR1. Significant progressive reduction of S1PR1 was detected in rat brains from 4 to 9 weeks following UCAO/JPD onset, which was also detected in cerebral vasculature in human SVD. S1PR1 activation by SEW2871 significantly reduced lesions in both white and grey matter and ameliorated cerebral blood flow. SEW2871 reversed the loss of endothelial S1PR1 and tight junction proteins, and significantly attenuated UCAO/JPD induced accumulation of neuronal phosphorylated tau. This protective role of SEW2871 is associated with promotion of Akt phosphorylation and inhibition of S1PR2/Erk1/2 activation. Our data suggest S1PR1 signalling as a potential molecular mechanistic basis that links hypoxic hypoperfusion with BBB damage in the neuropathological cascades in SVD. The reversal of BBB disruption through pharmacological intervention of S1PR1 signalling likely reveals a novel therapeutic target for SVD.

Blood-brain barrier disruption measured by albumin index correlates with inflammatory fluid biomarkers.

Blood-brain barrier (BBB) permeability can be measured by the ratio of albumin in cerebrospinal fluid (CSF) and blood and by dynamic contrast-enhanced MRI (DCEMRI). Albumin is a large molecule measured in CSF and blood to form the albumin index (Qalb), which is a global measure of BBB permeability, while the smaller Gadolinium molecule measures regional transfer (Ktrans); few studies have directly compared them in the same patients. We used both methods as part of a study of mechanisms of white matter injury in patients with different forms of dementia. In addition, we also measured biomarkers for inflammation, including proteases, angiogenic growth factors, and cytokines, and correlated them with the BBB results. We found that there was no correlation between Qalb and Ktrans. The Qalb was associated with the matrix metalloproteinases (MMP-2, MMP-3, and MMP-10), the angiogenic factors (VEGF-C and PlGF), and the cytokines (IL-6, IL-8 and TNF-α). On the other hand, Ktrans was associated with the diffusion measures, mean free water and PSMD, which indicate white matter injury. Our results show that the Qalb and Ktrans measure different aspects of BBB permeability, with albumin being a measure of inflammatory BBB opening and Ktrans indicating white matter injury.

Inflammatory Biomarkers Aid in Diagnosis of Dementia.

Dual pathology of Alzheimer's disease (AD) and vascular cognitive impairment and dementia (VCID) commonly are found together at autopsy, but mixed dementia (MX) is difficult to diagnose during life. Biological criteria to diagnose AD have been defined, but are not available for vascular disease. We used the biological criteria for AD and white matter injury based on MRI to diagnose MX. Then we measured multiple biomarkers in CSF and blood with multiplex biomarker kits for proteases, angiogenic factors, and cytokines to explore pathophysiology in each group. Finally, we used machine learning with the Random forest algorithm to select the biomarkers of maximal importance; that analysis identified three proteases, matrix metalloproteinase-10 (MMP-10), MMP-3 and MMP-1; three angiogenic factors, VEGF-C, Tie-2 and PLGF, and three cytokines interleukin-2 (IL-2), IL-6, IL-13. To confirm the clinical importance of the variables, we showed that they correlated with results of neuropsychological testing.

MMP-9 inhibitors impair learning in spontaneously hypertensive rats.

Vascular cognitive impairment dementia (VCID) is a major cause of cognitive loss in the elderly. Matrix metalloproteinases (MMPs) are a family of proteases involved in remodeling the extracellular matrix in development, injury and repair. Blood-brain barrier (BBB) disruption due to inflammation mediated by MMPs is a mechanism of white matter injury. Currently there are no treatments besides the control of vascular risk factors. We tested two MMP-9 inhibitors that improved outcome in acute stroke: DP-460 and SB-3CT. We hypothesized that these inhibitors would have a beneficial effect in chronic stroke by reducing edema in white matter and improving behavioral outcomes. Spontaneously hypertensive stroke-prone rats (SHRSPs) with unilateral carotid artery occlusion (UCAO) fed a Japanese Permissive Diet (JPD) were used as a model of VCID. JPD was begun in the 12th week of life. Rats were treated with DP-460 (500 mg/kg) for 4 weeks, or SB-3CT (10 mg/kg) for 8 weeks, beginning at the UCAO/JPD onset. Rats treated with a dextrose or DMSO solution served as vehicle controls. Naïve SHRSPs on a standard diet served as sham control. Magnetic resonance imaging (MRI) analyses of the corpus callosum, external capsule, hippocampus and Morris water maze behavioral tests were conducted. We found an increase in body weight (p = 0.004) and blood pressure (p = 0.007) at 15 weeks with the DP-460 drug. SB-3CT increased body weight at 14 weeks (p = 0.015) and had significant but variable effects on blood pressure. Neither drug affected imaging parameters. Behavioral studies showed an impaired ability to learn with DP-460 (p<0.001) and no effect on learning with SB-3CT. Unchanged MMP-9 levels were detected in DP-460-treated rats via gel zymography. Our findings suggest that MMPs are not major factors in white matter damage in the SHRSP model of VCID and that drugs that are relatively selective for MMP-9 can interfere with learning.