Dynamic Volumetric Imaging of Mouse Cerebral Blood Vessels In Vivo with an Ultralong Anti-Diffracting Beam.

Volumetric imaging of a mouse brain in vivo with one-photon and two-photon ultralong anti-diffracting (UAD) beam illumination was performed. The three-dimensional (3D) structure of blood vessels in the mouse brain were mapped to a two-dimensional (2D) image. The speed of volumetric imaging was significantly improved due to the long focal length of the UAD beam. Comparing one-photon and two-photon UAD beam volumetric imaging, we found that the imaging depth of two-photon volumetric imaging (80 µm) is better than that of one-photon volumetric imaging (60 µm), and the signal-to-background ratio (SBR) of two-photon volumetric imaging is two times that of one-photon volumetric imaging. Therefore, we used two-photon UAD volumetric imaging to perform dynamic volumetric imaging of mouse brain blood vessels in vivo, and obtained the blood flow velocity.

Subtle vascular and astrocytic changes in the brain of coronavirus disease 2019 (COVID-19) patients.

BACKGROUND AND PURPOSE: In the central nervous system, a multitude of changes have been described associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, such as microglial activation, perivascular lymphocyte cuffing, hypoxic-ischaemic changes, microthrombosis, infarcts or haemorrhages. It was sought here to assess the vascular basement membranes (vBMs) and surrounding perivascular astrocytes for any morphological changes in acute respiratory syndrome (coronavirus disease 2019, COVID-19) patients. METHODS: The light microscopy morphology of the vBMs and perivascular astrocytes from brains of 14 patients with confirmed SARS-CoV-2 infection was analysed and compared to four control patients utilizing fluorescent immunohistochemistry for collagen IV and astrocytes (GFAP), endothelia (CD31), tight junction 1 (TJ1) adhesion protein, as well as the aquaporin 4 (AQP4) water channel. On 2D and 3D deconvoluted images from the cortex and white matter, vessel densities, diameters, degree of gliosis, collagen IV/GFAP and GFAP/AQP4 colocalizations were calculated, as well as the fractal dimension of astrocytes and vBMs viewed in tangential planes. RESULTS: Fractal dimension analysis of the GFAP-stained astrocytes revealed lower branching complexities and decreased GFAP/collagen IV colocalization for COVID-19 patients. Interestingly, vBMs showed significantly increased irregularities (fractal dimension values) compared to controls. Vessel diameters were increased in COVID-19 cases, especially for the white matter, TJ1 protein decreased its colocalization with the endothelia, and AQP4 reduced its co-expression in astrocytes. CONCLUSIONS: Our data on the irregularity of the basement membranes, loss of endothelial tight junction, reduction of the astrocyte end-feet and decrease of AQP4 suggest subtle morphological changes of the blood-brain barrier in COVID-19 brains that could be linked with indirect inflammatory signalling or hypoxia/hypercapnia.

Neurodevelopmental toxicity of pyrazinamide to larval zebrafish and the restoration after intoxication withdrawal.

To investigate the neurotoxicity of pyrazinamide (PZA) to larval zebrafish, the PZA effects were assessed followed by its mechanism being explored. Same as isoniazid (INH), this compound is a first-line anti-tuberculosis drug and is suggested to be a risk that inducing nerve injury with long-term intoxication. Our findings indicated that zebrafish larvae obtained severe nerve damage secondary to constant immersion in various concentrations of PZA (i.e., 0.5, 1.0, and 1.5 mM) from 4 hpf (hours post fertilization) onwards until 120 hpf. The damage presented as dramatically decrease of locomotor capacity and dopaminergic neuron (DAN)-rich region length in addition to defect of brain blood vessels (BBVs). Moreover, PZA-administrated zebrafish showed a decreased dopamine (DA) level and downregulated expression of neurodevelopment-related genes, such as shha, mbp, neurog1, and gfap. However, secondary to 48-h restoration in fish medium (i.e., at 168 hpf), the neurotoxicity described above was prominently ameliorated. The results showed that PZA at the concentrations we tested was notably neurotoxic to larval zebrafish, and this nerve injury was restorable after PZA withdrawing. Therefore, this finding will probably provide a reference for clinical medication.

Muramyl dipeptide-mediated immunomodulation on monocyte subsets exerts therapeutic effects in a mouse model of Alzheimer's disease.

BACKGROUND: Muramyl dipeptide (MDP) is a component derived from minimal peptidoglycan motif from bacteria, and it is a ligand for the NOD2 receptor. Peripheral administration of MDP converts Ly6Chigh into Ly6Clow monocytes. Previously, we have shown that Ly6Clow monocytes play crucial roles in the pathology of a mouse model of Alzheimer's disease (AD). However, medications with mild immunomodulatory effects that solely target specific monocyte subsets, without triggering microglial activation, are rare. METHODS: Three months old APPswe/PS1 transgenic male mice and age-matched C57BL/6 J mice were used for high frequency (2 times/week) over 6 months and low frequency (once a week) over 3 months of intraperitoneally MDP (10 mg/kg) administrations. Flow cytometry analysis of monocyte subsets in blood, and behavioral and postmortem analyses were performed. RESULTS: Memory tests showed mild to a strong improvement in memory function, increased expression levels of postsynaptic density protein 95 (PSD95), and low-density lipoprotein receptor-related protein 1 (LRP1), which are involved in synaptic plasticity and amyloid-beta (Aß) elimination, respectively. In addition, we found monocyte chemoattractant protein-1(MCP-1) levels significantly increased, whereas intercellular adhesion molecule-1(ICAM-1) significantly decreased, and microglial marker (Iba1) did not change in the treatment group compared to the control. In parallel, we discovered elevated cyclooxygenase-2 (COX2) expression levels in the treated group, which might be a positive factor for synaptic activity. CONCLUSIONS: Our results demonstrate that MDP is beneficial in both the early phase and, to some extent, later phases of the pathology in the mouse model of AD. These data open the way for potential MDP-based medications for AD.

Circulating Ouabain Modulates Expression of Claudins in Rat Intestine and Cerebral Blood Vessels.

The ability of exogenous low ouabain concentrations to affect claudin expression and therefore epithelial barrier properties was demonstrated previously in cultured cell studies. We hypothesized that chronic elevation of circulating ouabain in vivo can affect the expression of claudins and tight junction permeability in different tissues. We tested this hypothesis in rats intraperitoneally injected with ouabain (1 µg/kg) for 4 days. Rat jejunum, colon and brain frontal lobes, which are variable in the expressed claudins and tight junction permeability, were examined. Moreover, the porcine jejunum cell line IPEC-J2 was studied. In IPEC-J2-cells, ouabain (10 nM, 19 days of incubation) stimulated epithelial barrier formation, increased transepithelial resistance and the level of cSrc-kinase activation by phosphorylation, accompanied with an increased expression of claudin-1, -5 and down-regulation of claudin-12; the expression of claudin-3, -4, -8 and tricellulin was not changed. In the jejunum, chronic ouabain increased the expression of claudin-1, -3 and -5 without an effect on claudin-2 and -4 expression. In the colon, only down-regulation of claudin-3 was observed. Chronic ouabain protected the intestine transepithelial resistance against functional injury induced by lipopolysaccharide treatment or by modeled acute microgravity; this regulation was most pronounced in the jejunum. Claudin-1 was also up-regulated in cerebral blood vessels. This was associated with reduction of claudin-3 expression while the expression of claudin-5 and occludin was not affected. Altogether, our results confirm that circulating ouabain can functionally and tissue-specifically affect barrier properties of epithelial and endothelial tissues via Na,K-ATPase-mediated modulation of claudins expression.

Assessment of the Effects of Bisphenol A on Dopamine Synthesis and Blood Vessels in the Goldfish Brain.

Bisphenol A (BPA) is an abundant contaminant found in aquatic environments. While a large number of toxicological studies have investigated the effects of BPA, the potential effects of BPA exposure on fish brain have rarely been studied. To understand how BPA impacts goldfish brains, we performed a transcriptome analysis of goldfish brains that had been exposed to 50 µg L-1 and 0 µg L-1 BPA for 30 days. In the analysis of unigene expression profiles, 327 unigenes were found to be upregulated and 153 unigenes were found to be downregulated in the BPA exposure group compared to the control group. Dopaminergic signaling pathway-related genes were significantly downregulated in the BPA exposure group. Furthermore, we found that serum dopamine concentrations decreased and TUNEL (terminal deoxynucleotidyl transferase 2-deoxyuridine, 5-triphosphate nick end labeling) staining was present in dopamine neurons enriched regions in the brain after BPA exposure, suggesting that BPA may disrupt dopaminergic processes. A KEGG analysis revealed that genes involved in the fluid shear stress and atherosclerosis pathway were highly significantly enriched. In addition, the qRT-PCR results for fluid shear stress and atherosclerosis pathway-related genes and the vascular histology of the brain showed that BPA exposure could damage blood vessels and induce brain atherosclerosis. The results of this work provide insights into the biological effects of BPA on dopamine synthesis and blood vessels in goldfish brain and could lay a foundation for future BPA neurotoxicity studies.

Mobilizing endogenous neuroprotection: the mechanism of the protective effect of acupuncture on the brain after stroke.

Given its high morbidity, disability, and mortality rates, ischemic stroke (IS) is a severe disease posing a substantial public health threat. Although early thrombolytic therapy is effective in IS treatment, the limited time frame for its administration presents a formidable challenge. Upon occurrence, IS triggers an ischemic cascade response, inducing the brain to generate endogenous protective mechanisms against excitotoxicity and inflammation, among other pathological processes. Stroke patients often experience limited recovery stages. As a result, activating their innate self-protective capacity [endogenous brain protection (EBP)] is essential for neurological function recovery. Acupuncture has exhibited clinical efficacy in cerebral ischemic stroke (CIS) treatment by promoting the human body's self-preservation and "Zheng Qi" (a term in traditional Chinese medicine (TCM) describing positive capabilities such as self-immunity, self-recovery, and disease prevention). According to research, acupuncture can modulate astrocyte activity, decrease oxidative stress (OS), and protect neurons by inhibiting excitotoxicity, inflammation, and apoptosis via activating endogenous protective mechanisms within the brain. Furthermore, acupuncture was found to modulate microglia transformation, thereby reducing inflammation and autoimmune responses, as well as promoting blood flow restoration by regulating the vasculature or the blood-brain barrier (BBB). However, the precise mechanism underlying these processes remains unclear. Consequently, this review aims to shed light on the potential acupuncture-induced endogenous neuroprotective mechanisms by critically examining experimental evidence on the preventive and therapeutic effects exerted by acupuncture on CIS. This review offers a theoretical foundation for acupuncture-based stroke treatment.

Super resolution imaging reconstruction reveals that gold standard methods may not correctly conclude neural/brain functional recovery.

The status of cerebral perfusion and its restoration level play a vital role in the prognosis and clinical decision making of many neurosurgical diseases. As such, gold standard methods including CT, MR and ICP monitoring, which can indicate and measure cerebral perfusion and restoration, have been widely adopted to evaluate whether or not a patient has recovered from neurofunctional disabilities. This robust combination of methods, however, is confronted with a growing number of contradictions in recent years due to its inability to measure the status of cerebral reperfusion in microvasculature level, even though this has been shown to determine neurofunctional restoration as well or even better. To this date, nevertheless, we have very limited imaging methods that could evaluate human cerebral microperfusion both safely and accurately under most neurosurgical conditions. We herein report a new method of acquiring a patient's cerebral microperfusion status noninvasively which could display the precise distribution of microvasculature in deep cerebral regions with a resolution of ∼30 µm, using everyday bed-side ultrasonography combined with a computerized super-resolution reconstruction algorithm. Using this imaging modality, we found that a patient's cerebral microperfusion might not be improved by some routine administrations even though the gold standard method had yielded the opposite conclusions. Our imaging modality retains the safe, portable feature of ordinary ultrasonography while possesses the extraordinary super-resolution nature, which enables an efficient, precise diagnosis of cerebral perfusion. Most importantly, the super resolution nature of this method may also facilitate early-stage evaluation of a patient's neurofunctional restoration level and avoid overoptimistic conclusions from conventional angiography or ICP monitoring.

Cystathionine gamma-lyase (CTH) inhibition attenuates glioblastoma formation.

PURPOSE: Glioblastoma (GBM) is the most common type of adult brain tumor with extremely poor survival. Cystathionine-gamma lyase (CTH) is one of the main Hydrogen Sulfide (H2S) producing enzymes and its expression contributes to tumorigenesis and angiogenesis but its role in glioblastoma development remains poorly understood. METHODS: and Principal Results: An established allogenic immunocompetent in vivo GBM model was used in C57BL/6J WT and CTH KO mice where the tumor volume and tumor microvessel density were blindly measured by stereological analysis. Tumor macrophage and stemness markers were measured by blinded immunohistochemistry. Mouse and human GBM cell lines were used for cell-based analyses. In human gliomas, the CTH expression was analyzed by bioinformatic analysis on different databases. In vivo, the genetic ablation of CTH in the host led to a significant reduction of the tumor volume and the protumorigenic and stemness transcription factor sex determining region Y-box 2 (SOX2). The tumor microvessel density (indicative of angiogenesis) and the expression levels of peritumoral macrophages showed no significant changes between the two genotypes. Bioinformatic analysis in human glioma tumors revealed that higher CTH expression is positively correlated to SOX2 expression and associated with worse overall survival in all grades of gliomas. Patients not responding to temozolomide have also higher CTH expression. In mouse or human GBM cells, pharmacological inhibition (PAG) or CTH knockdown (siRNA) attenuates GBM cell proliferation, migration and stem cell formation frequency. MAJOR CONCLUSIONS: Inhibition of CTH could be a new promising target against glioblastoma formation.

In vivo detection of penetrating arteriole alterations in cerebral white matter in patients with diabetes with 7 T MRI.

Cerebral small vessel disease (SVD) is responsible for primary intracerebral hemorrhages, lacunar infarcts and white matter hyperintensity in T2 weighted images. While the brain lesions attributed to small vessel disease can be characterized by conventional MRI, it remains challenging to noninvasively measure the early pathological changes of the small underlying vessels. We evaluated the feasibility of detecting alterations in white matter penetrating arterioles (PA) in patients with diabetes with ultra-high field 7 T MRI. 19 participants with diabetes mellitus (DM) and 19 age- and sex-matched healthy controls were scanned with whole brain T2 and susceptibility weighted MRI and a single slice phase contrast MRI 15 mm above the corpus callosum. The PC-MRI scans were repeated three times. PA masks were manually drawn on the first images after anonymization or automatically segmented on all three images. For each PA, lumen diameter, flow velocity and volume flow rate were derived by model-based analyses of complex difference images. Quasi-Poisson regression was performed for PA count using disease condition, age, and sex as independent variables. Linear mixed effect model analyses were performed for the other measurements using disease condition and age as fixed effect and participant pair specific disease condition as random effect. No severe radiological features of SVD were observed in T2 and susceptibility weighted images in any of the participants except for white matter hyperintensities with Fazekas score of 1 or 2 in 68% and 26% of patients and controls, respectively. The minimum diameter of visible PA was 78 µm and the majority had diameters <250 µm. Among the manually segmented PA with tilt angle less than 30o from the slice normal direction, flow velocities were lower in the DM group (1.9 ± 0.6 vs. 2.2 ± 0.6; p = 0.022), while no significant difference was observed in count, diameter, or volume flow rate. Similar results were observed in the automatically segmented PA. We also observed significantly increased diameter or decreased velocity with age in some of the scans. This study suggests that early PA alterations that are discriminative of disease state and age might be detectable in human cerebral white matter with 7 T MRI in vivo.

Using temporal and structural data to reconstruct 3D cerebral vasculature from a pair of 2D digital subtraction angiography sequences.

PURPOSE: The purpose of this work is to present a new method for reconstructing patient-specific three-dimensional (3D) vasculature of the brain from a pair of digital subtraction angiography (DSA) image sequences from different viewpoints, e.g., from bi-plane angiography. Our long-term goal is to provide high resolution visualization of 3D vasculature with dynamic flow of contrast agent from limited data that is readily available during surgical procedures. The proposed method is the second of a three-stage process composed of 1) augmenting vessel segmentation with vessel radii and timing of the arrival of a bolus of contrast agent, 2) reconstructing a volumetric representation of the augmented vessel data from the augmented 2D segmentations, and 3) generating a 3D model of vessels and flow of contrast agent from the volumetric reconstruction. Unlike previous methods, which are either limited to relatively simple vessel structures or rely on multiple views and/or prior models of the vasculature, our method requires only a single pair of 2D DSA sequences taken from different view directions. METHODS: We developed a new mathematical algorithm that augments vessel centerlines with vessel radii and bolus arrival times derived directly from the 2D DSA sequences to constrain the 3D reconstruction. We validated this method on digital phantoms derived from clinical data and from fractal models of branching tree structures. RESULTS: In standard reconstruction methods, reconstruction by projection of two views into 3D space results in 'ghosting' artifacts, i.e., false 3D structure that occurs where vessels or vessel segments overlap in the 2D images. For the complex vascular of the brain, this ghosting is severe and is a major hurdle for methods that attempt to generate 3D structure from 2D images. We show that our approach reduces ghosting by up to 99% in digital phantoms derived from clinical data. CONCLUSION: Our dramatic reduction in ghosting artifacts in 3D reconstructions from a pair of 2D image sequences is an important step towards generating high resolution 3D vasculature with dynamic flow information from a single DSA sequence acquired using bi-plane angiography.

Dual U-Net-Based Conditional Generative Adversarial Network for Blood Vessel Segmentation with Reduced Cerebral MR Training Volumes.

Segmenting vessels in brain images is a critical step for many medical interventions and diagnoses of illnesses. Recent advances in artificial intelligence provide better models, achieving a human-like level of expertise in many tasks. In this paper, we present a new approach to segment Time-of-Flight Magnetic Resonance Angiography (TOF-MRA) images, relying on fewer training samples than state-of-the-art methods. We propose a conditional generative adversarial network with an adapted generator based on a concatenated U-Net with a residual U-Net architecture (UUr-cGAN) to carry out blood vessel segmentation in TOF-MRA images, relying on data augmentation to diminish the drawback of having few volumes at disposal for training the model, while preventing overfitting by using regularization techniques. The proposed model achieves 89.52% precision and 87.23% in Dice score on average from the cross-validated experiment for brain blood vessel segmentation tasks, which is similar to other state-of-the-art methods while using considerably fewer training samples. UUr-cGAN extracts important features from small datasets while preventing overfitting compared to other CNN-based methods and still achieve a relatively good performance in image segmentation tasks such as brain blood vessels from TOF-MRA.

A neural network approach to segment brain blood vessels in digital subtraction angiography.

BACKGROUND AND OBJECTIVE: Cerebrovascular diseases (CVDs) affect a large number of patients and often have devastating outcomes. The hallmarks of CVDs are the abnormalities formed on brain blood vessels, including protrusions, narrows, widening, and bifurcation of the blood vessels. CVDs are often diagnosed by digital subtraction angiography (DSA) yet the interpretation of DSA is challenging as one must carefully examine each brain blood vessel. The objective of this work is to develop a computerized analysis approach for automated segmentation of brain blood vessels. METHODS: In this work, we present a U-net based deep learning approach, combined with pre-processing, to track and segment brain blood vessels in DSA images. We compared the results given by the deep learning approach with manually marked ground truth using accuracy, sensitivity, specificity, and Dice coefficient. RESULTS: Our results showed that the proposed approach achieved an accuracy of 0.978, with a standard deviation of 0.00796, a sensitivity of 0.76 with a standard deviation of 0.096, a specificity of 0.994 with a standard deviation of 0.0036, and an average Dice coefficient was 0.8268 with a standard deviation of 0.052. CONCLUSIONS: Our findings show that the deep learning approach can achieve satisfactory performance as a computer-aided analysis tool to assist clinicians in diagnosing CVDs.