Enhanced capillary delivery with nanobubble-mediated blood-brain barrier opening and advanced high resolution vascular segmentation.

Overcoming the blood-brain barrier (BBB) is essential to enhance brain therapy. Here, we utilized nanobubbles with focused ultrasound for targeted and improved BBB opening in mice. A microscopy technique method assessed BBB opening at a single blood vessel resolution employing a dual-dye labeling technique using green fluorescent molecules to label blood vessels and Evans blue brain-impermeable dye for quantifying BBB extravasation. A deep learning architecture enabled blood vessels segmentation, delivering comparable accuracy to manual segmentation with a significant time reduction. Segmentation outcomes were applied to the Evans blue channel to quantify extravasation of each blood vessel. Results were compared to microbubble-mediated BBB opening, where reduced extravasation was observed in capillaries with a diameter of 2-6 µm. In comparison, nanobubbles yield an improved opening in these capillaries, and equivalent efficacy to that of microbubbles in larger vessels. These results indicate the potential of nanobubbles to serve as enhanced agents for BBB opening, amplifying bioeffects in capillaries while preserving comparable opening in larger vessels.

Protocol to assess extravasation of fluorescent molecules in mice after ultrasound-mediated blood-brain barrier opening.

Blood-brain barrier disruption (BBBD) using focused ultrasound (FUS) and microbubbles (MBs) is an effective tool for therapeutic delivery to the brain. Here, we present an optimized protocol for quantifying fluorescent molecules extravasation in mice. We describe steps for ultrasound treatment, injection of MBs and fluorescent dyes, brain harvesting, microscopy imaging, and image postprocessing algorithm. Our protocol has proven to successfully conduct a diameter-dependent analysis that measures vascular leakage following FUS-mediated BBBD at a single blood vessel resolution. For complete details on the use and execution of this protocol, please refer to Katz et al.1.

Diameter-dependent assessment of microvascular leakage following ultrasound-mediated blood-brain barrier opening.

Blood brain barrier disruption (BBBD) using focused ultrasound (FUS) and microbubbles (MB) is an effective tool for therapeutic delivery to the brain. BBBD depends to a great extent on MB oscillations. Because the brain vasculature is heterogenic in diameter, reduced MB oscillations in smaller blood vessels, together with a lower number of MBs in capillaries, can lead to variations in BBBD. Therefore, evaluating the impact of microvasculature diameter on BBBD is of great importance. We present a method to characterize molecules extravasation following FUS-mediated BBBD, at a single blood vessel resolution. Evans blue (EB) leakage was used as marker for BBBD, whereas blood vessels localization was done using FITC labeled Dextran. Automated image processing pipeline was developed to quantify the extent of extravasation as function of microvasculature diameter, including a wide range of vascular morphological parameters. Variations in MB vibrational response were observed in blood vessel mimicking fibers with varied diameters. Higher peak negative pressures (PNP) were required to initiate stable cavitation in fibers with smaller diameters. In vivo in the treated brains, EB extravasation increased as a function of blood vessel diameter. The percentage of strong BBBD blood vessels increased from 9.75% for 2-3 µm blood vessels to 91.67% for 9-10 µm. Using this method, it is possible to conduct a diameter-dependent analysis that measures vascular leakage resulting from FUS-mediated BBBD at a single blood vessel resolution.

Low frequency nanobubble-enhanced ultrasound mechanotherapy for noninvasive cancer surgery.

Scaling down the size of microbubble contrast agents to the nanometer level holds the promise for noninvasive cancer therapy. However, the small size of nanobubbles limits the obtained bioeffects as a result of ultrasound cavitation, when operating near the nanobubble resonance frequency. Here we show that coupled with low energy insonation at a frequency of 80 kHz, well below the resonance frequency of these agents, nanobubbles serve as noninvasive therapeutic warheads that trigger potent mechanical effects in tumors following a systemic injection. We demonstrate these capabilities in tissue mimicking phantoms, where a comparison of the acoustic response of micro- and nano-bubbles after insonation at a frequency of 250 or 80 kHz revealed that higher pressures were needed to implode the nanobubbles compared to microbubbles. Complete nanobubble destruction was achieved at a mechanical index of 2.6 for the 250 kHz insonation vs. 1.2 for the 80 kHz frequency. Thus, the 80 kHz insonation complies with safety regulations that recommend operation below a mechanical index of 1.9. In vitro in breast cancer tumor cells, the cell viability was reduced to 17.3 ± 1.7% of live cells. In vivo, in a breast cancer tumor mouse model, nanobubble tumor distribution and accumulation were evaluated by high frequency ultrasound imaging. Finally, nanobubble-mediated low frequency insonation of breast cancer tumors resulted in effective mechanical tumor ablation and tumor tissue fractionation. This approach provides a unique theranostic platform for safe, noninvasive and low energy tumor mechanotherapy.

Acoustically Detonated Microbubbles Coupled with Low Frequency Insonation: Multiparameter Evaluation of Low Energy Mechanical Ablation.

Noninvasive ultrasound surgery can be achieved using focused ultrasound to locally affect the targeted site without damaging intervening tissues. Mechanical ablation and histotripsy use short and intense acoustic pulses to destroy the tissue via a purely mechanical effect. Here, we show that coupled with low-frequency excitation, targeted microbubbles can serve as mechanical therapeutic warheads that trigger potent mechanical effects in tumors using focused ultrasound. Upon low frequency excitation (250 kHz and below), high amplitude microbubble oscillations occur at substantially lower pressures as compared to higher MHz ultrasonic frequencies. For example, inertial cavitation was initiated at a pressure of 75 kPa for a center frequency of 80 kHz. Low frequency insonation of targeted microbubbles was then used to achieve low energy tumor cell fractionation at pressures below a mechanical index of 1.9, and in accordance with the Food and Drug Administration guidelines. We demonstrate these capabilities in vitro and in vivo. In cell cultures, cell viability was reduced to 16% at a peak negative pressure of 800 kPa at the 250 kHz frequency (mechanical index of 1.6) and to 10% at a peak negative pressure of 250 kPa at a frequency of 80 kHz (mechanical index of 0.9). Following an intratumoral injection of targeted microbubbles into tumor-bearing mice, and coupled with low frequency ultrasound application, significant tumor debulking and cancer cell death was observed. Our findings suggest that reducing the center frequency enhances microbubble-mediated mechanical ablation; thus, this technology provides a unique theranostic platform for safe low energy tumor fractionation, while reducing off-target effects.

Prenatal Diagnosis of Vasa Previa: Outpatient versus Inpatient Management.

OBJECTIVE: The aim of this study was to compare the pregnancy outcome of two different management strategies: outpatient versus inpatient in women with prenatal diagnosis of vasa previa. MATERIALS AND METHODS: This is a retrospective cohort study conducted at a single tertiary center. Women with a prenatally diagnosed vasa previa between January 2007 and June 2017 were included. Obstetric and neonatal outcomes were compared between two management strategies: elective admission at 34 weeks of gestation or outpatient management unless there were signs of labor or premature contractions. RESULTS: A total of 109 women met the inclusion criteria: 75 (68.8%) women in the inpatient group and 34 (31.2%) in the outpatient group. Women in the inpatient group were more likely to receive antenatal steroids (57.3 vs. 26.4%, p = 0.002) and were less likely to have an urgent cesarean section (34.6 vs. 58.8%, respectively, p < 0.001) compared with outpatient group. There was no difference in the rate of neonatal complications (inpatient: 64.6% vs. outpatient: 52.7%, p = 0.27) or neonatal anemia requiring transfusion (2.7 vs. 5.8%, respectively, p = 0.5) between the groups. CONCLUSION: The rate of elective cesarean section and exposure to antenatal steroids was higher in patients with vasa previa who were admitted electively at 34 weeks of gestation compared with patients who were managed as outpatient.

Gender disparities in the functional significance of anemia among apparently healthy adults.

BACKGROUND: Data on the functional impact of anemia on cardiorespiratory fitness (CRF) and survival in healthy individuals are limited. Our aim was to evaluate the association between anemia thresholds, low CRF, and survival in otherwise healthy adults. METHODS: Study population included 16 334 apparently healthy subjects attending annual periodic health screening examinations (71 200 annual visits), including exercise stress testing (EST). Anemia was defined by the World Health Organization (WHO) or Beutler and Waalens' (BW) criteria. Low CRF was defined as the lowest fitness quintile according to the Bruce protocol. RESULTS: The mean age was 46±10 years, and 70% were men. Mean Hb levels were 13±1 and 15±1 among women and men, respectively, with higher proportion of anemia among women. The majority of anemic subjects had mild anemia. When analyzing repeated annual visits, anemia was associated with a significant 39% and 64% increased risk of low CRF according the WHO and BW criteria only in women (n=18 672). Baseline anemia at first visit was associated with 2.6- and 1.9-fold increased risk of all-cause mortality using the WHO and BW criteria, exclusively in men (n=11 511). CONCLUSIONS: Overall, the functional and prognostic impact of anemia is gender dependent, based on the WHO and BW arbitrary criteria, suggesting differing mechanism and responses.

Cardiac failure in very long chain acyl-CoA dehydrogenase deficiency requiring extracorporeal membrane oxygenation (ECMO) treatment: A case report and review of the literature.

Fatty acid oxidation (FAO) defects often present with multi-system involvement, including several life-threatening cardiac manifestations, such as cardiomyopathy, pericardial effusion and arrhythmias. We report herein a fatal case of cardiac dysfunction and rapid-onset tamponade following an acute illness in a neonate with molecularly proven very long chain acyl-CoA dehydrogenase (VLCAD) deficiency (harboring the known del799_802 mutation), requiring 15 days of extracorporeal membrane oxygenation (ECMO) treatment. As data regarding the use of ECMO in FAO defects in general, and VLCAD in particular, are scarce, we review the literature and discuss insights from in vitro models and several successful reported cases.

Biomagnification of perfluorinated compounds in a remote terrestrial food chain: Lichen-Caribou-wolf.

The biomagnification behavior of perfluorinated carboxylates (PFCAs) and perfluorinated sulfonates (PFSAs) was studied in terrestrial food webs consisting of lichen and plants, caribou, and wolves from two remote northern areas in Canada. Six PFCAs with eight to thirteen carbons and perfluorooctane sulfonate (PFOS) were regularly detected in all species. Lowest concentrations were found for vegetation (0.02-0.26 ng/g wet weight (ww) sum (Σ) PFCAs and 0.002-0.038 ng/g ww PFOS). Wolf liver showed highest concentrations (10-18 ng/g ww ΣPFCAs and 1.4-1.7 ng/g ww PFOS) followed by caribou liver (6-10 ng/g ww ΣPFCAs and 0.7-2.2 ng/g ww PFOS). Biomagnification factors were highly tissue and substance specific. Therefore, individual whole body concentrations were calculated and used for biomagnification and trophic magnification assessment. Trophic magnification factors (TMF) were highest for PFCAs with nine to eleven carbons (TMF = 2.2-2.9) as well as PFOS (TMF = 2.3-2.6) and all but perfluorooctanoate were significantly biomagnified. The relationship of PFCA and PFSA TMFs with the chain length in the terrestrial food chain was similar to previous studies for Arctic marine mammal food web, but the absolute values of TMFs were around two times lower for this study than in the marine environment. This study demonstrates that challenges remain for applying the TMF approach to studies of biomagnification of PFCAs and PFSAs, especially for terrestrial animals.

Hypocatalasemic fibroblasts accumulate hydrogen peroxide and display age-associated pathologies.

Human epidemiological studies point to an association of hypocatalasemia and an increased risk of age-related disease. Unfortunately, the cellular and molecular manifestations of hypocatalasemia are only poorly understood. In this analysis, we have extensively characterized hypocatalasemic human fibroblasts and report that they amass hydrogen peroxide and are oxidatively damaged. Protein and DNA alike are affected, as are functioning and biogenesis of peroxisomes - the subcellular organelles which normally house catalase. Despite these pathologies and their relative inability to grow, the cells do not appear to be intrinsically senescent. With the goal of restoring oxidative balance and perhaps reversing some of the accumulated damage to critical cellular components, we transduced hypocatalasemic fibroblasts with a form of catalase specifically designed to efficiently traffic to peroxisomes. We show the strategy is extremely effective, with dramatic reductions seen in cellular hydrogen peroxide levels. Future longitudinal studies aimed at examining the effects of a more continuous and long-term protein therapy may now commence.