Hyperspectral absorption microscopy using photoacoustic remote sensing.

An improved method of remote optical absorption spectroscopy and hyperspectral optical absorption imaging is described which takes advantage of the photoacoustic remote sensing detection architecture. A wide collection of photoacoustic excitation wavelengths ranging from 210 nm to 1550 nm was provided by a nanosecond tunable source allowing access to various salient endogenous chromophores such as DNA, hemeproteins, and lipids. Sensitivity of the device was demonstrated by characterizing the infrared absorption spectrum of water. Meanwhile, the efficacy of the technique was explored by recovering cell nuclei and oxygen saturation from a live chicken embryo model and by recovering adipocytes from freshly resected murine adipose tissue. This represents a continued investigation into the characteristics of the hyperspectral photoacoustic remote sensing technique which may represent an effective means of non-destructive endogenous contrast characterization and visualization.

Different Parts of the Chicken Embryo Egg Improve D-Galactose-Induced Aging in a Mice Model.

Chick (CE) or duck embryo eggs are known for nutritional supplement foods in traditional East countries for physical fitness enhancement and postpartum conditioning for many years. In this study, we evaluated the effects of different parts of the 10-day CE (embryo: CEr, yolk: CEw, and chorioallantoic membrane: CEp) on the antifatigue and antiaging activities in a D-galactose- (D-gal) induced aging mice model. The results showed CEp obviously increased the muscle weight and the liver and muscle glycogen content and enhanced exercise performance. In the antiaging assay, CEp significantly increased the activity of superoxide dismutase (SOD) and Glutathione Peroxidase (GPx). Moreover, the immunohistochemistry results of NRF-2 and HO-1 were also detected in the livers of mice in the D-gal/CEp group. The only partially potential such as CEr might improve OFT function with TG level, and CEw had strange grip strength. Therefore, we suggest that CEp has a potent antifatigue ability and could minimize the occurrence of age-associated disorders, more than other parts of the 10 days chicken embryo egg.

Comparison between HET-CAM protocols and a product use clinical study for eye irritation evaluation of personal care products including cosmetics according to their surfactant composition.

The hen's egg test on chorioallantoic membrane (HET-CAM) is one of the most frequently used alternative tests for prediction of ocular irritation of cosmetic products. There are different HET-CAM protocols widely accepted, but there is no information about which of the protocols better correlates with the results obtained in product use clinical study under the conditions of use. Two Fix Time Methods (FTM) -Lüepke and the ICCVAM guideline - and two Reaction Time Methods (RTM) -ECVAM DBALM Prot. No. 47 and No. 96- were employed to test 18 cosmetic products. Simultaneously, they were evaluated by an ophthalmological clinical test. A unified classification system was used, and products were classified into four irritation levels: non-irritant, weak, moderate and severe irritant. The duration of use (rinse-off or leave-on), and the concentration and type of surfactants were taken into account in the analysis. All the products that were classified as non-irritant by any HET-CAM protocols were also safe in the product use clinical study. The product that was found to be non-safe in the product use clinical evaluation was also unsuitable by most of the HET-CAM protocols. These results were employed to develop an algorithm that allows selecting the appropriate HET-CAM protocol for each type of product to be tested.

The Preparation of Chicken Ex Ovo Embryos and Chorioallantoic Membrane Vessels as In Vivo Model for Contrast-Enhanced Ultrasound Imaging and Microbubble-Mediated Drug Delivery Studies.

The chicken embryo and the blood-vessel rich chorioallantoic membrane (CAM) is a valuable in vivo model to investigate biomedical processes, new ultrasound pulsing schemes, or novel transducers for contrast-enhanced ultrasound imaging and microbubble-mediated drug delivery. The reasons for this are the accessibility of the embryo and vessel network of the CAM as well as the low costs of the model. An important step to get access to the embryo and CAM vessels is to take the egg content out of the eggshell. In this protocol, three methods for taking the content out of the eggshell between day 5 and 8 of incubation are described thus allowing the embryos to develop inside the eggshell up to these days. The described methods only require simple tools and equipment and yield a higher survival success rate of 90% for 5-day, 75% for 6-day, 50% for 7-day, and 60% for 8-day old incubated eggs in comparison to ex ovo cultured embryos (~50%). The protocol also describes how to inject cavitation nuclei, such as microbubbles, into the CAM vascular system, how to separate the membrane containing the embryo and CAM from the rest of the egg content for optically transparent studies, and how to use the chicken embryo and CAM in a variety of short-term ultrasound experiments. The in vivo chicken embryo and CAM model is extremely relevant to investigate novel imaging protocols, ultrasound contrast agents, and ultrasound pulsing schemes for contrast-enhanced ultrasound imaging, and to unravel the mechanisms of ultrasound-mediated drug delivery.

Thyroxine impregnated chitosan-based dressings stimulate angiogenesis and support fast wounds healing in rats: Potential clinical candidates.

This research paper demonstrates efficacy of chitosan and thyroxine loaded chitosan (CS) dressings for their angiogenic and wound healing potential. The dressings were prepared by freeze gelation method. Thyroxine was loaded by physical adsorption into chitosan membranes. The porosity was analyzed by scanning electron microscopy (SEM) and chemical structures were investigated by Fourier transform infra-red spectroscopy (FTIR). Cell culture studies showed materials were non-toxic and chorioallantoic membranes assay (CAM) confirmed that the thyroxine loaded chitosan stimulated angiogenesis much higher than simple chitosan dressings. In addition, thyroxine loaded dressings showed excellent wound healing potential when tested on full thickness rats wounds. A good epithelialization was obtained along with robust wound closure. Overall, as compared to chitosan, thyroxine containing membranes showed high level of angiogenesis and fast wound healing.

First experience of chick chorioallantoic membrane (CAM) assay in the clinical work flow with oral squamous cell carcinoma patients.

INTRODUCTION: The oral squamous cell carcinoma (OSCC) is a leading cause of death in human malignancies. The aim of this study is to integrate the CAM Assay as a reliable and good working in vivo model for the evaluation of OSCC tumor samples and its growth into the clinical work flow. MATERIAL AND METHODS: Fresh human Tumor samples (OSCCs) 1×1 cm in size were cut into 350-450µm thick slices by a Vibratome and put on the prepared CAM model.After growth of the tumor tissue on the CAM, we started with topical induction of proinflammatory cytokines (TNFα) and growth factors (TGFß). After further growth of the tumor on the assay, we explanted the tumor tissue and first performed microscopic and then immunohistochemical examinations. E-cadherin and vimentin were used as Epithelial-to-mesenchymal transition (EMT) -makers and the histologic preparations were evaluated histomorphometrically. The results were correlated with clinical parameters of the patients. RESULTS: Under TNFα, the small tumors (T1 / T2) show higher E-cadherin expression than larger tumors (T3 / T4). The vimentin expression under TNFα behaved in the opposite direction, at T1 / T2 the expression decreased in T3 / T4 increased. Furthermore, an increased E-cadherin expression in N0 and diminished E-cadherin expression in N1 / N2b patients could be detected depending on the N-stage of the patients. Vimentin, on the other hand, was reduced in the N0 group and expressed more frequently in the N1 / N2b group. TGFß induction also led to increased expression of vimentin in the T3 / T4 tumors and N1 / N2b stages. CONCLUSION: By integrating a CAM assay into the clinical workflow, tumors with preserved tumor architecture can be cultured and subjected to histological and molecular biology studies. Effects on biological behavior are recognizable and demonstrable in this model. The key markers E-cadherin and vimentin alone are not sufficient to represent the complexity of the EMT in this model. Further molecular biology and signaling pathway analyzes are necessary.

A glycan-based approach to therapeutic angiogenesis.

Angiogenesis, the sprouting of new blood vessels from existing vasculature, involves multiple complex biological processes, and it is an essential step for hemostasis, tissue healing and regeneration. Angiogenesis stimulants can ameliorate human disease conditions including limb ischemia, chronic wounds, heart disease, and stroke. The current strategies to improve the bioavailability of pro-angiogenic growth factors, including VEGF and FGF2, have remained largely unsuccessful. This study demonstrates that small molecules, termed click-xylosides, can promote angiogenesis in the in vitro matrigel tube formation assay and the ex ovo chick chorioallantoic membrane assay, depending on their aglycone moieties. Xyloside treatment enhances network connectivity and cell survivability, thereby, maintaining the network structures on matrigel culture for an extended period of time. These effects were achieved via the secreted xyloside-primed glycosaminoglycans (GAG) chains that in part, act through an ERK1/2 mediated signaling pathway. Through the remodeling of GAGs in the extracellular matrix of endothelial cells, the glycan approach, involving xylosides, offers great potential to effectively promote therapeutic angiogenesis.

Angiogenic activity in vivo of the particulate matter (PM10).

BACKGROUND: Particulate matter (PM) is the most efficient vehicle for the inhalation and absorption of toxic substances into the body. METHOD: The present study was aimed at testing the hypothesis that PM10 samples collected on quartz filters exert an angiogenic activity in vivo in the chick embryo chorioallantoic membrane (CAM) assay. RESULTS: When the low, medium, and high PM10 concentrations filters were tested in the CAM assay, an increasing number of microvessels was detectable after 4 days of applications of the filters. Moreover, at histological level, numerous microvessels and a dense inflammatory infiltrate were recognizable in the CAM mesenchyme. CONCLUSION: Our data show a clear dose-response relationship between the dose variable (PM10 and Bap) and the outcome variable. So far, the PM10 target value is determined on the basis of regulatory agreements and is not health-based. In addition, the mere gravimetric measure of PM10 cannot be considered a fully reliable surrogate of the overall toxicity of the mixture.

Measurement of pO2 by luminescence lifetime spectroscopy: A comparative study of the phototoxicity and sensitivity of [Ru(Phen)3 ]2+ and PdTCPP in vivo.

Dysfunctions in tissue metabolism can be detected at early stages by oxygen partial pressure (pO2 ) measurement. The measurement of emission lifetimes offers very promising and non-invasive approach to estimate pO2 in vivo. This study compares two extensively used oxygen sensors and assesses their in vivo oxygen sensitivity and phototoxic effect. Luminescence lifetime of Ru-polypyridyl complex and of Pd-porphyrin is measured in the Chick's Chorioallantoic Membrane (CAM) model with a dedicated optical fiber-based, time-resolved spectrometer. The Pd-porphyrin luminescence lifetimes measured in the CAM model exposed to different pO2 levels are longer and have a broader dynamic range (10-100 µs) than those of Ru-polypyridyl complex (0.6-1 µs). The combined statistical analysis based on an estimate of the kurtosis and skewness, bootstrapping method and routine normality tests is performed. The indicators of the averages and signal to noise ratio stability are also calculated. The combination of several data processing allows selection of the better sensor for a given application. In particular, it is found that the advantage of Ru-polypyridyl complex over Pd-porphyrin is two-fold: i) Ru-polypyridyl complex datasets have consistently better statistical characteristics, ii) Ru-polypyridyl exhibits lower cytotoxicity.

Dual-targeting anti-angiogenic cyclic peptides as potential drug leads for cancer therapy.

Peptide analogues derived from bioactive hormones such as somatostatin or certain growth factors have great potential as angiogenesis inhibitors for cancer applications. In an attempt to combat emerging drug resistance many FDA-approved anti-angiogenesis therapies are co-administered with cytotoxic drugs as a combination therapy to target multiple signaling pathways of cancers. However, cancer therapies often encounter limiting factors such as high toxicities and side effects. Here, we combined two anti-angiogenic epitopes that act on different pathways of angiogenesis into a single non-toxic cyclic peptide framework, namely MCoTI-II (Momordica cochinchinensis trypsin inhibitor-II), and subsequently assessed the anti-angiogenic activity of the novel compound. We hypothesized that the combination of these two epitopes would elicit a synergistic effect by targeting different angiogenesis pathways and result in improved potency, compared to that of a single epitope. This novel approach has resulted in the development of a potent, non-toxic, stable and cyclic analogue with nanomolar potency inhibition in in vitro endothelial cell migration and in vivo chorioallantoic membrane angiogenesis assays. This is the first report to use the MCoTI-II framework to develop a 2-in-1 anti-angiogenic peptide, which has the potential to be used as a form of combination therapy for targeting a wide range of cancers.