Functional photoacoustic remote sensing microscopy using a stabilized temperature-regulated stimulated Raman scattering light source.

Stimulated Raman scattering (SRS) has been widely used in functional photoacoustic microscopy to generate multiwavelength light and target multiple chromophores inside tissues. Despite offering a simple, cost-effective technique with a high pulse repetition rate; it suffers from pulse-to-pulse intensity fluctuations and power drift that can affect image quality. Here, we propose a new technique to improve the temporal stability of the pulsed SRS multiwavelength source. We achieve this by lowering the temperature of the SRS medium. The results suggest that a decrease in temperature causes an improvement of temporal stability of the output, considerable rise in the intensity of the SRS peaks, and significant increase of SRS cross section. The application of the method is shown for in vivo functional imaging of capillary networks in a chicken embryo chorioallantois membrane using photoacoustic remote sensing microscopy.

Codelivery of BV6 and anti-IL6 siRNA by hyaluronate-conjugated PEG-chitosan-lactate nanoparticles inhibits tumor progression.

AIMS: Increased expression of inhibitor of apoptosis (IAP) genes has been associated with progressive cancer and chemoresistance. Accordingly, blockade of IAPs by BV6 has resulted in ameliorative outcomes. Interleukin (IL)-6 is another important mediator involved in the growth and survival of tumor cells. Therefore, we hypothesized that simultaneous inhibition of IAPs and IL-6 could be a new promising anti-tumor treatment strategy. MATERIALS AND METHODS: In this study, we generated and characterized hyaluronate-PEG-Chitosan-Lactate (H-PCL) nanoparticles (NPs) to simultaneously deliver IL6-specific siRNA and BV6 to 4T1 (breast cancer) and CT26 (colon cancer) cells, and investigate the anti-tumor properties of this combination therapy both in vitro and in vivo. KEY FINDINGS: H-PCL NPs exhibited good physicochemical properties leading to efficient transfection of cancer cells and suppression of target molecules. Moreover, combination therapy synergistically increased apoptosis, as well as decreased cell migration, proliferation, colony formation, and angiogenesis in both 4T1 and CT26 cell lines and suppressed cancer progression in tumor-bearing mice that was associated with enhanced survival time. SIGNIFICANCE: These findings imply the effectiveness of cancer combination therapy by using H-PCL NPs loaded with anti-IL-6 siRNA and BV6.

Use of animal models for the imaging and quantification of angiogenesis.

Angiogenesis is the process of developing new blood vessels from the original vascular network; it is necessary for normal physiological processes, such as embryonic development and wound healing. Angiogenesis is also involved in pathological events, including myocardial ischemia and tumor growth. To investigate the molecular mechanisms of this important process, a variety of methods and models are employed. These strategies can also be used to provide insight into the etiology of angiogenesis-related diseases, thereby contributing to the development of new diagnostics and treatments. Commonly used animal models include the chorioallantoic membrane and yolk sac membrane of chick embryos, the mouse retina and aortic ring, and angiogenesis reactors implanted into mice. These animal models have been instrumental in the study of the angiogenic process. For example, the chorioallantoic membrane undergoes robust angiogenesis during the development of chick embryos, and, because its surface is easily accessible, this membrane provides a convenient model for experimentation. Here, we discuss the methods that employ animal models for the imaging and quantification of angiogenesis. In addition, we propose potential novel directions for future investigations in this area.

3D Reconstruction of Chick Embryo Vascular Geometries Using Non-invasive High-Frequency Ultrasound for Computational Fluid Dynamics Studies.

Recent animal studies have provided evidence that prenatal blood flow fluid mechanics may play a role in the pathogenesis of congenital cardiovascular malformations. To further these researches, it is important to have an imaging technique for small animal embryos with sufficient resolution to support computational fluid dynamics studies, and that is also non-invasive and non-destructive to allow for subject-specific, longitudinal studies. In the current study, we developed such a technique, based on ultrasound biomicroscopy scans on chick embryos. Our technique included a motion cancelation algorithm to negate embryonic body motion, a temporal averaging algorithm to differentiate blood spaces from tissue spaces, and 3D reconstruction of blood volumes in the embryo. The accuracy of the reconstructed models was validated with direct stereoscopic measurements. A computational fluid dynamics simulation was performed to model fluid flow in the generated construct of a Hamburger-Hamilton (HH) stage 27 embryo. Simulation results showed that there were divergent streamlines and a low shear region at the carotid duct, which may be linked to the carotid duct's eventual regression and disappearance by HH stage 34. We show that our technique has sufficient resolution to produce accurate geometries for computational fluid dynamics simulations to quantify embryonic cardiovascular fluid mechanics.

Prenatal tolbutamide treatment alters plasma glucose and insulin concentrations and negatively affects the postnatal performance of chickens.

To examine the relationship of insulin and glucose, broiler embryos were subjected to acute or prolonged hypoglycemia during the late embryonic phase by, respectively, injecting once (at embryonic day [ED] 16 or 17) or on 3 consecutive days (ED 16, 17, and 18) with tolbutamide (80 µg/g embryo weight), a substance that stimulates insulin secretion from the pancreas. After 1 tolbutamide injection, a prolonged (32 h) decrease of plasma glucose and a profound acute increase in plasma insulin were observed. The 3 consecutive tolbutamide injections induced hypoglycemia for 4 days (from ED 16 to ED 19). The postnatal performance after 3 consecutive tolbutamide injections in broiler embryos was also investigated. Body weight was lower in tolbutamide-treated chickens from hatch to 42 d compared with sham (P = 0.001) and control (P < 0.001) chickens. Feed intake was lower in the tolbutamide group from hatch to 42 d as compared with sham (P = 0.007) and control (P = 0.017) animals. In addition, at 42 d, plasma glucose concentrations, after an insulin injection challenge (50 µg/kg body weight), were higher in tolbutamide-treated chickens compared with the sham and the control group as were their basal glucose levels (P value of group effect <0.001). In conclusion, tolbutamide treatment during the late embryonic development in broilers resulted in prolonged hypoglycemia in this period and negatively influenced the posthatch performance.

Effect of silver nanoparticles and hydroxyproline, administered in ovo, on the development of blood vessels and cartilage collagen structure in chicken embryos.

It has been considered that concentrations of certain amino acids in the egg are not sufficient to fully support embryonic development of modern broilers. In this study we evaluated embryo growth and development with particular emphasis on one of the major components of connective tissue, collagen. Experiments were performed on Ross 308 chicken embryos from 160 fertilised eggs. Experimental solutions of silver nanoparticles (Ag), hydroxyproline solution (Hyp) and a complex of silver nanoparticles with hydroxyproline (AgHyp) were injected into albumen, and embryos were incubated until day 20. An assessment of the mass of embryo and selected organs was carried out followed by measurements of the expression of the key signalling factors' fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-A (VEGF-A). Finally, an evaluation of collagen microstructure using scanning electron microscopy was performed. Our results clearly indicate that Hyp, Ag and AgHyp administered in ovo to chicken embryos did not harm embryos. Comparing to the control group, Hyp, Ag and the AgHyp complex significantly upregulated expression of the FGF-2 at the mRNA and protein levels. Moreover, Hyp, Ag and, in particular, the complex of AgHyp significantly increased blood vessel size, cartilage collagen fibre lattice size and bundle thickness. The general conclusion from this study is that AgHyp treatment may help to build a stronger and longer lasting form of collagen fibres.

Effect of embryonic development on the chicken egg yolk plasma proteome after 12 days of incubation.

To better appreciate the dynamics of yolk proteins during embryonic development, we analyzed the protein quantitative changes occurring in the yolk plasma at the day of lay and after 12 days of incubation, by comparing unfertilized and fertilized chicken eggs. Of the 127 identified proteins, 69 showed relative abundance differences among conditions. Alpha-fetoprotein and two uncharacterized proteins (F1NHB8 and F1NMM2) were identified for the first time in the egg. After 12 days of incubation, five proteins (vitronectin, α-fetoprotein, similar to thrombin, apolipoprotein B, and apovitellenin-1) showed a major increase in relative abundance, whereas 15 proteins showed a significant decrease in the yolks of fertilized eggs. In unfertilized/table eggs, we observed an accumulation of proteins likely to originate from other egg compartments during incubation. This study provides basic knowledge on the utilization of egg yolk proteins by the embryo and gives some insight into how storage can affect egg quality.

[Choline promotes angiogenesis in chick embryo chorioallantoic membrane].

OBJECTIVE: To investigate choline promoting angiogenesis on chick embryo chorioallantoic membrane (CAM). METHODS: CAM model was prepared, the choline chloride, human vascular endothelial growth factor (hVEGF) and normal saline were added respectively onto the carrier on the CAM, the state of angiogenesis was observed and the number of new blood vessels was counted. RESULTS: Choline chloride was tested at the concentrations of 0.5 nmol/L - 1 mmol/L in this experiment, when its concentrations were increased to 0.01 micromol/L - 1 000 micromol/L, it could stimulate angiogenesis, the minimum effective concentration was tested as 0.01 micromol/L, and its effect for promoting the angiogenesis was equivalent to that of hVEGF, the potent stimulator for angiogenesis. CONCLUSION: The result shows that choline can promote angiogenesis in the chick embryo chorioallantoic membrane.

In vitro polyphenolics erythrocyte model and in vivo chicken embryo model revealed gallic acid to be a potential hemorrhage inducer: physicochemical action mechanisms.

The in vivo chicken embryo model (CEM) demonstrated that gallic acid (GA) induced dysvascularization and hypoxia. Inflammatory edema, Zenker's necrosis, hemolysis, and liposis of cervical muscles were the common symptoms. Levels of the gene hif-1α, HIF-1α, TNF-α, IL-6, and NFκB in cervical muscles were all significantly upregulated, while the vascular endothelial growth factor (VEGF) was downregulated in a dose-responsive manner. Consequently, the cervical muscle inflammation and hemolysis could have been stimulated en route to the tissue TNF-α-canonical and the atypical pathways. We hypothesized that GA could deplete the dissolved oxygen (DO) at the expense of semiquinone and quinone formation, favoring the reactive oxygen species (ROS) production to induce RBC disruption and Fe(2+) ion release. To explore this, the in vitro polyphenolics-erythrocyte model (PEM) was established. PEM revealed that the DO was rapidly depleted, leading to the release of a huge amount of Fe (II) ions and hydrogen peroxide (HPO) in a two-phase kinetic pattern. The kinetic coefficients for Fe (II) ion release ranged from 0.347 h(-1) to 0.774 h(-1); and those for Fe (III) ion production were from 6.66 × 10(-3) h(-1) to 8.93 × 10(-3) h(-1). For phase I HPO production, they ranged from 0.236 h(-1) to 0.774 h(-1) and for phase II HPO production from 0.764 h(-1) to 2.560 h(-1) at GA within 6 µM to 14 µM. Thus, evidence obtained from PEM could strongly support the phenomena of CEM. To conclude, GA tends to elicit hypoxia-related inflammation and hemolysis in chicken cervical muscles through its extremely high prooxidant activity.

Direct and indirect roles of Fgf3 and Fgf10 in innervation and vascularisation of the vertebrate hypothalamic neurohypophysis.

The neurohypophysis is a crucial component of the hypothalamo-pituitary axis, serving as the site of release of hypothalamic neurohormones into a plexus of hypophyseal capillaries. The growth of hypothalamic axons and capillaries to the forming neurohypophysis in embryogenesis is therefore crucial to future adult homeostasis. Using ex vivo analyses in chick and in vivo analyses in mutant and transgenic zebrafish, we show that Fgf10 and Fgf3 secreted from the forming neurohypophysis exert direct guidance effects on hypothalamic neurosecretory axons. Simultaneously, they promote hypophyseal vascularisation, exerting early direct effects on endothelial cells that are subsequently complemented by indirect effects. Together, our studies suggest a model for the integrated neurohemal wiring of the hypothalamo-neurohypophyseal axis.

Decorin developmental expression and function in the early avian embryo.

Decorin, a proteoglycan, interacts with extracellular matrix proteins, growth factors and receptors. Decorin expression and spatio-temporal distribution were studied by RT-PCR and immunofluorescence, while decorin function was examined by blocking antibodies in the early chick embryo. Decorin was first detectable at stage XIII (late blastula). During gastrulation (stage HH3-4), decorin fluorescence was intense in epiblast cells immediately adjacent to the streak, and in migrating cells. Decorin fluorescence was intense in endoderm and strong at mesoderm-neural plate surfaces at stage HH5-6 (neurula). At stage HH10-11 (12 somites), decorin fluorescence was intense in myelencephalon and then showed distinct expression patterns along the myelencephalon axes by stage HH17. Decorin fluorescence was intense in neural crest cells, dorsal aorta, heart, somite and neuroepithelial cells apposing the somite, nephrotome, gut and in pancreatic and liver primordia. Antibody-mediated inhibition of decorin function affected the head-to-tail embryonic axis extension, indicating that decorin is essential for convergent extension cell movements during avian gastrulation. Decorin was also essential for retinal progenitor cell polarization, neural crest migration, somite boundary formation and cell polarization, mesenchymal cell polarization and primary endoderm displacement to the embryo periphery. The embryonic blood vessels were deformed, the dorsal mesocardium was thinned and the cardiac jelly was abnormally thickened in the heart. Decorin is known to modulate collagen fibrillogenesis, a key mechanism of matrix assembly, and cell proliferation. Decorin also appears to be essential for the coordination of cell and tissue polarization, which is an important feature in organ patterning of the embryo.

The potential of spatially resolved spectroscopy for monitoring angiogenesis in the chorioallantoic membrane.

Over the last decade, the poultry sector has sought to develop ways to monitor chicken embryonic development as to optimize the incubation conditions. One of the parameters of development which may change under different incubation conditions is the angiogenesis in the chorioallantoic membrane (CAM). To be able to quantify these changes in the angiogenesis and detect long-term effects on health, a non-destructive technique is necessary. In this article, the first steps toward such a non-destructive technique are successfully taken. A spatially resolved spectroscopy set-up is built and tested for its potential to measure changes in angiogenesis with incubation time, and differences between a normal and hypercapnic incubation. In this first study, reflectance measurements are performed directly on the CAM as the eggshell considerably complicates the analysis. This issue should be addressed in future research to come to a really non-destructive technique. An experiment was conducted in which one group was incubated under normal conditions, and another under early prenatal hypercapnic conditions (i.e., increased CO(2) concentrations). The angiogenesis in the CAM was measured at embryonic day (ED) 10, 13, and 16. The measurements showed a clear blood spectrum with an increasing amount of blood in time, and significant differences in the reflectance as function of the source-detector distances. However, no significant differences between the hypercapnia and the control group could be detected.

Yolk sac endoderm is the major source of serum proteins and lipids and is involved in the regulation of vascular integrity in early chick development.

An important function of the vascular system is nutrient delivery. In adult animals, this is mediated through a close contact of the mesoderm-derived vasculature with the endoderm-derived enterocytes and hepatocytes. During embryonic development, the yolk sac (YS) endoderm has been suggested to play a similar role. Physiological and molecular nature of the contact between the YS endoderm and the vasculature is not well-understood. To understand roles of the YS endoderm in early development, we used the avian model and carried out a gene expression profiling analysis of isolated area vasculosa YS endoderm tissues from embryonic day 2-4 chick embryos, covering the first 48 hr of postcirculation development. Genes involved in lipid metabolism are highly enriched, indicating an active modification of lipid components during their transfer from the yolk to the circulatory system. We also uncovered genes encoding major serum proteins and key regulators of vascular integrity. In particular, PTGDS, an enzyme controlling the last step of prostaglandin D2 production, shows high expression in the YS endoderm. Experimental introduction of prostaglandin D2 into embryonic circulation led to intraembryonic vessel rupture. These data suggest that the YS endoderm is the major, if not exclusive, source of lipid and protein constituents of the early embryonic serum and plays an important role in the regulation of vascular integrity in developing embryo.

Intravascular pillars and pruning in the extraembryonic vessels of chick embryos.

To investigate the local mechanical forces associated with intravascular pillars and vessel pruning, we studied the conducting vessels in the extraembryonic circulation of the chick embryo. During the development days 13-17, intravascular pillars and blood flow parameters were identified using fluorescent vascular tracers and digital time-series video reconstructions. The geometry of selected vessels was confirmed by corrosion casting and scanning electron microscopy. Computational simulations of pruning vessels suggested that serial pillars form along pre-existing velocity streamlines; blood pressure demonstrated no obvious spatial relationship with the intravascular pillars. Modeling a Reynolds number of 0.03 produced 4 pillars at approximately 20-µm intervals matching the observed periodicity. In contrast, a Reynolds number of 0.06 produced only 2 pillars at approximately 63-µm intervals. Our modeling data indicated that the combination of wall shear stress and gradient of shear predicted the location, direction, and periodicity of developing pillars.

Aortic remodelling during hemogenesis: is the chicken paradigm unique?

Since the era of the ancient Egyptians and Greeks, the avian embryo has been a subject of intense interest to visualize the first steps of development. It has served as a pioneer model to scrutinize the question of hematopoietic development from the beginning of the 20th century. It's large size and easy accessibility have permitted the development of techniques dedicated to following the origins and fates of different cell populations. Here, we shall review how the avian model has brought major contributions to our understanding of the development of the hematopoietic system in the past four decades and how these discoveries have influenced our knowledge of mammalian hematopoietic development. The discovery of an intra-embryonic source of hematopoietic cells and the developmental link between endothelial cells and hematopoietic cells will be presented. We shall then point to the pivotal role of the somite in the construction of the aorta and hematopoietic production and demonstrate how two somitic compartments cooperate to construct the definitive aorta. We shall finish by showing how fate-mapping experiments have allowed the identification of the tissue which gives rise to the sub-aortic mesenchyme. Taken together, this review aims to give an overview of how and to what extent the avian embryo has contributed to our knowledge of developmental hematopoiesis.

The quest for hematopoietic stem cells in the embryo. An interview with Françoise Dieterlen-Lièvre. Interview by Thierry Jaffredo and Charles Durand.

Françoise Dieterlen-Lièvre is probably the scientist who has most contributed to our basic knowledge of developmental hematopoiesis. She has dedicated her career to answering cutting edge questions on the origin of hematopoietic stem cells in the embryo. Her seminal contributions, widely recognized by the scientific community, have paved the way for generations of developmental hematologists questioning the origins of hematopoietic stem cells. After having demonstrated the intra-embryonic origin of hematopoietic stem cells, established the dual origin of the endothelial network in the embryo and revealed the hematopoietic function of the allantois in birds, she has switched to mammals and contributed to demonstrating that the aorta and allantois/placenta are new sites of hematopoietic production in the mouse embryo. The manifold insights generated by the pivotal work of Françoise Dieterlen-Lièvre have created multiple paradigm shifts which continue to challenge the field of developmental hematopoiesis.

Allantois and placenta as developmental sources of hematopoietic stem cells.

While the aortic region, the para-aortic splanchnopleura/aorta-gonads-mesonephros (P-Sp/AGM) is currently considered as the source of definitive hematopoietic stem cells during development, the mouse placenta has been found to generate large numbers of these cells and to remain functional in this respect for a longer period than the P-Sp/AGM. The fetal component, which derives from the fused allantois and chorion, is responsible for this activity. We and others have shown that the pre-fusion allantois (before the stage of 6 pairs of somites) is able to yield clonogenic progenitors, provided that it is pre-cultured in toto before it is dissociated into single cells and seeded in semi-solid medium. Thus placental hematopoiesis can be concluded to derive from intrinsic precursors. It is similar in this regard to the yolk sac which both produces hematopoietic progenitors and supports their multiplication and differentiation. Hematopoietic activity, detected by in vitro colony assays, has also been recently uncovered in the human placenta. According to the data available, this newly identified source probably provides a large number of HSC during development and must play a foremost role in founding the definitive hematopoietic system.

In vivo photoacoustic imaging of transverse blood flow by using Doppler broadening of bandwidth.

A method is proposed to measure transverse blood flow by using photoacoustic Doppler broadening of bandwidth. By measuring bovine blood flowing through a plastic tube, the linear dependence of the broadening on the flow speed was validated. The blood flow of the microvasculature in a mouse ear and a chicken embryo (stage 16) was also studied.

[Angiogenesis promoting effect of Morinda officinalis oligosaccharides on chicken embryo chorioallantoic membrane].

OBJECTIVE: To study the angiogenesis promoting effect of Morinda officinalis oligosaccharides(MOO) on chick embryo chorioallantoic membrane (CAM). METHOD: Rats blood serum containing low, medium and high doses of MOO was prepared using Chinese herbs serum pharmacology method. 60 chick embryoes were randomly divided into low, medium and high doses of MOO groups, as well as NS group, blank serum group and bFGF group. Each group included 10 embryoes. CAM model was prepared after 7 days incubation. Then NS, blank serum, bFGF (2500 U x mL(-1)), three doses of serum containing MOO were added respectively onto the carriers on the CAM. CAM sample was prepared after 3 days incubation. The state of angiogenesis was observed and the number of new blood vessels was counted. RESULT: Compared with blank serum and NS group, a more specific CAM angiogenesis appearance could be observed in each MOO group and bFGF group. Compared with blank serum group, the number of new blood vessels in each MOO group increased significantly (P < 0.05). But the drug had a lower efficacy than bFGF (P < 0.05). Compared with low dose group, the number of new blood vessels increased significantly in medium and high doses groups (P < 0.05). But there was no significant difference between the latter two groups. The number of new blood vessels showed no significant difference between NS group and blank serum group. CONCLUSION: MOO can obviously promote angiogenesis of CAM.

Hypoxia sensing in the fetal chicken femoral artery is mediated by the mitochondrial electron transport chain.

Vascular hypoxia sensing is transduced into vasoconstriction in the pulmonary circulation, whereas systemic arteries dilate. Mitochondrial electron transport chain (mETC), reactive O(2) species (ROS), and K(+) channels have been implicated in the sensing/signaling mechanisms of hypoxic relaxation in mammalian systemic arteries. We aimed to investigate their putative roles in hypoxia-induced relaxation in fetal chicken (19 days of incubation) femoral arteries mounted in a wire myograph. Acute hypoxia (Po(2) approximately 2.5 kPa) relaxed the contraction induced by norepinephrine (1 microM). Hypoxia-induced relaxation was abolished or significantly reduced by the mETC inhibitors rotenone (complex I), myxothiazol and antimycin A (complex III), and NaN(3) (complex IV). The complex II inhibitor 3-nitroproprionic acid enhanced the hypoxic relaxation. In contrast, the relaxations mediated by acetylcholine, sodium nitroprusside, or forskolin were not affected by the mETC blockers. Hypoxia induced a slight increase in ROS production (as measured by 2,7-dichlorofluorescein-fluorescence), but hypoxia-induced relaxation was not affected by scavenging of superoxide (polyethylene glycol-superoxide dismutase) or H(2)O(2) (polyethylene glycol-catalase) or by NADPH-oxidase inhibition (apocynin). Also, the K(+) channel inhibitors tetraethylammonium (nonselective), diphenyl phosphine oxide-1 (voltage-gated K(+) channel 1.5), glibenclamide (ATP-sensitive K(+) channel), iberiotoxin (large-conductance Ca(2+)-activated K(+) channel), and BaCl(2) (inward-rectifying K(+) channel), as well as ouabain (Na(+)-K(+)-ATPase inhibitor) did not affect hypoxia-induced relaxation. The relaxation was enhanced in the presence of the voltage-gated K(+) channel blocker 4-aminopyridine. In conclusion, our experiments suggest that the mETC plays a critical role in O(2) sensing in fetal chicken femoral arteries. In contrast, hypoxia-induced relaxation appears not to be mediated by ROS or K(+) channels.