Ultra high frequency ultrasound enables real-time visualization of blood supply from chorioallantoic membrane to human autosomal dominant polycystic kidney tissue.

Ultra high frequency (UHF) ultrasound enables the visualization of very small structures that cannot be detected by conventional ultrasound. The utilization of UHF imaging as a new imaging technique for the 3D-in-vivo chorioallantoic membrane (CAM) model can facilitate new insights into tissue perfusion and survival. Therefore, human renal cystic tissue was grafted onto the CAM and examined using UHF ultrasound imaging. Due to the unprecedented resolution of UHF ultrasound, it was possible to visualize microvessels, their development, and the formation of anastomoses. This enabled the observation of anastomoses between human and chicken vessels only 12 h after transplantation. These observations were validated by 3D reconstructions from a light sheet microscopy image stack, indocyanine green angiography, and histological analysis. Contrary to the assumption that the nutrient supply of the human cystic tissue and the gas exchange happens through diffusion from CAM vessels, this study shows that the vasculature of the human cystic tissue is directly connected to the blood vessels of the CAM and perfusion is established within a short period. Therefore, this in-vivo model combined with UHF imaging appears to be the ideal platform for studying the effects of intravenously applied therapeutics to inhibit renal cyst growth.

Improved Ultrasound Localization Microscopy Based on Microbubble Uncoupling via Transmit Excitation.

Ultrasound localization microscopy (ULM) demonstrates great potential for visualization of tissue microvasculature at depth with high spatial resolution. The success of ULM heavily depends on robust localization of isolated microbubbles (MBs), which can be challenging in vivo especially within larger vessels where MBs can overlap and cluster close together. While MB dilution alleviates the issue of MB overlap to a certain extent, it drastically increases the data acquisition time needed for MBs to populate the microvasculature, which is already on the order of several minutes using recommended MB concentrations. Inspired by optical super-resolution imaging based on stimulated emission depletion (STED), here we propose a novel ULM imaging sequence based on MB uncoupling via transmit excitation (MUTE). MUTE "silences" MB signals by creating acoustic nulls to facilitate MB separation, which leads to robust localization of MBs especially under high concentrations. The efficiency of localization accomplished via the proposed technique was first evaluated in simulation studies with conventional ULM as a benchmark. Then, an in-vivo study based on the chorioallantoic membrane (CAM) of chicken embryos showed that MUTE could reduce the data acquisition time by half, thanks to the enhanced MB separation and localization. Finally, the performance of MUTE was validated in an in vivo mouse brain study. These results demonstrate the high MB localization efficacy of MUTE-ULM, which contributes to a reduced data acquisition time and improved temporal resolution for ULM.

Utilisation of Chick Embryo Chorioallantoic Membrane as a Model Platform for Imaging-Navigated Biomedical Research.

The fertilised chick egg and particularly its chorioallantoic membrane (CAM) have drawn continuing interest in biomedicine and bioengineering fields, especially for research on vascular study, cancer, drug screening and development, cell factors, stem cells, etc. This literature review systemically introduces the CAM's structural evolution, functions, vascular features and the circulation system, and cell regulatory factors. It also presents the major and updated applications of the CAM in assays for pharmacokinetics and biodistribution, drug efficacy and toxicology testing/screening in preclinical pharmacological research. The time course of CAM applications for different assays and their advantages and limitations are summarised. Among these applications, two aspects are emphasised: (1) potential utility of the CAM for preclinical studies on vascular-disrupting agents (VDAs), promising for anti-cancer vascular-targeted therapy, and (2) modern imaging technologies, including modalities and their applications for real-time visualisation, monitoring and evaluation of the changes in CAM vasculature as well as the interactions occurring after introducing the tested medical, pharmaceutical and biological agents into the system. The aim of this article is to help those working in the biomedical field to familiarise themselves with the chick embryo CAM as an alternative platform and to utilise it to design and optimise experimental settings for their specific research topics.

Effects of topical prostaglandin drops on angiogenesis in an in ovo chick chorioallantoic membrane model.

BACKGROUND: To investigate the effects of bimatoprost, latanoprost and travoprost on angiogenesis in the chick chorioallantoic membrane (CAM) model in ovo. MATERIALS AND METHODS: Fifty fertilized specific-pathogen-free chick eggs were used in this preclinical, prospective, experimental embryo study. Eggs were randomly distributed into 5 groups of ten eggs. Eggs were placed in the incubator after disinfection of their shells with alcohol and monitored appropriate temperature and humidity. On the 3rd day of incubation, a small window was opened on the eggshell. Bimatoprost in group 1, latanoprost in group 2, travoprost in group 3, bevacizumab in group 4, phosphate-buffered-saline (PBS) used in group 5 was applied by injection to CAM. The sterile film was glued onto the broken part of the shell and the eggs were placed in the incubator again. On the 8th day of incubation, eggs were opened and vascular structures on CAMs were examined. Digital photographs were taken, analysed in the ImageJ open source image processing software and differences between groups were evaluated. Thereafter, VEGF (Vascular endothelial growth factor) levels were measured appropriately in the embryo samples. RESULTS: All embryos in the prostaglandin groups and the PBS control group were observed to have life signs confirmed by heart rate. In 8 embryos in the bevacizumab group, no life signs were confirmed, while 2 embryos with life signs showed severe hypoplasia. Vascular density, number of vessels and VEGF levels in the bimatoprost, latanoprost and travoprost groups, there were statistically significantly higher than the PBS control group. CONCLUSION: This study demonstrates that topical prostaglandin drops increase angiogenesis in the chick CAM model in ovo.

Tumor grafts grown on the chicken chorioallantoic membrane are distinctively characterized by MRI under functional gas challenge.

Recently, a tumor model based on the chorioallantoic membrane (CAM) was characterized structurally with Magnetic Resonance Imaging (MRI). Yet, capability of MRI to assess vascular functional reserve and potential of oxygenation-sensitive MRI remain largely unexplored in this model. For this purpose, we compared MC-38 colon and A549 lung adenocarcinoma cell grafts grown on the CAM, using quantitative T1 and T2* MRI readouts as imaging markers. These are associated with vascular functionality and oxygenation status when compared between periods of air and carbogen exposure. Our data show that in A549 lung adenocarcinoma cell grafts T2* values increased significantly upon carbogen exposure (p < 0.004, Wilcoxon test; no change in T1), while MC-38 grafts displayed no changes in T1 and T2*), indicating that the grafts differ in their vascular response. Heterogeneity with regard to T1 and T2* distribution within the grafts was noted. MC-38 grafts displayed larger T1 and T2* in the graft centre, while in A549 they were distributed more towards the graft surface. Finally, qualitative assessment of gadolinium-enhancement suggests that A549 grafts display more prominent enhancement compared to MC-38 grafts. Furthermore, MC-38 grafts had 65% larger volumes than A549 grafts. Histology revealed distinct underlying phenotypes of the two tumor grafts, pertaining to the proliferative status (Ki-67) and cellularity (H&E). In sum, a functional gas challenge with carbogen is feasible through gas exchange on the CAM, and it affects MRI signals associated with vascular reactivity and oxygenation status of the tumor graft planted on the CAM. Different grafts based on A549 lung adenocarcinoma and MC-38 colon carcinoma cell lines, respectively, display distinct phenotypes that can be distinguished and characterized non-invasively in ovo using MRI in the living chicken embryo.

In Vivo Confocal Imaging of Fluorescently Labeled Microbubbles: Implications for Ultrasound Localization Microscopy.

We report the time kinetics of fluorescently labeled microbubbles (MBs) in capillary-level microvasculature as measured via confocal microscopy and compare these results to ultrasound localization microscopy (ULM). The observed 19.4 ± 4.2 MBs per confocal field-of-view ( [Formula: see text]) are in excellent agreement with the expected count of 19.1 MBs per frame. The estimated time to fully perfuse this capillary network was 193 s, which corroborates the values reported in the literature. We then modeled the capillary network as an empirically determined discrete-time Markov chain with adjustable MB transition probabilities though individual capillaries. The Monte Carlo random walk simulations found perfusion times ranging from 24.5 s for unbiased Markov chains up to 182 s for heterogeneous flow distributions. This pilot study confirms a probability-derived explanation for the long acquisition times required for super-resolution ULM.

The CAM cancer xenograft as a model for initial evaluation of MR labelled compounds.

Non-invasive assessment of the biodistribution is of great importance during the development of new pharmaceutical compounds. In this contribution, the applicability of in ovo MRI for monitoring the biodistribution of MR contrast agent-labelled compounds was investigated in mamaria carcinomas xentotransplanted on the chorioallantoic membrane (CAM) exemplarily for Gd-DOTA and cHSA-PEO (2000)16-Gd after systemic injection of the compounds into a chorioallantoic capillary vein. MRI was performed directly prior and 30 min, 3 h, 5 h, 20 h, and 40 h after injection of the compound. The biodistribution of injected compounds could be assessed by MRI in different organs of the chicken embryo as well as in xenotransplanted tumors at all time points. A clearly prolonged enhancement of the tumor substrate could be shown for cHSA-PEO (2000)16-Gd. In conclusion, high-resolution in ovo MR imaging can be used for assessment of the in vivo biodistribution of labelled compounds, thus enabling efficient non-invasive initial testing.

High Resolution Ultrasound Superharmonic Perfusion Imaging: In Vivo Feasibility and Quantification of Dynamic Contrast-Enhanced Acoustic Angiography.

Mapping blood perfusion quantitatively allows localization of abnormal physiology and can improve understanding of disease progression. Dynamic contrast-enhanced ultrasound is a low-cost, real-time technique for imaging perfusion dynamics with microbubble contrast agents. Previously, we have demonstrated another contrast agent-specific ultrasound imaging technique, acoustic angiography, which forms static anatomical images of the superharmonic signal produced by microbubbles. In this work, we seek to determine whether acoustic angiography can be utilized for high resolution perfusion imaging in vivo by examining the effect of acquisition rate on superharmonic imaging at low flow rates and demonstrating the feasibility of dynamic contrast-enhanced superharmonic perfusion imaging for the first time. Results in the chorioallantoic membrane model indicate that frame rate and frame averaging do not affect the measured diameter of individual vessels observed, but that frame rate does influence the detection of vessels near and below the resolution limit. The highest number of resolvable vessels was observed at an intermediate frame rate of 3 Hz using a mechanically-steered prototype transducer. We also demonstrate the feasibility of quantitatively mapping perfusion rate in 2D in a mouse model with spatial resolution of ~100 µm. This type of imaging could provide non-invasive, high resolution quantification of microvascular function at penetration depths of several centimeters.

Ex Vivo Porcine Arterial and Chorioallantoic Membrane Acoustic Angiography Using Dual-Frequency Intravascular Ultrasound Probes.

The presence of blood vessels within a developing atherosclerotic plaque has been found to be correlated with increased plaque vulnerability and ensuing cardiac events, however, detection of coronary intraplaque neovascularization poses a significant challenge in the clinic. We describe here a new in vivo intravascular ultrasound imaging method using a dual-frequency transducer to visualize contrast flow in microvessels with high specificity. This method uses a specialized transducer capable of exciting contrast agents at a low frequency (5.5 MHz) while detecting their nonlinear superhamonics at a much higher frequency (37 MHz). In vitro evaluation of the approach was performed in a microvascular phantom to produce 3-D renderings of simulated vessel patterns and to determine image quality metrics as a function of depth. Furthermore, we describe the ability of the system to detect microvessels both ex vivo using porcine arteries and in vivo using the chorioallantoic membrane of a developing chicken embryo with optical confirmation. Dual-frequency contrast-specific imaging was able to resolve vessels similar in size to those found in vulnerable atherosclerotic plaques at clinically relevant depths. The results of this study add to the support for further evaluation and translation of contrast-specific imaging in intravascular ultrasound for the detection of vulnerable plaques in atherosclerosis.

Methods for Analyzing Tumor Angiogenesis in the Chick Chorioallantoic Membrane Model.

Models of tumor angiogenesis have played a critical role in understanding the mechanisms involved in the recruitment of vasculature to the tumor mass, and have also provided a platform for testing antiangiogenic potential of new therapeutics that combat the development of malignant growth. In this regard, the chorioallantoic membrane (CAM) of the developing chick embryo has proven to be an elegant model for investigation of angiogenic processes. Here, we describe methods for effectively utilizing the preestablished vascular network of the chick CAM to investigate and quantify tumor-associated angiogenesis in a breast tumor model.

Noncontact photoacoustic tomography of in vivo chicken chorioallantoic membrane based on all-fiber heterodyne interferometry.

We present three-dimensional (3-D) in vivo photoacoustic (PA) images of the blood vasculature of a chicken chorioallantoic membrane (CAM) obtained by using a fiber-based noncontact PA tomography system. With a fiber-optic heterodyne interferometer, the system measures the surface displacement of a sample, induced by the PA wave, which overcomes the disadvantage of physical-contact of ultrasonic transducer in a conventional system. The performance of an implemented system is analyzed and its capability of in vivo 3-D bioimaging is presented. At a depth of 2.5 mm in a phantom experiment, the lateral and axial resolutions were measured as 100 and 30 µm, respectively. The lateral resolution became doubled at a depth of 7.0 mm; however, interestingly, the axial resolution was not noticeably deteriorated with the depth. With the CAM experiment, performed under the American National Standards Institute laser safety standard condition, blood vessel structures placed as deep as 3.5 mm were clearly recognized.

A new in vivo magnetic resonance imaging method to noninvasively monitor and quantify the perfusion capacity of three-dimensional biomaterials grown on the chorioallantoic membrane of chick embryos.

Adequate vascularization in biomaterials is essential for tissue regeneration and repair. Current models do not allow easy analysis of vascularization of implants in vivo, leaving it a highly desirable goal. A tool that allows monitoring of perfusion capacity of such biomaterials noninvasively in a cheap, efficient, and reliable in vivo model would hence add great benefit to research in this field. We established, for the first time, an in vivo magnetic resonance imaging (MRI) method to quantify the perfusion capacity of a model biomaterial, DegraPol(®) foam scaffold, placed on the embryonic avian chorioallantoic membrane (CAM) in ovo. Perfusion capacity was assessed through changes in the longitudinal relaxation rate before and after injection of a paramagnetic MRI contrast agent, Gd-DOTA (Dotarem(®); Guerbet S.A.). Relaxation rate changes were compared in three different regions of the scaffold, that is, at the interface to the CAM, in the middle and on the surface of the scaffold (p<0.05). The highest relaxation rate changes, and hence perfusion capacities, were measured in the interface region where the scaffold was attached to the CAM, whereas the surface of the scaffold showed the lowest relaxation rate changes. A strong positive correlation was obtained between relaxation rate changes and histologically determined vessel density (R(2) = 0.983), which corroborates our MRI findings. As a proof-of-principle, we measured the perfusion capacity in different scaffold materials, silk fibroin either with or without human dental pulp stem cells. For these, three to four times larger perfusion capacities were obtained compared to DegraPol; demonstrating that our method is sensitive to reveal such differences. In summary, we present a novel in vivo method for analyzing the perfusion capacity in three-dimensional-biomaterials grown on the CAM, enabling the determination of the perfusion capacity of a large variety of bioengineered materials.

In vivo PET/CT in a human glioblastoma chicken chorioallantoic membrane model: a new tool for oncology and radiotracer development.

UNLABELLED: For many years the laboratory mouse has been used as the standard model for in vivo oncology research, particularly in the development of novel PET tracers, but the growth of tumors on chicken chorioallantoic membrane (CAM) provides a more rapid, low cost, and ethically sustainable alternative. For the first time, to our knowledge, we demonstrate the feasibility of in vivo PET and CT imaging in a U87 glioblastoma tumor model on chicken CAM, with the aim of applying this model for screening of novel PET tracers. METHODS: U87 glioblastoma cells were implanted on the CAM at day 11 after fertilization and imaged at day 18. A small-animal imaging cell was used to maintain incubation and allow anesthesia using isoflurane. Radiotracers were injected directly into the exposed CAM vasculature. Sodium (18)F-fluoride was used to validate the imaging protocol, demonstrating that image-degrading motion can be removed with anesthesia. Tumor glucose metabolism was imaged using (18)F-FDG, and tumor protein synthesis was imaged using 2-(18)F-fluoro-l-tyrosine. Anatomic images were obtained by contrast-enhanced CT, facilitating clear delineation of the tumor, delineation of tracer uptake in tumor versus embryo, and accurate volume measurements. RESULTS: PET imaging of tumor glucose metabolism and protein synthesis was successfully demonstrated in the CAM U87 glioblastoma model. Catheterization of CAM blood vessels facilitated dynamic imaging of glucose metabolism with (18)F-FDG and demonstrated the ability to study PET tracer uptake over time in individual tumors, and CT imaging improved the accuracy of tumor volume measurements. CONCLUSION: We describe the novel application of PET/CT in the CAM tumor model, with optimization of typical imaging protocols. PET imaging in this valuable tumor model could prove particularly useful for rapid, high-throughput screening of novel radiotracers.

Enhancement of vascular permeability with low-frequency contrast-enhanced ultrasound in the chorioallantoic membrane model.

PURPOSE: To characterize the effect of low-frequency contrast material-enhanced ultrasound on the vascular endothelium and to determine the parameters and techniques required to deliver a therapeutic agent by using the chorioallantoic membrane (CAM) model. MATERIALS AND METHODS: All in vivo animal procedures were conducted with institutional Animal Care and Use Committee approval. Extravasation of 8.5-nm-diameter fluorescein isothiocyanate-labeled dextran was evaluated in the vasculature of a chick CAM model. Intravital microscopy was performed during contrast-enhanced ultrasound exposure (1.00 or 2.25 MHz); results were compared with results of electron microscopy of the insonated regions. Data acquired after insonation with greater mechanical stress (n = 30 animals) (mechanical index [MI] > 1.3) and with lower mechanical stress (n = 86 animals) (MI < 1.13) were compared with measurements in control conditions (n = 46 animals). The diameter of affected vessels; number of extravasation sites; extravasation rate, area, and location; and changes in endothelial cells and basement membrane were evaluated. Differences were tested with analysis of variance or the Student t test. RESULTS: After ultrasound application, convective transport of the model drug was observed through micron-sized openings with a mean fluid velocity of 188.6 microm/sec in the low-stress class and 362.5 microm/sec in the high-stress class. Electron microscopy revealed micron-sized focal endothelial gaps and disseminated blebs, vacuoles, and filopodia extending across tens of microns. The threshold pressure for extravasation was 0.5 MPa for a transmitted center frequency of 1.00 MHz (MI = 0.5) and 1.6 MPa for a frequency of 2.25 MHz (MI = 1.06); thus, the frequency dependence of the threshold was not predicted simply by the MI. CONCLUSION: Low-frequency contrast-enhanced ultrasound can increase vascular permeability and result in convective extravasation of an 8.5-nm-diameter model drug.