Name: novel in vivo micro-computed tomography imaging techniques for assessing the progression of non-alcoholic fatty liver disease
Uploaded: 2023-03-24T13:25:00
Description: Watch this Scientific Journal Video about Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease at JoVE.com

Figure 1 was created with BioRender.com. This work was supported by the Hellenic Foundation for Research and Innovation (#3222 to A.C.). Anna Hadjihambi is funded by The Roger Williams Institute of Hepatology, Foundation for Liver Research.

All procedures were carried out by BIOEMTECH&#39;s personnel in accordance with European and national welfare regulations and were approved by national authorities (license number EL 25 BIOexp 45/PN 49553 21/01/20). All experiments were designed and reported with adherence to ARRIVE guidelines26. The mice were purchased from the Hellenic Pasteur Institute, Athens, Greece.
NOTE: Animals were group-housed in individually ventilated cages enriched with rails and cardboard ...

The current recommended method for NAFLD diagnosis and staging in humans is liver biopsy, which harbors the risk of bleeding complexities, as well as sampling inaccuracies40. On the contrary, in animal models, such diagnosis is performed by histology post-mortem, although protocols for survivable liver biopsy are now available and are recommended when the study design allows41. The use of post-mortem histology means that a large number of animals are required to investigate...

Non-alcoholic fatty liver disease (NAFLD) is a metabolic disease that affects approximately 25% of the population and &#62;80% of morbidly obese people1. An estimated one-third of these individuals progress to non-alcoholic steatohepatitis (NASH), which is characterized by hepatic steatosis, inflammation, and fibrosis2. NASH is a disease stage with a significantly higher risk for the development of cirrhosis and hepatocellular carcinoma (HCC)3,4. For this reason, NASH is currently the second most common cause of liver transplantation, and it is also expected to soon become the most important predictor of liver transplantation5,6,7. Despite its prevalence and severity, no disease-specific therapy is available for NAFLD, and the existing treatments only aim to tackle disease-associated pathologies such as insulin resistance and hyperlipidemia5,6.
In recent years, the pathophysiological role and adaptations of the endothelium and, in general, of the vascular network of metabolic tissues, such as the adipose tissue and the liver, have been gaining more importance in research, especially during obesity and metabolic dysregulation7,8. The endothelium is a cellular monolayer that lines the vascular network internally, acting as a functional and structural barrier. It also contributes to various physiological and pathological processes, such as thrombosis, metabolite transport, inflammation, and angiogenesis9,10. In the case of the liver, the vascular network is, among other features, characterized by the presence of highly specialized cells, defined as liver sinusoidal endothelial cells (LSECs). These cells lack a basement membrane and have multiple fenestrae, allowing for the easier transfer of substrates between the blood and liver parenchyma. Due to their distinctive anatomical location and characteristics, LSECs likely have a crucial role in the pathophysiological processes of the liver, including the development of liver inflammation and fibrosis during NAFLD/NASH. Indeed, the pathological, molecular, and cellular adaptations that LSECs undergo in the course of NAFLD contribute to the disease progression11. Specifically, the LSEC-dependent hepatic angiogenesis that takes place during NAFLD is significantly associated with the development of inflammation and the progression of the disease to NASH or even HCC12. Besides, obesity-related early NAFLD is characterized by the development of insulin resistance in LSECs, which precedes the development of hepatic inflammation or other advanced NAFLD signs13.
Additionally, LSECs have recently emerged as central regulators of hepatic blood flow and vascular network adaptations during liver disease of several etiologies14,15. Indeed, chronic liver disease is characterized by prominent intra-hepatic vasoconstriction and increased resistance to blood flow, which contribute to the development of portal hypertension16. In the case of NAFLD, several LSEC-related mechanisms contribute to this phenomenon. For instance, LSEC-specific insulin resistance, as mentioned above, is associated with reduced insulin-dependent vasodilation of the hepatic vasculature13. Besides, over the course of the disease, the liver vasculature becomes more sensitive to vasoconstrictors, further contributing to impairment of the hepatic blood flow and leading to the emergence of shear stress, which both result in a disruption of the sinusoidal microcirculation17. These facts suggest that the vasculature is a key target in liver disease. Nevertheless, limiting factors hindering the timely diagnosis and monitoring of NAFLD/NASH, as well as the development of potential therapies, are the inadequacies in the consistent characterization of the hepatic microenvironment and (micro)vascular structure, as well as the scoring of the disease stage in a spatiotemporal and non-invasive manner.
Micro-computed tomography (CT) imaging is currently the gold-standard non-invasive imaging method for accurately depicting anatomical information within a living organism. Micro-CT and MRI represent two complementary imaging methods that can cover a vast range of pathologies and provide exceptional resolution and detail in the imaged structures and tissues. Micro-CT, in particular, is a very fast and accurate tool that is often used for studying pathologies such as bone diseases and associated bone surface changes18, assessing the progression of pulmonary fibrosis over time19, diagnosing lung cancer and its staging20, or even examining dental pathologies21, without any special preparation (or destruction) of the samples being imaged.
The imaging technology of micro-CT is based on the different attenuation properties of various organs in terms of the interaction of X-rays with matter. Organs presenting high X-ray attenuation differences are depicted with high contrast in CT images (i.e., the lungs appear dark and the bones light). Organs presenting very similar attenuation properties (different soft tissues), are challenging to distinguish on CT images22. To address this limitation, specialized contrast agents based on iodine, gold, and bismuth have been extensively investigated for in vivo use. These agents alter the attenuation properties of the tissues in which they accumulate, are cleared slowly from the circulation, and enable the uniform and stable opacification of the entire vascular system or chosen tissues23.
In human diagnostics, CT imaging and comparable techniques, such as MRI-derived proton density fat fraction, are already in use for the determination of hepatic fat content24,25. In the context of NAFLD, high soft tissue contrast is essential to accurately distinguish pathological lesions or small vessels. For this purpose, contrast agents providing enhanced contrast of the liver tissue characteristics are utilized. Such tools and materials allow for the study of multiple liver characteristics and possible pathology expressions, such as the architecture and density of the vascular network, lipid deposition/steatosis, and functional tissue uptake/lipid (chylomicron) transfer in the liver. Additionally, hepatic relative blood volume and portal vein diameter can also be evaluated. In a very short scan time, all these parameters provide different and complementary information on the evaluation and progression of NAFLD, which can be used to develop a non-invasive and detailed diagnosis.
In this article, we provide a step-by-step protocol for the use of novel in vivo micro-CT imaging techniques as a non-invasive method to assess the progression stages of NAFLD. Using this protocol, the longitudinal analysis of liver steatosis and functional tissue uptake, as well as the evaluation of the relative blood volume, portal vein diameter, and density of the vascular network, can be performed and applied in mouse models of liver disease.

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			<td>eXIA160</td>
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			<td>High fat diet with 60% of kilocalories from fat</td>
			<td>Research Diets, New Brunswick, NJ, USA</td>
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			<td>High-fructose corn syrup&#160;</td>
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			<td>Lacrinorm ophthalmic ointment&#160;</td>
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			<td>Normal diet with 10% of kilocalories from fat&#160;</td>
			<td>Research Diets, New Brunswick, NJ, USA</td>
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			<td>Viscover ExiTron nano 12000&#160;</td>
			<td>Milteny Biotec, Bergisch Gladbach, Germany</td>
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novel in vivo micro-computed tomography imaging techniques for assessing the progression of non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is a growing global health issue, and the impact of NAFLD is compounded by the current lack of effective treatments. Considerable limiting factors hindering the timely and accurate diagnosis (including grading) and monitoring of NAFLD, as well as the development of potential therapies, are the current inadequacies in the characterization of the hepatic microenvironment structure and the scoring of the disease stage in a spatiotemporal and non-invasive manner. Using a diet-induced NAFLD mouse model, we investigated the use of in vivo micro-computed tomography (CT) imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation. This imaging methodology allows for longitudinal analysis of liver steatosis and functional tissue uptake, as well as the evaluation of the relative blood volume, portal vein diameter, and density of the vascular network. Understanding the adaptations of the hepatic vascular network during NAFLD progression and correlating this with other ways of characterizing the disease progression (steatosis, inflammation, fibrosis) using the proposed method can pave the way toward the establishment of new, more efficient, and reproducible approaches for NAFLD research in mice. This protocol is also expected to upgrade the value of preclinical animal models for investigating&#160;the development of novel therapies against disease progression.

In this representative study, micro-CT imaging without any contrast agent indicated a higher percentage of liver fat in mice with NAFLD compared to controls (Table 2), confirming the pathology. Using the ExiTron contrast agent and the hepatic vascular network architecture and density analysis described above, the total volume density of the hepatic vascular network was found to be higher in mice with NAFLD compared to healthy controls (Figure 6, Table 2). Mi...

Watch this Scientific Journal Video about Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease at JoVE.com

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Using a diet-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we describe the use of novel in vivo micro-computed tomography imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation.

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is a growing global health issue, and the impact of NAFLD is compounded by the current lack of effective ...

We consider this protocol significant because it demonstrates a non-invasive simple and easy method to assess over time key indicators for the diagnosis of liver disease. This imaging methodology allows for analysis over time of liver statuses, evaluation of the relative blood volume, portal vein diameter, intensity of the vascular network, all important parameters of liver pathology. Understanding the adaptations of the hepatic vascular network during NAFLD progression and correlating this with specific markers such as steatosis, inflammation, and fibrosis can help establish new efficient therapy schemes.This technique aids in the identification of such markers and also upgrades the value of mice in preclinical studies focusing on the development of novel therapies against disease progression. To begin, place the anesthetized animal in the CT scanner cradle. Apply ophthalmic ointment, secure the nose cone, and set the scanning parameters on the CT scanner.Prepare the tail vein catheter and place the mouse on a bed to warm the tail in warm water. Insert the catheter into the tail vein and administer the first contrast agent via an injection, performed slowly and manually with a duration of one to three minutes. Then, acquire whole body and liver scans at different time points.Follow the same procedure for the administration of the second contrast agent, 10 days following the final reading with the first contrast agent. After loading the DICOM file of the pre-contrast scan, adjust the contrast to see the liver, spleen, and white adipose tissue clearly. Under the 3D ROI tool, select add ROI to generate multiple ROIs to perform sampling in the areas where the liver, spleen, and adipose tissue appear clear with no apparent blood vessels and fat.Under paint mode, choose Sphere. Tick the box 2D only and select a diameter of eight pixels from the dropdown menu to paint ROI over data. From the erode dilate feature, choose minus one erode.Perform sampling by segmenting the 2D ROIs on the areas of interest using the Sphere tool on the transverse plane. Go to navigation and select show table to display the quantification table containing the calculated hounds field unit values for each ROI. Then, plug the values into this equation.Calculate the percentage of liver fat. Load the eXIA scan DICOM file, and adjust the contrast to see the liver, spleen, and left ventricle clearly. Under the 3D ROI tool, select add ROI to segment multiple ROIS for the liver.Under paint mode, select 2D only, and choose Sphere add a diameter of eight pixels to paint ROI over data. From the erode dilate feature, choose minus one erode. Specify a name and color for each ROI and select show table to display the quantification table containing the calculated HU values for each ROI.After repeating these steps to obtain PV organ t0, insert the values into the equation shown on the screen to extract the percentage contrast corresponding to the functional tissue uptake lipid transfer. Load the ExiTron scan DICOM file, and adjust the contrast to see the liver vascular network clearly. Select add ROI to generate a 3D ROI for the liver.Define the liver ROI by marking the desired area. Then choose the background ROI from the ROI selector. Click on the Perform Cut icon, to remove the background without changing the liver ROI.Activate the maximum intensity projection viewer. Then, under segmentation algorithms denoted by the magic wand icon, select connected thresholding to resegment the liver ROI. Set the thresholds by entering the minimum and maximum values and obtain the vascular network.Again, remove the background liver tissue to get a clear representation of the vascular network. After loading the ExiTron scan DICOM file, adjust the contrast to see the liver, spleen, and left ventricle clearly. Select add ROI and segment ROIs for the liver and a large blood vessel.Define the segmentation layers to generate 2D ROIs for each tissue. Repeat the steps for the pre-contrast DICOM file to obtain the mean brightness of the liver before the contrast agent injection. For the portal vein diameter, locate the transversal planes of three to four slices above the junction of the superior mesenteric and splenic veins, and then go to navigation followed by distance sanitation, select line to measure the exact distance between two points.The average contrast values are presented by this grouped data. The functional tissue uptake assay indicated a higher accumulation and slower clearance of the eXIA contrast agent in mice with non-alcoholic fatty liver disease compared to healthy controls. These representative results indicate the differences in the percentage of liver fat, hepatic vascular network volume, portal vein diameter, and hepatic relative blood volume between mice with non-alcoholic fatty liver disease and healthy controls.Micro-CT imaging with without any contrast agent, indicated a higher percentage of liver fat in mice with non-alcoholic fatty liver disease compared to controls. Similarly, a higher hepatic vascular network volume, hepatic relative blood volume, and a larger portal vein diameter were found in mice with non-alcoholic fatty liver disease compared to healthy controls. The most important thing to remember while attempting this technique is to constantly be accurate on the anatomical region selected.Choosing the wrong anatomical spot can lead in non accurate results. By following this methodology and protocol, an accurate evaluation of liver disease staging can be performed. Thus, it can act as a specified platform for evaluating new drug efficiency.This technique paves the way for new, easier, and more accurate ways to evaluate liver pathologies and initiate relevant drug development assays.

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Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography (Micro-CT) Imaging Method

Reports show that prolonged exposure to a spaceflight environment produces morphologic and functional ophthalmic changes in astronauts during and after an International Space Station (ISS) mission. However, the underlying mechanisms of these spaceflight-induced changes are currently unknown. The purpose of the present study was to determine the impact of the spaceflight environment on ocular structures by evaluating the thickness of the mouse retina, the retinal pigment epithelium (RPE), the choroid and the sclera layer using micro-CT imaging. Ten-week-old C57BL/6 male mice were housed aboard the ISS for a 35-day mission and then returned to Earth alive for tissue analysis. For comparison, ground control (GC) mice on Earth were maintained in identical environmental conditions and hardware. Ocular tissue samples were collected for micro-CT analysis within 38(&#177;4) hours after splashdown. The images of the cross-section of the retina, the RPE, the choroid, and the sclera layer of the fixed eye was recorded in an axial and sagittal view using a micro-CT imaging acquisition method. The micro-CT analysis showed that the cross-section areas of the retina, RPE, and choroid layer thickness were changed in spaceflight samples compared to GC, with spaceflight samples showing significantly thinner cross-sections and layers compared to controls. The findings from this study indicate that micro-CT evaluation is a sensitive and reliable method to characterize ocular structure changes. These results are expected to improve the understanding of the impact of environmental stress on global ocular structures.

Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography Micro-CT Imaging Method

We present a protocol using high-resolution micro-computed tomography imaging to determine whether spaceflight induced damage on ocular structures. The protocol shows the micro-CT-derived measurement of ex vivo rodent ocular structures. We demonstrate the ability to assess ocular morphological changes following spaceflight using a non-destructive tridimensional technique to evaluate ocular damage.

Reports show that prolonged exposure to a spaceflight environment produces morphologic and functional ophthalmic changes in astronauts during and after an ...

Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro

DILI is a major cause of attrition in drug development with over 1000 FDA-approved drugs known to potentially cause DILI in humans. Unfortunately, DILI is often not detected until drugs have reached clinical stages, risking patients' safety and leading to substantial losses for the pharma industry. Taking into account that standard 2D models have limitations in detecting DILI it is essential to develop in vitro models that are more predictive to improve data translatability. To understand causality and mechanistic aspects of DILI in detail, a human liver MPS consisting of human primary liver parenchymal and non-parenchymal cells (NPCs) and cultured in 3D microtissues on an engineered scaffold under perfusion has been developed. Cryopreserved primary human hepatocytes (PHHs) and Kupffer cells (HKCs) were cocultured as microtissues in the MPS platform for up to two weeks, and each compound of interest was repeatably dosed onto liver microtissues at seven test concentrations for up to four days. Functional liver-specific endpoints were analyzed (including clinical biomarkers such as alanine aminotransferase, ALT) to evaluate liver function. Acute and chronic exposure to compounds of various DILI severities can be assessed by comparing responses to single and multi-dosed microtissues. The methodology has been validated with a broad set of severe and mildly hepatotoxic compounds. Here we show the data for pioglitazone and troglitazone, well-known hepatotoxic compounds withdrawn from the market for causing liver failures. Overall, it has been shown that the liver MPS model can be a useful tool to assess DILI and its association with changes in hepatic function. The model can additionally be used to assess how novel compounds behave in distinct patient subsets and how toxicity profiles may be affected by liver disease states (e.g., viral hepatitis, fatty liver disease).

Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro

Drug-induced liver injury (DILI) is a major cause of drug failure. A protocol has been developed to accurately predict the DILI liability of a compound using a liver microphysiological system (MPS). The liver model uses the coculture of primary hepatic cells and translationally relevant endpoints to assess cellular responses to treatment.

DILI is a major cause of attrition in drug development with over 1000 FDA-approved drugs known to potentially cause DILI in humans. Unfortunately, DILI is ...

Assessment of Bone Fracture Healing Using Micro-Computed Tomography

Micro-computed tomography (&#181;CT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed bone during fracture healing in translational science investigations. Studies of long bone fracture healing in rodents typically involve secondary healing and the formation of a mineralized callus. The shape of the callus formed and the density of the newly formed bone may vary substantially between timepoints and treatments. Whereas standard methodologies for quantifying parameters of intact cortical and trabecular bone are widely used and embedded in commercially available software, there is a lack of consensus on procedures for analyzing the healing callus. The purpose of this work is to describe a standardized protocol that quantitates bone volume fraction and callus mineral density in the healing callus. The protocol describes different parameters that should be considered during imaging and analysis, including sample alignment during imaging, the size of the volume of interest, and the number of slices that are contoured to define the callus.

Micro-computed tomography (&#181;CT) is a non-destructive imaging tool that is instrumental in assessing bone structure in preclinical studies, however there is a lack of consensus on &#181;CT procedures for analyzing the bone healing callus. This study provides a step-by-step &#181;CT protocol that allows the monitoring of fracture healing.

Micro-computed tomography (&#181;CT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed ...