This work has received funding from the Carlsberg Foundation (CF17-0778; CF18-0658), the Lundbeck Foundation (R324-2019-1470; R346-2020-1210), the Velux Foundations (00022458), The A.P. M&#248;ller Foundation for the Advancement of Medical Science, the European Union&#39;s Horizon 2020 research and innovation program under the Marie Sk&#322;odowska-Curie grant agreement (No. 754513), and The Aarhus University Research Foundation.

The protocol below was performed with permission from the Danish Inspectorate for Animal Experimentation within the Danish Ministry of Food, Agriculture, and Fisheries, Danish Veterinary and Food Administration (Permit number 2016-15-0201-00835).
1. Anesthesia and ultrasound medium
<ol>
	<li>Anesthetize the research animal. 
	NOTE: Type and dose of appropriate anesthesia are highly species-dependent. In general, immersion-based anesthetics such as MS-222 (ethyl 3-aminobenzoate methanesulfonic acid), benzocaine (ethyl 4-aminobenzoate), and propofol (2,6-diisopropyl phenol) are useful in fish and amphibians which readily absorbs the anesthetic over gills or skin (e.g., 0.05 mg&#183;L-1 benzocaine in rainbow trout). A range of dissolved compounds that can be administered intravenously, intramuscularly, intraperitoneally is available for amniotes, as are gas-based anesthetics. Alfaxalon administered intramuscularly is useful in reptiles (e.g., 30 mg&#183;kg-1 in lizards), and isoflurane administered as gas is useful in birds (e.g., 2% in air for pigeons). Refer to the published literature30,31,32 for a full overview of available anesthetics across species.</li>
	<li>Test reflexes in the animal to confirm an optimal level of anesthesia. Ensure that the animal is completely motionless during the procedure as the flow-enhanced ultrasound procedure is sensitive to motion noise.
	<ol>
		<li>Too deep anesthesia can alter blood flow patterns, so conduct a dose titration in the start-up phase of an experiment.</li>
		<li>Increase the anesthesia dosage in steps and observe blood flow in the eye aided by simple brightness mode (B-mode) ultrasound. 
		NOTE: An optimal level of anesthesia is obtained when the animal is motionless (except respiration) with visible ocular blood flow.</li>
	</ol>
	</li>
	<li>If the type/dose of anesthetic is not permissive for respiratory movements, then ensure adequate ventilation of the animal, e.g., using an air pump to oxygenate the water for aquatic species or a ventilator for air-breathing species.</li>
	<li>Position the animal in a posture that allows direct access from above to the eye. 
	NOTE: Depending on species, this can be in either a supine or lateral position. It can be useful to construct a simple holding device using a small piece of non-reactive metal (e.g., stainless steel) and loose rubber bands (see Figure 1).</li>
	<li>Place appropriate ultrasound medium on the eye of the animal. If scaled eyelids (ultrasound impermeable) cover the eye, then displace these gently with a cotton swab. 
	&#8203;NOTE: For aquatic species, the best ultrasound medium is clean tank water in which the animal usually lives. For terrestrial species, a generous amount of ultrasound gel ensures free movements and imaging of the ultrasound transducer (i.e., linear array probe) across the entire surface of the eye. Vet ointment on the contralateral eye is required for terrestrial species.</li>
</ol>
2. 2D and 3D ocular ultrasound image acquisition
<ol>
	<li>Position the ultrasound transducer medial to the eye in either a dorsal/ventral or rostral/caudal orientation depending on desired image orientation.</li>
	<li>In B-mode, with a maximum depth of field, image the medial and deepest portion of the eye and make sure that all structures of interest are visible in the image field. 
	NOTE: In some species, the crystalline lens takes up a comparatively large proportion of the vitreous humor, which may absorb the ultrasound, especially at higher frequencies.</li>
	<li>Slowly translate the transducer to each side while inspecting the real-time images. Make sure all structures of interest are visible in the image field; if not, switch to a transducer with a lower frequency and larger depth of field. 
	NOTE: The following center frequencies allow for the following maximum depth of field: 21 MHz: 3 cm, 40 MHz: 1.5 cm, 50 MHz: 1 cm (see Table 1). However, these maximum depth of field values can be markedly lower if the eye contains calcified or other ultrasound impermeable structures.</li>
	<li>Adjust image depth, depth offset (distance from the top of the image to the structure of interest), image width, as well as number and position of focal zones to cover the desired region of interest in all three spatial dimensions (e.g., 1 cm image depth, 2 mm depth offset, 1 cm image width, one focal zone). 
	NOTE: Although specific naming of buttons that adjust these parameters may vary between ultrasound systems, most systems will have buttons with logical names for these adjustments. These image parameter settings usually affect the range of possible temporal resolutions of the ultrasound acquisition.</li>
	<li>Set frame rate in the range of 50-120 frames&#183;s-1. 
	NOTE: The temporal resolution (i.e., the time interval between successive B-scans) must be adequate to display large pixel intensity variability in imaged blood vessels, i.e., the temporal resolution must not be too high. On the other hand, to complete a full 3D recording of the eye in a reasonable time, temporal resolution cannot be too low. A temporal resolution ranging from 50-120 frames&#183;s-1 is usually adequate for the flow-enhanced procedure in most species. On some ultrasound systems, this desired temporal resolution can be obtained by switching between the &#34;general imaging&#34; (high spatial/low temporal resolution) and &#34;cardiology&#34; (low spatial/high temporal resolution) modes.</li>
	<li>Adjust 2D gain to a level (~5 dB), so anatomical structures are only just visible in the B-mode acquisition to increase the signal-to-noise ratio in the subsequent flow-enhanced reconstruction.</li>
	<li>To acquire a 2D flow-enhanced image at a single slice position, translate the transducer to this position and continue at step 3.1.</li>
	<li>To acquire a 3D recording of an entire region of interest, e.g., the retina, translate the transducer to one extreme of the region of interest.
	<ol>
		<li>To determine the exact position of the extreme end of the region of interest, increase the 2D gain briefly.</li>
		<li>After correct transducer placement is complete, lower the 2D gain before recording to ensure maximal signal-to-noise ratio in the subsequent flow-enhanced reconstruction.</li>
	</ol>
	</li>
	<li>For each step (slice) in the 3D recording, acquire &#8805;100 frames (optimally &#8805;1000 frames).</li>
	<li>Using a micromanipulator or build-in transducer motor, translate the transducer across the entire region of interest in steps of, e.g., 25 &#181;m or 50 &#181;m (remember to note the step size) and repeat the &#8805;100 frames acquisition for each step.</li>
	<li>Euthanize the research animal according to the animal care guidelines of the institution.</li>
</ol>
3. Flow-enhanced image reconstruction
<ol>
	<li>Export the recordings into digital imaging and communications in medicine (DICOM) file format (little-endian).</li>
	<li>To produce a single flow-enhanced image based on a &#8805;100 frames (T) cine recording, calculate the standard deviation on pixel level (STD(x,y)) using the formula: 
	<img alt="Equation 1" src="/files/ftp_upload/62986/62986eq01.jpg" /> 
	Where It(x,y) is the intensity of the pixel at the (x,y) pixel coordinate at time t, and &#298;t(x,y) is the arithmetic mean value of I over time.</li>
	<li>Repeat step 3.2 for each slice in the 3D recording.</li>
	<li>To automate the STD-calculation and image reconstruction process for multiple slices in a 3D recording, conduct this operation in batch mode using, e.g., ImageJ and the supplementary macro script (Supplementary File 1).</li>
	<li>Combine all reconstructed slices into one image stack (Images to Stack command in ImageJ).</li>
	<li>Specify slice thickness from the step size used during acquisition (Properties command in ImageJ).</li>
	<li>Save the image stack as a 3D TIF file. 
	NOTE: Flow-weighted three-dimensional recordings of ocular blood vessels can subsequently be used to create volume renderings and build digital and physical anatomical models of vascular structures of the eye. These image processing options are outside the scope of this protocol; refer to the previously published articles for more details33,34,35.</li>
</ol>

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The authors declare that no completing interests exists.

Vascular imaging using flow-enhanced ultrasound provides a new method for non-invasive imaging of the vasculature of the eye that offers several advantages over present techniques but has its intrinsic limitations. The primary advantage of flow-enhanced ultrasound is the ability to generate ocular angiographies with a depth of field that exceeds the retinal pigment epithelium, which limits the depth of field in optical techniques. In ultrasound imaging, spatial resolution and depth of field are ultimately determined by t...

The high metabolism on the vertebrate retina imposes an intrinsic tradeoff between two contrasting needs; high blood flow rates and a light path devoid of blood vessels. To avoid visual disturbance of perfusing red blood cells, the retina of all vertebrates receives oxygen and nutrients via a sheet of capillaries behind the photoreceptors, the choriocapillaris1,2,3. However, this single source of nutrients and oxygen imposes a diffusion limitation to the thickness of the retina4,5, so many visually active species possess a variety of elaborate vascular networks to provide additional blood supply to this metabolically active organ6. These vascular beds include blood vessels perfusing the internal retinal layers in mammals and some fishes4,7,8,9,10, blood vessels on the inner (light-facing) side of the retina found in many fishes, reptiles, and birds4,11,12,13, and countercurrent vascular arrangements of the fish choroid, the choroid rete mirabile, that allows for the generation of super-atmospheric oxygen partial pressures14,15,16,17,18,19,20. Despite that these additional non-choroidal paths for retinal nutrient supply play an essential role in fueling the metabolic requirements of superior vision4, the three-dimensional anatomy of these vascular structures is poorly understood, limiting our understanding of the morphological evolution of the vertebrate eye.
Traditionally, retinal blood supply has been studied using optical techniques, such as fundus ophthalmoscopy. This category of techniques provides high-throughput non-destructive information on non-choroidal blood vessel anatomy in high-resolution21 and is therefore readily used in clinical diagnosis of abnormalities in retinal vessel structure22. However, the retinal pigment epithelium absorbs the transmitted light and limits the depth of view in these optical techniques, providing reduced information on choroidal structure and function without the use of contrast agent21. Similar depth limitations are experienced in optical coherence tomography (OCT). This technique can generate high-resolution fundus angiographies using light waves at the technical expense of depth penetration23, while the enhanced depth imaging OCT can visualize the choroid at the expense of retinal imaging quality24. Magnetic resonance imaging overcomes the optical limitations of ophthalmoscopy and OCT and can map vascular layers in the retina, albeit at a low resolution25. Histology and microcomputed tomography (&#181;CT) maintain the high-resolution of the optical techniques and provide information on whole-eye vascular morphology4, but both techniques require ocular sampling and are therefore not possible in the clinic or rare or endangered species. To overcome some of the limitations of these established retinal imaging techniques, the study here presents an ultrasound protocol on anesthetized animals, where blood movement is mapped in silico on a series of equally-spaced two-dimensional ultrasound scans spanning a whole eye by applying a comparable technique as described previously for embryonic and cardiovascular imaging26,27,28 and in OCT angiography29. This approach allows for the generation of non-invasive three-dimensional deep ocular angiographies without using a contrast agent and opens up new avenues for mapping blood flow distribution within the eye across species.

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deep vascular imaging in the eye with flow-enhanced ultrasound

The retina within the eye is one of the most energy-demanding tissues in the body and thus requires high rates of oxygen delivery from a rich blood supply. The capillary lamina of the choroid lines the outer surface of the retina and is the dominating source of oxygen in most vertebrate retinas. However, this vascular bed is challenging to image with traditional optical techniques due to its position behind the highly light-absorbing retina. Here we describe a high-frequency ultrasound technique with subsequent flow-enhancement to image deep vascular beds (0.5-3 cm) of the eye with a high spatiotemporal resolution. This non-invasive method works well in species with nucleated red blood cells (non-mammalian and fetal animal models). It allows for the generation of non-invasive three-dimensional angiographies without the use of contrast agents, and it is independent of blood flow angles with a higher sensitivity than Doppler-based ultrasound imaging techniques.

The flow-enhanced ultrasound technique to image vascular beds of the eye can be applied in a range of species and has currently been used in 46 different vertebrate species (Figure 1, Table 1). The presence of nucleated red blood cells in non-adult-mammalian vertebrates provides positive contrast of flowing blood compared to static tissue in cine recordings (Supplementary File 2). However, when analyzed on a frame-by-frame basis, the clear distinction betwee...

Watch this Scientific Journal Video about Deep Vascular Imaging in the Eye with Flow-Enhanced Ultrasound at JoVE.com

Deep Vascular Imaging in the Eye with Flow-Enhanced Ultrasound

We present a non-invasive ultrasound technique for generating three-dimensional angiographies in the eye without the use of contrast agents.

The retina within the eye is one of the most energy-demanding tissues in the body and thus requires high rates of oxygen delivery from a rich blood ...

deep-vascular-imaging-in-the-eye-with-flow-enhanced-ultrasound

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JoVE Journal

Biology

2.3K Views.  Aarhus University. We present a non-invasive ultrasound technique for generating three-dimensional angiographies in the eye without the use of contrast agents. 

Video: Deep Vascular Imaging in the Eye with Flow-Enhanced Ultrasound

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

In vivo Imaging of Deep Cortical Layers using a Microprism

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size and have a reflective coating on the hypotenuse to allow internal reflection of excitation and emission light.  The microprism probe simultaneously images multiple cortical layers with a perspective typically seen only in slice preparations.  Images are collected with a large field-of-view (~900 &mu;m).  In addition, we provide details on the non-survival surgical procedure and microscope setup.  Representative results include images of layer V pyramidal neurons from Thy-1 YFP-H mice showing their apical dendrites extending through the superficial cortical layer and extending into tufts.  Resolution was sufficient to image dendritic spines near the soma of layer V neurons.  A tail-vein injection of fluorescent dye reveals the intricate network of blood vessels in the cortex.  Line-scanning of red blood cells (RBCs) flowing through the capillaries reveals RBC velocity and flux rates can be obtained. This novel microprism probe is an elegant, yet powerful new method of visualizing deep cellular structures and cortical function in vivo.

In vivo Imaging of Deep Cortical Layers using a Microprism

Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 &mu;m) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby the permeability of the endothelium to blood cells, plasma macromolecules, and water can be adapted according to the physiological need. In certain diseases, cytokines and growth factors are released that target the endothelial barrier to transiently increase vascular permeability; however, their prolonged presence may cause chronic vascular hyperpermeability and thereby tissue-damaging edema. The Miles assay is an in vivo technique that allows researchers to study vascular hyperpermeability through the proxy measurement of vascular leakage. Here, we provide a detailed protocol on how to perform this procedure in the mouse, which is the most widely used model organism to study mammalian physiology and pathology. The procedure involves the intravenous injection of Evans blue dye to label the circulating albumin followed by multiple intradermal injections of permeability-inducing agents and vehicle control solutions into opposing flanks of the mouse. Consequently, Evans blue dye gradually leaks into the dermis, where it accumulates and can be extracted for quantification as leakage induced by the permeability-inducing agent relative to the vehicle. The Miles assay can be performed in wild type or genetically modified mouse models and may be combined with drug administration to study molecular mechanisms that regulate vascular permeability and identify agents/targets capable of inducing or blocking hyperpermeability.

Here, we present a protocol to measure the vascular leakage induced by intradermal administration of permeability promoting agents into the murine skin. This technique can be used to determine the ability of molecules to promote or inhibit vascular leakage or to study the molecular mechanisms that regulate vascular permeability.

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby ...

Application of Ultrasound and Shear Wave Elastography Imaging in a Rat Model of NAFLD/NASH

Nonalcoholic Steatohepatitis (NASH) is a condition within the spectrum of Non-Alcoholic Fatty Liver Disease (NAFLD), which is characterized by liver fat accumulation (steatosis) and inflammation leading to fibrosis. Preclinical models closely recapitulating human NASH/NAFLD are essential in drug development. While liver biopsy is currently the gold standard for measuring NAFLD/NASH progression and diagnosis in the clinic, in the preclinical space, either collection of whole liver samples at multiple timepoints during a study or biopsy of liver is needed for histological analysis to assess the disease stage.
Conducting a liver biopsy mid-study is an invasive and labor-intensive procedure, and collecting liver samples to assess disease level increases the number of research animals needed for a study. Thus, there is a need for a reliable, translatable, non-invasive imaging biomarker to detect NASH/NAFLD in these preclinical models. Non-invasive ultrasound-based B-mode images and Shear Wave Elastography (SWE) can be used to measure steatosis as well as liver fibrosis. To assess the utility of SWE in preclinical rodent models of NASH, animals were placed on a pro-NASH diet&#160;and underwent non-invasive ultrasound B-mode and shear wave elastography imaging to measure hepatorenal (HR) index and liver elasticity, measuring progression of both liver fat accumulation and tissue stiffness, respectively, at multiple time points over the course of a given NAFLD/NASH study.
The HR index and elasticity numbers were compared to histological markers of steatosis and fibrosis. The results showed strong correlation between the HR index and percentage of Oil Red O (ORO) staining, as well as between elasticity and Picro-Sirius Red (PSR) staining of livers. The strong correlation between classic ex vivo methods and in vivo imaging results provides evidence that shear wave elastography/ultrasound-based imaging can be used to assess disease phenotype and progression in a preclinical model of NAFLD/NASH.

This protocol describes the use of an enhanced ultrasound technique to non-invasively observe and quantify liver tissue changes in rodent models of nonalcoholic fatty liver disease.

Nonalcoholic Steatohepatitis (NASH) is a condition within the spectrum of Non-Alcoholic Fatty Liver Disease (NAFLD), which is characterized by liver fat ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...

Alkali Burn-Induced Corneal Neovascularization in Mice

A Mouse Model for Corneal Neovascularization by Alkali Burn

Corneal neovascularization (CoNV), a pathological form of angiogenesis, involves the growth of blood and lymph vessels into the avascular cornea from the limbus&#160;and adversely&#160;affects transparency and vision. Alkali burn is one of the most common forms of ocular trauma that leads to CoNV. In this protocol, CoNV is experimentally induced using sodium hydroxide solution in a controlled manner to ensure reproducibility. The alkali burn model is useful for understanding the pathology of CoNV and can be extended to study angiogenesis in general because of the avascularity, transparency, and accessibility of the cornea. In this work, CoNV was analyzed by direct examination under a dissecting microscope and by immunostaining flat-mount corneas using anti-CD31 mAb. Lymphangiogenesis was detected on flat-mount corneas by immunostaining using anti-LYVE-1 mAb. Corneal edema was visualized and quantified using optical coherence tomography (OCT). In summary, this model will help to advance existing neovascularization assays and discover new treatment strategies for pathologic ocular and extraocular angiogenesis.

Author Spotlight: Decoding Corneal Neovascularization with Alkali Burn Model for Future Therapeutic Strategies 

This protocol focuses on alkali burn-induced corneal neovascularization in mice. The method generates a reproducible and controllable corneal disease model to study pathological angiogenesis and the associated molecular mechanisms and to test new pharmacological agents to prevent corneal neovascularization.

Corneal neovascularization (CoNV), a pathological form of angiogenesis, involves the growth of blood and lymph vessels into the avascular cornea from the ...