Name: use of ultra-high field mri in small rodent models of polycystic kidney disease for in vivo phenotyping and drug monitoring
Uploaded: 2015-06-23T14:00:00.000+00:00
Duration: 7 min 35 s
Description: Watch this Scientific Journal Video about Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring at JoVE.com

We thank Drs. Xiaofang Wang and Katharina Hopp for their invaluable help with the animal models. This work has been supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (DK090728, DK058816).

Before starting any procedures with live animals, experimental protocols should be approved by the institutional animal care and use committee (IACUC).
1. Scanner Configuration
<ol>
	<li>Before starting, make sure the heater is in OFF position.</li>
	<li>Select the mini imaging gradient and 38 mm RF coil and mini imaging holder.</li>
	<li>In the central bore of the holder install the variable temperature assembly.</li>
</ol>
2. Animal Preparation
<ol>
	<li>For MRI experiments, achieve optimal anesthesia using vaporized isoflurane. For induction of anesthesia, place animal in an induction chamber lined with an absorbent tissue. Adjust the flowmeter of the isoflurane vaporizer to 2.0-2.5 L/min, and the isoflurane to 3% in oxygen.</li>
	<li>Remove any metal tag or other metallic object at this stage. Apply vet ointment on animal&#8217;s eyes to prevent dryness while under anesthesia.</li>
	<li>Once the animal has reached the surgical plane of anesthesia (i.e., loss of withdrawal reflex to toe pinch), place the animal on a holder with its nose inserted into a nose cone. Set the anesthesia air flow in the probe to 2.0-2.5 ml/min and the isoflurane concentration to 1.5-2.0% in oxygen. Anesthesia will be delivered through the nose cone during the procedure. Periodically adjust isoflurane concentration depending on animal&#8217;s age and weight to maintain a respiration rate of ~40 bpm.</li>
	<li>Use animal holders to secure the animal in place and prevent motion during the MRI experiment. Vary the type of animal holder depending on the body region to be scanned. 
	Note: Customized holders from laboratory plastics (polypropylene, Teflon, polystyrene, polycarbonate) can be made to accommodate specific experiment and to fit the animal size (from newborn mouse to 160 g rat).</li>
	<li>Place the rectal thermometer in the animal to monitor animal&#8217;s body temperature. During the experiment, keep the animal at 35-37 &#176;C, using a stream of warm air. Adjust air temperature (30-38 &#176;C) and flow (1,200-2,000 L/hr) based on animal&#8217;s body temperature feedback.</li>
	<li>Attach a balloon respiratory pressure sensor to animal&#8217;s abdomen to monitor respiration rate.</li>
	<li>Secure the animal at the center of the RF coil and carefully place the RF coil with animal into MRI scanner.</li>
</ol>
3. MRI Experiment
<ol>
	<li>Tune and match the RF coil before starting the experiments to minimize RF power used and to maximize signal-to-noise ratio. To start the matching/tuning:
	<ol>
		<li>Open the spectrometer control tool by clicking the tools icon.</li>
		<li>In the spectrometer control tool click Acquisition&#160;&#8594; Wobble. An Acq/Reco window will open displaying the wobble curve.</li>
		<li>Alternatively adjust the tuning and matching capacitors (using the tuning and matching rods) in small steps until the reflected RF power is minimized. The goal is to see a curve with a minimum at the vertical axis positioned at zero on the horizontal axis.</li>
		<li>When the calibration of the coil has been successfully achieved, hit the Stop button in the Acq/Reco window.</li>
	</ol>
	</li>
	<li>Acquire scout images in the three orthogonal planes to create axial, coronal and sagittal images. Use a fast image sequence such as Intra Gate Fast Low Angle Shot (IG-FLASH) to acquire the scout images12. Use the scout images to set the proper geometry for the actual imaging.</li>
	<li>Depending on the specific research aims, select proper image sequence and parameters and start the scan with a traffic light. This will calibrate RF channel, shim the magnet, set carrier frequency on-resonance for water and adjust receiver gain, all automatically.
	<ol>
		<li>For anatomic studies and T2 weighted images, acquire in 2D multi slice or 3D mode. To shorten the experiment time for a given spatial resolution, keep the field-of-view (FOV) as small as possible but large enough to avoid wrap-around artifacts (2.56-3.2 cm).</li>
	</ol>
	</li>
	<li>Keep the cycle of selected sequence slightly shorter than the animal respiration cycle by proper selection of repetition time (TR) and/or number of slices. This ensures that the data are collected during animals&#39; quiet period.
	<ol>
		<li>For example, for abdominal images, keep animal&#8217;s respiratory rate at ~30 bpm; that is about 2,000 msec per breath. Use a Turbo Rapid Acquisition with Relaxation Enhancement (RARE) sequence and acquire 11-19 coronal slices, with TR/TE 1500/9 msec, RARE factor 8 and (matrix 256 x 256, FOV 2.56 x 2.56 cm, slice thickness 0.75 mm). 
		Note: By adjusting the TR to 1,500 msec, and keeping the animal&#8217;s respiratory rate ~30 bpm (2,000 msec per breath), we ensure that the data are collected during animals&#39; quiet period.</li>
	</ol>
	</li>
	<li>After all image acquisition has been completed, place the scanned animal on heated pad and monitor until ambulatory. After recovery, return the animal to the cage and monitored at least for 1 hr before returning to the animal facility.</li>
</ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiographic+findings+in+autosomal+dominant+polycystic+kidney+disease.">Echocardiographic findings in autosomal dominant polycystic kidney disease.</a> N Engl J Med. 319, 907-912 (1988).</li><li>Lumiaho, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitral+valve+prolapse+and+mitral+regurgitation+are+common+in+patients+with+polycystic+kidney+disease+type+1.">Mitral valve prolapse and mitral regurgitation are common in patients with polycystic kidney disease type 1.</a> American journal of kidney diseases : the official journal of the National Kidney Foundation. 38, 1208-1216 (2001).</li><li>Vallee, J. P., Ivancevic, M. K., Nguyen, D., Morel, D. R., Jaconi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+cardiac+MRI+in+small+animals.">Current status of cardiac MRI in small animals.</a> Magma. 17, 149-156 (2004).</li><li>Epstein, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MR+in+mouse+models+of+cardiac+disease.">MR in mouse models of cardiac disease.</a> NMR Biomed. 20, 238-255 (2007).</li><li>Bloomgarden, D. 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G., Bolon, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+with+morphine+is+a+suitable+anaesthetic+regimen+for+embryo+transfer+in+the+production+of+transgenic+rats.">Isoflurane with morphine is a suitable anaesthetic regimen for embryo transfer in the production of transgenic rats.</a> Laboratory animals. 38, 38-43 (2004).</li><li>Ahrens, E. T., Srinivas, M., Capuano, S., Simhan, H. N., Schatten, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+of+embryonic+and+fetal+development+in+model+systems.">Magnetic resonance imaging of embryonic and fetal development in model systems.</a> Methods Mol Med. 124, 87-101 (2006).</li><li>Zhou, R., Pickup, S., Glickson, J. D., Scott, C. H., Ferrari, V. 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The authors have nothing to disclose.

This manuscript shows the feasibility of using UHF MRI as a tool for in vivo phenotypical characterization or drug monitoring in rodent models for PKD.
We describe experiments done at 16.4 T with a wide bore Avance III high resolution NMR spectrometer equipped with micro and mini imaging accessories. The spectrometer was driven by the acquisition and processing software TopSpin2.0PV controlled by Paravision 5.1 imaging software. Because the rodent size varies in longitudinal studies, ...

Polycystic Kidney Disease (PKD) includes a group of monogenic disorders characterized by the development of renal cysts. Among them are autosomal-dominant polycystic kidney disease (ADPKD) and autosomal-recessive polycystic kidney disease (ARPKD), which represent the most common types1,2. ADPKD, the most frequent form of hereditary renal cystic diseases, is originated by mutations in the PKD1 or PKD2 genes. It is characterized by late-onset, multiple bilateral renal cysts, accompanied by variable extra-renal cysts, as well as cardiovascular and muscle skeletal abnormalities. ARPKD, most commonly affecting newborns and young children, is caused by mutations in PKHD1 and is characterized by enlarged echogenic kidneys and congenital hepatic fibrosis3.
Importantly, ADPKD is characterized by heterogeneity, both at the gene (genic) and mutation (allelic) levels, which results in substantial phenotypic variability. Mutations in the PKD1 gene are associated with severe clinical presentation (numerous cysts, early diagnosis, hypertension, and hematuria), as well as rapid progression to end-stage renal disease (20 years earlier than patients with PKD2 mutations)4. Severe polycystic liver disease (PLD) and vascular abnormalities can be associated with mutations in both PKD1 and PKD25. The majority of renal complications of ADPKD arise mainly as a consequence of the cyst expansion along with associated inflammation and fibrosis. Cyst development starts in utero and continues through the patient lifetime. Kidneys usually maintain their reniform shape even though they could reach more than 20 times the normal kidney volume. Most of the patients present bilateral distribution of renal cysts, but in some unusual cases, cyst may develop in a unilateral or asymmetric pattern.
A major challenge for nephrologists following patients with ADPKD or implementing therapies is the natural history of the disease. During most of its course, the renal function remains normal and by the time the renal function starts to decline, most of the kidneys have been replaced by cysts. When therapies are implemented at later stages, it is less likely to be successful since the patient may already have reached a point of no return in chronic kidney disease. In contrast, when therapies are started at early stages, it is difficult to identify a response based solely on glomerular filtration rate. As a result, the notion of kidney volume as a marker of disease progression gained attention.
The Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease (CRISP) study has shown that in patients with ADPKD the increase in kidney and cyst volumes directly correlates with renal function deterioration, underscoring the potential of Total Kidney Volume (TKV) as a surrogate marker for disease progression6,7. Consequently, TKV is currently used as primary or secondary endpoint in multiple clinical trials for ADPKD2,8,9.
Multiple murine models including spontaneous mutations and genetically engineered have shed light on the pathogenesis of PKD10,11. Pkd1 or Pkd2 models (mutations in either Pkd1 or Pkd2) have become the most popular ones, as they perfectly mimic human disease. In addition, rodent models with mutations in genes other than Pkd1 or Pkd2 genes have been used as an experimental platform to elucidate signaling pathways related to the disease. In addition, several of these models have been used to test potential therapies. However, a limiting factor in many rodent studies for PKD is often the lack of efficient non-invasive methods to sequentially analyze the anatomical and functional changes in the kidney.
Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow ADPKD patients, providing excellent soft tissue contrast and anatomic detail, and allowing TKV measurements. Even though MRI is well established for anatomical imaging in larger animals and humans, imaging small rodents in vivo entails additional technical challenges, where the ability to acquire high resolution images may limit its usefulness. With the introduction of Ultra-high field (UHF) MRI (7-16.4 T) and the development of stronger gradients, it is now possible to achieve higher signal-to noise ratios and spatial resolution of MRI images with a diagnostic quality similar to that obtained in humans. Consequently, the use of UHF MRI for in vivo imaging of small rodent models for PKD has become a powerful tool for researchers.

<table><tbody><tr><td>AVANCEIII-700 (16.4 T)</td><td>Bruker</td><td>BH067206</td><td>Wide-bore two channel multinuclear spectrometer equipped with mini and micro-imaging accessories for &lt;em&gt;in vivo&lt;/em&gt; small rodent imaging</td></tr><tr><td>TopSpin2.0PV</td><td>Bruker</td><td>H9088TA2</td><td>Spectrometer processing software</td></tr><tr><td>Paravision 5.1</td><td>Bruker</td><td>T10314L5</td><td>Imaging sofware</td></tr><tr><td>VTU BVT 3000 digital</td><td>Bruker</td><td>W1101095</td><td>Temperature controller</td></tr></tbody></table>

use of ultra-high field mri in small rodent models of polycystic kidney disease for in vivo phenotyping and drug monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

In this manuscript, we aim to show the usefulness of UHF MRI as a tool for in vivo phenotypical characterization or drug monitoring in rodent models for PKD and other kidney diseases. All of the experiments were part of experimental protocols approved by the IACUC.
In vivo phenotyping of small rodent models for PKD using UHF MRI:
All imaging studies were performed on live animals under isoflurane anesthesia, with a Bruker AVANCEIII-700 (16.4 T) vertic...

Watch this Scientific Journal Video about Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring at JoVE.com

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

use-ultra-high-field-mri-small-rodent-models-polycystic-kidney

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

11.4K Views.  Mayo Clinic. The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

Video: Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Utilizing High Resolution Ultrasound to Monitor Tumor Onset and Growth in Genetically Engineered Pancreatic Cancer Models

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel therapies in pancreatic cancer. Tumor onset is variable in the KPC model between 8 weeks and several months. Therefore, non-invasive imaging tools are required to screen for tumor onset and monitor for response to treatment. To address this issue, different approaches have emerged over the last years. High resolution ultrasound has major advantages such as non-invasiveness, fast session times and a high image resolution without radiation exposure. However, ultrasound in mice is not trivial and sufficient anatomical knowledge and practical skills are required to successfully perform high resolution ultrasound in preclinical pancreatic cancer models. With the following article, a detailed hands-on guide for abdominal ultrasound in murine models with a particular focus on endogenous pancreatic cancer models is presented. Furthermore, a summary of common mistakes and how to avoid them is provided.

This article describes the utilization of high-resolution ultrasound in genetically engineered pancreatic cancer mice. The primary aim is to provide a detailed instruction for detection and evaluation of endogenous pancreatic tumors.

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel ...

Cancer Research

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Neuroscience

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is estimated to contribute to 19- 25% of all clinical cases of acute kidney injury (AKI) in critically ill patients. AKI detection has historically relied on metrics such as serum creatinine (sCr) or blood urea nitrogen (BUN) which are demonstrably inadequate in full assessment of nephrotoxicity in the early phase of renal dysfunction. Currently, there is no robust diagnostic method to accurately detect hemodynamic alteration in the early phase of AKI while such alterations might actually precede the rise in serum biomarker levels. Such early detection can help clinicians make an accurate diagnosis and help in in decision making for therapeutic strategy. Rats were treated with Cisplatin to induce AKI. Nephrotoxicity was assessed for six days using high-frequency sonography, sCr measurement and upon histopathology of kidney. Hemodynamic evaluation using 2D and Color-Doppler images were used to serially study nephrotoxicity in rats, using the sonography. Our data showed successful drug-induced kidney injury in adult rats by histological examination. Color-Doppler based sonographic assessment of AKI indicated that resistive-index (RI) and pulsatile-index (PI) were increased in the treatment group; and peak-systolic velocity (mm/s), end-diastolic velocity (mm/s) and velocity-time integral (VTI, mm) were decreased in renal arteries in the same group. Importantly, these hemodynamic changes evaluated by sonography preceded the rise of sCr levels. Sonography-based indices such as RI or PI can thus be useful predictive markers of declining renal function in rodents. From our sonography-based observations in the kidneys of rats that underwent AKI, we showed that these noninvasive hemodynamic measurements may consider as an accurate, sensitive and robust method in detecting early stage kidney dysfunction. This study also underscores the importance of ethical issues associated with animal use in research.

Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model.

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Use of 3D Robotic Ultrasound for In Vivo Analysis of Mouse Kidneys

Common modalities for in vivo imaging of rodents include positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Each method has limitations and advantages, including availability, ease of use, cost, size, and the use of ionizing radiation or magnetic fields. This protocol describes the use of 3D robotic US for in vivo imaging of rodent kidneys and heart, subsequent data analysis, and possible research applications. Practical applications of robotic US are the quantification of total kidney volume (TKV), as well as the measurement of cysts, tumors, and vasculature. Although the resolution is not as high as other modalities, robotic US allows for more practical high throughput data collection. Furthermore, using US M-mode imaging, cardiac function may be quantified. Since the kidneys receive 20%-25% of the cardiac output, assessing cardiac function is critical to the understanding of kidney physiology and pathophysiology.

Use of 3D Robotic Ultrasound for In Vivo Analysis of Mouse Kidneys

This protocol demonstrates robotic ultrasound (US) as a practical, cost-effective, and quick alternative to traditional non-invasive image modalities.

Common modalities for in vivo imaging of rodents include positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), ...

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations.
In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Fr&#246;mter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p &#60; 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 &#177; 0.002 in untreated MWFs to 0.045 &#177; 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.

Here, we present a protocol using 2-photon microscopy in Munich Wistar Fromter rats with surface glomeruli to quantifythe effects of prolonged ureteral obstruction on glomerular dynamics and function.

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface ...

High-Resolution Cardiac Positron Emission Tomography/Computed Tomography for Small Animals

Positron emission tomography (PET) and computed tomography (CT) are among the most employed diagnostic imaging techniques, and both serve in understanding cardiac function and metabolism. In preclinical research, dedicated scanners with high sensitivity and high spatio-temporal resolution are employed, designed to cope with the demanding technological requirements posed by the small heart size and very high heart rates of mice and rats. In this paper, a bimodal cardiac PET/CT imaging protocol for experimental mouse and/or rat models of cardiac diseases is described, from animal preparation and image acquisition and reconstruction to image processing and visualization.
In particular, the 18F-labeled fluorodeoxyglucose ([18F]FDG)-PET scan allows for the measurement and visualization of glucose metabolism in the different segments of the left ventricle (LV). Polar maps are convenient tools to display this information. The CT part consists of a time-resolved 3D reconstruction of the entire heart (4D-CT) using retrospective gating without electrocardiography (ECG) leads, allowing the morphofunctional evaluation of the LV and the subsequent quantification of the most important cardiac function parameters, such as ejection fraction (EF) and stroke volume (SV). Using an integrated PET/CT scanner, this protocol can be executed within the same anesthesia induction without the need to reposition the animal between different scanners. Hence, PET/CT can be seen as a comprehensive tool for the morphofunctional and metabolic evaluation of the heart in several small animal models of cardiac diseases.

Here, we present an experimental imaging protocol for the quantification of cardiac function and morphology using high-resolution positron emission tomography/computed tomography for small animals. Both mice and rats are considered, discussing the different requirements of computed tomography contrast agents for the two species.

Positron emission tomography (PET) and computed tomography (CT) are among the most employed diagnostic imaging techniques, and both serve in understanding ...

Ultrafast Doppler Ultrasound for Hepatic Microvasculature Analysis in Mice

Imaging and Quantification of the Hepatic Vasculature of Mice Using Ultrafast Doppler Ultrasound

Non-invasive in vivo imaging of the vasculature is a powerful tool for studying disease mechanisms in rodents. To achieve high sensitivity imaging of the microvasculature using Doppler ultrasound methods, imaging modalities employing the concept of ultrafast imaging are preferred. By increasing the frame rate of the ultrasound scanner to thousands of frames per second, it becomes possible to improve the sensitivity of the blood flow down to 2 mm/s and to obtain functional information about the microcirculation in comparison to a sensitivity of around 1 cm/s in conventional Doppler modes. While Ultrafast Doppler ultrasound (UFUS) imaging has become adopted in neuroscience, where it can capture brain activity through neurovascular coupling, it presents greater challenges when imaging the vasculature of abdominal organs due to larger motions linked to breathing. The liver, positioned anatomically under the diaphragm, is particularly susceptible to out-of-plane movement and oscillating respiratory motion. These artifacts not only adversely affect Doppler imaging but also complicate the anatomical analysis of vascular structures and the computation of vascular parameters. Here, we present a qualitative and quantitative imaging analysis of the hepatic vasculature in mice by UFUS. We identify major anatomical vascular structures and provide graphical illustrations of the hepatic macroscopical anatomy, comparing it to an in-depth anatomical assessment of the hepatic vasculature based on Doppler readouts. Additionally, we have developed a quantification protocol for robust measurements of hepatic blood volume of the microvasculature over time. To contemplate further research, qualitative vascular analysis provides a comprehensive overview and suggests a standardized terminology for researchers working with mouse models of liver disease. Furthermore, it offers the opportunity to apply ultrasound as a non-invasive complementary method to inspect hepatic vascular defects in vivo and measure functional microvascular alterations deep within the organ before unraveling blood vessel anomalies at the micron scale levels using ex vivo staining on tissue sections.

Ultrafast Doppler ultrasound (UFUS) with high spatial resolution (100 &#956;m) and sensitivity represents a suitable noninvasive imaging modality for obtaining a comprehensive qualitative overview of the hepatic vasculature. UFUS also facilitates quantitative measurements of the microvasculature, aiming to enhance our understanding of vascular disease mechanisms.

Non-invasive in vivo imaging of the vasculature is a powerful tool for studying disease mechanisms in rodents. To achieve high sensitivity imaging of the ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

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Chapters in this video

Utilizing High Resolution Ultrasound to Monitor Tumor Onset and Growth in Genetically Engineered Pancreatic Cancer Models

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

Transcutaneous Assessment of Renal Function in Conscious Rodents

Assessment of Kidney Function in Mouse Models of Glomerular Disease

Use of 3D Robotic Ultrasound for In Vivo Analysis of Mouse Kidneys

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

High-Resolution Cardiac Positron Emission Tomography/Computed Tomography for Small Animals

Imaging and Quantification of the Hepatic Vasculature of Mice Using Ultrafast Doppler Ultrasound