Name: early detection of drug-induced renal hemodynamic dysfunction using sonographic technology in rats
Uploaded: 2016-03-11T13:00:00.000+00:00
Duration: 6 min 38 s
Description: Watch this Scientific Journal Video about Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats at JoVE.com

We thank Fred Roberts for exemplary technical support. We thank Brigham Women&#8217;s Hospital Cardiovascular Physiology Core for providing with the instrumentation and the funds for this work. This work was supported in part by NHLBI grants HL093148, HL086967, and Departmental Funds from (SUNDRY).

Perform all procedures in male Sprague Dawley rats purchased from Charles River Laboratories in accordance with American Veterinary Medical Association (AVMA) guidelines and using approved Institutional Animal Care and Use Committee (IACUC) protocols.
1. Animal Preparation and Surgical Procedures
<ol>
	<li>Acclimate all animals for one week before any experimental procedure.</li>
	<li>Anesthetize animal using isoflurane (2-3% to induce, and 1.0% to maintain) and apply eye ointment to both eyes to prevent desiccation, irritation or ulceration.</li>
	<li>Remove hair from the animal&#8217;s chest using #40 blade and depilatory cream as necessary.&#160;&#160;It may have to remove hair form the animal&#8217;s back if we cannot have good image data obtained from ventral side imaging.</li>
</ol>
2. Nephrotoxicity Rat Model
<ol>
	<li>For Cisplatin-induced nephrotoxicity model, administer Cisplatin, using protocol as described previously. 15</li>
	<li>Perform sonography at baseline, 24 hr prior to Cisplatin administration (Day 0). (See step 3, Imaging Protocol)</li>
	<li>Randomize rats (n=6) into two groups. At Day 1, administer Cisplatin (10 mg/ml) (10 mg/Kg body weight, single dose nephrotoxicity induction), injection volume (1ml/Kg body weight) calculated by animal&#8217;s body weight),&#160;intraperitoneally in study group and normal saline (NS) in control group.</li>
	<li>Anesthetize animal as step 1.2 at 24, 48, 72, 96, 120, 144 hr after Cisplatin administration.</li>
	<li>Take image using high resolution ultrasound system (See Materials and Equipment Table) under stable anesthesia stage of animal. Continue to monitor the animal&#8217;s basic physiological function during imaging from anesthesia induction through full recovery.</li>
	<li>Monitor animal&#8217;s vital signs during imaging procedures: rat-temperature: 35.9-37.5, respiratory rate: 66-144/min, heart rate: 250-600/min. The optimal vital sign reading in our proposed study is: temperature: 36.5-37.0, respiratory rate: 80-100/min, heart rate: 450-550/min. 
	NOTE: Use Intravenous fluid infusion, and heating lamp to maintain animal&#8217;s normal physiological condition to minimize the effects of surgery and anesthesia. Assist respiration with mechanical ventilator during the procedure if necessary. However, mechanical ventilation is rarely needed in this experiment.</li>
</ol>
3. Imaging Protocol
Note: The ultrasound machine provider provides the heated platform for long imaging procedure. However, we do not use the heated platform in our demonstrated experiment because it only takes 5 to 15 min. Its controlled for body temperature which is monitored with a rectal thermometer connected to the physiology control switch.&#160;
<ol>
	<li>Transverse image of Kidney (B Mode):
	<ol>
		<li>Using MS 250 ultrasound with center frequency of 21 MHz connected to the active-port, set the application preset to &#8220;General Imaging&#8221;.</li>
		<li>With the animal supine on the platform, position the 21 MHz ultrasound probe using the rail system, midline on animal and isolate the aorta. In this position the probe angle is 90 degrees to the left parasternal line (transverse axis) (Figure 1A,B).</li>
		<li>From this position slide the platform with the animal such that the probe is now at the level of the renal artery (either left or right, can image one at a time).</li>
		<li>By using the micromanipulators, view either the right or the left renal artery.</li>
		<li>Adjust the probe angle by tilting slightly along the y axis of the probe to obtain a full kidney view in the center of the screen.</li>
		<li>Once the proper landmarks (renal pelvis, renal artery) are identified as illustrated in Figure 1C and D, cine store the image using the highest frame-rate allowed with the probe used.</li>
	</ol>
	</li>
	<li>Transverse image of Kidney (Color-Doppler view):
	<ol>
		<li>Using the Color-Doppler key on the keyboard, turn on Color Doppler acoustic window. This helps to isolate renal artery and renal vein (Figure 1D). (Blue color indicates arterial flow; and red color indicates venous flow).</li>
		<li>Ensure that the focus depth (indicated by and yellow arrowhead on the Y axis) lies in the center of kidney. Record the data with cine store.</li>
		<li>Make sure record the data at the highest possible frame rate possible (&#62;200 frames/sec).</li>
	</ol>
	</li>
	<li>Transverse image of Kidney (Pulsed-wave or PW view):
	<ol>
		<li>Click on the PW key, while in Color-Doppler mode, to bring up a yellow indicator line (Pulsed-wave Doppler sample volume) on the screen (Figure 1F).</li>
		<li>Place the yellow line in the renal artery, at an angle that parallels the directionality of the flow through the vessel by using the PW angle key.</li>
		<li>For flow assessment in the right renal artery, place the PW yellow indicator line along the renal artery in the direction of the flow (this is shown in blue in Figure 1D and E) ensuring the Doppler angle is 60 degrees or less.</li>
		<li>In this mode, the accoustic window splits up into upper and lower sections.</li>
		<li>Use Cine store to capture the image of the wave forms that indicate the velocity of the arterial flow at peak systole and diastole.</li>
	</ol>
	</li>
</ol>
4. Animal Handling After Imaging
<ol>
	<li>From Day 0 to Day 5, place animal into a clean recovery area (with clean paper towel on bedding) in sternal recumbency position after imaging.&#160;Note that we handle all animals with extreme care with &#8220;Tail Holding&#8221; method for aggressive animals such as animals recover from anesthesia.</li>
	<li>During anesthetic recovery, keep animal&#8217;s body temperature with an external heat source&#160;and monitor animal&#8217;s vital sign with electrophysiological probes until animal fully recovers from anesthesia.</li>
	<li>Return recovered animals to the facility housing room when they are alert and active.</li>
	<li>Euthanize all rats according to institutional guidelines on Day 6 and harvest kidneys (see step 4.7) for histological assessment as well as step 4.5.</li>
	<li>Collect animal&#8217;s urine from collection tubes attached in the metabolic cage for creatinine test to check kidney function.</li>
	<li>Perform paraffin section of animal kidney, and carry out HE (Hematoxylin and Eosin) staining to check nephrotoxicity (see step 4.7 for detail).</li>
	<li>Sacrifice animals and exsanguinate with 0.9% NaCl solution, followed by 10% buffered formalin fixation through the left ventricle. After exsanguination with 0.9% NaCl solution, remove the rat kidneys for histological assessment16.
	<ol>
		<li>Paraffin embed 6-mm sections to observe the kidney morphology and nephrotoxicity. Dehydrate kidney tissue in 30% sucrose in phosphate-buffered saline (PBS) for 48 hr at 4 &#186;C. Then fix the sections in 10% buffered formalin for 24&#8211;48 hr at 4 &#186;C.</li>
		<li>Next, embed the kidney tissue in paraffin, and store the tissue paraffin blocks at RT until sectioning. Further section the tissue blocks using a paraffin section machine and place the sections on a coated glass slide.</li>
		<li>Deparrafinize the section and rehydrated and stained with Hematoxylin for 10 min followed by Eosin for 3 min. Mount the sections on a slide and have them evaluated by a rodent pathologist.</li>
	</ol>
	</li>
</ol>
5. Data Calculation and Analysis
<ol>
	<li>Calculate renal arterial peak velocities from the Color Doppler images obtained from step 3.2. Select Velocity Time Integral (VTI) tool to trace the peaks of systolic and diastolic velocity.</li>
	<li>Calculate Resistive Index (RI) and Pulsatile Index (PI) using the equations below. 
	RI= (peak systolic velocity-end diastolic velocity)/peak systolic velocity 
	PI= (peak systolic velocity-end diastolic velocity)/mean velocity.</li>
	<li>Perform statistical analysis of RI and PI results with standard deviations from the average of three cycle measurements. For other standard parameters, please refer to the manuals from the manufacturer to perform data analysis using proprietary software (see Materials and Equipment Table).</li>
</ol>

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The authors have nothing to disclose.

In the past decade, many advancements have occurred in sonographic technology including the development of high-frequency mechanical probes, which offer sonographic data with high quality, sensitivity, and accuracy. These probes can provide approximately 50 &#181;m axial resolution at a penetration depth of 5 to 12 mm and high frame rates (greater than 200 frame/sec), thus it can serve as a robust tool to study small animals such as rats and mice18,19. Furthermore, it also allows to collect sonographic images on lightly sedated or conscious animals with vital signs at physiological levels. In addition, this non-invasive modality also provides opportunity to perform longitudinal assessment of structural and functional changes during disease progression without sacrificing animals19.
In 1959, Drs. Rusell and Brush first described the three &#8220;R&#8221; rules (Replacement, Reduction, and Refinement) to raise awareness of ethical issues in animal use in research. The proposed protocol shows for the first time that non-invasive small animal sonography can utilizes minimal number of animals under least pain, suffering or distress in Nephorotoxicity study. Therefore, it is a potential effective modality to meet the three &#8220;R&#8217; rules for experimental animals.
Many sonographic studies have focused in cardiac applications; the kidney function assessments were often derived from measurements of cardiac status rather than a direct study of kidney 20-25. We have established an imaging methodology to visualize anatomical and functional changes in kidney in real time. We used a pre-selected set of complementary acoustic windows, gray-scale/B Mode and Color-Doppler, specific for kidney view. We used the RI and PI indices to evaluate the relationship between these indices and the changes of renal function in the Cisplatin induced toxicity model.
However, there are few challenges and limitations to the proposed imaging methods as follows: 1) Appropriate choice of anesthetic agent and the degree of anesthesia are crucial for cardiac and respiratory stability. Inconsistent physiological phenomenon (including respiratory and heart rate fluctuations) affect renal artery flow, quality of imaging and kidney function assessment. We will use IACUC approved injectable anesthetic agent, Pentobarbital (50 mg/kg body weight, i.p.), as our backup anesthetic agent to ensure proper normal physiological function during the imaging and kidney function assessment; 2) Depilation is a critical step, as presence of chest hair affects the quality of the sonographic images; 3) While sonography of kidneys is relatively straightforward for a trained operator; for the average operator, it is critical to adapt technique to individual animal&#8217;s unique anatomy and make minor manipulations; 4) If the size of rats is rather large (above 350 grams), a lower frequency probe (less than used in this study 21 MHz) may be required for optimal imaging. It might be prudent to take a training course before the proposed operation of the imaging system.
The novelty in detecting drug-induced nephrotoxicity using the proposed sonographic methodology and derived protocol is its early robust detection of hemodynamic changes in the event of kidney injury. The results indicate that the intra-renal vascular hemodynamic changes in fact precede the rising sCr. These data is benchmarked against the conventional gold standard using histological analysis and demonstrate that small animal sonography is a noninvasive, sensitive, and reproducible modality, which has minimal requirement of animal use. It is thus an effective tool for early detection of drug-induced nephrotoxicity using rat model.

Serum creatinine (sCr) has been the gold standard metric to assess kidney function for more than two decades. Recently, many studies have reported that renal injury occurs much earlier than the changes in sCr1. However, there are no robust methods for detection of hemodynamic changes that occur early in the course of renal injury including drug-induced nephrotoxicity.
Drug-induced acute renal hemodynamic dysfunction leads to renal tissue damage and further progression to renal failure2,3. In the past couple decades, studies indicate that imaging tools such as computed tomography (CAT), Functional magnetic resonance imaging (fMRI) and sonography play a role in hemodynamic assessment4. In the current imaging tools, gray scale sonography coupled with Color-Doppler techniques, are the most commonly used to establish and assess anatomical status of kidney3,5,6. Sullivan et. al. and Bonnin et. al. recently reported that sonography is an effective, powerful and non-invasive tool in analysis hemodynamic changes in vasoconstriction and hypoxia stress animal models7,8. This technique is also commonly used to detect arterial stenosis9,10.
Latest technical advances in the field of high-resolution ultrasound imaging have allowed investigators to address cardiovascular toxicity using high-frequency (25-80 MHz) and high-resolution (&#60; 0.03 mm resolution) probes, in vivo11. We hypothesize that using this high-resolution sonography to study kidney will provide an unprecedented opportunity for a non-invasive and sensitive method for early detection of nephrotoxicity.
Cisplatin is used to treat testicular, ovarian, bladder, head, lung, and neck cancers in combination with other drugs12-14. Cisplatin has had well-documented nephrotoxicity due to cell necrosis of proximal tubules (PT) and collecting ducts resulted in rising blood urea nitrogen (BUN) and sCr15. Herein, we provide a detailed step-by-step methodology of using non-invasive renal sonography to characterize kidney dysfunction using the rat model of Drug (Cisplatin)-induced nephrotoxicity.

<table><tbody><tr><td>Depilatory cream</td><td>Miltex, Inc.</td><td>Surgi-Prep</td><td>Apply 24 hours prior to imaging</td></tr><tr><td>cis-Diamineplatinum(II) dichloride</td><td>Sigma</td><td>479306</td><td>To induce acute kidney injury at small animals.</td></tr><tr><td>Isoflurane</td><td>Baxter International Inc.</td><td>NDC 10019-773-40</td><td>2-3% for induction, and 1-1.5 % for maintenance; heart beats will be maintained at above 500 beats per minute</td></tr><tr><td>High Frequency Ultrasound</td><td>FUJIFILM VisualSonics, Inc.</td><td>Vevo 2100</td><td></td></tr><tr><td>High-frequency Mechanical Transducer</td><td>FUJIFILM VisualSonics, Inc.</td><td>MS250, MS550D, MS400</td><td></td></tr></tbody></table>

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.

The images presented in this study were taken by a single operator. All images were collected using a high frequency ultrasound machine (please see Materials and Equipment Table). All imaging data was analyzed by a single investigator. The results showed that Cisplatin-treated animals had sCr ranging from 0.5 to 2.1 (normal range &#60;1.1) at day 6 (Figure 2A). However, the histological studies demonstrated consistent patterns of acute tubule interstitial injuries when compared to normal saline treated animals.
Using high-resolution ultrasound imaging to measure hemodynamic changes of kidney, data showed that there was no change of morphology in animals treated with NS between day 0 and day 6, while pulsus parvus morphology was detected in animals at day six after Cisplain treatment. The upper limit of normal RI and PI are 0.7 and 1.15, respectively, in rats 17. Using the above indices to assess hemodynamic changes of kidney, which demonstrated that there is significant increase of RI and PI in Cisplatin-treated animals at day 6.

<img alt="Figure 1" src="/files/ftp_upload/52409/52409fig1.jpg" /> 
Figure 1. Ultrasound apparatus settings for detecting kidney images in rats. Graphical illustrations of imaging system with the setting of animal stage (A) and imaging probe position (B) during the operation of rat kidney sonographic imaging. The sample sonographic images obtained from rat kidney using the high-frequency, high-resolution ultrasound systems (see Material andand Equipment Table). (C-F). The data demonstrate clear kidney anatomic structure and blood flow in the renal vessels with sufficient information for further hemodynamic parameter measurement and analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52409/52409fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52409/52409fig2.jpg" /> 
Figure 2. Histology and Kidney sonographic images of rats under Cisplatin treatment.&#160; Serum creatinine (sCr) and histology study presents normal kidney tissue in vehicle treated rat and severe proximal tubular kidney injury (yellow arrow) in Cisplatin treated rat (A, B). sCr increased marginally after Cisplatin treatment, but remained within the normal range (&#60;1.1). Sonographic images of right kidney of rats in Color-Doppler Mode at day 0, 3, and 6 on vehicle and Cisplatin treated rats (C); hemodynamic parameters, RI and PI, were significant increased, assessed by Color-Doppler ultrasound (D, E). The upper limit of normal RI is 0.7 and 1.15 for PI. Importantly, the above data show those hemodynamic changes preceded the rising of sCr. Pulse wave velocity measurement show a slow and weak pulse (pulsus parvus sign, yellow circle) after Cisplatin treatment which correlate with histology study results. This phenomenon indicates renal artery stenosis, obstruction and further kidney dysfunction. Histological data showed successful drug-induced proximal tubular kidney injury and sonographic assessment showed significant changes in RI, PI and pulse wave velocity using Color-Doppler technology. N=3, *, p&#60;0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52409/52409fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/52409/52409table1.jpg" /> 
Table 1. Renal hemodynamic parameters for Drug-induced AKI

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

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 ...

early-detection-drug-induced-renal-hemodynamic-dysfunction-using

Nanyang Technological University

Research

JoVE Journal

Medicine

12.0K Views.  Brigham and Women's Hospital, Harvard Medical School. 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. 

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

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 ...

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

Ultrasound Assessment of Flow-Mediated Dilation of the Brachial and Superficial Femoral Arteries in Rats

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of endothelial function can be achieved by employing the flow-mediated dilation technique, typically performed in the brachial or superficial femoral artery. Briefly, a blood pressure cuff placed distal to an ultrasound probe is inflated to a suprasystolic pressure, which results in limb ischemia. After 5 min of occlusion the cuff is deflated, resulting in reactive hyperemia and increases in wall shear rate that signal vasodilatory molecules to be released from the endothelium eliciting vasodilation. Despite the thousands of studies performing flow-mediated dilation in humans, surprisingly, no studies have performed this technique non-invasively in living rats. Considering the recent shift in focus to translational research, the establishment of guidelines for non-invasive measurement of flow-mediated dilation in rats and other rodents would be extremely valuable. In the following article, a protocol is presented for the non-invasive measurement of flow-mediated dilation in brachial and superficial femoral arteries of rats, as those sites are most commonly measured in humans.

Non-invasive assessment of endothelial function in humans can be determined by the flow-mediated dilation technique. Although thousands of studies have used this technique, no study has performed this technique non-invasively in rats. The following article describes non-invasive measurement of flow-mediated dilation in the brachial and superficial femoral arteries of rats.

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of ...

Ultrasonography in Experimental Reproductive Investigations on Rats

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in reproductive medicine. In the past, the study of reproductive system development in rodents has been based on one-time histological examination of excised tissues. Recently, with the development of high-resolution transabdominal ultrasound, high-quality sonography can now be performed to evaluate the reproductive organs of rats, allowing a new method for studying the reproductive system. Images were obtained using a high-resolution ultrasonographic system. Gynecological ultrasonography was performed on 28 eight-week-old non-pregnant rats and 5 pregnant Sprague-Dawley rats. We describe how to recognize organs of the reproductive system and associated structures in typical views during different phases of the estrus cycle. Color flow Doppler was used to measure uterine artery blood flow and evaluate uterine blood flow pattern changes during different stages of pregnancy. We have demonstrated that ultrasound exploration is a useful method for evaluating changes in internal reproductive organs. Its use raises the possibility of conducting additional experiments, including medical or surgical procedures, and provides the ability to monitor sonographic changes to internal organs without sacrificing animals.

This manuscript describes the utility of ultrasound performed on female rats to design experimental models for reproductive and gynecological investigation. A step-by-step explanation of how to perform ultrasonographic evaluation is shown.

With the development of assisted reproductive technology and the ethical limitations of research on humans, rat animal models have been widely used in ...

Noninvasive and Invasive Renal Hypoxia Monitoring in a Porcine Model of Hemorrhagic Shock

Up to 50% of patients with trauma develop acute kidney injury (AKI), in part due to poor renal perfusion after severe blood loss. AKI is currently diagnosed based on a change in serum creatinine concentration from baseline or prolonged periods of decreased urine output. Unfortunately, baseline serum creatinine concentration data is unavailable in most patients with trauma, and current estimation methods are inaccurate. In addition, serum creatinine concentration may not change until 24-48 h after the injury. Lastly, oliguria must persist for a minimum of 6 h to diagnose AKI, making it impractical for early diagnosis. AKI diagnostic approaches available today are not useful for predicting risk during the resuscitation of patients with trauma. Studies suggest that urinary partial pressure of oxygen (PuO2) may be useful for assessing renal hypoxia. A monitor that connects the urinary catheter and the urine collection bag was developed to measure PuO2 noninvasively. The device incorporates an optical oxygen sensor that estimates PuO2 based on luminescence quenching principles. In addition, the device measures urinary flow and temperature, the latter to adjust for confounding effects of temperature changes. Urinary flow is measured to compensate for the effects of oxygen ingress during periods of low urine flow. This article describes a porcine model of hemorrhagic shock to study the relationship between noninvasive PuO2, renal hypoxia, and AKI development. A key element of the model is the ultrasound-guided surgical placement in the renal medulla of an oxygen probe, which is based on an unsheathed optical microfiber. PuO2 will also be measured in the bladder and compared to the kidney and noninvasive PuO2 measurements. This model can be used to test PuO2 as an early marker of AKI and assess PuO2 as a resuscitative endpoint after hemorrhage that is indicative of end-organ rather than systemic oxygenation.

Presented here is a protocol to measure renal oxygenation in the medulla and noninvasive urine oxygen partial pressure in a hemorrhagic shock porcine model to establish urine oxygen partial pressure as an early indicator of acute kidney injury (AKI) and a novel resuscitative endpoint.

Up to 50% of patients with trauma develop acute kidney injury (AKI), in part due to poor renal perfusion after severe blood loss. AKI is currently ...

Bioengineering

Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional imaging to study the cardiac anatomy of the left ventricle and M-mode echo to assess its dimensions. However, this could limit a comprehensive study. Herein, we describe a protocol that allows an assessment of the heart chamber size, left ventricular function (systolic and diastolic) and valvular function. A conventional medical ultrasound machine was used in this protocol and different echo views were obtained through left parasternal, apical and suprasternal windows. In the left parasternal window, the long and short axis were acquired to analyze left chamber dimensions, right ventricle and pulmonary artery dimensions, and mitral, pulmonary and aortic valve function. The apical window allows the measurement of heart chamber dimensions and evaluation of systolic and diastolic parameters. It also allows Doppler assessment with detection and quantification of heart valve disturbances (regurgitation or stenosis). Different segments and walls of the left ventricle are visualized throughout all views. Finally, the ascending aorta, aortic arch, and descending aorta can be imaged through the suprasternal window. A combination of ultrasound imaging, Doppler flow and tissue Doppler assessment have been obtained to study cardiac morphology and function. This represents an important contribution to improve the assessment of cardiac function in adult rats with impact for research using these animal models.

A non-invasive protocol for transthoracic echocardiography assessment of cardiac anatomy and function for adult rats is presented in the current study. The heart valves, all four cardiac chambers and the ascending aorta, aortic arch and descending aorta are studied in detail.

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional ...

A Large Animal Model for Acute Kidney Injury by Temporary Bilateral Renal Artery Occlusion

Acute kidney injury (AKI) is associated with higher risk for morbidity and mortality post-operatively. Ischemia-reperfusion injury (IRI) is the most common cause of AKI. To mimic this clinical scenario, this study presents a highly reproducible large animal model of renal IRI in swine using temporary percutaneous bilateral balloon-catheter occlusion of the renal arteries. The renal arteries are occluded for 60 min by introducing the balloon-catheters through the femoral and carotid artery and advancing them into the proximal portion of the arteries. Iodinated contrast is injected in the aorta to assess any opacification of the kidney vessels and confirm the success of the artery occlusion. This is furtherly confirmed by the flattening of the pulse waveform at the tip of the balloon catheters. The balloons are deflated and removed after 60 min of bilateral renal artery occlusion, and the animals are allowed to recover for 24 h. At the end of the study, plasma creatinine and blood urea nitrogen significantly increase, while eGFR and urine output significantly decrease. The need for iodinated contrast is minimal and does not affect renal function. Bilateral renal artery occlusion better mimics the clinical scenario of perioperative renal hypoperfusion, and the percutaneous approach minimizes the impact of the inflammatory response and the risk of infection seen with an open approach, such as a laparotomy. The ability to create and reproduce this clinically relevant swine model eases the clinical translation to humans.

This study presents a highly reproducible large animal model of renal ischemia-reperfusion injury in swine using temporary percutaneous bilateral balloon-catheter occlusion of the renal arteries for 60 min and reperfusion for 24 h.

Acute kidney injury (AKI) is associated with higher risk for morbidity and mortality post-operatively. Ischemia-reperfusion injury (IRI) is the most ...

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), ...

Long-Term Continuous Measurement of Renal Blood Flow in Conscious Rats

The kidneys play a crucial role in maintaining the homeostasis of body fluids. The regulation of renal blood flow (RBF) is essential to the vital functions of filtration and metabolism in kidney function. Many acute studies have been carried out in anesthetized animals to measure RBF under various conditions to determine mechanisms responsible for the regulation of kidney perfusion. However, for technical reasons, it has not been possible to measure RBF continuously (24 h/day) in unrestrained unanesthetized rats over prolonged periods. These methods allow the continuous determination of RBF over many weeks while also simultaneously recording blood pressure (BP) with implanted catheters (fluid-filled or by telemetry). RBF monitoring is carried out with rats placed in a circular servo-controlled rat cage that enables the unrestrained movement of the rat throughout the study. At the same time, the tangling of cables from the flow probe and arterial catheters is prevented. Rats are first instrumented with an ultrasonic flow probe placement on the left renal artery and an arterial catheter implanted in the right femoral artery. These are routed subcutaneously to the nape of the neck, and connected to the flowmeter and pressure transducer, respectively, to measure RBF and BP. Following surgical implantation, rats are immediately placed in the cage to recover for at least one week and stabilize the ultrasonic probe recordings. Urine collection is also feasible in this system. The surgical and post-surgical procedures for continuous monitoring are demonstrated in this protocol.

The present protocol describes a long-term continuous measurement of renal blood flow in conscious rats and simultaneously recording blood pressure with implanted catheters (fluid-filled or by telemetry).

The kidneys play a crucial role in maintaining the homeostasis of body fluids. The regulation of renal blood flow (RBF) is essential to the vital ...

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 ...

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

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