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>

<ol>
	<li>Bonventre, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+acute+kidney+injury:+from+classic+parameters+to+new+biomarkers.">Diagnosis of acute kidney injury: from classic parameters to new biomarkers.</a> Contrib Nephrol. 156, 213-219 (2007).</li><li>Lerman, L. O., Textor, S. C., Grande, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+tissue+injury+in+renal+artery+stenosis:+ischemia+and+beyond.">Mechanisms of tissue injury in renal artery stenosis: ischemia and beyond.</a> Progress in cardiovascular diseases. 52 (3), 196-203 (2009).</li><li>Karas, M. G., Kizer, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiographic+assessment+of+the+right+ventricle+and+associated+hemodynamics.">Echocardiographic assessment of the right ventricle and associated hemodynamics.</a> Progress in cardiovascular diseases. 55 (2), 144-160 (2012).</li><li>Milman, Z., 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=Hemodynamic+response+magnetic+resonance+imaging:+application+for+renal+hemodynamic+characterization.">Hemodynamic response magnetic resonance imaging: application for renal hemodynamic characterization.</a> Nephrol Dial Transplant. 28, 1150-1156 (2013).</li><li>Anavekar, N. S., 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=Usefulness+of+right+ventricular+fractional+area+change+to+predict+death,+heart+failure,+and+stroke+following+myocardial+infarction+(from+the+VALIANT+ECHO+Study).">Usefulness of right ventricular fractional area change to predict death, heart failure, and stroke following myocardial infarction (from the VALIANT ECHO Study).</a> Am J Cardiol. 101 (5), 607-612 (2008).</li><li>Lindqvist, P., Calcutteea, A., Henein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiography+in+the+assessment+of+right+heart+function.">Echocardiography in the assessment of right heart function.</a> Eur J Echocardiogr. 9 (2), 225-234 (2008).</li><li>Sullivan, J. 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S., Ferguson, M. A., Bonventre, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+of+acute+kidney+injury.">Biomarkers of acute kidney injury.</a> Annu Rev Pharmacol Toxicol. 48, 463-493 (2008).</li><li>Hye Khan, M. A., Abdul Sattar, M., Abdullah, N. A., Johns, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cisplatin-induced+nephrotoxicity+causes+altered+renal+hemodynamics+in+Wistar+Kyoto+and+spontaneously+hypertensive+rats:+role+of+augmented+renal+alpha-adrenergic+responsiveness.">Cisplatin-induced nephrotoxicity causes altered renal hemodynamics in Wistar Kyoto and spontaneously hypertensive rats: role of augmented renal alpha-adrenergic responsiveness.</a> Exp Toxicol Pathol. 59, 253-260 (2007).</li><li>Vaidya, V. S., Bonventre, J. 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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=High+frame+rate+Doppler+echocardiography+in+the+rat:+an+evaluation+of+the+method.">High frame rate Doppler echocardiography in the rat: an evaluation of the method.</a> European journal of echocardiography : the journal of the Working Group on Echocardiography of the European Society of Cardiology. 2 (2), 78-87 (2001).</li><li>Zhang, L., 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=A+high-frequency,+high+frame+rate+duplex+ultrasound+linear+array+imaging+system+for+small+animal+imaging.">A high-frequency, high frame rate duplex ultrasound linear array imaging system for small animal imaging.</a> IEEE transactions on ultrasonics, ferroelectrics, and frequency. 57, 1548-1557 (2010).</li><li>Frea, S., 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=Echocardiographic+evaluation+of+right+ventricular+stroke+work+index+in+advanced+heart+failure:+a+new+index?.">Echocardiographic evaluation of right ventricular stroke work index in advanced heart failure: a new index?.</a> J Card Fail. 18 (12), 886-893 (2012).</li><li>Marwick, T. H., Raman, S. V., Carrio, I., Bax, J. 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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.

&#160;
<table border="0" cellpadding="0" cellspacing="0">
	<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></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td colspan="2">Table of equipments</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>High Frequency Ultrasound</td>
			<td>FUJIFILM VisualSonics, Inc.</td>
			<td>Vevo 2100</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="3">High-frequency Mechanical Transducer</td>
			<td rowspan="3">FUJIFILM VisualSonics, Inc.</td>
			<td rowspan="3">MS250, MS550D, MS400</td>
			<td rowspan="3"></td>
		</tr>
		<tr>
		</tr>
		<tr>
		</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 ...

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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

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Using Continuous Data Tracking Technology to Study Exercise Adherence in Pulmonary Rehabilitation

Pulmonary rehabilitation (PR) is an important component in the management of respiratory diseases. The effectiveness of PR is dependent upon adherence to exercise training recommendations. The study of exercise adherence is thus a key step towards the optimization of PR programs. To date, mostly indirect measures, such as rates of participation, completion, and attendance, have been used to determine adherence to PR. The purpose of the present protocol is to describe how continuous data tracking technology can be used to measure adherence to a prescribed aerobic training intensity on a second-by-second basis.
In our investigations, adherence has been defined as the percent time spent within a specified target heart rate range. As such, using a combination of hardware and software, heart rate is measured, tracked, and recorded during cycling second-by-second for each participant, for each exercise session. Using statistical software, the data is subsequently extracted and analyzed. The same protocol can be applied to determine adherence to other measures of exercise intensity, such as time spent at a specified wattage, level, or speed on the cycle ergometer. Furthermore, the hardware and software is also available to measure adherence to other modes of training, such as the treadmill, elliptical, stepper, and arm ergometer. The present protocol, therefore, has a vast applicability to directly measure adherence to aerobic exercise.

Pulmonary rehabilitation is widely recognized in the management of respiratory diseases. A key component to successful pulmonary rehabilitation is adherence to the recommended exercise training. The purpose of the present protocol is to describe how continuous data tracking technology can be used to precisely measure adherence to a prescribed aerobic training intensity.

Pulmonary rehabilitation (PR) is an important component in the management of respiratory diseases. The effectiveness of PR is dependent upon adherence to ...

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory&#160;rate&#160;and heart&#160;rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure.
This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41&#177;2.94 cmH2O which was significantly higher (P&#60;0.05) than before resection (6.87&#177;2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in mice.

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. ...

Invasive Hemodynamic Characterization of the Portal-hypertensive Syndrome in Cirrhotic Rats

This is a detailed protocol describing invasive hemodynamic measurements in cirrhotic rats for the characterization of portal hypertensive syndrome. Portal hypertension (PHT) due to cirrhosis is responsible for the most severe complications in patients with liver disease. The full picture of the portal hypertensive syndrome is characterized by increased portal pressure (PP) due to the increased intrahepatic vascular resistance (IHVR), hyperdynamic circulation, and increased splanchnic blood flow. Progressive splanchnic arterial vasodilation and increased cardiac output with elevated heart rate (HR) but low arterial pressure characterizes the portal hypertensive syndrome.
Novel therapies are currently being developed that aim to decrease PP by either targeting IHVR or increased splanchnic blood flow &#8212; but side effects on systemic hemodynamics may occur. Thus, a detailed characterization of portal venous, splanchnic, and systemic hemodynamic parameters, including measurement of PP, portal venous blood flow (PVBF), mesenteric arterial blood flow, mean arterial pressure (MAP), and HR is needed for preclinical evaluation of the efficacy of novel treatments for PHT. Our video article provides the reader with a structured protocol for performing invasive hemodynamic measurements in cirrhotic rats. In particular, we describe the catheterization of the femoral artery and the portal vein via an ileocolic vein and the measurement of portal venous and splanchnic blood flow via perivascular Doppler-ultrasound flow probes. Representative results of different rat models of PHT are shown.

Here we describe a detailed protocol for invasive measurements of hemodynamic parameters including portal pressure, splanchnic blood flow, and systemic hemodynamics in order to characterize the portal hypertensive syndrome in rats.

This is a detailed protocol describing invasive hemodynamic measurements in cirrhotic rats for the characterization of portal hypertensive syndrome. ...

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 ...

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis (NASH) Resolution

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and assessment of improvement in disease endpoints, often reflected as a percent of each treatment arm that improved, worsened or remained unchanged. Traditional preclinical rodent studies for putative NASH therapies are often limited by not knowing the level of liver disease/NASH present at the start of therapeutic intervention, instead of randomizing treatment groups on easily measurable endpoints such as body weight, metabolic status or similar. Here, we describe a liver biopsy technique in a diet-induced NASH mouse model, for the assessment of baseline liver disease in order to exclude mice that do not exhibit fibrosis and to equally distribute animals with similar fibrosis between treatment groups. These levels can then be compared to the terminal, post-intervention levels for a truer understanding of in vivo pharmacological effects and thus more accurately reflect clinical trial design strategies. The mouse is properly anesthetized and prepared for the surgery using sterile conditions. A small incision is made in the upper abdomen and the left lateral lobe of the liver is exposed. A wedge of the liver is surgically removed, and a similar-sized piece of absorbable gelatin is put in its place to stop any bleeding. The mouse is surgically sutured and stapled closed and will recover back to normal within 1 day. The entire process takes 5-10 min per mouse. Here we exemplify the utility of this procedure by leveraging the pre-study biopsy to assess the impact of the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide on NASH endpoints in mice.

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis NASH Resolution

In clinical settings, a liver biopsy is used to assess stages of non-alcoholic steatohepatitis (steatosis, inflammation, hepatocyte ballooning, and fibrosis). Here, we illustrate a survivable liver biopsy in mice which can be used for histological analyses to enable assessment of therapeutic agents in a manner more aligned with clinical trials.

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and ...

An In Vitro Hemodynamic Loop Model to Investigate the Hemocytocompatibility and Host Cell Activation of Vascular Medical Devices

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive conjugates was compared to uncoated PVC tubes, latex tubes, and a stent for intravascular application that was placed inside the PVC tubes. Evaluation of hemocompatibility was done using an in vitro hemodynamic loop model that is recommended by the ISO standard 10993-4. The tubes were cut into segments of identical length and closed to form loops avoiding any gap at the splice, then filled with human blood and rotated in a water bath at 37 &#176;C for 3 hours. Thereafter, the blood inside the tubes was collected for the analysis of whole blood cell count, hemolysis (free plasma hemoglobin), complement system (sC5b-9), coagulation system (fibrinopeptide A), and leukocyte activation (polymorphonuclear elastase, tumor necrosis factor and interleukin-6). Host cell activation was determined for platelet activation, leukocyte integrin status and monocyte platelet aggregates using flow cytometry. The effect of inaccurate loop closure was examined with x-ray microtomography and scanning electron microscopy, that showed thrombus formation at the splice. Latex tubes showed the strongest activation of both plasma and cellular components of the blood, indicating a poor hemocompatibility, followed by the stent group and uncoated PVC tubes. The coated PVC tubes did not show a significant decrease in platelet activation status, but showed an increased in complement and coagulation cascade compared to uncoated PVC tubes. The loop model itself did not lead to the activation of cells or soluble factors, and the hemolysis level was low. Therefore, the presented in vitro hemodynamic loop model avoids excessive activation of blood components by mechanical forces and serves as a method to investigate in vitro interactions between donor blood and vascular medical devices.

Presented here is a protocol for a standardized in vitro hemodynamic loop model. This model allows to test the hemocompatibility of perfusion tubes or vascular stents to be in accordance with ISO (International Organization for Standardization) standard 10993-4.

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

Direct Drug Delivery to Kidney via the Renal Artery

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research field. Accumulation of drugs in certain organs may give rise to adverse and unwanted effects, depending on the nature of injectate. To minimize spillover and/or accumulation in other tissues, the herein described method directs the formulation into the renal artery bloodstream by inserting a catheter in the infra renal aorta, just below where it branches into the renal artery, resulting in the kidney as first reached organ and distributing of formulation throughout the kidney.
This manuscript provides a detailed description of the method, as well as its challenges and difficulties. It guides the experimenter to become skillful with this type of microsurgery that requires accuracy under sterile conditions. Speed is crucial for minimizing the ischemia and practicing the procedure will increase the chance of successful injections without adverse effects. By modulating the time between injection and reperfusion as well as the injected volume, the risk of spillover to other organs is mitigated. 
Note that this technique is suitable for single dosing strategies.

This manuscript describes a method for targeted delivery to a single kidney via a catheter placed in the infrarenal abdominal aorta in the mouse.

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research ...

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).