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

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

1. Acclimate all animals for one week before any experimental procedure.
2. 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.
3. Remove hair from the animal’s chest using #40 blade and depilatory cream as necessary.  It may have to remove hair form the animal’s back if we cannot have good image data obtained from ventral side imaging.

2. Nephrotoxicity Rat Model

1. For Cisplatin-induced nephrotoxicity model, administer Cisplatin, using protocol as described previously. ¹⁵
2. Perform sonography at baseline, 24 hr prior to Cisplatin administration (Day 0). (See step 3, Imaging Protocol)
3. 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’s body weight), intraperitoneally in study group and normal saline (NS) in control group.
4. Anesthetize animal as step 1.2 at 24, 48, 72, 96, 120, 144 hr after Cisplatin administration.
5. Take image using high resolution ultrasound system (See Materials and Equipment Table) under stable anesthesia stage of animal. Continue to monitor the animal’s basic physiological function during imaging from anesthesia induction through full recovery.
6. Monitor animal’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’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.

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. 

1. Transverse image of Kidney (B Mode):
   1. Using MS 250 ultrasound with center frequency of 21 MHz connected to the active-port, set the application preset to “General Imaging”.
   2. 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).
   3. 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).
   4. By using the micromanipulators, view either the right or the left renal artery.
   5. 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.
   6. 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.
2. Transverse image of Kidney (Color-Doppler view):
   1. 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).
   2. 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.
   3. Make sure record the data at the highest possible frame rate possible (>200 frames/sec).
3. Transverse image of Kidney (Pulsed-wave or PW view):
   1. 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).
   2. 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.
   3. 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.
   4. In this mode, the accoustic window splits up into upper and lower sections.
   5. Use Cine store to capture the image of the wave forms that indicate the velocity of the arterial flow at peak systole and diastole.

4. Animal Handling After Imaging

1. 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. Note that we handle all animals with extreme care with “Tail Holding” method for aggressive animals such as animals recover from anesthesia.
2. During anesthetic recovery, keep animal’s body temperature with an external heat source and monitor animal’s vital sign with electrophysiological probes until animal fully recovers from anesthesia.
3. Return recovered animals to the facility housing room when they are alert and active.
4. 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.
5. Collect animal’s urine from collection tubes attached in the metabolic cage for creatinine test to check kidney function.
6. Perform paraffin section of animal kidney, and carry out HE (Hematoxylin and Eosin) staining to check nephrotoxicity (see step 4.7 for detail).
7. 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.
   1. 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 ºC. Then fix the sections in 10% buffered formalin for 24–48 hr at 4 ºC.
   2. 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.
   3. 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.

5. Data Calculation and Analysis

1. 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.
2. 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.
3. 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).