The work in this grant is funded by the University of Utah Clinical and Translational Science Institute through the Translational and Clinical Studies Pilot Program and the Department of Defense office of&#160;the Congressionally Directed Medical Research Programs (PR192745).

The Institutional Animal Care and Use Committee of the University of Utah approved all experimental protocols described here. Prior to the experiment, a total of 12 castrated male or non-pregnant female Yorkshire swine weighing 50-75 kg and between 6-8 months old were acclimated in their enclosures for at least 7 days. During this period, all care is directed by a veterinarian and in accordance with the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act Regulations and Standards. The animals are fasted overnight prior to induction of anesthesia but are allowed free access to water.
1. Sensor assembly
<ol>
	<li>Cut a 6 cm piece of 3/8 in thermoplastic elastomer (TPE) tubing, 25 mm pieces of 1/8 in and 3/16 in PVC tubing, and 31 mm pieces of 1/8 in and 3/16 in PVC tubing.</li>
	<li>Drill a hole in the top of the non-vented cap to fit the exposed tip of the temperature probe; start with a 3/32 in drill bit, then use a 1/8 in drill bit.</li>
	<li>Use a 5/32 in drill bit to drill out the top part of the T-connector to fit the oxygen sensor.</li>
	<li>Slide the shorter piece of the 1/8 in PVC tubing over the inlet side of the flow sensor. Slide the longer 1/8 in piece of PVC tubing over the outlet side (as designated by the arrow on the flow sensor itself) of the flow sensor. Slide the shorter and longer 3/16 in pieces of PVC tubing over the corresponding lengths of the 1/8 in PVC tubing. Insert the barbed end of the male luer lock connector into the open end of the 1/8 in PVC tubing. 
	NOTE: If necessary, use a heat gun to heat the tubing prior to sliding over barbed fittings. It is also possible to use isopropyl alcohol to lubricate the barbed end to make it easier to slide the tubing over the barbed connector.</li>
	<li>Mix the biocompatible glue.</li>
	<li>Expose the tip of the temperature probe by removing any protective sheathing or tubing. Fill the inside of the tubing of the thermistor with biocompatible glue but do not cover the exposed tip.</li>
	<li>Assemble the parts as shown in Figure 1. Use the glue to secure each luer lock connection, when inserting the thermistor in the non-vented cap, and prior to sliding the 3/8 in TPE tubing over the barbed end.</li>
	<li>Prior to sterilization, ensure the blue cap on the oxygen stick is not twisted too tightly, or it will be difficult to undo after sterilization. 
	NOTE: An image of an assembled device is shown in Figure 1 for reference. For this experiment, the fiber optic cable was connected to an electro-optical module that contains software that is designed to work with the specific oxygen sensors used in the device. Any luminescence quenching-based oxygen sensor and compatible data collection device will work. In addition, a custom module and a printed circuit board were designed to connect the flow sensor and temperature probe. Custom software was used to collect and display data in real-time.</li>
</ol>
2. Experimental procedure
<ol>
	<li>Induction of anesthesia and monitoring.
	<ol>
		<li>Sedate the animal with a combined intramuscular injection of Ketamine (2.2 mg/kg) and Xylazine (2.2 mg/kg) &#160;and Telazol (4.4 mg/kg).</li>
		<li>Depending on the size of the animal, place an appropriately sized (most likely between 7 mm and 8 mm) cuffed endotracheal tube with the assistance of a laryngoscope.</li>
		<li>Apply eye lubricant to both eyes.</li>
		<li>Following induction, mechanically ventilate the animal with the maintenance of anesthesia with 1.5%-3.0% gaseous isoflurane mixed in oxygen. Set the fraction of inspired oxygen between 40%-100%, the positive end-expiratory pressure to 4 cm H2O, the tidal volume to 6-8 mL/kg, and adjust the respiratory rate and tidal volume to maintain end-tidal CO2 of 35-45 mmHg.</li>
		<li>Monitor and confirm the proper depth of anesthesia by assessing jaw tone, palpebral reflex approximately every 15 min, and absence of spontaneous movement throughout the experiment. Additionally, monitor clinical parameters of tissue perfusion (mucous membrane color, capillary refill time, heart rate), pulse oximetry, end-tidal CO2, core body temperature, and electrocardiogram.</li>
		<li>Position the animal in dorsal recumbency on a warming blanket and secure each leg to the table.</li>
		<li>The protocol is a non-survival procedure with euthanasia of the animal at the end of the experiment, as described in section 5.</li>
	</ol>
	</li>
	<li>Prepare the animal for the experiment.
	<ol>
		<li>Prepare all puncture sites (which are listed in steps 2.2.3-2.2.7) by scrubbing the skin with three alternating scrubs of chlorhexidine followed by alcohol. After the third scrub, apply chlorhexidine and allow to dry completely, then drape the surgical site in a sterile fashion.</li>
		<li>Locally infiltrate all puncture and incision sites with 2% lidocaine for local pain relief.</li>
		<li>Using ultrasound guidance and the Seldinger technique, place a 9 Fr catheter in the right external jugular vein for medication infusion and central venous pressure monitoring and a 7 Fr catheter in the right femoral vein for resuscitation.</li>
		<li>Under ultrasound guidance, place a 7 Fr sheath in the right brachial artery.</li>
		<li>Under ultrasound guidance, place a 7 Fr sheath in the right femoral artery.</li>
		<li>Under ultrasound guidance, place a 7 Fr sheath in the left femoral artery.</li>
		<li>Under ultrasound guidance, place a 5 Fr sheath in the right or left carotid artery.</li>
		<li>Monitor the pressure distal to the balloon of the resuscitative endovascular occlusion of the aorta (REBOA) catheter via the left femoral artery sheath.
		<ol>
			<li>Connect a disposable pressure transducer to the arterial catheter that is distal to the REBOA balloon.</li>
		</ol>
		</li>
		<li>Monitor the pressure proximal to the balloon of the REBOA catheter via the carotid artery sheath.
		<ol>
			<li>Connect a disposable pressure transducer to the arterial catheter that is proximal to the REBOA balloon.</li>
		</ol>
		</li>
		<li>Perform a midline laparotomy by making an incision along the midline of the abdomen, starting at the inferior part of the sternum and ending at the pubis.</li>
		<li>With the abdomen open, identify the bladder and perform a cystotomy, or make a small incision, to insert the tip of a 20 Fr urinary catheter in the bladder. Close the cystotomy with the urinary catheter in place using a purse string suture. After the catheter is in place, secure it to the skin with sutures.</li>
		<li>Prior to connecting the outlet of the catheter to the urinary collection bag, insert the cone-shaped end of the noninvasive PuO2 monitor into the outlet of the catheter.</li>
		<li>Place the open tubing at the end of the novel PuO2 monitor over the cone-shaped connector on the tubing that is connected to the urine collection bag.</li>
		<li>Remove the spleen to eliminate hemorrhage-induced auto-transfusion.
		<ol>
			<li>Locate the spleen. Identify the hilum of the spleen or the site where the splenic artery and vein enter the spleen. Clamp and transect each vessel.</li>
			<li>After transection, ligate each vessel using modified Miller knots using 2-0 sutures.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Place the instrument to measure bladder PuO2 and tissue oxygenation.
	<ol>
		<li>Measure PuO2&#160;at the outlet of&#160;the bladder.
		<ol>
			<li>Identify the balloon on the catheter. Just below the balloon make an incision along the long axis of the catheter, ensuring that you do not cut the lumen that connects to the balloon.</li>
			<li>After making the incision, insert a t-connector that contains the sensing material into the incision.</li>
			<li>Use tissue glue to secure the t-connector in place.</li>
			<li>Connect the fiber optic cable from the bladder data collection device to the connector that contains the sensing material.</li>
			<li>Create a new file on the data collection device and note the time difference between the stand-alone collection device and other devices used in the experiment.
			<ol>
				<li>For the data collection device used in this study: push the back arrow to reach the main menu.</li>
				<li>Go to measurement settings and click on Ok. Use the arrows to highlight the measurement browser box and push Ok.</li>
				<li>Push the right arrow to create a new file. Type in the name of the new file and select Done.</li>
				<li>Highlight the new file name and select Ok. Navigate to the measurement screen and click on Ok to start recording.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Measure medullary renal tissue oxygenation.
		<ol>
			<li>Identify the location of the kidney internally.</li>
			<li>Move the bowel so that you have a clear line of site and access to the entire kidney.&#160;</li>
			<li>Insert the sensor into 2&#34; 18 gauge catheter. Adjust the luer lock connector on the sensor so that the tip of the sensor is exposed. Remove the catheter and place it over an 18 gauge needle.</li>
			<li>Place the 18 gauge needle and 2 in catheter into the renal medulla under ultrasound guidance.</li>
			<li>Remove the needle, keeping the catheter in place. Thread the tissue sensor through the catheter and use the luer lock to connect the sensor to the catheter.&#160;</li>
			<li>Use tissue glue to secure the catheter in place.&#160;</li>
			<li>Connect the tissue sensor to the data collection box.</li>
			<li>Wait for 10 min before beginning the experimental protocol after preparing the instrumentation and animal. This will be considered a baseline period.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Experimental protocol
	<ol>
		<li>Prior to starting the experimental procedure, ensure the mean arterial pressure (MAP) is &#8805;65 mmHg. If MAP is below the threshold, give up to two 5 mL/kg boluses of isotonic crystalloid solution. If MAP remains below 65 mmHg, infuse norepinephrine (0.02 &#181;g/kg/min) until target MAP is achieved.</li>
		<li>Induce hemorrhagic shock.
		<ol>
			<li>Remove 25% (estimated as 60 mL/kg) of the animal&#39;s estimated blood volume through the right brachial artery sheath over 30 min into gently agitated citrated blood collection bags. Mark the beginning of blood removal as t = 0 min.</li>
			<li>Store the removed blood in a warm water bath at 37 &#176;C.</li>
			<li>Then perform randomization to assign animals to either the REBOA with whole blood or REBOA with crystalloids group (n = 6 for each group).</li>
		</ol>
		</li>
		<li>Place the REBOA catheter.
		<ol>
			<li>Insert a 7 Fr REBOA catheter in the right femoral artery sheath. Place the balloon of the catheter immediately superior to the diaphragm and confirm the location using fluoroscopy.</li>
			<li>At t = 30 min, inflate the REBOA balloon and completely occlude the aorta for 45 min.</li>
		</ol>
		</li>
		<li>Initiate resuscitation and administer critical care.
		<ol>
			<li>At t = 70 min, transfuse each animal with their shed blood over 15 min.</li>
			<li>Infuse intravenous calcium over 10 min to prevent citrate-induced hypocalcemia.</li>
			<li>At t = 75 min, deflate the REBOA balloon over 10 min.</li>
			<li>Until t = 360 min, resuscitate the animal with fluids and norepinephrine to maintain a MAP &#62; 65 mmHg.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>End of experiment and euthanasia
	<ol>
		<li>Collect any remaining blood or urine samples.</li>
		<li>Euthanize the animal by injecting a combination of Pentobarbital Sodium (390 mg) and Phenytoin Sodium (50 mg) (1 mL/10 lbs).</li>
	</ol>
	</li>
</ol>
3. Data processing
<ol>
	<li>Time-sync all data files.
	<ol>
		<li>Based on the times that were noted on each device relative to each other and the start of the experiment, align all data files such that t = 0 indicates the start of the experiment.</li>
	</ol>
	</li>
	<li>Remove any data points associated with error flags from the flow sensor. 
	NOTE: The error types are High Flow Rate and Air-in-Line. The High Flow Rate error indicates the flow rate exceeded the sensor&#39;s output limit. The Air-in-Line error flag is raised when the sensor detects air in the flow channel.</li>
	<li>Discard the data associated with the negative flow.
	<ol>
		<li>Once flow becomes negative, track the volume that flows past the sensor in the backward direction.</li>
		<li>After the flow becomes positive, track the volume and compare it to the volume of negative flow to only include measurements from recently voided urine.</li>
	</ol>
	</li>
</ol>

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B., 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=Trauma+induced+acute+kidney+injury.">Trauma induced acute kidney injury.</a> Plos One. 14 (1), 0211001 (2019).</li><li>Lai, W. 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N. Silverton, K. Kuck, and L. Lofgren are inventors of a patent and patent application surrounding the noninvasive monitor used in this study. This prototype is under development for commercial consideration by N. Silverton and K. Kuck, but as of yet, no commercial activity has occurred. The other authors declare no competing interests. The interpretation and reporting of these data are the responsibility of the authors alone.

AKI is a common complication in patients with trauma, and currently, there is no validated bedside monitor for kidney tissue oxygenation, which could enable earlier AKI detection and guide potential interventions. This manuscript describes the use and instrumentation of a porcine hemorrhagic shock model to establish noninvasive PuO2 as an early indicator of AKI and a novel resuscitation endpoint in trauma settings.
One of the distinct advantages of this porcine model is the ability ...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64461">Noninvasive and Invasive Renal Hypoxia Monitoring in a Porcine Model of Hemorrhagic Shock</a>. The Protocol&#160;section was updated.
Step 2.3.1&#160;-&#160;2.3.1.2&#160;of the Protocol was updated from:
<ol>
	<li>Measure PuO2&#160;in the bladder.
	<ol>
		<li>Remove all air from the bladder by slowly squeezing the bladder while ensuring urine does not leak out.</li>
		<li>Place the tip of the luminescence quenching-based PuO2&#160;sensor in the bladder&#160;via&#160;a cystotomy, similar to the catheter.</li>
		<li>Connect the fiber optic cable from the bladder sensor to the data collection device.</li>
	</ol>
	</li>
</ol>
to:
<ol>
	<li>&#160;Measure PuO2 at the outlet of the bladder
	<ol>
		<li>Identify the balloon on the catheter. Just below the balloon make an incision along the long axis of the catheter, ensuring that you do not cut the lumen that connects to the balloon.&#160;</li>
		<li>After making the incision, insert a t-connector that contains the sensing material into the incision.&#160;</li>
		<li>Use tissue glue to secure the t-connector in place.&#160;</li>
		<li>Connect the fiber optic cable from the bladder data collection device to the connector that contains the sensing material.&#160;</li>
	</ol>
	</li>
</ol>
Step 2.3.2.2&#160;-&#160;2.3.2.7&#160;of the Protocol was updated from:
<ol start="2">
	<li>Make a flank incision large enough to expose the kidney (approx. 2-3 in) on the side of the pig at approximately the same location where the kidney was identified.</li>
	<li>With the tips of a retractor together, introduce the retractor into the incision and then spread the tips of the retractor to expose the kidney.</li>
	<li>Use a micro-manipulator or similar tool to hold the oxygen probe steady. If possible, attach this tool to the end of an articulating arm.</li>
	<li>Attach the other end of the articulating arm to the surgical table so that the other end that will hold the oxygen probe is near the opened incision. If the tool that is used to hold the oxygen probe is not connected to an articulating arm, position the tool so the oxygen sensor is near the opened incision and is stable.</li>
	<li>Unlock all articulating joints of the arm. Using ultrasound, place the tip of the oxygen probe in the medulla region of the kidney. Lock all articulating joints on the arm.</li>
	<li>After confirming placement of the tip of the sensor in the medulla with ultrasound, use the micromanipulator to retract the needle housing the luminescence-based oxygen sensor. Connect the other end of the sensor to the data collection device connected to the computer running the data collection software. Start recording.</li>
</ol>
to:
<ol start="2">
	<li>Move the bowel so that you have a clear line of site and access to the entire kidney.&#160;</li>
	<li>Insert the sensor into 2&#34; 18 gauge catheter. Adjust the luer lock connector on the sensor so that the tip of the sensor is exposed. Remove the catheter and place it over an 18 gauge needle.</li>
	<li>Place the 18 gauge needle and 2 in catheter into the renal medulla under ultrasound guidance.</li>
	<li>Remove the needle, keeping the catheter in place. Thread the tissue sensor through the catheter and use the luer lock to connect the sensor to the catheter.&#160;</li>
	<li>Use tissue glue to secure the catheter in place.&#160;</li>
	<li>Connect the tissue sensor to the data collection box.</li>
</ol>
The Table of Materials was updated from:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>1/8&#34; PVC tubing</td>
			<td>Qosina</td>
			<td>SKU: T4307</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>3/16&#34; PVC tubing</td>
			<td>Qosina</td>
			<td>SKU: T4310</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>3/32&#34; (1), 1/8&#34; (1), 5/32&#34; (1) drill bit</td>
			<td>Dewalt</td>
			<td>N/A</td>
			<td>For building noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>3/8&#34; TPE tubing&#160;</td>
			<td>Qosina</td>
			<td>SKU: T2204</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>Biocompatible Glue</td>
			<td>Masterbond</td>
			<td>EP30MED</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>Bladder oxygen measurement device</td>
			<td>Presens</td>
			<td>Fibox 4</td>
			<td>Stand-alone fiber optic oxygen meter</td>
		</tr>
		<tr>
			<td>Bladder PuO2 sensor</td>
			<td>Presens</td>
			<td>DP-PSt3</td>
			<td>Oxygen dipping probe</td>
		</tr>
		<tr>
			<td>Chlorhexidine 4% scrub</td>
			<td>Vetone</td>
			<td>N/A</td>
			<td>For scrubbing insertion or puncture sites</td>
		</tr>
		<tr>
			<td>Conical connector with female luer lock</td>
			<td>Qosina</td>
			<td>SKU: 51500</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>Cuffed endotracheal tube</td>
			<td>Vetone</td>
			<td>600508</td>
			<td>For sedating the subject and providing respiratory support</td>
		</tr>
		<tr>
			<td>Euthanasia solution (pentobarbital sodium|pheyntoin sodium)</td>
			<td>Vetone</td>
			<td>11168</td>
			<td>For euthanasia after completion of experiment</td>
		</tr>
		<tr>
			<td>General purpose temperature probe, 400 series thermistor</td>
			<td>Novamed</td>
			<td>10-1610-040</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>Hemmtop Magic Arm 11 inch</td>
			<td>Amazon</td>
			<td>B08JTZRKYN</td>
			<td>Holding invasive oxygen sensor in place</td>
		</tr>
		<tr>
			<td>HotDog veterinary warming system</td>
			<td>HotDog</td>
			<td>V106</td>
			<td>For controlling subject temperature during experiment</td>
		</tr>
		<tr>
			<td>Invasive tissue oxygen measurement device</td>
			<td>Presens</td>
			<td>Oxy-1 ST&#160;</td>
			<td>Compact oxygen transmitter</td>
		</tr>
		<tr>
			<td>Invasive tissue oxygen sensor</td>
			<td>Presens</td>
			<td>PM-PSt7</td>
			<td>Profiling oxygen microsensor</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Vetone</td>
			<td>501017</td>
			<td>To maintain sedation throughout the experiment</td>
		</tr>
		<tr>
			<td>Isotonic crystalloid solution</td>
			<td>HenrySchein</td>
			<td>1537930 or 1534612</td>
			<td>Used during resuscitation in the critical care period</td>
		</tr>
		<tr>
			<td>Liquid flow sensor</td>
			<td>Sensirion</td>
			<td>LD20-2600B</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>Male luer lock to barb connector</td>
			<td>Qosina</td>
			<td>SKU: 11549</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>Male to male luer connector</td>
			<td>Qosina</td>
			<td>SKU: 20024</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>Noninvasive oxygen measurement device</td>
			<td>Presens</td>
			<td>EOM-O2-mini</td>
			<td>Electro optical module transmitter for contactless oxygen measurements</td>
		</tr>
		<tr>
			<td>Non-vented male luer lock cap</td>
			<td>Qosina</td>
			<td>SKU: 65418</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>Norepinephrine</td>
			<td>HenrySchein</td>
			<td>AIN00610</td>
			<td>Infusion during resuscitation</td>
		</tr>
		<tr>
			<td>O2 sensor stick</td>
			<td>Presens</td>
			<td>SST-PSt3-YOP</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>PowerLab data acquisition platform</td>
			<td>AD Instruments</td>
			<td>N/A</td>
			<td>For data collection</td>
		</tr>
		<tr>
			<td>REBOA catheter</td>
			<td>Certus Critical Care</td>
			<td>N/A</td>
			<td>Used in experimental protocol</td>
		</tr>
		<tr>
			<td>Super Sheath arterial catheters (5 Fr, 7 Fr, 9 Fr)</td>
			<td>Boston Scientific</td>
			<td>C1894</td>
			<td>For intravascular access</td>
		</tr>
		<tr>
			<td>Suture</td>
			<td>Ethicon</td>
			<td>C013D</td>
			<td>For securing catheter to skin and closing incisions</td>
		</tr>
		<tr>
			<td>T connector, all female luer locks</td>
			<td>Qosina</td>
			<td>SKU: 88214</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
	</tbody>
</table>
to:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>1/8&#34; PVC tubing</td>
			<td>Qosina</td>
			<td>SKU: T4307</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>3/16&#34; PVC tubing</td>
			<td>Qosina</td>
			<td>SKU: T4310</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>3/8&#34; TPE tubing&#160;</td>
			<td>Qosina</td>
			<td>SKU: T2204</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>3/32&#34; (1), 1/8&#34; (1), 5/32&#34; (1) drill bit</td>
			<td>Dewalt</td>
			<td>N/A</td>
			<td>For building noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>Biocompatible Glue</td>
			<td>Masterbond</td>
			<td>EP30MED</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>Bladder PuO2 sensor</td>
			<td>Presens</td>
			<td>DP-PSt3</td>
			<td>Oxygen dipping probe</td>
		</tr>
		<tr>
			<td>Bladder oxygen measurement device</td>
			<td>Presens</td>
			<td>Fibox 4</td>
			<td>Stand-alone fiber optic oxygen meter</td>
		</tr>
		<tr>
			<td>Chlorhexidine 4% scrub</td>
			<td>Vetone</td>
			<td>N/A</td>
			<td>For scrubbing insertion or puncture sites</td>
		</tr>
		<tr>
			<td>Conical connector with female luer lock</td>
			<td>Qosina</td>
			<td>SKU: 51500</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>Cuffed endotracheal tube</td>
			<td>Vetone</td>
			<td>600508</td>
			<td>For sedating the subject and providing respiratory support</td>
		</tr>
		<tr>
			<td>Euthanasia solution (pentobarbital sodium|pheyntoin sodium)</td>
			<td>Vetone</td>
			<td>11168</td>
			<td>For euthanasia after completion of experiment</td>
		</tr>
		<tr>
			<td>General purpose temperature probe, 400 series thermistor</td>
			<td>Novamed</td>
			<td>10-1610-040</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>HotDog veterinary warming system</td>
			<td>HotDog</td>
			<td>V106</td>
			<td>For controlling subject temperature during experiment</td>
		</tr>
		<tr>
			<td>Invasive tissue oxygen measurement device</td>
			<td>Optronix</td>
			<td>N/A</td>
			<td>OxyLite&#8482; oxygen monitors</td>
		</tr>
		<tr>
			<td>Invasive tissue oxygen sensor</td>
			<td>Optronix</td>
			<td>NX-BF/OT/E</td>
			<td>Oxygen/Temperature bare-fibre sensor</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Vetone</td>
			<td>501017</td>
			<td>To maintain sedation throughout the experiment</td>
		</tr>
		<tr>
			<td>Isotonic crystalloid solution</td>
			<td>HenrySchein</td>
			<td>1537930 or 1534612</td>
			<td>Used during resuscitation in the critical care period</td>
		</tr>
		<tr>
			<td>Liquid flow sensor</td>
			<td>Sensirion</td>
			<td>LD20-2600B</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>Male luer lock to barb connector</td>
			<td>Qosina</td>
			<td>SKU: 11549</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>Male to male luer connector</td>
			<td>Qosina</td>
			<td>SKU: 20024</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>Norepinephrine</td>
			<td>HenrySchein</td>
			<td>AIN00610</td>
			<td>Infusion during resuscitation</td>
		</tr>
		<tr>
			<td>Noninvasive oxygen measurement device</td>
			<td>Presens</td>
			<td>EOM-O2-mini</td>
			<td>Electro optical module transmitter for contactless oxygen measurements</td>
		</tr>
		<tr>
			<td>Non-vented male luer lock cap&#160;</td>
			<td>Qosina</td>
			<td>SKU: 65418</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>O2 sensor stick</td>
			<td>Presens</td>
			<td>SST-PSt3-YOP</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>PowerLab data acquisition platform</td>
			<td>AD Instruments</td>
			<td>N/A</td>
			<td>For data collection</td>
		</tr>
		<tr>
			<td>REBOA catheter</td>
			<td>Certus Critical Care</td>
			<td>N/A</td>
			<td>Used in experimental protocol</td>
		</tr>
		<tr>
			<td>Super Sheath arterial catheters (5 Fr, 7 Fr, 9 Fr)</td>
			<td>Boston Scientific</td>
			<td>C1894</td>
			<td>for intravascular access</td>
		</tr>
		<tr>
			<td>Suture</td>
			<td>Ethicon</td>
			<td>C013D</td>
			<td>For securing catheter to skin and closing incisions</td>
		</tr>
		<tr>
			<td>T connector, all female luer locks</td>
			<td>Qosina</td>
			<td>SKU: 88214</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
	</tbody>
</table>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64461">Noninvasive and Invasive Renal Hypoxia Monitoring in a Porcine Model of Hemorrhagic Shock</a>. The Protocol and Table of Materials were&#160;updated.

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

Acute kidney injury (AKI) affects up to 50% of patients with trauma admitted to the intensive care unit1. Patients who develop AKI tend to have longer hospital and intensive care unit lengths of stay and a threefold greater risk of mortality2,3,4. Currently, AKI is most commonly defined by the Kidney Disease Improving Global Outcomes (KDIGO) guidelines, which are based on changes in serum creatinine concentration from baseline or periods of prolonged oliguria5. Baseline creatinine concentration data are unavailable in most patients with trauma, and estimation equations are unreliable and have not been validated in patients with trauma6. In addition, serum creatinine concentration may not change until at least 24 h after the injury, precluding early identification and intervention7. While research suggests that urine output is an earlier indicator of AKI than serum creatinine concentration, the KDIGO criteria require a minimum of 6 h of oliguria, which precludes interventions targeting injury prevention8. The optimal hourly urine output threshold and appropriate duration of oliguria for defining AKI are also debated, which limit its effectiveness as an early marker of the disease9,10. Thus, current diagnostic measures for AKI are not useful in trauma settings, lead to delayed diagnosis of AKI, and do not provide real-time information regarding a patient&#39;s risk status for developing AKI.
While the development of AKI in a trauma setting is complex and likely associated with several causes such as poor renal perfusion due to hypovolemia, reduced renal blood flow due to vasoconstriction, trauma-related inflammation, or ischemia-reperfusion injury, renal hypoxia is a common factor among most forms of AKI11,12. In particular, the medulla region of the kidney is highly susceptible to an imbalance between oxygen demand and supply in the trauma setting due to reduced oxygen delivery and high metabolic activity associated with sodium reabsorption. Thus, if it were possible to measure renal medulla oxygenation, it may be possible to monitor a patient&#39;s risk status for developing AKI. While this is not clinically feasible, urinary partial pressure of oxygen (PuO2) at the outlet of the kidney strongly correlates with medullary tissue oxygenation13,14. Other studies have shown that it is possible to measure bladder PuO2 and that it changes in response to stimuli that alter medullary oxygen and renal pelvis PuO2 levels, such as a decrease in renal blood flow15,16,17. These studies suggest that PuO2 may indicate end-organ perfusion and could be useful for monitoring the impact of interventions in trauma settings on renal function.
To monitor PuO2 noninvasively, a noninvasive PuO2 monitor was developed that can easily connect to the end of a urinary catheter outside the body. The noninvasive PuO2 monitor consists of three main components: a temperature sensor, a luminescence quenching oxygen sensor, and a thermal-based flow sensor. Since each oxygen sensor is optically based and relies on the Stern-Volmer relationship to quantify the relationship between luminescence and oxygen concentration, a temperature sensor is necessary to offset any potential confounding effects of changes in temperature. The flow sensor is important to quantify urine output and to determine the direction and magnitude of urine flow. All three components are connected by a combination of male, female, and t-shaped luer lock connectors and poly-vinyl chloride (PVC) flexible tubing. The end with the conical connector connects to the outlet of the urinary catheter, and the end with tubing over the conical connector connects slides over the connector on the urine collection bag.
Despite measuring distally to the bladder, a recent study showed that low urinary PuO2 during cardiac surgery is associated with an increased risk of developing AKI18,19. Similarly, current animal models have primarily focused on the early detection of AKI during cardiac surgery and sepsis14,20,21,22. Thus, questions remain about the use of this novel device in settings of trauma. The aim of this research is to establish PuO2 as an early marker of AKI and investigate its use as a resuscitative endpoint in patients with trauma. This manuscript describes a porcine model of hemorrhagic shock that includes the placement of the noninvasive PuO2 monitor, a bladder PuO2 sensor, and a tissue oxygen sensor in the renal medulla. Data from the noninvasive monitor will be compared to bladder PuO2 and invasive tissue oxygen measurements. The noninvasive monitor also includes a flow sensor which will be useful for understanding the relationship between urine flow rate and oxygen ingress, which reduces the ability to infer renal medullar tissue oxygenation from noninvasive PuO2 as urine traverses the urinary tract. Additionally, data from the three oxygen sensors will be compared to systemic vital signs, such as mean arterial pressure. The central hypothesis is that noninvasive PuO2 data will strongly correlate with invasive medullary oxygen content and will reflect medullary hypoxia during resuscitation. Noninvasive PuO2 monitoring has the potential to improve trauma-related outcomes by identifying AKI earlier and serving as a novel resuscitative endpoint after hemorrhage that is indicative of end-organ rather than systemic oxygenation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1/8&#34; PVC tubing</td>
			<td>Qosina</td>
			<td>SKU: T4307</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>3/16&#34; PVC tubing</td>
			<td>Qosina</td>
			<td>SKU: T4310</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>3/8&#34; TPE tubing&#160;</td>
			<td>Qosina</td>
			<td>SKU: T2204</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>3/32&#34; (1), 1/8&#34; (1), 5/32&#34; (1) drill bit</td>
			<td>Dewalt</td>
			<td>N/A</td>
			<td>For building noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>Biocompatible Glue</td>
			<td>Masterbond</td>
			<td>EP30MED</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>Bladder PuO2 sensor</td>
			<td>Presens</td>
			<td>DP-PSt3</td>
			<td>Oxygen dipping probe</td>
		</tr>
		<tr>
			<td>Bladder oxygen measurement device</td>
			<td>Presens</td>
			<td>Fibox 4</td>
			<td>Stand-alone fiber optic oxygen meter</td>
		</tr>
		<tr>
			<td>Chlorhexidine 4% scrub</td>
			<td>Vetone</td>
			<td>N/A</td>
			<td>For scrubbing insertion or puncture sites</td>
		</tr>
		<tr>
			<td>Conical connector with female luer lock</td>
			<td>Qosina</td>
			<td>SKU: 51500</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>Cuffed endotracheal tube</td>
			<td>Vetone</td>
			<td>600508</td>
			<td>For sedating the subject and providing respiratory support</td>
		</tr>
		<tr>
			<td>Euthanasia solution (pentobarbital sodium|pheyntoin sodium)</td>
			<td>Vetone</td>
			<td>11168</td>
			<td>For euthanasia after completion of experiment</td>
		</tr>
		<tr>
			<td>General purpose temperature probe, 400 series thermistor</td>
			<td>Novamed</td>
			<td>10-1610-040</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>HotDog veterinary warming system</td>
			<td>HotDog</td>
			<td>V106</td>
			<td>For controlling subject temperature during experiment</td>
		</tr>
		<tr>
			<td>Invasive tissue oxygen measurement device</td>
			<td>Optronix</td>
			<td>N/A</td>
			<td>OxyLite&#8482; oxygen monitors</td>
		</tr>
		<tr>
			<td>Invasive tissue oxygen sensor</td>
			<td>Optronix</td>
			<td>NX-BF/OT/E</td>
			<td>Oxygen/Temperature bare-fibre sensor</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Vetone</td>
			<td>501017</td>
			<td>To maintain sedation throughout the experiment</td>
		</tr>
		<tr>
			<td>Isotonic crystalloid solution</td>
			<td>HenrySchein</td>
			<td>1537930 or 1534612</td>
			<td>Used during resuscitation in the critical care period</td>
		</tr>
		<tr>
			<td>Liquid flow sensor</td>
			<td>Sensirion</td>
			<td>LD20-2600B</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>Male luer lock to barb connector</td>
			<td>Qosina</td>
			<td>SKU: 11549</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>Male to male luer connector</td>
			<td>Qosina</td>
			<td>SKU: 20024</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>Norepinephrine</td>
			<td>HenrySchein</td>
			<td>AIN00610</td>
			<td>Infusion during resuscitation</td>
		</tr>
		<tr>
			<td>Noninvasive oxygen measurement device</td>
			<td>Presens</td>
			<td>EOM-O2-mini</td>
			<td>Electro optical module transmitter for contactless oxygen measurements</td>
		</tr>
		<tr>
			<td>Non-vented male luer lock cap&#160;</td>
			<td>Qosina</td>
			<td>SKU: 65418</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>O2 sensor stick</td>
			<td>Presens</td>
			<td>SST-PSt3-YOP</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
		<tr>
			<td>PowerLab data acquisition platform</td>
			<td>AD Instruments</td>
			<td>N/A</td>
			<td>For data collection</td>
		</tr>
		<tr>
			<td>REBOA catheter</td>
			<td>Certus Critical Care</td>
			<td>N/A</td>
			<td>Used in experimental protocol</td>
		</tr>
		<tr>
			<td>Super Sheath arterial catheters (5 Fr, 7 Fr, 9 Fr)</td>
			<td>Boston Scientific</td>
			<td>C1894</td>
			<td>for intravascular access</td>
		</tr>
		<tr>
			<td>Suture</td>
			<td>Ethicon</td>
			<td>C013D</td>
			<td>For securing catheter to skin and closing incisions</td>
		</tr>
		<tr>
			<td>T connector, all female luer locks</td>
			<td>Qosina</td>
			<td>SKU: 88214</td>
			<td>Part of noninvasive PuO2 monitor</td>
		</tr>
	</tbody>
</table>

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.

Figure 1 shows an image of the noninvasive PuO2 monitor described in this manuscript. Figure 2 shows a plot of MAP and noninvasive PuO2 measurements in a single subject during an experiment similar to the described porcine hemorrhage model. At the start of the experiment, as hemorrhage was initiated, there was a drop in MAP and PuO2. Following the initial decline in PuO2 it gradually increased until after the REBOA bal...

Watch this Scientific Journal Video about Noninvasive and Invasive Renal Hypoxia Monitoring in a Porcine Model of Hemorrhagic Shock at JoVE.com

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

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 ...

noninvasive-invasive-renal-hypoxia-monitoring-porcine-model

Research

JoVE Journal

Bioengineering

1.1K Views.  University of Utah. 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. 

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

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Shock Wave Application to Cell Cultures

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to target cells or tissue without any subsequent damage. Therefore, in vitro experiments are of increasing interest. Various methods of applying shock waves onto cell cultures have been described. In general, all existing models focus on how to best apply shock waves onto cells.
However, this question remains: What happens to the waves after passing the cell culture? The difference of the acoustic impedance of the cell culture medium and the ambient air is that high, that more than 99% of shock waves get reflected! We therefore developed a model that mainly consists of a Plexiglas built container that allows the waves to propagate in water after passing the cell culture. This avoids cavitation effects as well as reflection of the waves that would otherwise disturb upcoming ones. With this model we are able to mimic in vivo conditions and thereby gain more and more knowledge about how the physical stimulus of shock waves gets translated into a biological cell signal (&#8220;mechanotransduction&#34;).

Shock waves nowadays are well known for their regenerative effects. Therefore in vitro experiments are of increasing interest. We therefore developed a model for in vitro shock wave trials (IVSWT) that enables us to mimic in vivo conditions thereby avoiding distracting physical effects.

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to ...

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

In this study, the use of an in situ loading device coupled with micro-X-ray computed tomography for fibrous joint biomechanics will be discussed. Experimental readouts identifiable with an overall change in joint biomechanics will include: 1) reactionary force vs. displacement, i.e. tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics, i.e.&#160;geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. concentric or eccentric loads.

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on F&#246;rster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.

A description of how to calibrate F&#246;rster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented.&#160;The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Quantification of Strain in a Porcine Model of Skin Expansion Using Multi-View Stereo and Isogeometric Kinematics

Tissue expansion is a popular technique in plastic and reconstructive surgery that grows skin in vivo for correction of large defects such as burns and giant congenital nevi. Despite its widespread use, planning and executing an expansion protocol is challenging due to the difficulty in measuring the deformation imposed at each inflation step and over the length of the procedure. Quantifying the deformation fields is crucial, as the distribution of stretch over time determines the rate and amount of skin grown at the end of the treatment. In this manuscript, we present a method to study tissue expansion in order to gain quantitative knowledge of the deformations induced during an expansion process. This experimental protocol incorporates multi-view stereo and isogeometric kinematic analysis in a porcine model of tissue expansion. Multi-view stereo allows three-dimensional geometric reconstruction from uncalibrated sequences of images. The isogeometric kinematic analysis uses splines to describe the regional deformations between smooth surfaces with few mesh points. Our protocol has the potential to bridge the gap between basic scientific inquiry regarding the mechanics of skin expansion and the clinical setting. Eventually, we expect that the knowledge gained with our methodology will enable treatment planning using computational simulations of skin deformation in a personalized manner.

This protocol uses multi-view stereo to generate three-dimensional (3D) models out of uncalibrated sequences of photographs, making it affordable and adjustable to a surgical setting. Strain maps between the 3D models are quantified with spline-based isogeometric kinematics, which facilitate representation of smooth surfaces over coarse meshes sharing the same parameterization.

Tissue expansion is a popular technique in plastic and reconstructive surgery that grows skin in vivo for correction of large defects such as burns and ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...