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

Summary

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.

Abstract

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.

Introduction

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 mortality^2,3,4. 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 disease^9,10. 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'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 AKI^11,12. 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'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 oxygenation^13,14. 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 flow^15,16,17. 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 AKI^18,19. Similarly, current animal models have primarily focused on the early detection of AKI during cardiac surgery and sepsis^14,20,21,22. 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.

Protocol

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

1. 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.
2. 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.
3. Use a 5/32 in drill bit to drill out the top part of the T-connector to fit the oxygen sensor.
4. 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.
5. Mix the biocompatible glue.
6. 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.
7. 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.
8. 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.

2. Experimental procedure

1. Induction of anesthesia and monitoring.
   1. Sedate the animal with a combined intramuscular injection of Ketamine (2.2 mg/kg) and Xylazine (2.2 mg/kg)  and Telazol (4.4 mg/kg).
   2. 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.
   3. Apply eye lubricant to both eyes.
   4. 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.
   5. 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.
   6. Position the animal in dorsal recumbency on a warming blanket and secure each leg to the table.
   7. The protocol is a non-survival procedure with euthanasia of the animal at the end of the experiment, as described in section 5.
2. Prepare the animal for the experiment.
   1. 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.
   2. Locally infiltrate all puncture and incision sites with 2% lidocaine for local pain relief.
   3. 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.
   4. Under ultrasound guidance, place a 7 Fr sheath in the right brachial artery.
   5. Under ultrasound guidance, place a 7 Fr sheath in the right femoral artery.
   6. Under ultrasound guidance, place a 7 Fr sheath in the left femoral artery.
   7. Under ultrasound guidance, place a 5 Fr sheath in the right or left carotid artery.
   8. Monitor the pressure distal to the balloon of the resuscitative endovascular occlusion of the aorta (REBOA) catheter via the left femoral artery sheath.
      1. Connect a disposable pressure transducer to the arterial catheter that is distal to the REBOA balloon.
   9. Monitor the pressure proximal to the balloon of the REBOA catheter via the carotid artery sheath.
      1. Connect a disposable pressure transducer to the arterial catheter that is proximal to the REBOA balloon.
   10. 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.
   11. 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.
   12. 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.
   13. 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.
   14. Remove the spleen to eliminate hemorrhage-induced auto-transfusion.
       1. 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.
       2. After transection, ligate each vessel using modified Miller knots using 2-0 sutures.
3. Place the instrument to measure bladder PuO₂ and tissue oxygenation.
   1. Measure PuO₂ at the outlet of the bladder.
      1. 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.
      2. After making the incision, insert a t-connector that contains the sensing material into the incision.
      3. Use tissue glue to secure the t-connector in place.
      4. Connect the fiber optic cable from the bladder data collection device to the connector that contains the sensing material.
      5. 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.
         1. For the data collection device used in this study: push the back arrow to reach the main menu.
         2. Go to measurement settings and click on Ok. Use the arrows to highlight the measurement browser box and push Ok.
         3. Push the right arrow to create a new file. Type in the name of the new file and select Done.
         4. Highlight the new file name and select Ok. Navigate to the measurement screen and click on Ok to start recording.
   2. Measure medullary renal tissue oxygenation.
      1. Identify the location of the kidney internally.
      2. Move the bowel so that you have a clear line of site and access to the entire kidney. 
      3. Insert the sensor into 2" 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.
      4. Place the 18 gauge needle and 2 in catheter into the renal medulla under ultrasound guidance.
      5. 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. 
      6. Use tissue glue to secure the catheter in place. 
      7. Connect the tissue sensor to the data collection box.
      8. Wait for 10 min before beginning the experimental protocol after preparing the instrumentation and animal. This will be considered a baseline period.
4. Experimental protocol
   1. Prior to starting the experimental procedure, ensure the mean arterial pressure (MAP) is ≥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 µg/kg/min) until target MAP is achieved.
   2. Induce hemorrhagic shock.
      1. Remove 25% (estimated as 60 mL/kg) of the animal'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.
      2. Store the removed blood in a warm water bath at 37 °C.
      3. Then perform randomization to assign animals to either the REBOA with whole blood or REBOA with crystalloids group (n = 6 for each group).
   3. Place the REBOA catheter.
      1. 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.
      2. At t = 30 min, inflate the REBOA balloon and completely occlude the aorta for 45 min.
   4. Initiate resuscitation and administer critical care.
      1. At t = 70 min, transfuse each animal with their shed blood over 15 min.
      2. Infuse intravenous calcium over 10 min to prevent citrate-induced hypocalcemia.
      3. At t = 75 min, deflate the REBOA balloon over 10 min.
      4. Until t = 360 min, resuscitate the animal with fluids and norepinephrine to maintain a MAP > 65 mmHg.
5. End of experiment and euthanasia
   1. Collect any remaining blood or urine samples.
   2. Euthanize the animal by injecting a combination of Pentobarbital Sodium (390 mg) and Phenytoin Sodium (50 mg) (1 mL/10 lbs).

3. Data processing

1. Time-sync all data files.
   1. 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.
2. 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's output limit. The Air-in-Line error flag is raised when the sensor detects air in the flow channel.
3. Discard the data associated with the negative flow.
   1. Once flow becomes negative, track the volume that flows past the sensor in the backward direction.
   2. 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.

Results

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...

Discussion

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 ...

Materials

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