This study was supported by the National Institutes of Health grants R01 DK120595 and R01 DK127625 awarded to Dr. Adebiyi. The content of this paper is solely the authors&#39; responsibility and does not necessarily represent the official views of the National Institutes of Health. Thanks to Dr. Daniel Schock-Kusch, site Director at MediBeacon GmbH, for his advice.

The procedures are written according to national standards for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Tennessee Health Science Center (UTHSC).
NOTE: Piglets in the experimental group are subjected to cecal ligation and puncture, while the sham group only undergoes opening of the abdomen without cecal ligation or puncture. Piglets in both groups are kept under anesthesia for 12 h post-procedure to allow enough time for sepsis and acute kidney injury (AKI) to ensue in the experimental group. Transdermal GFR measurement will ensue at 8 h post-procedure for a total of 12 h.
1. Piglet supply and housing
<ol>
	<li>Identify a local hog farm that can provide neonatal piglets aged 3-5 days. Schedule the delivery early in the week to complete the experimentation before any piglets are older than 7 days. 
	NOTE: The supplier provided three to five piglets on Mondays for this experiment; by Friday, the piglets would have undergone the experiment. Using the same sex and near similar age is essential to avoid confounding factors.</li>
	<li>Upon the piglet&#39;s arrival, ensure they have an individual identification (e.g., an ear tag and a record that includes weight and age).</li>
	<li>House the piglets in a lab animal care unit (LACU) under the care of a licensed veterinarian. The animals are housed as a group in a spacious pen with a solid concrete floor that is easily washed with water to maintain good sanitation.</li>
	<li>Add a piece of furniture such as a heavy ball to allow for environmental enrichment and stimulation.</li>
	<li>Ensure that the LACU provides optimal environmental conditions, including the following key elements: sanitation, nutrition, temperature control, ventilation, and day-night cycle by controlling illumination.</li>
	<li>Have the veterinarian check on the piglet daily, including weight measurement, to inform the investigator if any piglet appears sick, which may necessitate exclusion from the experiment.</li>
	<li>Leave the piglets for at least 1 day to acclimatize to the environment, which helps minimize the stress.</li>
</ol>
2. Pre-operative preparation
<ol>
	<li>Prepare the surgical station before initiating the experiment. This includes a heating pad, catheters, a ventilator, an endotracheal tube, heparinized saline, and a bag of ringer lactate fluid. 
	NOTE: Piglets have poor thermoregulatory capacitance and are prone to hypothermia which alters hemodynamics22,23. Therefore, allowing enough time for the heating pad to warm up is essential.</li>
	<li>Prepare 10 mg/mL of &#945;-chloralose by mixing it with saline at 60 &#730;C until the mixture is clear. Do not overheat the solution to avoid crystallizing of the medication upon cooling. Filter with a syringe filter (size 0.22 &#181;m) before administering to the piglets.</li>
	<li>Draw up anesthetic medication based on animal weight-Ketamine: 20 mg/kg and Xylazine: 2.2 mg/kg. Use &#945; -chloralose (5 mL/kg) to maintain anesthesia. 
	NOTE:&#160;&#945; -chloralose is used due&#160;to the ease of IV administration when compared to inhaled&#160; 
	anesthetics, as the latter require an anesthetic machine and an appropriate scavenging system to be delivered via an endotracheal tube.</li>
</ol>
3. Anesthesia
<ol>
	<li>Perform induction of anesthesia in the pig pen, an environment familiar for piglets, to avoid undue stress.</li>
	<li>Gently pick the piglet by the back legs and administer Ketamine: 20 mg/kg and Xylazine: 2.2 mg/kg into the rear leg at the semimembranosus/semitendinosus muscle, using a 23 G &#190; needle.</li>
	<li>Allow a few minutes for the medications to take effect. Check for the adequate anesthesia level by ensuring that the animal is relaxed enough to be immobile, with loss of palpebral reflex and jaw tone to allow ease and safe transportation to the surgical station. Assess the palpebral reflex by touching the inner corner of the eye; absence of blinking indicates adequate anesthesia.</li>
</ol>
4. Tracheostomy
NOTE: This experiment is non-survival, so a tracheotomy is performed to establish an airway for mechanical ventilation. Tracheostomy is a quick and easy procedure, as opposed to endotracheal intubation, which is challenging in piglets given their head and upper airway anatomy24,25. Additionally, laryngospasm is commonly reported during intubation, resulting in a prolonged period of hypoxia and hypercarbia that may compromise results26.
<ol>
	<li>Position the piglet in dorsal recumbency. Identify the cricothyroid cartilage by palpating the prominence of the thyroid cartilage which feels firm. Sterilize the area using povidone-iodine and 70% ethanol before applying a sterile drape.</li>
	<li>Using a a surgical blade, make a 2-3 cm ventral midline incision inferior to the caudal end of the thyroid cartilage.</li>
	<li>Using a curved mosquito hemostat, bluntly dissect the overlying subcutaneous tissues and muscles (sternohyoideus and cutaneous coli) until the cricothyroid membrane and the first few tracheal rings are visualized. When dissecting, be cautious to avoid injuring any blood vessels.</li>
	<li>Obtain a clear view of the cricothyroid membrane and tracheal rings24, then use a pair of long mixter right angle forceps to elevate the structures.
	<ol>
		<li>With a pair of small scissors, make a small cut at the cricothyroid membrane or the first tracheal ring. Extend the cut horizontally to ~0.5 cm to pass a 3.0 mm endotracheal tube.</li>
		<li>Insert the tube to the 5 cm mark. Ensure bilateral chest expansion and breath sounds prior to securing the tube.</li>
	</ol>
	</li>
	<li>Pass umbilical tape around the trachea to secure it in place. Additional tape is used to secure the tube to the base of the jaw.</li>
	<li>Switch on the ventilator, connect the endotracheal tube, and roll the specific knobs (eg. SIMV knobs, PEEP knobs, etc) to select the following baseline settings. Pressure Control Mode: synchronized intermittent mechanical ventilation (SIMV); peak inspiratory pressure (PIP) - 15; positive end-expiratory pressure (PEEP) - 5; Rate- 20; I-time - 0.6. Following the first blood gas analysis, adjust the ventilator settings according to the blood gas results, with the goal of maintaining adequate oxygenation and ventilation.</li>
</ol>
5. Femoral vessel cannulation
<ol>
	<li>Establish the airway and ventilation, before switching attention to the femoral vessels for venous access and invasive blood pressure monitoring. The femoral artery is identified by feeling a pulse at the groove between the sartorius and gracilis muscles, and the vein can be found just medial to the artery.</li>
	<li>While the piglet is lying in a dorsal recumbent position, sterilize the groin area using povidone-iodine and ethanol, and apply an appropriately sized drape.</li>
	<li>Use a surgical blade to create a 3-4 cm longitudinal incision, starting cranially at the inguinal crease and extending distally along the femoral canal.</li>
	<li>Apply blunt and sharp dissection, using mosquito curved forceps and scissors, respectively, to dissect down to the level of the femoral neurovascular bundle. The bundle can be found deep in the body of the gracillis muscle27. Circumferentially dissect the femoral artery and vein over the course of 2-3 cm to allow for cannulation. Ligate small side branches if necessary.</li>
	<li>Apply a 3.0 silk tie at both the artery and vein&#39;s proximal and distal ends to apply traction. Tie the distal silk suture on both the vein and artery, ligating the vessels.</li>
	<li>Beginning with the femoral vein, maintain distal and proximal traction on the silk ties and then use a pair of micro scissors to create a venotomy.</li>
	<li>Next, use a vein pick catheter introducer to open the vessel while inserting a pre-measured polyurethane catheter with an internal diameter x outer diameter of 0.86 mm x 1.32 mm. Once inserted, tie the proximal 3.0 silk suture to fixate the catheter. Flush the catheter with 3 mL of heparinized saline solution (1 U/mL). This solution can be made by adding 0.5 mL of heparin to 50 mL of normal saline.</li>
	<li>Insert an invasive blood pressure catheter using the same approach above to create an arteriotomy and pass the catheter. 
	NOTE: Maintaining distal and proximal traction is essential to minimize blood loss when accessing the artery.</li>
	<li>Once the catheters are secured, cover the site with saline-soaked gauze, and if necessary, the skin may be sutured using a 3.0 silk suture to prevent infection.</li>
</ol>
6. Maintenance of anesthesia, fluid and blood gas
<ol>
	<li>Monitor the depth of anesthesia throughout the experiment, using jaw tone and palpebral reflex, and administer&#160; &#945;-chloralose, intravenously, as needed to maintain the animal under deep anesthesia. Use an&#160;initial loading dose of 50 mg/kg, and&#160;20 mg/kg for further boluses.</li>
	<li>Infuse ringer lactate at a rate of 4 mL/kg/h throughout the experiment as maintenance fluid. For example, if the piglet weight is 3 kg, then the fluid infusion rate is 12 mL/h.</li>
	<li>For bedside gas analysis, draw an arterial blood sample in a heparinized blood gas syringe and present the sample to the analyzer machine. Select the option arterial blood gas, and wait for ~2-3 s for the the analyzer to present the blood-draw needle.
	<ol>
		<li>Carefully insert the needle into the end of the syringe containing the blood sample. Wait for the analyzer to aspirate the required sample and withdraw the syringe. Allow the machine to analyze the blood gas and present the results.</li>
		<li>Based on the results, adjust the ventilator to maintain the pH between 7.35--7.45, partial pressure of carbon dioxide (PCO2) between 35-45 mmHg, and partial pressure of oxygen (PaO2) between 80-150 mmHg. The settings differ based on the ventilator type, but largely involves increasing or reducing the respiratory rate using appropriate knobs to compensate for hypoxia and/or hypercapnia.</li>
	</ol>
	</li>
	<li>Draw 3 mL of blood into a light green tube (Lithium Heparin). Centrifuge the sample at 2000 xg for 15 min, maintained at 4 &#730;C to extract plasma. Once completed, the plasma can be analysed immediately for serum creatinine level with the bedside chemistry analyzer or stored at -80 &#730;C for later analysis.</li>
	<li>Monitor the temperature continuously&#160;using a rectal probe thermometer and adjust the heating pad temperature to maintain piglet temperature between 101 to 103 &#730;F.</li>
</ol>
7. Experiment group; cecal ligation and perforation (CLP) 25,28,29
NOTE: For piglets in the experiment group, perform CLP to induce polymicrobial sepsis28 and monitor the animal for 12 h post-surgery to allow enough time for severe sepsis to ensue. Transdermal GFR recording starts at 8 h post-cecal ligation to allow for 4 h of recording.
<ol>
	<li>Use a surgical blade to create a 5-6 cm left paramedian vertical incision, as the cecum in pigs lies in the left paralumbar fossa30. Dissect down the abdominal wall layers, avoiding injury to the superficial epigastric vessels.</li>
	<li>Once the peritoneal layer is incised, use a retractor to improve access to intrabdominal structures.</li>
	<li>Identify the spiral colon in the upper left quadrant of the abdomen. Trace the spiral colon, caudally and dorsally, to locate the cecum. The ileum is seen joining the spiral colon at the base of the cecum.</li>
	<li>Ligate the cecum just distal to the ileocecal junction (Figure 1).</li>
	<li>Using a 18 G needle, make seven punctures in the cecum and extrude feces into the peritoneal area.</li>
	<li>Close the abdomen in layers with a 3.0 silk suture using either simple interrupted or continuous stitches. A stapler may also be used to close the skin layer if available.</li>
</ol>
8. Sham group
<ol>
	<li>Follow the steps 7.2-7.4 as above. After identifying the cecum, place it back untouched and close the abdominal wall similarly.</li>
	<li>Monitor piglets in the sham group for 12 h to eliminate any confounding bias attributed to prolonged exposure to anesthesia.</li>
</ol>
9. Transdermal GFR device setup
<ol>
	<li>After 8 h of cecal ligation, get ready to initiate transdermal measurement of GFR.</li>
	<li>Use the MB service software version 3.0 to adjust the sampling rate on the GFR device. Briefly, connect the transdermal GFR device to the computer software using the USB connector. Open the software, click connect, and adjust the timing to 4000 ms. Click write to save the settings. 
	NOTE: This gives up to 6 h of total sampling time. In the pigs, transdermal GFR is completed in 4 h. For experiments that require sampling up to 12 h, choose the 8000 ms option.</li>
	<li>Attach the dual-sided adhesive patches with a clear window to the device. Attach the device to one side, ensuring the light-emitting diode overlies the clear window to allow tracer detection.</li>
	<li>Shave the area overlying the lateral thoracic wall. Attach the battery to the device and immediately stick the adhesive patch with the device in place and make sure it is well secured (Figure 2). Since the piglets are deeply anesthetized, tape might be unnecessary to hold the device in place. 
	NOTE: The adhesive patch alone is enough to secure. However, in procedures where the animal would be manipulated, become active, or where anesthesia might be disrupted, it might be important to apply a tape. A bandage might also be an alternative approach31.</li>
	<li>A baseline recording of 3-5 min is required before administering FITC-sinistrin.</li>
</ol>
10. FITC-sinistrin preparation and injection
<ol>
	<li>Prepare a mixture of FITC-sinistrin with saline solution to a final concentration of 50 mg/mL. The dose administered to the piglet is 20 mg/kg. FITC-sinistrin is supplied in powder form. 
	NOTE: The FITC-sinistrin may also be administered through a peripheral venous catheter inserted in the auricular vein. It is essential to achieve a high peak level by administering FITC-sinistrin as a push bolus through the femoral vein venous catheter.</li>
	<li>Attach the syringe with medication to one side of a three-way stop cock and a saline flush on the other side of the stop cock. Push the FITC-sinistrin and immediately follow with a 5 mL saline bolus before closing the three-way stop cock to the piglet vein.</li>
</ol>
11. Transdermal GFR recording
<ol>
	<li>Keep the device attached to the piglet for 4 h. During this time, keep the piglet under anesthesia using intermittent doses of &#945;-chloralose at a concentration of 20 mg/kg to avoid any motion artifact.</li>
	<li>At the end of the 4 h, remove the device and immediately disconnect the battery.</li>
</ol>
12. GFR measurement
<ol>
	<li>Connect the transdermal GFR device to the computer using the USB connector provided by the supplier.</li>
	<li>Open the reading software to retrieve data from the device. Save the raw data by clicking the sequence: connect, read, re-name, and save. As instructed in the manual, process and evaluate the saved data in the analysis software.</li>
	<li>Briefly, open the software ver. 3.0 and import the data. Adjust the offset, start, and end positions using the automated markers. Remove artifacts if necessary, and click fit. This gives a readout that shows FITC-sinistrin clearance in minutes (t1/2). The t1/2 is subsequently used to calculate the tGFR32,33 as below: 
	<img alt="Equation 1" src="/files/ftp_upload/64413/64413eq01.jpg" style="margin: auto;" /> 
	NOTE: In consultation with the manufacturer, the conversion factor used for pigs is 20 (indicating that 20% of the body weight is extracellular space), as opposed to 21.33 in rats (tGFR in mL/min) and 14,616.8 in mice (tGFR in &#181;L/min). This is because GFR is accurately measured as a function of extracellular fluid34,35, which in turn is dependent on body weight36.</li>
</ol>
13. Piglet euthanasia
<ol>
	<li>Collect 3 mL of blood after 12 h of CLP for further biochemical analysis.</li>
	<li>Euthanize the piglet by administering 0.2 mL/kg of pre-mixed mixture of 20% sodium pentobarbital and Phenytoin Sodium intravenously.</li>
	<li>Harvest the right kidney for histopathologic study before taking the piglet to the morgue.</li>
</ol>

This paper describes practical steps to determining kidney function in pigs using the miniaturized transdermal GFR monitors and FITC-sinistrin in a mechanically ventilated, anesthetized neonatal pig model. Previous papers have established experimental transdermal GFR protocols in rodents11,12,14, but no protocols exist in pigs.
Recently, there has been a drive to explore alternative animal models to s...

A practical and quantitative evaluation of renal function is the glomerular filtration rate (GFR) measurement, which tells how well the kidneys filter blood based on the clearance principle1. An earlier method of measuring GFR entails the intravenous injection of exogenous compounds such as inulin or sinistrin, conducting serial measurements of plasma/urinary levels to detect their clearance2,3. This method is cumbersome, requiring the serial collection of plasma and urine samples4. An alternative is the measurement of endogenous metabolic end-products such as creatinine. However, this is time-consuming and, at times, inaccurate, as it is not only filtered by the glomerulus but also secreted by the tubules5,6. Furthermore, creatinine level is influenced by gender, age, diet, and muscle mass7,8,9.
A more precise, minimally invasive, and widely used measure of GFR is the use of transdermal GFR monitors, which measure real-time GFR in animals4,10. Sinistrin, a highly soluble and freely filtered exogenous renal marker, is labeled with fluorescein-isothiocyanate (FITC). This conjugated compound is injected intravenously, and real-time kidney function can be assessed without collecting blood and urine samples11. The use of transdermal GFR measurement has been validated in rodents12, dogs13, and cats14, but not in swine.
Porcine species share several anatomical and physiological characteristics with humans, making them ideal animals for studying various human diseases15. The use of pigs in translational biomedical research has become increasingly popular and preferred over rodent models because it mimics human physiology and pathophysiology16. Neonatal pigs are of interest in understanding the mechanisms of diseases unique to pediatric patients17. Moreover, the recent advancement in pig to human organ transplantation puts an urge to expand the diagnostic tools for preclinical and clinical trials18,19,20,21. This paper, for the first time, provides a guide for the use of the transdermal device in measuring GFR in neonatal pigs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alpha - Chloralose</td>
			<td>Sigma-Aldrich</td>
			<td>C0128-25G</td>
			<td>Used for maintanining anesthesia</td>
		</tr>
		<tr>
			<td>Black braided silk&#160; 3-0</td>
			<td>Surgical Specialties</td>
			<td>SP117</td>
			<td>Silk tie for blood vessel traction and ligation</td>
		</tr>
		<tr>
			<td>Centrifugation machine AccuSpin 8C</td>
			<td>Fischer Scientific</td>
			<td>75-008-821</td>
			<td>Used to extract plasma from whole blood sample</td>
		</tr>
		<tr>
			<td>Endotracheal Tube 3.0 uncuffed</td>
			<td>Progressive Medical International</td>
			<td>1109021995</td>
			<td>Inserted through tracheostomy</td>
		</tr>
		<tr>
			<td>FITC-Sinistrin 1.0 g</td>
			<td>MediBeacon Inc.</td>
			<td>FTCF S001</td>
			<td>Store at room temp and protect from light</td>
		</tr>
		<tr>
			<td>GEM Premier 3000 Blood gas analyzer</td>
			<td>Instrumentation Laboratory</td>
			<td>5700</td>
			<td>For bedside blood gas analysis</td>
		</tr>
		<tr>
			<td>Heating Pad medium size 20 in x 29 in</td>
			<td>Adroit Medical Systems</td>
			<td>V029</td>
			<td>Connects to heat therapy pump</td>
		</tr>
		<tr>
			<td>HTP-Heat Therapy Pump</td>
			<td>Adroit Medical Systems</td>
			<td>HTP</td>
			<td>Allows you to set temperature as needed.</td>
		</tr>
		<tr>
			<td>IDEXX Catalyst One</td>
			<td>IDEXX Laboratories</td>
			<td>89-92525-00</td>
			<td>Plasma creatinine analysis</td>
		</tr>
		<tr>
			<td>Invasive blood pressure catheter 3.5Fr</td>
			<td>Millar</td>
			<td>SPR-524</td>
			<td>Inserted in femoral artery</td>
		</tr>
		<tr>
			<td>IV adminstration set with flow regulator</td>
			<td>True Care</td>
			<td>TCRTCBINF033G</td>
			<td>Used to connect IV fluid bag to vein catheter</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Covetrus</td>
			<td>68317</td>
			<td>Used for induction of Anesthesia</td>
		</tr>
		<tr>
			<td>MediBeacon analysis software version 3.0</td>
			<td>MediBeacon Inc.</td>
			<td>N/A</td>
			<td>Software program used for analysing data to obtain sinistrin clearance half life and curve</td>
		</tr>
		<tr>
			<td>Millex-GV Syringe Filter Unit 0.22 &#181;m</td>
			<td>Millipore Sigma</td>
			<td>SLGVR33RS</td>
			<td>Syringe filter for chloralose injection</td>
		</tr>
		<tr>
			<td>Neonate/Infant Ventilator</td>
			<td>Sechrist Millennium</td>
			<td>20409</td>
			<td>Connected to air supply to provide ventilation through endotracheal tube</td>
		</tr>
		<tr>
			<td>Phenobarbital Sodium + Phenytoin Sodium (Euthasol)</td>
			<td>Covetrus</td>
			<td>72934</td>
			<td>Used for euthanasia</td>
		</tr>
		<tr>
			<td>Ringer Lactate 500 mL bag</td>
			<td>Baxter</td>
			<td>2B2323Q</td>
			<td>Maintanence fluid infusion</td>
		</tr>
		<tr>
			<td>Sterile Gloves</td>
			<td>Henry Schein</td>
			<td>104-5920</td>
			<td>Used by operator during surgery</td>
		</tr>
		<tr>
			<td>Sterile Gown</td>
			<td>Halyard Health</td>
			<td>95021</td>
			<td>Used by operator during surgery</td>
		</tr>
		<tr>
			<td>Steril Towel</td>
			<td>Medline</td>
			<td>42131704</td>
			<td>Used as drape to maintaine sterile field when operating</td>
		</tr>
		<tr>
			<td>Suture 3-0 silk reverse cutting needle</td>
			<td>Ethicon</td>
			<td>NC1842168</td>
			<td>Used for suturing abdominal wall layers</td>
		</tr>
		<tr>
			<td>Transdermal Mini GFR Monitor</td>
			<td>MediBeacon Inc.</td>
			<td>TDM004</td>
			<td>Battery and USB connector included in package</td>
		</tr>
		<tr>
			<td>Transdermal monitor adhesive patch</td>
			<td>MediBeacon Inc.</td>
			<td>PTC-SM001</td>
			<td>Doubl sided adhesive patch for GFR probe</td>
		</tr>
		<tr>
			<td>Umbilical Tape 1/8 in x 20 yds</td>
			<td>Fisher Scientific</td>
			<td>NC9303017</td>
			<td>To secure endotracheal tube</td>
		</tr>
		<tr>
			<td>Venous Catheter size PE/5</td>
			<td>Micro medical tubing</td>
			<td>BB31695</td>
			<td>For femoral vein cannulation</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Covetrus</td>
			<td>61035</td>
			<td>Used for induction of anesthesia</td>
		</tr>
	</tbody>
</table>

transdermal measurement of glomerular filtration rate in mechanically ventilated piglets

Transdermal measurement of glomerular filtration rate (GFR) has been used to evaluate kidney function in conscious animals. This technique is well established in rodents to study acute kidney injury and chronic kidney disease. However, GFR measurement using the transdermal system has not been validated in pigs, a species with a similar renal system to humans. Hence, we investigated the effect of sepsis on transdermal GFR in anesthetized and mechanically ventilated neonatal pigs. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP). The transdermal GFR measurement system consisting of a miniaturized fluorescence sensor was attached to the pig's shaved skin to determine the clearance of fluorescein-isothiocyanate (FITC) conjugated sinistrin, an intravenously injected GFR tracer. Our results show that at 12 h post-CLP, serum creatinine increased with a decrease in GFR. This study demonstrates, for the first time, the utility of the transdermal GFR approach in determining renal function in mechanically ventilated, neonatal pigs.

In this section, we present for the first time, the representative data from the use of transdermal GFR in neonatal pigs. We used a cecal ligation and puncture model which has previously been shown to decrease kidney function28. Accordingly, we hypothesized that in our CLP pigs, there should be an acute drop in GFR corresponding to AKI, and this should be detected on the transdermal GFR device as increased clearance time (t1/2), thereby validating its use in pigs. Seven male piglets wer...

Watch this Scientific Journal Video about Transdermal Measurement of Glomerular Filtration Rate in Mechanically Ventilated Piglets at JoVE.com

Transdermal Measurement of Glomerular Filtration Rate in Mechanically Ventilated Piglets

Glomerular filtration rate (GFR) is the ideal marker for assessing kidney function. However, the standard measurement method using inulin injection with serial blood and urine analysis is impractical. This article delineates a practical method to measure GFR transdermally in piglets.

Transdermal measurement of glomerular filtration rate (GFR) has been used to evaluate kidney function in conscious animals. This technique is well ...

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2.1K Views.  College of Medicine University of Tennessee Health Science Center. Glomerular filtration rate (GFR) is the ideal marker for assessing kidney function. However, the standard measurement method using inulin injection with serial blood and urine analysis is impractical. This article delineates a practical method to measure GFR transdermally in piglets.

Video: Transdermal Measurement of Glomerular Filtration Rate in Mechanically Ventilated Piglets

Biochemical Measurement of Neonatal Hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment 1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded 2. This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances 4, 5. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation 4, 6. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) 7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.
We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators 10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al 7. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere 14, 15. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. 16, 17. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin 18. This approach proved to be sufficiently sensitive and reproducible.

A method is described to measure biochemical markers of neonatal hypoxia-ischemia. The approach utilizes high pressure liquid chromatography (HPLC) and Gas Chromatography Mass Spectrometry (GC/MS).

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

Kidney diseases involving urinary loss of large essential macromolecules, such as serum albumin, have long been thought to be caused by alterations in the permeability barrier comprised of podocytes, vascular endothelial cells, and a basement membrane working in unison. Data from our laboratory using intravital 2-photon microscopy revealed a more permeable glomerular filtration barrier (GFB) than previously thought under physiologic conditions, with retrieval of filtered albumin occurring in an early subset of cells called proximal tubule cells (PTC)1,2,3.
Previous techniques used to study renal filtration and establishing the characteristic of the filtration barrier involved micropuncture of the lumen of these early tubular segments with sampling of the fluid content and analysis4. These studies determined albumin concentration in the luminal fluid to be virtually non-existent; corresponding closely to what is normally detected in the urine. However, characterization of dextran polymers with defined sizes by this technique revealed those of a size similar to serum albumin had higher levels in the tubular lumen and urine; suggesting increased permeability5.
Herein is a detailed outline of the technique used to directly visualize and quantify glomerular fluorescent albumin permeability in vivo. This method allows for detection of filtered albumin across the filtration barrier into Bowman's space (the initial chamber of urinary filtration); and also allows quantification of albumin reabsorption by proximal tubules and visualization of subsequent albumin transcytosis6. The absence of fluorescent albumin along later tubular segments en route to the bladder highlights the efficiency of the retrieval pathway in the earlier proximal tubule segments. Moreover, when this technique was applied to determine permeability of dextrans having a similar size to albumin virtually identical permeability values were reported2. These observations directly support the need to expand the focus of many proteinuric renal diseases to included alterations in proximal tubule cell reclamation.

A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states.

Kidney  diseases involving urinary loss of large essential macromolecules, such as  serum albumin, have long been thought to be caused by alterations in ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease and injury regional estimates, World Health Organization, 2004). Surgeries that often compromise the quality of life are required to correct heart defects, reminding us of the importance of finding the causes of CHD. Mutant mouse models and live imaging technology have become essential tools to study the etiology of this disease. Although advanced methods allow live imaging of abnormal hearts in embryos, the physiological and hemodynamic states of the latter are often compromised due to surgical and/or lengthy procedures. Noninvasive ultrasound imaging, however, can be used without surgically exposing the embryos, thereby maintaining their physiology. Herein, we use simple M-mode ultrasound to assess heart rates of embryos at E18.5 in utero. The detection of abnormal heart rates is indeed a good indicator of dysfunction of the heart and thus constitutes a first step in the identification of developmental defects that may lead to heart failure.

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Ultra-high frequency ultrasound is a powerful live imaging tool to examine cardiac abnormalities in small animals. Its noninvasiveness allows maintaining the physiologic state of embryos. Herein, we show the use of M-mode ultrasound to measure the heart rates of embryos at E18.5 in utero.

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular failure. Current treatments are limited. Physiologically, pulsatile blood flow is detrimental to the vasculature. In response to sustained pulsatile stress, vessels release nitric oxide (NO) to induce vasodilation for self-protection. Based on this observation, this study developed a protocol to assess whether an artificial pulmonary pulsatile blood flow could induce an NO-dependent decrease in pulmonary artery pressure. One group of piglets was exposed to chronic hypoxia for 3 weeks and compared to a control group of piglets. Once a week, the piglets underwent echocardiography to assess PAH severity. At the end of hypoxia exposure, the piglets were subjected to a pulsatile protocol using a pulsatile catheter. After being anesthetized and prepared for surgery, the jugular vein of the piglet was isolated and the catheter was introduced through the right atrium, the right ventricle and the pulmonary artery, under radioscopic control. Pulmonary artery pressure (PAP) was measured before (T0), immediately after (T1) and 30 min after (T2) the pulsatile protocol. It was demonstrated that this pulsatile protocol is a safe and efficient method of inducing a significant reduction in mean PAP via an NO-dependent mechanism. These data open up new avenues for the clinical management of PAH.

Pulmonary hypertension is associated with a significant reduction in pulmonary artery pulsatility, contractility and elasticity, contributing to an increase in pulmonary artery pressure and pulmonary resistance. Using a hypoxic piglet model, this study demonstrated that improving pulmonary artery plasticity using a newly developed pulsatile catheter improves hypoxic pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

Objective Nociceptive Assessment in Ventilated ICU Patients: A Feasibility Study Using Pupillometry and the Nociceptive Flexion Reflex

The concept of objective nociceptive assessment and optimal pain management have gained increasing attention. Despite the known negative short- and long-term consequences of unresolved pain or excessive analgosedation, adequate nociceptive monitoring remains challenging in non-communicative, critically ill adults. In the intensive care unit (ICU), routine nociceptive evaluation is carried out by the attending nurse using the Behavior Pain Scale (BPS) in mechanically ventilated patients. This assessment is limited by medication use (e.g., neuromuscular blocking agents) and the inherent subjective character of nociceptive evaluation by third parties.
Here, we describe the use of two nociceptive reflex testing devices as tools for objective pain evaluation: the pupillary dilation reflex (PDR) and nociception flexion reflex (NFR). These measurement tools are non-invasive and well tolerated, providing clinicians and researchers with objective information regarding two different nociceptive processing pathways: (1)&#160;the pain-related autonomic reactivity&#160;and (2) the ascending component of the somatosensory system. The use of PDR and NFR measurements are currently limited to specialized pain clinics and research institutions because of impressions that these are technically demanding or time-consuming procedures, or even because of a lack of knowledge regarding their existence.
By focusing on the two abovementioned nociceptive reflex assessments, this study evaluated their feasibility as a physiological pain measurement method in daily practice. Pursuing novel technologies for evaluating the analgesia level in unconscious patients may further improve individual pharmacological treatment and patient related outcome measures. Therefore, future research must include large well-designed clinical trials in a real-life environment.

Pain assessment in anesthetized patients who cannot communicate with the outside world in any way remains challenging despite the development of innovative objective pain evaluation tools. In this project, the pupillary dilation reflex and the nociception flexion reflex are assessed in critically ill, mechanically ventilated adult patients.

The concept of objective nociceptive assessment and optimal pain management have gained increasing attention. Despite the known negative short- and ...

Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV diastolic function is rarely quantified in experimental studies. This might be due to technical difficulties in the visualization of the RV in the apical four-chamber view in rodents. Here we describe two positions facilitating the visualization of the apical four-chamber view in mice to assess the RV diastolic function.
The apical four-chamber view is enabled by tilting the mouse fixation platform to the left and caudally (LeCa) or to the right and cranially (RiCr). Both positions provide images of comparable quality. The results of the RV diastolic function obtained from two positions are not significantly different. Both positions are comparably easy to perform. This protocol can be incorporated into published protocols and enables detailed investigations of the RV function.

Here we describe and compare two positions for obtaining the apical four-chamber view in mice. These positions enable the quantification of the right ventricular function, provide comparable results, and can be used interchangeably.

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV ...

Transdermal Measurement of Glomerular Filtration Rate in Mice

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of acute kidney injury and chronic kidney disease. The measurement system consists of a miniaturized fluorescence detector that is directly attached to the skin on the back of conscious, freely moving animals, and measures the excretion kinetics of the exogenous GFR tracer, fluorescein-isothiocyanate (FITC) conjugated sinistrin (an inulin analog). This system has been described in detail in rats. However, because of their smaller size, measurement of transcutaneous GFR in mice presents additional technical challenges. In this paper we therefore provide the first detailed practical guide to the use of transdermal GFR monitors in mice based on the combined experience of three different investigators who have been performing this assay in mice over a number of years.

Here we describe a protocol to measure glomerular filtration rate (GFR) in conscious, freely moving mice using a transdermal GFR monitor.

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of ...