Name: transdermal measurement of glomerular filtration rate in mechanically ventilated piglets
Uploaded: 2022-09-13T14:30:00
Description: Watch this Scientific Journal Video about Transdermal Measurement of Glomerular Filtration Rate in Mechanically Ventilated Piglets at JoVE.com

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 ...

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.

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			<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>
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		<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>
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		<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>
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		<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>
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		<tr>
			<td>Phenobarbital Sodium + Phenytoin Sodium (Euthasol)</td>
			<td>Covetrus</td>
			<td>72934</td>
			<td>Used for euthanasia</td>
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		<tr>
			<td>Ringer Lactate 500 mL bag</td>
			<td>Baxter</td>
			<td>2B2323Q</td>
			<td>Maintanence fluid infusion</td>
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			<td>Sterile Gloves</td>
			<td>Henry Schein</td>
			<td>104-5920</td>
			<td>Used by operator during surgery</td>
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		<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>
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		<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>
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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 ...

transdermal-measurement-glomerular-filtration-rate-mechanically

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Unit 1

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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 ...

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 ...

Highly Sensitive Measurement of Glomerular Permeability in Mice with Fluorescein Isothiocyanate-polysucrose 70

The loss of albumin in urine (albuminuria) predicts cardiovascular outcome. Under physiological conditions, small amounts of albumin are filtered by the glomerulus and reabsorbed in the tubular system up until the absorption limit is reached. Early increases in pathological albumin filtration may, thus, be missed by analyzing albuminuria. Therefore, the use of tracers to test glomerular permselectivity appears advantageous. Fluorescently labeled tracer fluorescein isothiocyanate (FITC)-polysucrose (i.e., FITC-Ficoll), can be used to study glomerular permselectivity. FITC-polysucrose molecules are freely filtered by the glomerulus but not reabsorbed in the tubular system. In mice and rats, FITC-polysucrose has been investigated in models of glomerular permeability by using technically complex procedures (i.e., radioactive measurements, high-performance liquid chromatography [HPLC], gel filtration). We have modified and facilitated a FITC-polysucrose tracer-based protocol to test early and small increases in glomerular permeability to FITC-polysucrose 70 (size of albumin) in mice. This method allows repetitive urine analyses with small urine volumes (5 &#181;L). This protocol contains information on how the tracer FITC-polysucrose 70 is applied intravenously and urine is collected via a simple urinary catheter. Urine is analyzed via a fluorescence plate reader and normalized to a urine concentration marker (creatinine), thereby avoiding technically complex procedures.

Here, we present a protocol to test glomerular permeability in mice using a highly sensitive, nonradioactive tracer. This method allows repetitive urine analyses with small urine volumes.

The loss of albumin in urine (albuminuria) predicts cardiovascular outcome. Under physiological conditions, small amounts of albumin are filtered by the ...

Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or bradycardia. Reliable identification of cardiac pacemaking defects requires the measurement of intrinsic heart rates by largely preventing the influence of the autonomic nervous system, which can mask rate deficits. Traditional methods to analyze intrinsic cardiac pacemaker function include drug-induced autonomic blockade to measure in vivo heart rates, isolated heart recordings to measure intrinsic heart rates, and sinoatrial strip or single-cell patch-clamp recordings of sinoatrial pacemaker cells to measure spontaneous action potential firing rates. However, these more traditional techniques can be technically challenging and difficult to perform. Here, we present a new methodology to measure intrinsic cardiac firing rate by performing microelectrode array (MEA) recordings of whole-mount sinoatrial node preparations from mice. MEAs are composed of multiple microelectrodes arranged in a grid-like pattern for recording in vitro extracellular field potentials. The method described herein has the combined advantage of being relatively faster, simpler, and more precise than previous approaches for recording intrinsic heart rates, while also allowing easy pharmacological interrogation.

This protocol aims to describe a new methodology to measure intrinsic cardiac firing rate using microelectrode array recording of the whole sinoatrial node tissue to identify pacemaking defects in mice. Pharmacological agents can also be introduced in this method to study their effects on intrinsic pacemaking.

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or ...