This work was supported by the Basic Science Research Programs that are managed by the National Research Foundation of Korea (NRF) (2015R1C1A2A01052419 and 2018R1D1A1B07042484).

All animal procedures were approved by the local committee for the Care and Use of Laboratory Animals, Kyung Hee University (license number: KHUASP(SE)-18-108) and conformed to the US National Institutes of Health Guide for the Care and Use of Laboratory Animals.
1. Experimental animals
<ol>
	<li>Keep all mice (39 mice, Balb/c, male, 7&#8210;9 weeks old) in a pathogen-free facility as per the guide for the care and use of laboratory animals.</li>
	<li>Maintain the mice on a 12 h light/dark cycle at constant temperature with free access to food and water.</li>
</ol>
2. Preparation of anesthetics
NOTE: Tribromoethanol is used over ketamine combinations and isoflurane, based on the stability of heart rate and the reproducibility of echocardiography in tribromoethanol-anesthetized mice1,5,6
<ol>
	<li>Make a stock solution of 2,2,2-tribromoethanol at a concentration of 1 g per 1 mL tertiary amyl alcohol. Warm at 40&#8210;45 &#176;C for 24 h. Store at 4 &#176;C for 12 months.</li>
	<li>For working solution, dilute 0.5 mL of stock solution in 19.5 mL of saline (0.9% NaCl) to 25 mg/mL. Warm at 40&#8210;45 &#176;C for 1 h. Store at 4 &#176;C for 1 month.</li>
</ol>
3. ECG system setup
<ol>
	<li>Make sure to set up the system such that there is no noise or vibration within 2 m since ECG signals in a mouse are sensitive to the environmental noise and movement.</li>
	<li>Prepare the hardware setup: a data acquisition system, a bio amplifier, and a computer that is installed with an ECG data analysis software.
	<ol>
		<li>Connect the data acquisition system to the mains (AC) using the power cable.</li>
		<li>Connect the data acquisition system to the computer using a USB cable.</li>
		<li>Connect the signal output on the rear panel of the bio amplifier to an analog input on the front panel of the data acquisition system using a cable.</li>
		<li>Connect the I2C output of the data acquisition system to the I2C input of the bio amplifier using the I2C cable.</li>
		<li>Connect the 3-lead bio amplifier cable to the 6-pin input socket on the front panel of the bio amplifier.</li>
		<li>Turn on the data acquisition system using the switch on the back panel. 
		NOTE: In brief, the signals are amplified through a bio amplifier and recorded using a computerized data acquisition and analysis system with the following channel settings: sampling rate of 2 k/s, range of 20 mV, and low-pass filter setting of 200 Hz.</li>
	</ol>
	</li>
	<li>Open the analysis software program and set it up for ECG data acquisition.
	<ol>
		<li>Go to Setup | Channel Settings. Set the Sample Rate to 2 k/s. Set the Range to 20 mV. Set the Input Amplifier to 200 Hz of Low Pass.</li>
		<li>Go to ECG Analysis | ECG Settings. Choose &#8220;Mouse&#8221; in the Preset of Detection and Analysis settings.</li>
		<li>In the Averaging panel, choose to concatenate N (e.g., 4 beats or 60 s) consecutive cardiac cycles into a single average signal for Averaging View and Table View.</li>
		<li>In the QTc panel, select &#8220;Bazett&#8221; method, which is defined as the heart rate-corrected value of QT interval: QTc = QT / (RR/100)0.5, RR interval = 60 / heart rate7.</li>
	</ol>
	</li>
</ol>
4. ECG measurement
<ol>
	<li>Place a mouse on a precision scale and record its weight.</li>
	<li>Induce anesthesia in the mouse by intraperitoneal (i.p.) injection of a working solution of tribromoethanol (18 mL of working solution per kg body weight (b.w.)).</li>
	<li>Place an anesthetized mouse in supine position. Ensure that the mouse is completely anesthetized (less than 2 min).</li>
	<li>Insert the electrodes with acupuncture needles subcutaneously into the right and left forelimbs and the left hindlimb according to the lead II ECG scheme and fix them with tape (Figure 1). Ensure that the depth and position of inserted electrodes are consistent throughout the experiments.</li>
	<li>Connect the other ends of the electrodes by clicking them into the three snap connectors at the other end of the lead wires of the 3-lead bio amplifier cable.</li>
	<li>Inject drugs (i.p.) 3 min after the anesthetics have been delivered (Figure 2).</li>
	<li>Begin recording the ECG 10 min after injecting anesthetics. Once the recording is completed, use the ECG data from 12 to 17 min after injection of anesthetics for analysis.</li>
	<li>At the end of the ECG recording session, carefully remove the electrodes.</li>
</ol>
5. ECG data analysis
<ol>
	<li>Go to ECG Analysis | Averaging View and ensure that the software correctly identifies the start and end of the P wave, QRS complex, and T wave in individual beats. If necessary, manual correction of these waves and intervals is possible by moving misplaced cursors to the appropriate positions. 
	NOTE: As depicted in Figure 3A, the PR interval spans the onset of the P wave to that of the QRS complex (mostly missing Q wave in a mouse ECG). The QRS duration extends from the onset of the Q wave (primarily an R wave in a mouse ECG) to the end of the S wave. The QT interval comprises the onset of the Q wave (mostly the R wave in a mouse ECG) to the end of the T wave. Note the shorter duration and absence of a Q wave and ST segment in the mouse ECG relative to the human ECG8.</li>
	<li>Go to ECG Analysis | Table View and select the correctly identified ECG data by checking individual beats in the Averaging View window. 
	NOTE: Figure 3 shows several examples of actual mouse ECG signals. Figure 3A represents a normal wild-type signal that has been correctly identified with regard to P wave, QRS complex, and T wave. Computerized selection of PQRS waves can incur erroneous misplacements, such as in Figure 3B a normal wild-type signal that misplaces the onset of the P wave. In Figure 3C an ECG signal that misplaces the end of the QRS complex, resulting in an overestimation of QRS duration. In Figure 3D an ECG signal that misplaces the end of the QRS complex, resulting in underestimation of the QRS complex due to the ambiguous T wave and Figure 3E an ECG signal with an unidentifiable T wave. Without exclusion or manual corrections, PQRS intervals can be over or underestimated. Be sure to select the ECG signals that have been correctly identified and the signals that do not miss the target peaks. Consequently, such cases, including B, C, D, and E (Figure 3), are excluded in accurately estimating ECG parameters in general.</li>
	<li>Select the ECG data of interest in Table View, and copy/paste them to a spreadsheet file.</li>
</ol>
6. Statistical analysis
<ol>
	<li>Perform the statistical analysis using a statistics program. Analyze the data with the experimental conditions blinded. Perform Student&#8217;s t-test and Mann-Whitney U-test for 2-group comparisons. The numbers in each figure indicate the number of mice that is used for each group. Report the results as mean &#177; SEM.</li>
	<li>Consider differences with p &#60; 0.05 by U-test to be statistically significant: *, p &#60; 0.05; **, p &#60; 0.01; and ***, p &#60; 0.005 versus respective controls.</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+physiology+in+mice:+Conscious+measurements+and+effects+of+anesthesia.">Cardiovascular physiology in mice: Conscious measurements and effects of anesthesia.</a> Cardiovascular Physiology in the Genetically Engineered Mouse. , 257-275 (2002).</li><li>Cesarovic, N., Jirkof, P., Rettich, A., Arras, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantation+of+radiotelemetry+transmitters+yielding+data+on+ecg,+heart+rate,+core+body+temperature+and+activity+in+free-moving+laboratory+mice.">Implantation of radiotelemetry transmitters yielding data on ecg, heart rate, core body temperature and activity in free-moving laboratory mice.</a> Journal of visualized experiments : JoVE. 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A., Ross, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+anesthesia+on+cardiac+function+during+echocardiography+in+mice.">Impact of anesthesia on cardiac function during echocardiography in mice.</a> American Journal of Physiology. Heart and Circulatory Physiology. 282 (6), 2134-2140 (2002).</li><li>Mitchell, G. F., Jeron, A., Koren, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+heart+rate+and+q-t+interval+in+the+conscious+mouse.">Measurement of heart rate and q-t interval in the conscious mouse.</a> The American Journal of Physiology. 274 (3), 747-751 (1998).</li><li>Farraj, A. K., Hazari, M. S., Cascio, W. 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O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Garem1+regulates+the+pr+interval+on+electrocardiograms.">Garem1 regulates the pr interval on electrocardiograms.</a> Journal of Human Genetics. 63 (3), 297-307 (2018).</li><li>Nam, J. M., Lim, J. E., Ha, T. W., Oh, B., Kang, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac-specific+inactivation+of+prdm16+effects+cardiac+conduction+abnormalities+and+cardiomyopathy-associated+phenotypes.">Cardiac-specific inactivation of prdm16 effects cardiac conduction abnormalities and cardiomyopathy-associated phenotypes.</a> American Journal of Physiology. Heart and Circulatory Physiology. 318 (4), 764-777 (2020).</li><li>Knollmann, B. 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No conflicts of interest, financial or otherwise, are declared by the authors.

There are several critical steps in the protocol. The surrounding environment should be free from noise and vibration. The ECG electrodes must be inserted under the skin stably and consistently of which the insertion step requires preliminary experiments until the researcher is technically experienced. Further, the anesthetic should be prepared and stored appropriately and used at the proper dose. Finally, the PQRS waves should be located appropriately in individual ECG beats in the Averaging View window.
<p class="j...

The electrocardiogram (ECG), a test that measures the electrical activity of one&#8217;s heartbeat, is a valuable tool for evaluating the cardiac conduction system. The parameters that are measured by an ECG include heart rate, PR interval, QRS duration, and QT interval. In brief, PR interval corresponds to the time that is required for an electrical impulse to travel from the atrial sinus node through the atrioventricular node to the Purkinje fibers; QRS duration is the time for ventricular depolarization to occur through the Purkinje system and ventricular myocardium; and QT interval is the duration of ventricular repolarization.
ECG recordings in mice have helped researchers examine cardiac function and determine the physiological and pathophysiological mechanisms of cardiac phenotypes, such as arrhythmia, atrial fibrillation, and heart failure. Most cardiovascular research has involved studies in genetically engineered mouse models. It is often challenging to obtain meaningful data on ECG recordings from small mice that have been genetically manipulated.
There are several methods for performing ECGs in mice1. Studies suggest that ECG recordings in conscious animals are preferred over anesthetized animals when possible since the effects of anesthesia on cardiac function have been well established2. Two protocols that record ECG in conscious mice are of note1. The ECG radiotelemetry system is the gold standard for continuous long term monitoring of ECG in conscious mice1,3. Despite their strength in being recorded in a conscious state, radiotelemetry-coupled ECG measurements have several limitations, including the high expense for setup and for the implant, its requirement of a highly experienced operator, a stabilization period of over 1 week, its need for large mice (&#62; 20 g), and acquisition of only a single lead of ECG recording1. Another system that uses paw-sized conductive electrodes embedded in a platform allows ECG recordings in conscious mice without anesthesia or implants1,4. This non-invasive system is an alternative method in situations in which radiotelemetry systems are unavailable since it has many advantages: no requirement of surgical treatment, no need of anesthesia, low cost per mouse (only the initial setup is expensive), short time for measurement, and affordability of neonates1,4. The main disadvantage of this system is that it is not suited for continuous long term monitoring1.
Here we introduce another inexpensive, simple, and fast ECG recording method in anesthetized mice and demonstrate its validity and sensitivity by performing an ECG after autonomic blockade/stimulation of the cardiac conduction system. We suggest this ECG method for screening the effects of pharmacological agents, genetic modifications, and disease models in mice.

<table>
	<tbody>
		<tr>
			<td>2,2,2-tribromoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>T48402-25G</td>
			<td>anesthetics, Avertin</td>
		</tr>
		<tr>
			<td>Animal</td>
			<td>Japan SLC, Inc., Shizuoka, Japan</td>
			<td></td>
			<td>Balb/c mice, male, aged 7-9 weeks</td>
		</tr>
		<tr>
			<td>Atropine</td>
			<td>Sigma-Aldrich</td>
			<td>A0123</td>
			<td>parasympathetic antagonist</td>
		</tr>
		<tr>
			<td>BioAmp</td>
			<td>AD Instruments, Bella Vista, Australia</td>
			<td>ML132</td>
			<td>bio amplifier</td>
		</tr>
		<tr>
			<td>Carbachol</td>
			<td>Sigma-Aldrich</td>
			<td>C4382</td>
			<td>parasympathetic agonist</td>
		</tr>
		<tr>
			<td>Electrodes with acupuncture needles</td>
			<td>DongBang Acupuncture Inc., Sungnam, Korea</td>
			<td>DB106</td>
			<td>0.20 x 15 mm</td>
		</tr>
		<tr>
			<td>Isoprenaline</td>
			<td>Sigma-Aldrich</td>
			<td>I2760</td>
			<td>sympathetic agonist</td>
		</tr>
		<tr>
			<td>LabChart 8</td>
			<td>AD Instruments, Bella Vista, Australia</td>
			<td></td>
			<td>data analysis software</td>
		</tr>
		<tr>
			<td>Mouse food</td>
			<td>LabDiet, St. Louis, MO, USA</td>
			<td>5L79</td>
			<td>Mouse diet</td>
		</tr>
		<tr>
			<td>PowerLab 2/28</td>
			<td>AD Instruments, Bella Vista, Australia</td>
			<td></td>
			<td>data acquisition system</td>
		</tr>
		<tr>
			<td>Propranolol</td>
			<td>Sigma-Aldrich</td>
			<td>P0884</td>
			<td>sympathetic antagonist</td>
		</tr>
		<tr>
			<td>SPSS Statistics program</td>
			<td>SPSS</td>
			<td>SPSS 25.0</td>
			<td>statistics program</td>
		</tr>
	</tbody>
</table>

electrocardiogram recordings in anesthetized mice using lead ii

The electrocardiogram is a valuable tool for evaluating the cardiac conduction system. Animal research has helped generate novel genetic and pharmacological information regarding the electrocardiogram. However, making electrocardiogram measurements in small animals in vivo, such as mice, has been challenging. To this end, we used an electrocardiogram recording method in anesthetized mice with many advantages: it is a technically simple procedure, is inexpensive, has short measuring time, and is affordable, even in young mice. Despite the limitations with using anesthesia, comparisons between control and experimental groups can be performed with enhanced sensitivity. We treated mice with agonists and antagonists of the autonomic nervous system to determine the validity of this protocol and compared our results with previous reports. Our ECG protocol detected increased heart rates and QTc intervals on treatment with atropine, decreased heart rates and QTc intervals after carbachol treatment, and higher heart rates and QTc intervals with isoprenaline but did not note any change in ECG parameters on administration of propranolol. These results are supported by previous reports, confirming the reliability of this ECG protocol. Thus, this method can be used as a screening approach to making ECG measurements that otherwise would not be attempted due to high cost and technical difficulties.

Pharmacological experiments
To determine whether our noninvasive ECG measurement reflects the influence of autonomic modulation on the cardiac conduction system, normal Balb/c mice were challenged with agonists and antagonists of the autonomic nervous system (ANS). Atropine and carbachol were used to effect parasympathetic autonomic blockade and stimulation, respectively, whereas propranolol and isoprenaline were administered to elicit sympathetic autonomic blockade and stimul...

Watch this Scientific Journal Video about Electrocardiogram Recordings in Anesthetized Mice using Lead II at JoVE.com

Electrocardiogram Recordings in Anesthetized Mice using Lead II

We present an ECG protocol that is technically easy, inexpensive, fast, and affordable in small mice, and can be performed with enhanced sensitivity. We suggest this method as a screening approach for studying pharmacological agents, genetic modifications, and disease models in mice.

The electrocardiogram is a valuable tool for evaluating the cardiac conduction system. Animal research has helped generate novel genetic and ...

electrocardiogram-recordings-in-anesthetized-mice-using-lead-ii

Research

JoVE Journal

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12.3K Views.  Kyung Hee University. We present an ECG protocol that is technically easy, inexpensive, fast, and affordable in small mice, and can be performed with enhanced sensitivity. We suggest this method as a screening approach for studying pharmacological agents, genetic modifications, and disease models in mice.

Video: Electrocardiogram Recordings in Anesthetized Mice using Lead II

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of isolated tracheal preparations has proven to be a useful assay of airway smooth muscle (ASM) contractile response in genetically modified mice. These in vitro tracheal preparations are relatively simple, provide a robust response, and retain both functional cholinergic nerve endings and muscle responses, even after long incubations.
Tracheal tensometry also provides a functional assay to study a variety of second messenger signaling pathways that affect contraction of smooth muscle. Contraction in trachea is primarily mediated by parasympathetic, cholinergic nerves that release acetylcholine onto ASM (Figure 1). The major ASM acetylcholine receptors are muscarinic M2 and M3 which are Gi/o and Gq coupled receptors, respectively3,4,5. M3 receptors evoke contraction by coupling to Gq to activate phospholipase C, increase IP3 production and IP3-mediated calcium release from the sarcoplasmic reticulum3,6,7. M2/Gi/o signaling is believed to enhance contractions by inhibition of adenylate cyclase leading to a decrease in cAMP levels5,8,9,10. These pathways constitute the so called "pharmaco-contraction coupling" of airway smooth muscle11. In addition, cholinergic signaling through M2 receptors (and modulated by M3 signaling) involves pathways that depolarize the ASM which in turn activate L-type, voltage-dependent calcium channels (Figure 1) and calcium influx (so called "excitation-contraction coupling")4,7. More detailed reviews on signaling pathways controlling airway constriction can be found4,12. The above pathways appear to be conserved between mice and other species. However, mouse tracheas differ from other species in some signaling pathways. Most prominent is their lack of contractile response to histamine and adenosine13,14, both well-known ASM modulators in humans and other species5,15.
Here we present protocols for the isolation of murine tracheal rings and the in vitro measurement of their contractile output. Included are descriptions of the equipment configuration, trachea ring isolation and contractile measurements. Examples are given for evoking contractions indirectly using high potassium stimulation of nerves and directly by depolarization of ASM muscle to activate voltage-dependent calcium influx (1. high K+, Figure 1). In addition, methods are presented for stimulations of nerves alone using electric field stimulation (2. EFS, Figure 1), or for direct stimulation of ASM muscle using exogenous neurotransmitter applied to the bath (3. exogenous ACH, Figure 1). This flexibility and ease of preparation renders the isolated trachea ring model a robust and functional assay for a number of signaling cascades involved in airway smooth muscle contraction.

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic mice have been extremely useful in ascribing physiological function to genes. As such, research in general, and functional studies of airway, in particular, have undergone a remarkable shift toward murine models. Here we provide protocols for in vitro trachea constriction studies to evaluate smooth muscle function in murine airway.

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Brain Source Imaging in Preclinical Rat Models of Focal Epilepsy using High-Resolution EEG Recordings

Electroencephalogram (EEG) has been traditionally used to determine which brain regions are the most likely candidates for resection in patients with focal epilepsy. This methodology relies on the assumption that seizures originate from the same regions of the brain from which interictal epileptiform discharges (IEDs) emerge. Preclinical models are very useful to find correlates between IED locations and the actual regions underlying seizure initiation in focal epilepsy. Rats have been commonly used in preclinical studies of epilepsy1; hence, there exist a large variety of models for focal epilepsy in this particular species. However, it is challenging to record multichannel EEG and to perform brain source imaging in such a small animal. To overcome this issue, we combine a patented-technology to obtain 32-channel EEG recordings from rodents2 and an MRI probabilistic atlas for brain anatomical structures in Wistar rats to perform brain source imaging. In this video, we introduce the procedures to acquire multichannel EEG from Wistar rats with focal cortical dysplasia, and describe the steps both to define the volume conductor model from the MRI atlas and to uniquely determine the IEDs. Finally, we validate the whole methodology by obtaining brain source images of IEDs and compare them with those obtained at different time frames during the seizure onset.

This video introduces the preparation, recording, and source analysis procedures of high-resolution EEG on sedated rats with a particular preclinical model of focal epilepsy under noninvasive conditions.

Electroencephalogram (EEG) has been traditionally used to determine which brain regions are the most likely candidates for resection in patients with ...

Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice

Osmotic pumps continuously deliver compounds at a constant rate into small animals. This article introduces a standard protocol used to induce aortic aneurysms via subcutaneous infusion of angiotensin II (AngII) from implanted osmotic pumps. This protocol includes calculation of AngII amount and dissolution, osmotic pump filling, implantation of osmotic pumps subcutaneously, observation after pump implantation, and harvest of aortas to visualize aortic aneurysms in mice. Subcutaneous infusion of AngII through osmotic pumps following this protocol is a reliable and reproducible technique to induce both abdominal and thoracic aortic aneurysms in mice. Infusion durations range from a few days to several months based on the purpose of the study. AngII 1,000 ng/kg/min is sufficient to provide maximal effects on abdominal aortic aneurysmal formation in male hypercholesterolemic mouse models such as apolipoprotein E deficient or low-density lipoprotein receptor deficient mice. Incidence of abdominal aortic aneurysms induced by AngII infusion via osmotic pumps is 5 - 10 times lower in female hypercholesterolemic mice and also lower in both genders of normocholesterolemic mice. In contrast, AngII-induced thoracic aortic aneurysms in mice are not hypercholesterolemia or gender-dependent. Importantly, multiple features of this mouse model recapitulate those of human aortic aneurysms.

Subcutaneous implantation of osmotic pumps provides a convenient approach for prolonged and consistent delivery of compounds. This approach has been used extensively to study both abdominal and thoracic aortic aneurysms in mice.

Osmotic pumps continuously deliver compounds at a constant rate into small animals.  This article introduces a standard protocol used to induce aortic ...

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

The use of personalized medicine to treat rare monogenic diseases like lysosomal storage disorders (LSDs) is challenged by complex clinical trial designs, high costs, and low patient numbers. Hundreds of mutant alleles are implicated in most of the LSDs. The diseases are typically classified into 2 to 3 different clinical types according to severity. Moreover, molecular characterization of the genotype can help predict clinical outcomes and inform patient care. Therefore, we developed a simple cell culture assay based on HEK293H cells heterologously over-expressing the mutations identified in Fabry and Pompe disease. A similar assay has recently been introduced as a preclinical test to identify amenable mutations for Pharmacological Chaperone Therapy (PCT) in Fabry disease. This manuscript describes an amended cell culture assay which enables rapid phenotypic assessment of allelic variants in Fabry and Pompe disease to identify eligible patients for PCT and may aid in the development of novel pharmacochaperones.

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

There is a demand to make pre-clinical testing for a novel class of "orphan" drugs called pharmacological chaperones reproducible, fast, and efficient. We developed a simple, highly standardized, and versatile cell culture-based assay to screen for eligible patients as well as novel pharmacological chaperone drugs.

The use of personalized medicine to treat rare monogenic diseases like lysosomal storage disorders (LSDs) is challenged by complex clinical trial designs, ...

A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence

Necrotic enteritis was studied in chickens using various in vivo infection models. Most of these use a combination of predisposing factors, such as coccidiosis and diet, with gavage or administration via the feed using Clostridium perfringens. In these models, the comparison of multiple C. perfringens strains for virulence studies requires a large number of hosts to obtain significant results. Mortality during the course of the study can be high depending on the experimental model, hence raising ethical concerns regarding animal welfare in research. The development of new infection models requiring fewer animals to study pathogenesis, yet providing statistically significant and valid results, is important in reducing animal use in research. Intestinal ligated loop models have been used to study clostridial infections in various species such as mice, rabbits and calves. Following surgical procedures to create ligated loop segments, C. perfringens strains are injected directly into the loops to establish a close contact between the bacteria and the intestinal mucosa. Samples of the small intestine and luminal contents are taken at the termination of the procedures after a few hours. Multiple bacterial strains can be inoculated in each animal, hence reducing the number of required subjects in the experiments. Also, procedures are performed under general anesthesia to reduce animal pain. In chickens, this model would be more appropriate than oral administration to compare C. perfringens strain pathogenicity because fewer animals are needed, no predisposing factors are required to induce the disease, and pain is controlled by analgesics. The intestinal ligated loop model is poorly described in chickens and standardization is essential for its optimal use. This manuscript provides all the necessary steps to create numerous intestinal ligated loops in chickens and brings information on the critical points to obtain valid results.

A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence

Here we present a protocol to surgically create &#39;intestinal ligated loops&#39; in chicken small intestines. This procedure allows for the comparison of multiple Clostridium perfringens strains&#39; virulence in situ in a single host. This method markedly decreases the number of chickens usually necessary for similar in vivo experiments.

Necrotic enteritis was studied in chickens using various in vivo infection models. Most of these use a combination of predisposing factors, such as ...

Patient Directed Recording of a Bipolar Three-Lead Electrocardiogram using a Smartwatch with ECG Function

Cardiac arrhythmias and cardiovascular diseases are a major public health problem in developed countries. A major goal in preventive medicine is the reduction of cardiovascular death by early detection of atrial fibrillation (AF), which may cause stroke, or early detection of life-threatening myocardial ischemia in acute coronary syndrome. Detection of arrhythmia is often challenging if symptoms occur when patients have no chance for immediate electrocardiogram (ECG) diagnostic testing, or if the observation time period is short or an immediate visit to their doctor is not possible. Smartwatches and other wearable devices are able to record a single lead ECG recording, but a single lead ECG is often not sufficient for diagnosis of cardiovascular disorders. Even diagnosis of AF can be difficult with only information from a single lead bipolar ECG. Some smart devices use photoplethysmography for detection of cardiac rhythm, but this technique can only give indirect hints of the underlying cardiac rhythm, is prone to interferences, and cannot be used for detection of myocardial ischemia. A three-lead bipolar ECG like the Einthoven leads used in regular ECGs can add useful information concerning arrhythmia detection or even diagnosis of other cardiovascular diseases like ischemia. Therefore, we describe a protocol for the patient-directed recording of an Einthoven three-lead ECG using a smartwatch.

We describe a protocol for the patient-directed registration of a three lead bipolar electrocardiogram by a smartwatch that functions identically to the Einthoven leads from standard electrocardiograms. This enables patients to record electrocardiograms on their own immediately after the onset of symptoms.

Cardiac arrhythmias and cardiovascular diseases are a major public health problem in developed countries. A major goal in preventive medicine is the ...