This video publication was made possible by funding from the National Institute of Diabetes and Digestive and Kidney diseases (NIDDK) for the Program Project Grant "Pathobiology of the Enteric System" DK 68055. Christopher T. Creedon was supported by the Rochester Public Schools Mentorship Program.
We thank Mr. Gary Stoltz for technical assistance, Ms. Kristy Zodrow for secretarial assistance and Dr. Douglas Baer from Los Gatos Research, Inc (Mountain View, CA).

1. Training and Habituation of the Mice
<ol>
 <li>Prior to analysis, put all mice in the testing chambers for 2-4 hr with constant airflow in order to habituate them to the testing conditions. This markedly reduces stress levels that might otherwise cause aberrant detection of delayed gastric emptying. Treat the mice the same way as if the gastric emptying experiment was running. Prepare egg yolk (see further) without adding octanoic acid and feed 0.2 g to each mouse. </li>
 <li>Repeat this process till the mice ar...

<ol>
	<li>Yeung, C. K., McCurrie, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+to+investigate+the+inhibitory+effects+of+drugs+on+gastric+emptying+in+the+mouse+in+vivo.">A simple method to investigate the inhibitory effects of drugs on gastric emptying in the mouse in vivo.</a> J. Pharmacol. Toxicol. Methods. 45, 235-240 (2001).</li><li>Osinski, M. A., Seifert, T. R., Cox, B. F., Gintant, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+of+evaluation+of+drug-evoked+changes+in+gastric+emptying+in+mice.">An improved method of evaluation of drug-evoked changes in gastric emptying in mice.</a> J. Pharmacol. Toxicol. Methods. 47, 115-120 (2002).</li><li>Ghoos, Y. F., 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=Measurement+of+gastric+emptying+rate+of+solids+by+means+of+a+carbon-labeled+octanoic+acid+breath+test.">Measurement of gastric emptying rate of solids by means of a carbon-labeled octanoic acid breath test.</a> Gastroenterology. 104, 1640-1647 (1993).</li><li>Symonds, E., Butler, R., Omari, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+breath+tests+can+detect+alterations+in+gastric+emptying+in+the+mouse.">Noninvasive breath tests can detect alterations in gastric emptying in the mouse.</a> Eur. J. Clin. Invest. 32, 341-344 (2002).</li><li>Symonds, E. L., Butler, R. N., Omari, T. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+gastric+emptying+in+the+mouse+using+the+[13C]-octanoic+acid+breath+test.">Assessment of gastric emptying in the mouse using the [13C]-octanoic acid breath test.</a> Clin. Exp. Pharmacol. Physiol. 27, 671-675 (2000).</li><li>Verhulst, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+ghrelin+in+the+relationship+between+hyperphagia+and+accelerated+gastric+emptying+in+diabetic+mice.">Role of ghrelin in the relationship between hyperphagia and accelerated gastric emptying in diabetic mice.</a> Gastroenterology. 135, 1267-1276 (2008).</li><li>Schoonjans, R., 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=The+13C-octanoic+acid+breath+test:+validation+of+a+new+noninvasive+method+of+measuring+gastric+emptying+in+rats.">The 13C-octanoic acid breath test: validation of a new noninvasive method of measuring gastric emptying in rats.</a> Neurogastroenterol. Motil. 14, 287-293 (2002).</li><li>Choi, K. M., 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=Determination+of+gastric+emptying+in+nonobese+diabetic+mice.">Determination of gastric emptying in nonobese diabetic mice.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 293, 1039-1045 (2007).</li><li>Atkinson, M. A., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NOD+mouse+model+of+type+1+diabetes:+as+good+as+it+gets?.">The NOD mouse model of type 1 diabetes: as good as it gets?.</a> Nat. Med. 5, 601-604 (1999).</li><li>Camilleri, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+practice.+Diabetic+gastroparesis.">Clinical practice. Diabetic gastroparesis.</a> N. Engl. J. Med. 356, 820-829 (2007).</li><li>Choi, K. M., 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=Heme+oxygenase-1+protects+interstitial+cells+of+Cajal+from+oxidative+stress+and+reverses+diabetic+gastroparesis.">Heme oxygenase-1 protects interstitial cells of Cajal from oxidative stress and reverses diabetic gastroparesis.</a> Gastroenterology. 135, 2055-2064 (2008).</li><li>Kashyap, P. C., 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=Carbon+monoxide+reverses+diabetic+gastroparesis+in+NOD+mice.">Carbon monoxide reverses diabetic gastroparesis in NOD mice.</a> Am. J. Physiol. GI. G298, G1013-G1019 (2010).</li></ol>

The described technique herein allows for repeated and non-invasive in vivo measurement of solid gastric emptying in mice. This system has the advantage that the animals are not restrained in the measurement chamber, allowing them to move and turn freely. Since this is an unfamiliar environment, the mice still need to be trained and habituated to the testing chambers to prevent effects of stress on gastric emptying. In general, we assume the gastric emptying data are reliable if the intra-mouse variability betwe...

This manuscript describes the technical and methodological considerations involved in non-invasive measurement of gastric emptying in mice. By following the protocol described here, investigators can reliably and reproducibly follow changes in gastric emptying due to development of disease, study the impact of pharmacological agents on gastric emptying and follow the response of gastric emptying to treatment of underlying diseases or defects6,8,11,12. In previous publications, the application of 13C octanoic acid breath tests was shown to be a useful way of measuring gastric emptying in humans and animals3,8.This paper describes in detail, the procedures necessary to obtain reliable data over the 6 to 8 months necessary for a longitudinal study of gastric emptying in mice with diabetes. The advantages of following this protocol when compared to previously published methods are that the investigator can be assured the data obtained will be reliable and reproducible. In addition, the automated system for collecting and analyzing the gas samples described here increases the number of animals that can be followed simultaneously in a study. Overall, the goal of this paper is to identify the key factors that maintain habituation of the mice to the test and that reduce variability in obtained results.
For the in vivo measurement of gastric emptying, mice are fasted overnight and put into the transparent plastic testing chambers with constant airflow. After the mice get habituated to the tubes, baseline exhaled 13CO2 levels are determined and airflow adjusted accordingly. Next, we administer a test meal consisting of egg yolk mixed with 13C-labeled octanoic acid. Because the mice are fasted and trained, they generally eat the test meal within 2 min. The administered octanoic acid is not absorbed in the stomach but will be taken up in the duodenum and will get metabolized in the liver into 13CO2, which is released and exhaled, resulting in an enrichment of 13CO2 in the surrounding air. Air samples are collected at determined time intervals and are analyzed by the carbon isotope analyzer. The rate-limiting step in this whole process is gastric emptying and the pulmonary excretion of 13CO2 directly corresponds with gastric emptying of the labeled meal. 
<img src="/files/ftp_upload/50301/50301fig1.jpg" alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50301/50301fig1highres.jpg"/> 
Figure 1. Schematic of gastric emptying apparatus. After overnight fasting, mice are placed in transparent chambers allowing them to move and turn freely. An inlet tube allows fresh and constant air influx and an outlet leads to the isotope analyzer to measure the 13C-to-12C ratio in the exhaled breath. The chamber also has a central port for delivery of food containing [13C]-octanoic acid.

<table border="1">
<tbody>
 <tr>
 <td>NAME</td>
 <td>COMPANY</td>
 <td>CAT NUMBER</td>
 <td>COMMENTS</td>
 </tr>
 <tr>
 <td>A source of constant air-supply, flow as well as composition</td>
 <td>central air supply in the research facility</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>130 ml sampling chamber that has air inlet, air outlet, and food administration opening 
Plastic tubes for air supply</td>
 <td>In-house built</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Octanoic acid</td>
 <td>Cambridge isotope laboratories (Andover, MA)</td>
 <td>CLM-293-1</td>
 <td></td>
 </tr>
 <tr>
 <td>To prepare the egg meal: 
 <ul>
 <li>small beaker</li>
 <li>50 ml plastic tube</li>
 <li>Bunsen burner</li>
 <li>egg</li>
 <li>spatula</li>
 </ul></td>
 <td>Any supplier</td>
 <td></td>
 <td>Try to be consistent with the egg supplier since the nutritional content and palatability of the eggs can affect ingestion and gastric emptying of the meal</td>
 </tr>
 <tr>
 <td>Carbon dioxide isotope analyzer</td>
 <td>Los Gatos Research Inc. (Mountain View, CA)</td>
 <td></td>
 <td></td>
 </tr>
</tbody>
</table>

assessment of gastric emptying in non-obese diabetic mice using a [13c]-octanoic acid breath test

Gastric emptying studies in mice have been limited by the inability to follow gastric emptying changes in the same animal since the most commonly used techniques require killing of the animals and postmortem recovery of the meal1,2. This approach prevents longitudinal studies to determine changes in gastric emptying with age and progression of disease. The commonly used [13C]-octanoic acid breath test for humans3 has been modified for use in mice4-6 and rats7 and we previously showed that this test is reliable and responsive to changes in gastric emptying in response to drugs and during diabetic disease progression8. In this video presentation the principle and practical implementation of this modified test is explained. As in the previous study, NOD LtJ mice are used, a model of type 1 diabetes9. A proportion of these mice develop the symptoms of gastroparesis, a complication of diabetes characterized by delayed gastric emptying without mechanical obstruction of the stomach10.
This paper demonstrates how to train the mice for testing, how to prepare the test meal and obtain 4 hr gastric emptying data and how to analyze the obtained data. The carbon isotope analyzer used in the present study is suitable for the automatic sampling of the air samples from up to 12 mice at the same time. This technique allows the longitudinal follow-up of gastric emptying from larger groups of mice with diabetes or other long-standing diseases.

A representative data set from three different mice is shown in Figure 2. The black graph represents the data points from a mouse with normal gastric emptying. It shows the fraction of 13C that is recovered in the exhaled air expressed as a percentage of the administered dose per hour expressed as a function of time. The blue curve is from a mouse with an accelerated gastric emptying with a T half value of 40 min and the red curve is from a mouse with a delayed gastric emptying with a T half v...

Watch this Scientific Journal Video about Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test at JoVE.com

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Determination of gastric emptying with a non-invasive [13C]-octanoic acid breath test for tracking gastroparesis in female NOD LtJ mice.

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Gastric emptying studies in mice have been limited by the inability to follow gastric emptying changes in the same animal since the most commonly used ...

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13.5K vues.  Mayo Clinic. Détermination de la vidange gastrique avec un non-invasive [ 13 C]-octanoïque test respiratoire à l'acide pour la gastroparésie suivi dans des souris femelles NOD LTJ.

Vidéo: Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Determination of gastric emptying with a non-invasive [13C]-octanoic acid breath test for tracking gastroparesis in female NOD LtJ...

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The sampler is relatively inexpensive and quite portable, but the monitor is neither. This limits options for some clinical studies and provides rational for future innovations.

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Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

Roux-en-Y gastric bypass (RYGB) is commonly performed for the treatment of severe obesity and type 2 diabetes. However, the mechanism of weight loss and metabolic changes are not well understood. Multiple factors are thought to play a role, including reduced caloric intake, decreased nutrient absorption, increased satiety, the release of satiety-promoting hormones, shifts in bile acid metabolism, and alterations in the gut microbiota.
The rat RYGB model presents an ideal framework to study these mechanisms. Prior work on mouse models have had high mortality rates, ranging from 17 to 52%, limiting their adoption. Rat models demonstrate more physiologic reserve to surgical stimulus and are technically easier to adopt as they allow for the use of surgical staplers. One challenge with surgical staplers, however, is that they often leave a large gastric pouch which is not representative of RYGB in humans. 
In this protocol, we present a RYGB protocol in rats that result in a small gastric pouch using surgical staplers. Utilizing two stapler fires which remove the forestomach of the rat, we obtain a smaller gastric pouch similar to that following a typical human RYGB. Surgical stapling also results in better hemostasis than sharp division. Additionally, the forestomach of the rat does not contain any glands and its removal should not alter the physiology of RYGB.
Weight loss and metabolic changes in the RYGB cohort were significant compared to the sham cohort, with significantly lower glucose tolerance at 14 weeks. Furthermore, this protocol has an excellent survival of 88.9% after RYGB. The skills described in this protocol can be acquired without previous microsurgical experience. Once mastered, this procedure will provide a reproducible tool for studying the mechanisms and effects of RYGB.

Roux-en-Y gastric bypass (RYGB) is performed to treat obesity and diabetes. However, the mechanisms underlying RYGB's efficacy are not fully understood, and studies are limited by technical difficulty leading to high mortality in animal models. This article provides instructions on how to perform RYGB in rats with high success rates.

Roux-en-Y gastric bypass (RYGB) is commonly performed for the treatment of severe obesity and type 2 diabetes. However, the mechanism of weight loss and ...

Providing Visual Biofeedback Using Brightness Mode Ultrasound During a Golf Swing

Using ultrasound biofeedback in conjunction with verbal cueing can increase muscle thickness more than verbal cueing alone and may augment traditional rehabilitation techniques in an athletic, physically active population. Brightness mode (B-mode) ultrasound can be applied using frame-by-frame analysis synchronized with video to understand muscle thickness changes during these dynamic tasks. Visual biofeedback with ultrasound has been established in static positions for the muscles of the lateral abdominal wall. However, by securing the transducer to the abdomen using an elastic belt and foam block, biofeedback can be applied during more specific tasks prevalent in lifetime sports, such as golf. To analyze muscle activity during a golf swing, muscle thickness changes can be compared. The thickness must increase throughout the task, indicating that the muscle is more active. This methodology allows clinicians to immediately replay ultrasound videos for patients as a visual tool to instruct proper activity of the muscles of interest. For example, ultrasound can be used to target the external and internal obliques, which play an important role in swinging a golf club or any other rotational sport or activity. This methodology aims to increase oblique muscle thickness during the golf swing. Additionally, the timing of muscle contraction can be targeted by instructing the patient to contract the abdominal muscles at specific time points, such as the beginning of the downswing, with the goal of improving muscle firing patterns during tasks.

Brightness mode ultrasound can be used to provide visual biofeedback of the muscles of the lateral abdominal wall during a golf swing. Post-swing visual and verbal instruction can increase the muscle activation and timing of the external and internal obliques.

Using ultrasound biofeedback in conjunction with verbal cueing can increase muscle thickness more than verbal cueing alone and may augment traditional ...

Assessing Pulmonary Alveolar-Capillary Reserve During Exercise

The combined single-breath measurement of the diffusing capacity of carbon monoxide (DL,CO) and nitric oxide (DL,NO) is a useful technique to measure pulmonary alveolar-capillary reserve in both healthy and patient populations. The measurement provides an estimate of the participant&#39;s ability to recruit and distend pulmonary capillaries. The method has recently been reported to exhibit a high test-retest reliability in healthy volunteers during exercise of light to moderate intensity. Of note, this technique permits up to 12 repeated maneuvers and only requires a single breath with a relatively short breath-hold time of 5 s. Representative data are provided showing the gradual changes in DL,NO and DL,CO from rest to exercise at increasing intensities&#160;of up to 60% of maximal workload. The measurement of diffusing capacity and evaluation of alveolar-capillary reserve is a useful tool to evaluate the lung&#39;s ability to respond to exercise both in the healthy population as well as in patient populations such as those with chronic lung disease.

This protocol presents a method to assess pulmonary alveolar-capillary reserve measured by combined single-breath measurement of the diffusing capacity to carbon monoxide (DL,CO) and nitric oxide (DL,NO) during exercise. Assumptions and recommendations for using the technique during exercise form the foundation of this article.

The combined single-breath measurement of the diffusing capacity of carbon monoxide (DL,CO) and nitric oxide (DL,NO) is a useful technique to measure ...