This research was supported by the Medical Scientific Fund of the Mayor of the City of Vienna and the &#214;sterreichische Gesellschaft f&#252;r Laboratoriumsmedizin und Klinische Chemie.

All methods described here have been approved by the Animal Care and Use Committee of the Medical University of Vienna and conducted according to the Federation of European Laboratory Animal Science Associations (FELASA).&#160;Please note that all procedures described in this protocol should only be performed after institutional and governmental approval as well as by staff that are technically proficient.
1. HFD-fed mice
NOTE: Maintain all C57BL/6J mice on a 12-h light/dark cycle with free access to food and water.
<ol>
	<li>At 6 weeks of age, place mice for 8-12 weeks on an HFD (40-60% fat calories) to induce obesity, while feeding the lean control-group a low-fat diet (LFD) (10% fat calories).</li>
	<li>Determine the body weight of the mice on a weekly basis. The weight curves should show similar patterns in both groups, with a higher slope in the HFD-fed group.</li>
</ol>
2. OGTT
NOTE: If blood sampling time points are chosen during OGTT every 15 min, the experiment should be performed with a maximum of 15 mice in parallel, in order to have at least 1 min handling-time per mouse.
<ol>
	<li>Preparations on the day before OGTT
	<ol>
		<li>Transfer the mice into a cage with fresh bedding and fast them overnight before testing (14 h), while ensuring that the mice have access to drinking water (e.g., remove the food at 6:00 pm for a start time on the next morning at 8:00 am). 
		NOTE: Fasting mice overnight is the standard approach, however a shorter fast (5-6 h) is more physiological for mice (see Discussion for details).</li>
	</ol>
	</li>
	<li>Preparations on the day of the experiment (but prior to the experiment)
	<ol>
		<li>Prepare 10 mL of 20% glucose solution (dissolve D-(+)-Glucose in distilled water). 
		NOTE: All reagents that are administered to the animals have to be pharmacological grade and sterile.</li>
		<li>Prepare a 96-well plate for plasma collection, by filling one well for each sampling time point and each mouse, with 5 &#181;L NaEDTA (0.5 M EDTA, pH 8.0 in 0.9% NaCl, storage at RT). During the experiment, store this plate on ice. 
		NOTE: See Supplementary Figure 1 for a detailed checklist.</li>
	</ol>
	</li>
	<li>Measure the body weight of all mice and mark their tails with a permanent marker in order to make the mice easily distinguishable (e.g., mouse 1 = 1 dash, mouse 2 = 2 dashes, etc.).</li>
	<li>Glucose measurement and blood sampling (Figure 2)
	<ol>
		<li>Carefully cut off 1-2 mm of the tail tip using sharp scissors (&#34;Variant A&#34; in Figure 2). Always wipe off the first drop of blood to avoid hemolysis or contamination with tissue fluid before taking new blood samples for blood glucose determination. Draw a small blood sample (~3 &#181;L) for the measurement of the basal blood glucose level (= time point 0) with the glucometer. 
		CAUTION: Check and adjust the charge number of test strips on a glucometer. 
		NOTE: As an alternative blood sampling method, nick the lateral tail vein of a mouse with a sharp scalpel blade (&#34;Variant B&#34; in Figure 2). The lateral tail vein is usually accessed approximately one-third along the length of the tail from the tail tip, moving towards the base of the tail for multiple samples. The use of a local anesthetic cream is recommended. Stop blood flow by applying finger pressure on the soft tissue for at least 30 s before the animal is returned to its cage.</li>
		<li>Collect a blood sample (around 30 &#181;L) using a fresh capillary tube (keep the capillary tube horizontal). Empty the capillary tube using a pipette by putting the pipette tip at the top of the capillary tube end and carefully pushing the collected blood into a well of the 96-well plate, while avoiding air bubbles. Repeat this procedure for all mice - one at a time. 
		NOTE: As an alternative for blood collection via a capillary tube, use a pipette adjusted to the correct volume (e.g., 30 &#181;L) to collect blood, or collect a drop of blood from the tail on paraffin film, and pipet it into the EDTA-solution. Strictly avoid the contact of petroleum jelly with blood or glucometer test strips, as it may influence subsequent glucose and insulin measurements. 
		CAUTION: The OGTT is very stressful for mice: lean mice can lose around 15% of their body weight during an overnight fast. Additionally, blood sampling at different time points leads to a considerable loss of blood. For easier blood sampling, it is possible to carefully massage the mouse-tail with petroleum jelly. 
		NOTE: Institutional guidelines may limit the allowable amount of blood collected within a set period. The sampling volumes and timepoints should be adjusted to not exceed the allowed maximums. The body weight of the mice should be used to calculate the total blood withdrawal permitted.</li>
	</ol>
	</li>
	<li>Calculate the required volume of glucose solution based on body weight (1 g glucose/kg body weight; this can be increased up to 3 g/kg) to be administered by oral gavage for every mouse. For example, a mouse with a body weight of 30 g would need 150 &#181;L of a 20% glucose solution to administer 30 mg of glucose. 
	NOTE: To base the dose of glucose on the weight of the mouse is the standard procedure. If body composition data are available, the dose of glucose for OGTT should be calculated based on the lean body mass (see Discussion for details).</li>
	<li>Glucose administration
	<ol>
		<li>Prepare everythingthat is needed during the whole experiment in advance (timer, experiment record sheet, glucose monitor and strips, capillaries, syringes, glucose solution, 96-well plate, scalpel, calculator, balance, permanent marker, bench papers, a pipette with a tip, and gloves).</li>
		<li>For glucose application, restrain the mouse by firmly grasping it by the scruff. Apply enough firmness to the skin around the neck to prevent the mouse from twisting out of the restrain and to properly tilt its head back. Also ensure that the mouse can breathe properly. 
		NOTE: Once glucose administration is started, good time management is very important.</li>
		<li>Carefully administer the glucose solution (based on step 2.5) directly into the stomach using a feeding needle. Cautiously direct the feeding needle through the mouth towards the esophagus. Allow the mouse to swallow the needle: the needle entirely sinks into the lower esophagus/stomach of the mouse. Then inject the glucose solution (Figure 3a).
		<ol>
			<li>If any resistance is met or if the animal struggles immediately, withdraw the needle and reposition it.Start the timer immediately after the first gavage and administer glucose to all other mice in 1 min intervals. 
			NOTE: It may be helpful to apply a drop of glucose solution directly from the feeding needle to the mouth of the mouse, which will stimulate licking and swallowing, thus facilitating easier insertion of the feeding needle. Do not apply pressure when inserting the feeding needle as this may seriously injure the animal.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>After 15 min, measure blood glucose levels with the glucometer and additionally take blood samples (~30 &#181;L) (as described in detail in step 2.4) of each mouse in the same order as they were injected. 
	NOTE: The time management is very important; follow as closely as possible using the same time intervals as for gavage. Let the mice move as freely as possible and limit restraining to a minimum during the whole procedure to reduce stress, which may modify the results. Milk tail with one hand and collect the blood with the other.</li>
	<li>Repeat step 2.7 at selected time points depending on the expected results (e.g., at 30, 45, 60, 90, 120, 150, and 180 min after glucose administration). If the selected time points are longer than 120 min, ensure that the mice have access to drinking water.&#160;Ensure that the mice have always access to drinking water. When finished with the experiment, return the mice to their home cages equipped with food and water. 
	CAUTION: The OGTT is very exhausting for the mice. Therefore wait at least 1 week before performing the next metabolic test, such as an ITT.</li>
	<li>After the experiment, centrifuge blood samples at 2,500 x g, 30 min, 4 &#176;C. Transfer the supernatant (plasma) to empty wells of the plate and store it at -20 &#176;C until analysis.
	<ol>
		<li>Record hemolysis of samples if present (see Section 3).</li>
	</ol>
	</li>
	<li>Determine plasma insulin levels using a commercially available ELISA kit (see the Table of Materials) following the manufacturer&#39;s instructions of the kit. 
	NOTE: Depending on the fasting state as well as on the metabolism of the investigated mice, difficulties during this assay may occur: overnight fasting insulin levels (time point 0) are very low and therefore close to the detection limit. To avoid this issue, double the quantity of recommended plasma volume and accordingly halve the result of the ELISA assay. On the other hand, if mice reach the insulin peak during OGTT, especially in HFD-fed mice, the insulin levels may exceed the detection limit: dilute the sample (e.g., 10-fold with 0.9% NaCl) and repeat the ELISA assay. Hemolysis in plasma samples may lead to the degradation of insulin, resulting in a decrease of the readout values. The degradation depends on time, temperature, and the hemoglobin concentration in the sample. Always keep hemolyzed samples cold or on ice to reduce insulin degradation.</li>
</ol>
3. ITT
NOTE: The same precautions described for OGTT (handling of mice, blood, glucometer, and petroleum jelly use) also have to be applied when performing the ITT. For example, all injections should be carried out within 15 min in 1 min intervals if 15 mice are tested in parallel. For the ITT, subsequent collection of blood samples with capillary tubes is optional.
<ol>
	<li>Preparations before the experiment
	<ol>
		<li>Fast mice for at least 2 h before insulin injection, while ensuring that the mice have access to drinking water (e.g., remove food at 8:00 am, test mice 2-5 h later).</li>
		<li>Dilute insulin 1:1,000 in 0.9% NaCl (Stock: 100 U/mL insulin; working concentration 0.1 U/mL) and prepare 20% glucose (D-(+)-Glucose solution dissolved in distilled water) to be administered if the mice become hypoglycemic. 
		NOTE: The ITT is typically performed after a short fast to avoid the hypoglycemia which may otherwise occur in overnight fasted animals.&#160;All reagents that are administered to the animals have to be pharmacological grade and sterile.</li>
	</ol>
	</li>
	<li>Measure the bodyweight of mice, mark tail, cut the tail tip using sharp scissors, and measure basal blood glucose levels as described previously for the OGTT in step 2.4.</li>
	<li>Insulin injection
	<ol>
		<li>To inject insulin intraperitoneally (0.75 U insulin/kg body weight, calculated beforehand), restrain the mouse by the scruff method.</li>
		<li>Use a fresh, sterile 27 or 30 gauge needle for each animal to avoid discomfort and the risk of any injection-site infection. 
		NOTE: Sterilization of the skin can prolong the duration of insulin administration, and thus can cause additional disturbances to the animal. Therefore, it is not recommended.</li>
		<li>Tilt the mouse head down at a slight angle to expose the ventral side of the animal. Place the sterile needle with the bevel up and at a 30 &#176; angle in the lower right quadrant of the animal&#39;s abdomen (Figure 3b). Start the timer immediately after the first mouse is injected. 
		NOTE: Low-dose ITTs (0.1 U/kg) may be performed to specifically assess hepatic insulin sensitivity. As for the OGTT, calculating the injection volume based on body weight is the standard procedure, while basing the dose on lean body mass is preferred if body composition data are available.</li>
	</ol>
	</li>
	<li>Measure blood glucose levels at selected time points (e.g., after 15, 30, 45, 60, and 90 min). 
	NOTE: As insulin has a short half-time of ~10 min in mice13, late differences after insulin administration (e.g., after 2 h) may not reflect a direct effect of insulin action. Administer 20% glucose solution in case a mouse becomes hypoglycemic (blood glucose levels below 35 mg/dL) and is in risk of dying.</li>
	<li>After the final time points, place the mice back into their home cages prepared with plenty of food and water.</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+experiment+in+centralized+mouse+phenotyping:+the+Vanderbilt+experience+and+recommendations+for+evaluating+glucose+homeostasis+in+the+mouse.">NIH experiment in centralized mouse phenotyping: the Vanderbilt experience and recommendations for evaluating glucose homeostasis in the mouse.</a> Am J Physiol Endocrinol Metab. 297 (4), E849-E855 (2009).</li><li>Pacini, G., Omar, B., Ahren, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+and+models+for+metabolic+assessment+in+mice.">Methods and models for metabolic assessment in mice.</a> J Diabetes Res. 2013, 986906 (2013).</li><li>Polonsky, K. S., Rubenstein, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-peptide+as+a+measure+of+the+secretion+and+hepatic+extraction+of+insulin.+Pitfalls+and+limitations.">C-peptide as a measure of the secretion and hepatic extraction of insulin. Pitfalls and limitations.</a> Diabetes. 33 (5), 486-494 (1984).</li><li>Hughey, C. C., Wasserman, D. H., Lee-Young, R. S., Lantier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approach+to+assessing+determinants+of+glucose+homeostasis+in+the+conscious+mouse.">Approach to assessing determinants of glucose homeostasis in the conscious mouse.</a> Mamm Genome. 25 (9-10), 522-538 (2014).</li></ol>

The authors have nothing to disclose.

With the high prevalence of diabetes and associated diseases in the world&#39;s population, there is a strong requirement for research addressing the molecular mechanism, prevention, and treatment of disease19. The presented protocol describes well-established methods for the generation of HFD mice, a robust animal model used for metabolic research, as well as the conduction of the OGTT and ITT, which are potent tools for the assessment of whole-body metabolic alterations such as insulin resistanc...

In the developed world, obesity and diabetes reached epidemic dimensions due to physical inactivity and the excess consumption of processed food, effects which are driven by rapid urbanization, industrialization as well as globalization. Although research on insulin resistance and it&#39;s co-morbidities, such as hyperlipidemia and atherosclerosis, has gained prominence during the last decades, the complex biological mechanisms which regulate metabolism in health and disease remain incompletely understood and there is still an urgent need for new treatment modalities to prevent and treat these diseases1.
Insulin, and it&#39;s counter-regulatory hormone glucagon serve as the principal regulators of cellular energy supply and macronutrient balance, thus also maintaining proper systemic blood glucose concentrations2. Glucose itself acts as one of the main stimulators of insulin secretion by pancreatic &#946;-cells, while other macronutrients, humoral factors as well as neural input further modify this response. Insulin consequently triggers the anabolic processes of the fed state by facilitating the diffusion of excess blood glucose into muscle and fat cells and further activating glycolysis as well as protein- or fatty acid synthesis, respectively. Additionally, insulin suppresses hepatic glucose output by inhibiting gluconeogenesis. Chronic excess energy consumption and meta-inflammation lead to hyperinsulinemia and peripheral insulin resistance due to the down-regulation of insulin receptor expression as well as alterations in downstream signaling pathways, thus resulting in impaired sensitivity to insulin-mediated glucose disposal as well as insufficient inhibition of hepatic glucose production3,4,5,6.
A wide range of animal models with genetic, nutritional, or experimental induction of disease have been proven to be excellent tools to study the molecular mechanisms of insulin resistance and various forms of diabetes as well as its accompanying illnesses7. A prime example is the widely used and well established HFD-induced mouse model, which is characterized by rapid weight gain due to increased dietary intake in combination with reduced metabolic efficiency, resulting in insulin resistance8,9. Both in animal models and humans, an elevation in fasting blood glucose and insulin levels, as well as an impaired tolerance to glucose administration are frequently used indicators of insulin resistance and other systemic alterations of glucose metabolism. Monitoring blood glucose and insulin levels at the basal state or after stimulation are therefore easily accessible readouts.
The present protocol outlines the generation of HFD-fed mice as well as two frequently used methods, the oral glucose tolerance test (OGTT) and the insulin resistance test (ITT), which are useful to characterize the metabolic phenotype and to investigate alterations in glucose metabolism. We describe the OGTT in detail, which assesses the disposal of an orally administered glucose load and insulin secretion over time. Further, we provide instructions on how to conduct the ITT to investigate whole-body insulin-action by monitoring blood glucose concentration in response to a bolus of insulin. The protocols described in this article are well-established and have been used in multiple studies10,11,12. In addition to slight modifications which may help to increase success, we provide guidelines for experimental design and data analysis, as well as useful hints to avoid potential pitfalls. The protocols described herein can be very powerful tools to investigate the influence of genetic, pharmacological, dietary, and other environmental factors on whole body glucose metabolism and on its associated disorders such as insulin resistance. In addition to stimulation with glucose or insulin, a variety of other compounds may be used for stimulation depending on the purpose of individual research. Although outside of the scope of this manuscript, many other downstream applications can be performed on the drawn blood samples, such as the analysis of blood values other than glucose and insulin (e.g., lipid and lipoprotein profiles) as well as detailed analysis of metabolic markers (e.g., by quantitative real time Polymerase Chain Reaction (PCR), Western blot analysis, and Enzyme-Linked Immunosorbent Assay (ELISA)). Further flow cytometry and Fluorescence Activated Cell Sorting (FACS) may be applied to investigate the effects in distinct single cell populations, while transcriptomic, proteomic, and metabolomic approaches may also be utilized for untargeted analysis.
Overall, we provide a simple protocol to generate an HFD-induced mouse model, while further describing two powerful approaches to study whole-body metabolic alterations, the OGTT and the ITT, which can be useful tools for studying disease pathogenesis and developing new therapies, especially in the field of metabolism-associated diseases such as insulin resistance &#38; diabetes.

<table>
	<tbody>
		<tr>
			<td>Mouse strain: C57BL/6J</td>
			<td>The Jackson Laboratory</td>
			<td>664</td>
			<td>LFD/HFD</td>
		</tr>
		<tr>
			<td>Accu Chek Performa - Glucometer</td>
			<td>Roche</td>
			<td>6870228</td>
			<td>OGTT/ITT</td>
		</tr>
		<tr>
			<td>Accu Chek Performa - Strips</td>
			<td>Roche</td>
			<td>6454038</td>
			<td>OGTT/ITT</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose solution</td>
			<td>Sigma-Aldrich</td>
			<td>G8769</td>
			<td>OGTT</td>
		</tr>
		<tr>
			<td>Actrapid - Insulin</td>
			<td>Novo Nordisk</td>
			<td>417642</td>
			<td>ITT</td>
		</tr>
		<tr>
			<td>Reusable Feeding Needles</td>
			<td>Fine Science Tools</td>
			<td>#18061-22</td>
			<td>OGTT; 22 gauge (-24 gauge for young mice)</td>
		</tr>
		<tr>
			<td>Omnifix-Fine dosing syringes</td>
			<td>Braun</td>
			<td>9161406V</td>
			<td>OGTT/ITT</td>
		</tr>
		<tr>
			<td>Sterican Insulin needle (30G x 1/3&#34;; &#248; 0.30 x 13 mm)</td>
			<td>Braun</td>
			<td>304000</td>
			<td>ITT; lean mice</td>
		</tr>
		<tr>
			<td>Sterican (G 27 x 3/4&#34;; &#248; 0.40 x 20 mm)&#160;&#160;</td>
			<td>Braun</td>
			<td>4657705</td>
			<td>ITT; mice on HFD</td>
		</tr>
		<tr>
			<td>96 Well PCR Plates, non-skirted, flexible</td>
			<td>Braintree Scientific, Inc.</td>
			<td>SP0016</td>
			<td>OGTT</td>
		</tr>
		<tr>
			<td>Ultrasensitive Mouse Insulin ELISA kit</td>
			<td>Crystam Chem</td>
			<td>90080</td>
			<td>OGTT</td>
		</tr>
		<tr>
			<td>Rodent Diet with 60% kcal% fat</td>
			<td>Research Diets Inc</td>
			<td>D12492</td>
			<td>mice on HFD</td>
		</tr>
		<tr>
			<td>Rodent Diet with 10% kcal% fat.</td>
			<td>Research Diets Inc</td>
			<td>D12450B</td>
			<td>mice on LFD</td>
		</tr>
		<tr>
			<td>BRAND micro haematocrit capillary</td>
			<td>Sigma-Aldrich</td>
			<td>BR749321</td>
			<td>OGTT/ITT</td>
		</tr>
		<tr>
			<td>Vaseline - creme</td>
			<td>Riviera</td>
			<td>P1768677</td>
			<td>OGTT/ITT</td>
		</tr>
	</tbody>
</table>

study of in vivo glucose metabolism in high-fat diet-fed mice using oral glucose tolerance test (ogtt) and insulin tolerance test (itt)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to insulin-stimulated glucose uptake and a gross decompensation of systemic glucose metabolism. Despite considerable progress in the understanding of glucose metabolism, the molecular mechanisms of its regulation in health and disease remain under-investigated, while novel approaches to prevent and treat diabetes are urgently needed. Diet derived glucose stimulates the pancreatic secretion of insulin, which serves as the principal regulator of cellular anabolic processes during the fed-state and thus balances blood glucose levels to maintain systemic energy status. Chronic overfeeding triggers meta-inflammation, which leads to alterations in peripheral insulin receptor-associated signaling and thus reduces the sensitivity to insulin-mediated glucose disposal. These events ultimately result in elevated fasting glucose and insulin levels as well as a reduction in glucose tolerance, which in turn serve as important indicators of insulin resistance. Here, we present a protocol for the generation and metabolic characterization of high-fat diet (HFD)-fed mice as a frequently used model of diet-induced insulin resistance. We illustrate in detail the oral glucose tolerance test (OGTT), which monitors the peripheral disposal of an orally administered glucose load and insulin secretion over time. Additionally, we present a protocol for the insulin tolerance test (ITT) to monitor whole-body insulin action. Together, these methods and their downstream applications represent powerful tools to characterize the general metabolic phenotype of mice as well as to specifically assess alterations in glucose metabolism. They may be especially useful in the broad research field of insulin resistance, diabetes and obesity to provide a better understanding of pathogenesis as well as to test the effects of therapeutic interventions.

Figure 1 illustrates a schematic time table for metabolic phenotyping of mice on diets. At an age of approximately 6 weeks, mice should be placed on an HFD, while an LFD-group may serve as the control group. Importantly, body weight should be determined weekly to observe if there is an expected increase in body weight. Any kind of stress (e.g., noise or aggressive male behavior) can interfere with body weight gain and should be eliminated immediately...

Watch this Scientific Journal Video about Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT at JoVE.com

Badanie metabolizmu glukozy in vivo u myszy karmionych dietą wysokotłuszczową przy użyciu doustnego testu tolerancji glukozy OGTT i testu tolerancji insuliny ITT | Medycyna | Wideo

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT

The current article describes the generation and metabolic characterization of high-fat diet-fed mice as a model of diet-induced insulin resistance and obesity. It further features detailed protocols to perform the oral glucose tolerance test and the insulin tolerance test, monitoring whole-body alterations of glucose metabolism in vivo.

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

Obesity represents the most important single risk factor in the pathogenesis of type 2 diabetes, a disease which is characterized by a resistance to ...

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67.3K Views.  Medical University of Vienna. The current article describes the generation and metabolic characterization of high-fat diet-fed mice as a model of diet-induced insulin resistance and obesity. It further features detailed protocols to perform the oral glucose tolerance test and the insulin tolerance test, monitoring whole-body alterations of glucose metabolism in vivo.

Video: Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test OGTT and Insulin Tolerance Test ITT

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

The &alpha;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time and temperature restrictions, technical sophistication, and cost of reagents, in particular monoclonal antibodies to measure CD4 on blood cells, the only currently acceptable method. A commonly used cost-saving and time-saving laboratory strategy is to calculate, rather than measure certain blood values. For example, LDL levels are calculated using the measured levels of total cholesterol, HDL, and triglycerides1. Thus, identification of cell-free correlates that directly regulate the number of CD4+ T cells could provide an accurate method for calculating CD4 counts due to the physiological relevance of the correlates.
The number of stem cells that enter blood and are destined to become circulating CD4+ T cells is determined by the chemokine CXCL12 and its receptor CXCR4 due to their influence on locomotion2. The process of stem cell locomotion into blood is additionally regulated by cell surface human leukocyte elastase (HLECS) and the HLECS-reactive active &alpha;1proteinase inhibitor (&alpha;1PI, &alpha;1antitrypsin, SerpinA1)3. In HIV-1 disease, &alpha;1PI is inactivated due to disease processes 4. In the early asymptomatic categories of HIV-1 disease, active &alpha;1PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 &mu;M, and to achieve normal levels during the symptomatic categories4, 5. This pattern has been attributed to immune inactivation, not to insufficient synthesis, proteolytic inactivation, or oxygenation. We observed that in HIV-1 subjects with &gt;220 CD4 cells/&mu;l, CD4 counts were correlated with serum levels of active &alpha;1PI (r2=0.93, p&lt;0.0001, n=26) and inactive &alpha;1PI (r2=0.91, p&lt;0.0001, n=26) 5. Administration of &alpha;1PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting &alpha;1PI participates in regulating the number of CD4+ T cells in blood 3.
With stimulation, whole saliva contains sufficient serous exudate (plasma containing proteinaceous material that passes through blood vessel walls into saliva) to allow measurement of active &alpha;1PI and the correlation of this measurement is evidence that it is an accurate method for calculating CD4 counts. Briefly, sialogogues such as chewing gum or citric acid stimulate the exudation of serum into whole mouth saliva. After stimulating serum exudation, the activity of serum &alpha;1PI in saliva is measured by its capacity to inhibit elastase activity. Porcine pancreatic elastase (PPE) is a readily available inexpensive source of elastase. PPE binds to &alpha;1PI forming a one-to-one complex that prevents PPE from cleaving its specific substrates, one of which is the colorimetric peptide, succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA3NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active &alpha;1PI in saliva. The resulting inhibition of PPE by active &alpha;1PI can be measured by adding the PPE substrate SA3NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/&mu;l), the concentration of &alpha;1PI in saliva is related to the concentration of serum in saliva, not to volume of saliva since volume can vary considerably during the day and person to person6. However, virtually all the protein in saliva is due to serum content, and the protein content of saliva is measurable7. Thus, active &alpha;1PI in saliva is calculated as a ratio to saliva protein content and is termed the &alpha;1PI Index. Results presented herein demonstrate that the &alpha;1PI Index provides an accurate and precise physiologic method for calculating CD4 counts.

The &#945;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

A CD4 enumeration method, the &#x03B1;-test, is described which uses whole saliva to provide rapid and accurate CD4 counts. The &alpha;-test costs pennies and eliminates the need for technical training, costly reagents such as monoclonal antibodies, instrumentation, refrigeration, transport of samples, as well as collection and handling of blood.

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time ...

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the arteries (&beta;2). When systemically administered, it increases cardiac demand. Thus, dobutamine unmasks abnormal rhythm or ischemic areas potentially at risk of infarction. 
Monitoring of heart function during a cardiac stress test can be performed by either ecocardiography or cardiac catheterization. The latter is an invasive but more accurate and informative technique that the former.
Cardiac stress test induced by dobutamine and monitored by cardiac catheterization accomplished as described here allows, in a single experiment, the measurement of the following hemodynamic parameters: heart rate (HR), systolic pressure, diastolic pressure, end-diastolic pressure, maximal positive pressure development (dP/dtmax) and maximal negative pressure development (dP/dtmin), at baseline conditions and under increasing doses of dobutamine.
As expected, in normal mice we observed a dobutamine dose-related increase in HR, dP/dtmax and dP/dtmin. Moreover, at the highest dose tested (12 ng/g/min) the cardiac decompensation of high fat diet-induced obese mice was unmasked.

We describe the protocol to perform a cardiac stress test induced by dobutamine and monitored by cardiac catheterization in normal mice. Also we show its application to unmask subclinical cardiac disease in high fat diet-induced obese mice.

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the ...

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

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.

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.

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 ...

Characterization of Metabolic Status in Nonhuman Primates with the Intravenous Glucose Tolerance Test

The intravenous glucose tolerance test (IVGTT) plays a key role in the characterization of glucose homeostasis. When taken together with serum biochemical profiles, inclusive of blood glucose levels in both the fed and fasted state, HbA1c, insulin levels, clinical history of diet, body composition, and body weight status, an assessment of normal and abnormal glycemic control can be made. Interpretation of an IVGTT is done through measurement of changes in glucose and insulin levels over time in relation to the dextrose challenge. Critical components to be considered are: peak glucose and insulin levels reached in relation to T0 (end of glucose infusion), the glucose clearance rate K derived from the slope of rapid glucose clearance in the first 20 min (T1 to T20), the time to return to glucose baseline, and the area under the curve (AUC). These IVGTT measures will show characteristic changes as glucose homeostasis moves from a healthy to a diseased metabolic state5. Herein we will describe the characterization of nonhuman primates (Rhesus and Cynomolgus macaques), which are the most relevant animal model of Type II diabetes (T2D) in humans and the IVGTT and clinical profiles of these animals from a lean healthy, to obese dysmetabolic, and T2D state 8, 10, 11.

The goal of this protocol is to present a standard method to perform intravenous glucose tolerance tests (IVGTTs) to assess glycemic control in nonhuman primates and assess their metabolic status from healthy to dysmetabolic.

The intravenous glucose tolerance test (IVGTT) plays a key role in the characterization of glucose homeostasis. When taken together with serum biochemical ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). Impaired uptake and metabolism of glucose in multiple organ systems due to insulin resistance is a hallmark of type 2 diabetes. The ability of this technique to extract an image-derived input function from the vena cava using an iterative deconvolution method eliminates the requirement of the collection of arterial blood samples. Fitting of tissue and vena cava time activity curves to a two-tissue, three compartment model permits the estimation of kinetic micro-parameters related to the 18F-FDG uptake from the plasma to the intracellular space, the rate of transport from intracellular space to plasma and the rate of 18F-FDG phosphorylation. This methodology allows for multiple measures of glucose uptake and metabolism kinetics in the context of longitudinal studies and also provides insights into the efficacy of therapeutic interventions.

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This study describes a protocol that uses 18F-FDG and positron emission tomography/computed tomography (PET/CT) imaging, together with kinetic modelling, to quantify the in vivo, real-time uptake of 18F-FDG into tissues.

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). ...

Improving Strength, Power, Muscle Aerobic Capacity, and Glucose Tolerance through Short-term Progressive Strength Training Among Elderly People

This protocol describes the simultaneous use of a broad span of methods to examine muscle aerobic capacity, glucose tolerance, strength, and power in elderly people performing short-term resistance training (RET). Supervised progressive resistance training for 1 h three times a week over 8 weeks was performed by RET participants (71&#177;1 years, range 65-80). Compared to a control group without training, the RET showed improvements on the measures used to indicate strength, power, glucose tolerance, and several parameters of muscle aerobic capacity. Strength training was performed in a gym with only robust fitness equipment. An isokinetic dynamometer for knee extensor strength permitted the measurement of concentric, eccentric, and static strength, which increased for the RET group (8-12% post- versus pre-test). The power (rate of force development, RFD) at the initial 0-30 ms also showed an increase for the RET group (52%). A glucose tolerance test with frequent blood glucose measurements showed improvements only for the RET group in terms of blood glucose values after 2 h (14%) and the area under the curve (21%). The blood lipid profile also improved (8%). From muscle biopsy samples prepared using histochemistry, the amount of fiber type IIa increased, and a trend towards a decrease in IIx in the RET group reflected a change to a more oxidative profile in terms of fiber composition. Western blot (to determine the protein content related to the signaling for muscle protein synthesis) showed a rise of 69% in both Akt and mTOR in the RET group; this also showed an increase in mitochondrial proteins for OXPHOS complex II and citrate synthase (both ~30%) and for complex IV (90%), in only the RET group. We demonstrate that this type of progressive resistance training offers various improvements (e.g., strength, power, aerobic capacity, glucose tolerance, and plasma lipid profile).

The effect of short-term resistance training on elderly people was investigated through the simultaneous use of several methods. Compared to a control group, many improvements were seen, including on muscle aerobic capacity, glucose tolerance, strength, power, and muscle quality (i.e., protein involved in cell signaling and muscle fiber type composition).

This protocol describes the simultaneous use of a broad span of methods to examine muscle aerobic capacity, glucose tolerance, strength, and power in ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

Study of In Vivo Glucose Metabolism in High-fat Diet-fed Mice Using Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT)

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