Name: body composition and metabolic caging analysis in high fat fed mice
Uploaded: 2018-05-24T14:00:00
Description: Watch this Scientific Journal Video about Body Composition and Metabolic Caging Analysis in High Fat Fed Mice at JoVE.com

Ringraziamo il personale dalla ricerca medica Alfred e team formazione Precinct Animal Services (AS AMREP) per la loro assistenza e cura i topi utilizzati in questo studio e per il sostegno del regime di supporto dell'infrastruttura operativa dello stato vittoriano Governo.

Tutti gli esperimenti descritti sono stati approvati da Alfred medica ricerca formazione Precinct animale comitato etico (AMREP AEC) e topi sono stati forniti assistenza umana in linea con il National Health and Medical Research Consiglio (NHMRC) delle linee guida Australia su Sperimentazione animale. Gli animali sono stati somministrati loro prescritto dieta e acqua ad libitum e ospitato in un ambiente a temperatura controllata (~ 21-22 ° C) con una luce di 12 h e 12 h-scuro ciclo. Sette settimane vecchio topo...

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J., 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=Vagal+tone+dominates+autonomic+control+of+mouse+heart+rate+at+thermoneutrality.">Vagal tone dominates autonomic control of mouse heart rate at thermoneutrality.</a> Am J Physiol Heart Circ Physiol. 294 (4), H1581-H1588 (2008).</li><li>Tian, X. Y., 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=Thermoneutral+housing+accelerates+metabolic+inflammation+to+potentiate+atherosclerosis+but+not+insulin+resistance.">Thermoneutral housing accelerates metabolic inflammation to potentiate atherosclerosis but not insulin resistance.</a> Cell Metab. 23 (1), 165-178 (2016).</li><li>Giles, D. A., 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=Thermoneutral+housing+exacerbates+nonalcoholic+fatty+liver+disease+in+mice+and+allows+for+sex-independent+disease+modeling.">Thermoneutral housing exacerbates nonalcoholic fatty liver disease in mice and allows for sex-independent disease modeling.</a> Nat Med. 23 (7), 829-838 (2017).</li><li>Lee, M. W., 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=Activated+type+2+innate+lymphoid+cells+regulate+beige+fat+biogenesis.">Activated type 2 innate lymphoid cells regulate beige fat biogenesis.</a> Cell. 160 (1-2), 74-87 (2015).</li><li>Kusminski, C. 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=MitoNEET-driven+alterations+in+adipocyte+mitochondrial+activity+reveal+a+crucial+adaptive+process+that+preserves+insulin+sensitivity+in+obesity.">MitoNEET-driven alterations in adipocyte mitochondrial activity reveal a crucial adaptive process that preserves insulin sensitivity in obesity.</a> Nat Med. 18 (10), 1539-1549 (2012).</li><li>Judex, S., 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=Quantification+of+adiposity+in+small+rodents+using+micro-CT.">Quantification of adiposity in small rodents using micro-CT.</a> Methods. 50 (1), 14-19 (2010).</li><li>Chaurasia, B., 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=Adipocyte+ceramides+regulate+subcutaneous+adipose+browning,+inflammation,+and+metabolism.">Adipocyte ceramides regulate subcutaneous adipose browning, inflammation, and metabolism.</a> Cell Metab. 24 (6), 820-834 (2016).</li><li>Matthews, V. B., 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=Interleukin-6-deficient+mice+develop+hepatic+inflammation+and+systemic+insulin+resistance.">Interleukin-6-deficient mice develop hepatic inflammation and systemic insulin resistance.</a> Diabetologia. 53 (11), 2431-2441 (2010).</li><li>Tschop, M., Smiley, D. L., Heiman, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ghrelin+induces+adiposity+in+rodents.">Ghrelin induces adiposity in rodents.</a> Nature. 407 (6806), 908-913 (2000).</li><li>Garcia, M. 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=Mature-onset+obesity+in+interleukin-1+receptor+I+knockout+mice.">Mature-onset obesity in interleukin-1 receptor I knockout mice.</a> Diabetes. 55 (5), 1205-1213 (2006).</li><li>Kowalski, G. M., Bruce, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+glucose+metabolism:+Implications+and+considerations+for+the+assessment+of+glucose+homeostasis+in+rodents.">The regulation of glucose metabolism: Implications and considerations for the assessment of glucose homeostasis in rodents.</a> Am J Physiol Endocrinol Metab. 307 (10), E859-E871 (2014).</li><li>McGuinness, O. P., Ayala, J. E., Laughlin, M. R., Wasserman, D. 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Fasi critiche
I protocolli descritti nel presente documento forniscono un esempio di modi in cui composizione corporea misura e vari parametri metabolici nei topi utilizzando un analizzatore di composizione corporea e un animale metabolico, sistema di monitoraggio. Per entrambe le tecniche, è criticamente importante garantire che le macchine stanno lavorando in modo ottimale, e per fare questo, è imperativo che il ricercatore esegue un test di sistema per l'analizzatore di c...

Metabolismo è alla base di molti aspetti della normale cellulare, organo e fisiologia del corpo intero. Di conseguenza, nella cornice di varie patologie, alterazioni al metabolismo possono contribuire direttamente allo stato di fondo o potrebbero essere negativamente influenzate come un effetto collaterale della patologia. Tradizionalmente, studi in equilibrio energetico e metabolico sono stati concentrati sul campo dell'obesità e patologie correlate come l'insulino-resistenza, pre-diabete, intolleranza al glucosio, malattia cardiovascolare ed il diabete. Questa ricerca è autorizzata data la crescente prevalenza di tali condizioni in tutto il mondo e dell'individuo, della società, e costi economici queste condizioni infliggono. Come tale, lo sviluppo di strategie di prevenzione e nuove terapie per l'obesità di destinazione è un obiettivo nei laboratori di ricerca del mondo e modelli preclinici del topo sono fortemente invocati per questi studi.
Mentre la pesatura topi fornisce una valutazione affidabile di aumento di peso o perdita, non fornisce una ripartizione delle diverse componenti che compongono la pagina di composizione del corpo intero (massa grassa, massa magra, acqua gratuita così come altri componenti quali pelliccia e artigli). La pesatura delle adiposità al completamento degli studi, una volta che il mouse è deceduto fornisca una misura precisa dei diversi depositi di grasso, ma può solo fornire dati per un unico punto di tempo. Di conseguenza, spesso è necessario iscriversi più coorti per studiare lo sviluppo dell'obesità nel tempo, significativamente crescente numero di animali, tempo e costi. L'uso del dual-energy x-ray absorptiometry (DEXA) fornisce un approccio per valutare il contenuto del corpo del tessuto grasso e magro e consente al ricercatore di ottenere dati in modo longitudinale. Tuttavia, la procedura richiede topi anestetizzati1e ripetuti attacchi di anestesia possono influenzare l'accumulo di tessuto adiposo o impatto altri aspetti della regolazione metabolica. EchoMRI utilizza la risonanza magnetica nucleare rilassometria per misurare la massa grassa e magra, acqua gratuita e contenuto d'acqua totale. Ciò è ottenibile grazie alla creazione di contrasto tra i componenti differenti del tessuto, con differenze nella durata, ampiezza e distribuzione spaziale delle radiofrequenze generati permettendo la delineazione e la quantificazione di ogni tipo di tessuto. Questa tecnica è vantaggiosa in quanto è semplice, rapido e non invasivo, non richiede alcuna anestesia o la radiazione e, soprattutto, è stato convalidato positivamente contro analisi chimica2.
Una considerazione chiave di ricerca correlata e l'obesità è l'equazione di equilibrio di energia. Accumulo di grasso è più complicato che puramente energetica (assunzione di cibo) contro energia fuori (dispendio energetico), ma sono fattori vitali per essere in grado di misurare. Dispendio energetico quotidiano è il totale di quattro diversi componenti: (1) dispendio energetico basale (tasso metabolico a riposo); (2) il dispendio energetico a causa dell'effetto termico del consumo alimentare; (3) l'energia necessaria per la termoregolazione; e (4) l'energia spesa sull'attività fisica. Come dispendio energetico genera calore, misurare la produzione di calore da un animale (conosciuto come calorimetria diretta) può essere utilizzato per valutare il dispendio energetico. In alternativa, misurazione di ispirato e scaduto concentrazioni di O2 e CO2, permettendo per la determinazione del corpo intero O2 consumo e produzione di CO2 , può essere utilizzata come un modo per misurare indirettamente (indiretta Calorimetria a) produzione di calore e di conseguenza calcolare il dispendio energetico. Un aumento nell'assunzione di cibo o una diminuzione nel dispendio energetico possa predisporre tali topi ad aumento di peso e osservazioni dei cambiamenti in questi parametri possono fornire utili informazioni di probabili meccanismi d'azione su particolari modelli di obesità. Un parametro metabolico correlato di interesse è il rapporto di cambio delle vie respiratorie (RER), un indicatore della percentuale di substrato/carburante (cioè, carboidrati o grassi) che è in fase di metabolismo e viene utilizzata per produrre energia. Misurazione dell'ingestione di cibo (energia consumata) combinato con livelli di attività fisica, consumo2 O, RER e dispendio energetico è pertanto in grado di garantire un'ampia comprensione del profilo metabolico dell'organismo. Un metodo per raccogliere tali dati consiste nell'utilizzare un animale di laboratorio completa sistema di monitoraggio (vongole), che si basa sul metodo calorimetria indiretta per misurare il dispendio energetico e ha la capacità aggiunte di determinazione dei livelli di attività fisica (fascio pause) e l'assunzione di cibo tramite scale incorporato nella camera di misura.
In questo protocollo forniamo una descrizione semplice dell'uso di un analizzatore di composizione corporea per valutare la composizione corporea nei topi e un sistema di monitoraggio metabolico animale per misurare aspetti del metabolismo. Considerazioni e limitazioni per queste tecniche saranno discusse così come suggeriti metodi di analisi, interpretazione e rappresentazione dei dati.

<table border="0" cellpadding="0" cellspacing="0" width="1053">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">4 in 1 system</td>
			<td style="width:157px;">EchoMRI</td>
			<td style="width:275px;">4 in 1 system</td>
			<td style="width:371px;">Whole body composition analyser</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Canola oil test sample (COSTS)</td>
			<td>EchoMRI</td>
			<td style="width:275px;">Mouse-specific (contact company for cat number)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Animal specimen holder&#160;</td>
			<td>EchoMRI</td>
			<td style="width:275px;">103-E56100R</td>
			<td style="width:371px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Delimiter&#160;</td>
			<td>EchoMRI</td>
			<td style="width:275px;">600-E56100D</td>
			<td style="width:371px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12 chamber system</td>
			<td>Columbus Instruments</td>
			<td>Custom built</td>
			<td>Metabolic Caging System; includes control program</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drierite</td>
			<td>Fisher Scientific</td>
			<td>238988</td>
			<td>CLAMS consumable</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Calibration gas tank</td>
			<td>Air Liquide</td>
			<td>Mixed to order</td>
			<td>Gas calibration (0.5% CO2, 20.5% O2, balance nitrogen).&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal chow diet</td>
			<td>Specialty Feeds</td>
			<td>Irradiated mouse and rat diet</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High fat diet</td>
			<td>Specialty Feeds</td>
			<td>SF04-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Balance</td>
			<td>Mettler Toledo</td>
			<td>PL202-S</td>
			<td>Balance for weighing mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TexQ Disinfectant spray</td>
			<td>TexWipe</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrogen Peroxide cleaning solution</td>
			<td>TexWipe</td>
			<td>TX684</td>
			<td></td>
		</tr>
	</tbody>
</table>

body composition and metabolic caging analysis in high fat fed mice

Alterazioni di composizione corporea (massa grassa o magra), parametri metabolici quali consumo di ossigeno del corpo intero, dispendio energetico e utilizzazione del substrato e comportamenti come l'ingestione di cibo e attività fisica in grado di fornire informazioni importanti per quanto riguarda i meccanismi di fondo di malattia. Data l'importanza della composizione corporea e il metabolismo allo sviluppo di obesità e le sue conseguenze successive, è necessario effettuare misure accurate di questi parametri in ambito di ricerca pre-clinica. I progressi della tecnologia nel corso degli ultimi decenni hanno reso possibile derivare queste misure nei modelli del roditore in modo non invasivo e longitudinale. Di conseguenza, queste misure metaboliche hanno dimostrato utile nel valutare la risposta di manipolazioni genetiche (ad esempio transgenici o knockout mice, virale knock-down o sovraespressione di geni), lo screening droga sperimentale/mescola e dietetici, interventi di attività fisiche o comportamentali. Qui, descriviamo i protocolli utilizzati per misurare la composizione corporea e parametri metabolici utilizzando un animale monitoraggio sistema nei topi di dieta-federazione grassi cibo-federazione e alti.

Visto nella Figura 3 vengono visualizzati una tipica variazione parametri di composizione corporea all'alta alimentazione grassa, come misurato tramite EchoMRI. Al basale non c'era alcuna differenza in qualsiasi parametro misurato (Figura 3A-F). Tuttavia, dopo appena 1 settimana di alimentazione alto di grassi, c'era un aumento significativo del peso corporeo, massa grassa e massa grassa nel gruppo HFD (<strong c...

Watch this Scientific Journal Video about Body Composition and Metabolic Caging Analysis in High Fat Fed Mice at JoVE.com

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Analisi metabolica ingabbiamento in topi alta Fed grasso e della composizione corporea

Questo protocollo descrive l'uso di un analizzatore di composizione corporea e sistema di monitoraggio degli animali metabolica per caratterizzare la composizione corporea e parametri metabolici nei topi. Un modello di obesità indotta alimentando grassi viene utilizzato come esempio per l'applicazione di queste tecniche.

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

The goal if this procedure is to collect body composition and metabolic measurements from mice such as fat and lean mass percentages and energy expenditure. This methods can help answer important questions regarding mice metabolism such as, how fat are my mice? And why are my mice so fat?The main advantage with this techniques is that they can be completed in living conscious mice and therefore can be repeated numerous times through out a study. Prior to scanning mice perform a system test on the body composition analyzer machine once the machine has warmed up for a few hours. Use the standards called Canola Oil System Test Sample or COSTS.Open the system software then click the System Test toolbar button or by pressing alt+Y on the keyboard. Before starting the test, put the correct standard inside the gantry of the system. Then run the diagnostic test following the system prompts as needed.After the system has passed the test proceed with the diagnosis of a mouse. Now put the mouse in a holder. A red opaque holder is the least stressful.Position the holder at a 45 degree angle and insert the mouse. Then slowly and carefully raise the holder until it is vertical. Next insert a delimiter into the holder to limit the movement of the mouse.In some circumstances, with extremely active mice, it may be necessary to manually secure the delimiter. Now within the software select a folder to save the data to and create a filename. Next if necessary, reduce the amount of random noise in the fat and lean measurements by increasing the number of Primary Accumulations of the scan.Usually however, the default settings will give the necessary level of precision. If data for free water and total water aren't needed, then turn off the water stage by using the appropriate toggle. This will significantly speed up the scan.Now initiate the scan my selecting Start Scan or by pressing F5 on the keyboard. Enter all relevant data about the animal and press OK or F5 to commence the scan which will take about a minute. After collecting the data return the animal back to its cage.Always safely clean the acrylic holder between uses such as with warm soapy water. Later, once all of the animals have been scanned export the data for further analysis and collation. Before beginning, make sure the machine has been on for over two hours.In the Animal Monitoring System, open the control program and select the Oxymax utility option from the tool menu to initiate the pumps. Then check the desiccant and replace it with fresh dry material if needed. Also, check the condition of the ammonia trap and soda lime and replace it if needed.Next, connect the calibration gas tank to the system using a regulator and hose. Then open the valve and output five to 10 PSI of gas. Next in the software, select Calibration from the Tools menu and check that the sample and reference flows are at precisely 0.4 liters per minute.Then check that the zirconia oxygen sensor is heated to 725 degrees Celsius give or take one degree Celsius. Next make certain that the sample and reference drier and air pumps are all ON and double check that the calibration gas is attached and tuned on. Now proceed with the calibration and as needed slightly adjust the OFFSET control on the front of the zirconia oxygen sensor to achieve an oxygen ratio value within acceptable limits.After successfully calibrating the oxygen and carbon dioxide sensors, detach the gas standard and read the oxygen levels of atmospheric gas which should be about 20.92. If measuring food intake, consider using powdered mouse diet. Then load the food hopper by depressing the spring loaded platform.Next fill the water bottles and organize the food. Now weigh and inspect the health of the mice before proceeding to assemble the chambers. Next place the food hopper on the balance.Above it, place the chamber with the perforated platform. Then carefully place the mouse in the chamber and attach a lid using the front and back clips. Then fasten on the water bottle to complete the set-up.Now set up the next mouse and after all the mice are ready, double check the chamber lids, mice, and water. To run the experiment, select Experimental File Open from the menu. Then select the appropriate template.Next, under Setup, define the parameters of the experiment that should be recorded and deselect any chambers not in use. Then select the location for the experiment to be saved. If food intake is to be measured, tare the scales.Then start the capturing of data by selecting Run in the Experiment menu. The first 24 hours can be used as an acclamation phase and the next 24 plus hours, used for data analysis. At least twice a day, check on the animals and the data collection.Make certain each has access to food and water and look for signs of distress such as digging of the perforated flooring. In the software check the real time data collection. Go to the Metabolic tab to look at oxygen consumption, RER and energy expenditure.O2IN should read between 20.90 and 20.94 CO2IN should read approximately 0.04 RER should read between 0.7 and one the flow rate should be at a constant 0.5 to 0.6 liters per minute. Under the Activity tab, real time data on beam breaks is displayed. Under the Feeding tabs, real time data on food consumption is displayed.To finish the experiment, select Stop from the Experiment menu and export the results as CSV files for analysis. Then inspect the health of the mice, weigh them, and then return them to their home cages. In the following graphs, the black squares represent mice fed the high fat diet and the black circles represent the mice fed the standard chow diet.Changes in body composition parameters caused by high fat feeding were measured via EchoMRI using the described procedures. After one week on the high fat diet, there was a significant weight gain which increased by three weeks. The weight change could be clearly attributed to a change in fat mass.It was not due to a change in lean mass, free water content or total water content. In the following bar graphs, the white columns represent the chow fed mice and the black columns represent the high fat fed mice. V02 measurements when normalized to animal mass showed no changes after the three week period.Similarly energy expenditure measurements when normalized to animal mass showed no changes. Physical activity measured by beam breaks, varied by time of day but was the same for both groups. Food intake was not different when normalized to body mass.This amount is significantly more energy intake with the high fat diet. Once established as it is non invasive the body composition analysis can be conducted on a daily basis if necessary. While extended animal metabolic monitoring sessions may be considered more stressful to the mice due to the social isolation, these analysis can also be performed numerous times within a The implications of these techniques extend towards identifying therapeutic targets for obesity and metabolic disorders such as type two diabetes by the careful metabolic phenotyping of mice in regards to genetic manipulations or drug treatments.Ultimately not only can these methods provide insights for obesity but also for other conditions such as mass models of sarcopenia, cancer cachexia, muscular dystrophy or widi dystrophy.

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Medicine

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

Iperinsulinemico-euglicemico Morsetti in Cosciente, Mouse sfrenato

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

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Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Protocollo per lungo Ipertermia Corpo intera durata in Mouse

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

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.

Infusi carotide di farmacocinetica e di farmacodinamica Analisi dei taxani nei topi

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 ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

Dissezione del Pancreas del Mouse per l'analisi istologica e Profiling metabolico

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p &#62; 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology.

Analisi semi-automatica del Mouse del muscolo scheletrico morfologia e composizione di fibra-tipo

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of ...

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.

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.

Studio In Vivo del metabolismo del glucosio nei topi di dieta-federazione di grassi utilizzando Test di tolleranza al glucosio orale (OGTT) e Test di tolleranza dell'insulina (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 ...

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis (NASH) Resolution

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and assessment of improvement in disease endpoints, often reflected as a percent of each treatment arm that improved, worsened or remained unchanged. Traditional preclinical rodent studies for putative NASH therapies are often limited by not knowing the level of liver disease/NASH present at the start of therapeutic intervention, instead of randomizing treatment groups on easily measurable endpoints such as body weight, metabolic status or similar. Here, we describe a liver biopsy technique in a diet-induced NASH mouse model, for the assessment of baseline liver disease in order to exclude mice that do not exhibit fibrosis and to equally distribute animals with similar fibrosis between treatment groups. These levels can then be compared to the terminal, post-intervention levels for a truer understanding of in vivo pharmacological effects and thus more accurately reflect clinical trial design strategies. The mouse is properly anesthetized and prepared for the surgery using sterile conditions. A small incision is made in the upper abdomen and the left lateral lobe of the liver is exposed. A wedge of the liver is surgically removed, and a similar-sized piece of absorbable gelatin is put in its place to stop any bleeding. The mouse is surgically sutured and stapled closed and will recover back to normal within 1 day. The entire process takes 5-10 min per mouse. Here we exemplify the utility of this procedure by leveraging the pre-study biopsy to assess the impact of the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide on NASH endpoints in mice.

Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Non-alcoholic Steatohepatitis NASH Resolution

In clinical settings, a liver biopsy is used to assess stages of non-alcoholic steatohepatitis (steatosis, inflammation, hepatocyte ballooning, and fibrosis). Here, we illustrate a survivable liver biopsy in mice which can be used for histological analyses to enable assessment of therapeutic agents in a manner more aligned with clinical trials.

Incorporazione di una procedura di biopsia del fegato Survivable in topi, per valutare la risoluzione di steatoepatite Non alcolica (NASH)

Clinical trials assessing therapies for the treatment of non-alcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and ...

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

Analisi quantitativa della composizione cellulare nelle lesioni aterosclerotiche avanzate del muscolo liscio delle cellule Lineage-Tracing topi

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

Vinyl Chloride and High-Fat Diet as a Model of Environment and Obesity Interaction

Vinyl chloride (VC), an abundant environmental contaminant, causes steatohepatitis at high levels, but is considered safe at lower levels. Although several studies have investigated the role of VC as a direct hepatotoxicant, the concept that VC modifies sensitivity of the liver to other factors, such as nonalcoholic fatty liver disease (NAFLD) caused by high-fat diet (HFD) is novel. This protocol describes an exposure paradigm to evaluate the effects of chronic, low-level exposure to VC. Mice are acclimated to low-fat or high-fat diet one week prior to the beginning of the inhalation exposure and remain on these diets throughout the experiment. Mice are exposed to VC (sub-OSHA level: &#60;1 ppm) or room air in inhalation chambers for 6 hours/day, 5 days/week, for up to 12 weeks. Animals are monitored weekly for body weight gain and food consumption. This model of VC exposure causes no overt liver injury with VC inhalation alone. However, the combination of VC and HFD significantly enhances liver disease. A technical advantage of this co-exposure model is the whole-body exposure, without restraint. Moreover, the conditions more closely resemble a very common human situation of a combined exposure to VC with underlying nonalcoholic fatty liver disease and therefore support the novel hypothesis that VC is an environmental risk factor for the development of liver damage as a complication of obesity (i.e., NAFLD). This work challenges the paradigm that the current exposure limits of VC (occupational and environmental) are safe. The use of this model can shed new light and concern on the risks of VC exposure. This model of toxicant-induced liver injury can be used for other volatile organic compounds and to study other interactions that may impact the liver and other organ systems.

The goal of this protocol was to develop a murine model of low-level toxicant exposure that does not cause overt liver injury but rather exacerbates pre-existing liver damage. This paradigm better recapitulates human exposure and the subtle changes that occur upon exposure to toxicant concentrations that are considered safe.

Cloruro di vinile e dieta ad alto contenuto di grassi come modello di interazione tra ambiente e obesità

Vinyl chloride (VC), an abundant environmental contaminant, causes steatohepatitis at high levels, but is considered safe at lower levels. Although ...

Segmentation and Linear Measurement for Body Composition Analysis using Slice-O-Matic and Horos

Body composition is associated with risk of disease progression and treatment complications in a variety of conditions. Therefore, quantification of skeletal muscle mass and adipose tissues on Computed Tomography (CT) and/or Magnetic Resonance Imaging (MRI) may inform surgery risk evaluation and disease prognosis. This article describes two quantification methods originally described by Mourtzakis et al. and Avrutin et al.: tissue segmentation and linear measurement of skeletal muscle. Patients' cross-sectional image at the midpoint of the third lumbar vertebra was obtained for both measurements. For segmentation, the images were imported into Slice-O-Matic and colored for skeletal muscle, intramuscular adipose tissue, visceral adipose tissue, and subcutaneous adipose tissue. Then, surface areas of each tissue type were calculated using the tag surface area function. For linear measurements, the height and width of bilateral psoas and paraspinal muscles at the level of the third lumbar vertebra are measured and the calculation using these four values yield the estimated skeletal muscle mass. Segmentation analysis provides quantitative, comprehensive information about the patients' body composition, which can then be correlated with disease progression. However, the process is more time-consuming and requires specialized training. Linear measurements are an efficient and clinic-friendly tool for quick preoperative evaluation. However, linear measurements do not provide information on adipose tissue composition. Nonetheless, these methods have wide applications in a variety of diseases to predict surgical outcomes, risk of disease progression and inform treatment options for patients.

Segmentation and linear measurements quantify skeletal muscle mass and adipose tissues using Computed Tomography and/or Magnetic Resonance Imaging images. Here, we outline the use of Slice-O-Matic software and Horos image viewer for rapid and accurate analysis of body composition. These methods can provide important information for prognosis and risk stratification.

Segmentazione e misurazione lineare per l'analisi della composizione corporea mediante Slice-O-Matic e Horos

Body composition is associated with risk of disease progression and treatment complications in a variety of conditions. Therefore, quantification of ...