Name: activating autophagy by aerobic exercise in mice
Uploaded: 2017-02-03T14:00:00
Description: Watch this Scientific Journal Video about Activating Autophagy by Aerobic Exercise in Mice at JoVE.com

We thank the Northwestern University Mouse Histology and Phenotyping Laboratoryfor technical support and assistance, and Noboru Mizushima (University of Tokyo) for providing GFP-LC3 transgenic mice. A. R. and C. H. were supported by the startup funds from Northwestern University and the grant from National Institutes of Health (DK094980).

Все процедуры с участием животных были проведены в соответствии с руководящими принципами, утвержденными Institutional уходу и использованию животных комитета Северо-западного университета (IACUC) путем. 1. Модели для мышей <ol><li> Используйте 8-12-недельных мышей в подготовке упражнений. Для...

<ol>
	<li>Mizushima, N., Yamamoto, A., Matsui, M., Yoshimori, T., Ohsumi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+analysis+of+autophagy+in+response+to+nutrient+starvation+using+transgenic+mice+expressing+a+fluorescent+autophagosome+marker.">In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker.</a> Mol Biol Cell. 15, 1101-1111 (2004).</li><li>Tracy, K., 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=BNIP3+is+an+RB/E2F+target+gene+required+for+hypoxia-induced+autophagy.">BNIP3 is an RB/E2F target gene required for hypoxia-induced autophagy.</a> Molecular and cellular biology. 27, 6229-6242 (2007).</li><li>Mizushima, N., Komatsu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy:+renovation+of+cells+and+tissues.">Autophagy: renovation of cells and tissues.</a> Cell. 147, 728-741 (2011).</li><li>Nikoletopoulou, V., Papandreou, M. E., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+the+physiology+and+pathology+of+the+central+nervous+system.">Autophagy in the physiology and pathology of the central nervous system.</a> Cell Death Differ. 22, 398-407 (2015).</li><li>Rocchi, A., He, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+roles+of+autophagy+in+metabolism+and+metabolic+disorders.">Emerging roles of autophagy in metabolism and metabolic disorders.</a> Frontiers in biology. 10, 154-164 (2015).</li><li>Mathew, R., Karantza-Wadsworth, V., White, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+autophagy+in+cancer.">Role of autophagy in cancer.</a> Nature reviews. Cancer. 7, 961-967 (2007).</li><li>He, 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=Exercise-induced+BCL2-regulated+autophagy+is+required+for+muscle+glucose+homeostasis.">Exercise-induced BCL2-regulated autophagy is required for muscle glucose homeostasis.</a> Nature. 481, 511-515 (2012).</li><li>He, C., Sumpter, R. J., Levine, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+induces+autophagy+in+peripheral+tissues+and+in+the+brain.">Exercise induces autophagy in peripheral tissues and in the brain.</a> Autophagy. 8, 4 (2012).</li><li>Kuramoto, K., 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=Autophagy+activation+by+novel+inducers+prevents+BECN2-mediated+drug+tolerance+to+cannabinoids.">Autophagy activation by novel inducers prevents BECN2-mediated drug tolerance to cannabinoids.</a> Autophagy. 12, 1460-1471 (2016).</li><li>Dougherty, J. P., Springer, D. A., Gershengorn, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Treadmill+Fatigue+Test:+A+Simple,+High-throughput+Assay+of+Fatigue-like+Behavior+for+the+Mouse.">The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse.</a> JoVE. , (2016).</li><li>Navone, S. E., 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=Isolation+and+expansion+of+human+and+mouse+brain+microvascular+endothelial+cells.">Isolation and expansion of human and mouse brain microvascular endothelial cells.</a> Nature protocols. 8, 1680-1693 (2013).</li><li>Liu, L., Cheung, T. H., Charville, G. W., Rando, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+skeletal+muscle+stem+cells+by+fluorescence-activated+cell+sorting.">Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting.</a> Nature protocols. 10, 1612-1624 (2015).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting:+sample+preparation+to+detection.">Western blotting: sample preparation to detection.</a> Journal of visualized experiments : JoVE. , (2010).</li><li>Bj&#248;rk&#248;y, G., 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=Chapter+12+Monitoring+Autophagic+Degradation+of+p62/SQSTM1.">Chapter 12 Monitoring Autophagic Degradation of p62/SQSTM1.</a> Methods Enzymol. 452, 181-197 (2009).</li><li>Klionsky, D. 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=Guidelines+for+the+use+and+interpretation+of+assays+for+monitoring+autophagy+(3rd+edition).">Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).</a> Autophagy. 12, 1-222 (2016).</li><li>Levine, B., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+the+Pathogenesis+of+Disease.">Autophagy in the Pathogenesis of Disease.</a> Cell. 132, 27-42 (2008).</li><li>Mizushima, N., Levine, B., Cuervo, A. M., Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+fights+disease+through+cellular+self-digestion.">Autophagy fights disease through cellular self-digestion.</a> Nature. 451, 1069-1075 (2008).</li><li>Fang, 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=Duration+of+rapamycin+treatment+has+differential+effects+on+metabolism+in+mice.">Duration of rapamycin treatment has differential effects on metabolism in mice.</a> Cell Metab. 17, 456-462 (2013).</li><li>Thomson, A. W., Turnquist, H. R., Raimondi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoregulatory+functions+of+mTOR+inhibition.">Immunoregulatory functions of mTOR inhibition.</a> Nature reviews. Immunology. 9, 324-337 (2009).</li><li>Miller, R. 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=Rapamycin-mediated+lifespan+increase+in+mice+is+dose+and+sex+dependent+and+metabolically+distinct+from+dietary+restriction.">Rapamycin-mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction.</a> Aging Cell. 13, 10 (2014).</li><li>Grumati, P., 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=Physical+exercise+stimulates+autophagy+in+normal+skeletal+muscles+but+is+detrimental+for+collagen+VI-deficient+muscles.">Physical exercise stimulates autophagy in normal skeletal muscles but is detrimental for collagen VI-deficient muscles.</a> Autophagy. 7, 1415-1423 (2011).</li><li>Lira, V. 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=Autophagy+is+required+for+exercise+training-induced+skeletal+muscle+adaptation+and+improvement+of+physical+performance.">Autophagy is required for exercise training-induced skeletal muscle adaptation and improvement of physical performance.</a> FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 27, 4184-4193 (2013).</li><li>Lo Verso, F., Carnio, S., Vainshtein, A., Sandri, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+is+not+required+to+sustain+exercise+and+PRKAA1/AMPK+activity+but+is+important+to+prevent+mitochondrial+damage+during+physical+activity.">Autophagy is not required to sustain exercise and PRKAA1/AMPK activity but is important to prevent mitochondrial damage during physical activity.</a> Autophagy. 10, 1883-1894 (2014).</li><li>Kregel, K. 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=Resource+book+for+the+design+of+animal+exercise+protocols.">Resource book for the design of animal exercise protocols.</a> American Physiological Society. , 152 (2006).</li><li>Lightfoot, J. T., Turner, M. J., Debated, K. S., Kleeberg, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstrain+variation+in+murine+aerobic+capacity.">Interstrain variation in murine aerobic capacity.</a> Med Sci Sports Exerc. 33, 5 (2001).</li><li>Rezende, E. L., Chappell, M. A., Gomes, F. R., Malisch, J. L., Garland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximal+metabolic+rates+during+voluntary+exercise,+forced+exercise,+and+cold+exposure+in+house+mice+selectively+bred+for+high+wheel-running.">Maximal metabolic rates during voluntary exercise, forced exercise, and cold exposure in house mice selectively bred for high wheel-running.</a> The Journal of experimental biology. 208, 2447-2458 (2005).</li><li>Kayatekin, B. M., Gonenc, S., Acikgoz, O., Uysal, N., Dayi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+sprint+exercise+on+oxidative+stress+in+skeletal+muscle+and+liver.">Effects of sprint exercise on oxidative stress in skeletal muscle and liver.</a> European journal of applied physiology. 87, 141-144 (2002).</li><li>Kawanaka, K., Tabata, I., Tanaka, A., Higuchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+high-intensity+intermittent+swimming+on+glucose+transport+in+rat+epitrochlearis+muscle.">Effects of high-intensity intermittent swimming on glucose transport in rat epitrochlearis muscle.</a> J Appl Physiol. 84, 4 (1998).</li><li>Fernando, P., Bonen, A., Hoffman-Goetz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+submaximal+oxygen+consumption+during+treadmill+running+in+mice.">Predicting submaximal oxygen consumption during treadmill running in mice.</a> Can J Physiol Pharmacol. 71, 4 (1993).</li></ol>

Аутофагия является катаболический процесс, который дает энергию и уменьшает цитотоксичность лизосомной деградации компонентов цитоплазмы или поврежденных органелл. Изучение аутофагию важно понимать регулирование клеточного гомеостаза и механизмов ответа на стресс. Новые модели и ...

Аутофагия является эволюционно консервативны путь деградации, который индуцируется в ответ на различные стрессовых условиях , таких как голод и гипоксии 1, 2. Во время аутофагии, двойные мембранные везикулы, называемые автофагосомы, включают ненужные или поврежденные субклеточных компонентов и транспортировать их в лизосомы для деградации 3. Базальный аутофагия имеет важное значение для клеточной функции и развития организма, а также ослабленным базальной аутофагии замешан во многих расстройств, в том числе нейродегенерации онкогенеза и диабета 2 -го типа 4, 5, 6. Наиболее известным физиологическим аутофагия индуктором является голодание. Тем не менее, он имеет два основных ограничения. Во- первых, голодание занимает длительный период , чтобы эффективно вызывать аутофагию у животных, например, 48 часов ограничения пищи у мышейв большинстве органов. Во-вторых, голодание едва индуцирует аутофагию мозга, из-за относительно стабильные поставки питательных веществ в головном мозге. На самом деле, это также трудно обнаружить индукцию аутофагии низкомолекулярных индукторов, поскольку многие лекарства не могут проходить через гематоэнцефалический барьер. Таким образом, чтобы лучше проанализировать функцию активации аутофагии в патогенезе заболевания, мы обнаружили , что в последнее время упражнения является более сильным физиологическим способом , чтобы вызвать автофагию в течение короткого периода времени , 7, 8, 9. По сравнению с голоданием, аутофагия эффективно индуцируется беговой дорожке так быстро, как 30 мин. Таким образом, физические упражнения является удобным и мощным физиологический подход к изучению механизма аутофагии в опосредовании пользы для здоровья и профилактики заболеваний. Есть несколько белковых маркеров для выявления аутофагии активности, в том числе LC3 и p62. LC3 (микротрубочки белок, ассоциированный с 1A / 1B-C светHain 3) является цитозольного белка (LC3-я форма), который конъюгирован с PE (фосфатидилэтаноламина) при индукции аутофагии. ПЭ-липоилироваться LC3 (LC3-II форма) набирается на autophagosomal мембран и могут быть использованы для визуализации автофагосомы когда метили GFP. Его транслокации из цитозоля в пунктате структуры автофагосомы под микроскопом является показателем индукции аутофагии. p62 является грузовой рецептор аутофагией субстратов (таких как Убиквитинирование белки), и включен в автофагосомы, а также. Так как белок разлагается в лизосомах вместе с автофагосомы, его уровни могут быть использованы для измерения потока аутофагии. Здесь мы покажем, как использовать эти маркеры для количественного определения аутофагию в различных тканях мышей, вызванных аэробных упражнений, включая принудительную упражнения (беговая дорожка) и добровольного упражнения (бег колес). Те же самые процедуры , также могут быть применены для измерения в естественных условиях аутофагии после лечения других индукторов.

<table border="0" cellpadding="0" cellspacing="0" width="863">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Treadmill</td>
			<td style="width:204px;">Columbus Instruments</td>
			<td style="width:195px;">150-RM Exer 3/6</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Mouse running wheel</td>
			<td style="width:204px;">Super Pet</td>
			<td style="width:195px;">100079365</td>
			<td>diameter 11.4 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Odometer</td>
			<td style="width:204px;">Bell</td>
			<td style="width:195px;">DASHBOARD 100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Syringe pump</td>
			<td style="width:204px;">KD Scientific</td>
			<td style="width:195px;">KDS100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Fluorescence microscope</td>
			<td style="width:204px;">Nikon</td>
			<td style="width:195px;">Model: inverted microscope ECLIPSE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Cryostat</td>
			<td style="width:204px;">Leica</td>
			<td style="width:195px;">CM 1850UV</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Homogenizer</td>
			<td style="width:204px;">IKA</td>
			<td style="width:195px;">003737001 / Model: T10 Basic S1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Chloroquine</td>
			<td style="width:204px;">CAYMAN CHEMICAL COMPANY</td>
			<td style="width:195px;">14194</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Parafolmaldehye</td>
			<td style="width:204px;">SIGMA-ALDRICH</td>
			<td style="width:195px;">P6148</td>
			<td>Personal protection equipment required. This product may release formaldehyde gas, a chemical known to cause cancer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Mounting media</td>
			<td style="width:204px;">Vector Laboratories</td>
			<td style="width:195px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">p62 antibody</td>
			<td style="width:204px;">BD Biosciences</td>
			<td style="width:195px;">610833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">LC3 antibody</td>
			<td style="width:204px;">Novus Biologicals</td>
			<td style="width:195px;">NB100-2220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2X Laemmli Sample Buffer</td>
			<td style="width:204px;">Bio-Rad Laboratories</td>
			<td style="width:195px;">161-0737</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">ImageJ</td>
			<td style="width:204px;">NIH</td>
			<td style="width:195px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

activating autophagy by aerobic exercise in mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

Этот протокол описывает два различных метода для индукции аутофагии в тканях мыши с помощью аэробных упражнений: в общей сложности 90 мин принудительного упражнений на многополосной беговой дорожке в протекала на два дня акклиматизации; или две недели добровольного ?...

Watch this Scientific Journal Video about Activating Autophagy by Aerobic Exercise in Mice at JoVE.com

Activating Autophagy by Aerobic Exercise in Mice

активация аутофагии является полезным в профилактике целого ряда заболеваний. Одним из физиологических подходов к индукции аутофагии в естественных условиях является физические упражнения. Здесь мы покажем, как активировать аутофагию с помощью аэробных упражнений и измерения уровней аутофагии у мышей.

Активация аутофагии аэробикой у мышей

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

The overall goal of this experiment is to physiologically activate autophagy in mice by forced or voluntary running, and then measure the levels of autophagy in specific tissues. This method can help answer key questions about autophagy, in particular, how autophagy and action contributes to the beneficial effects mediated by aerobic exercise. The main advantage of this technique is that physical exercise is a fast aerobic sway between autophagy in vivo using mice.For this experiment, start with eight to twelve week old C57 black six mice, harboring a trans gene to detect exercise induced autophagy in vivo, known as GFP-LC3. For details on the use of mice treadmills, see the following JoVE publication. Set the electrical stimulus of the treadmill to low intensity.On their first two sessions, acclimate the mice to a ten degree uphill open treadmill. On the first day, exercise the mice for five minutes with the treadmill running at eight meters per minute. On the second day, run the mice for ten minutes, initially at eight meters per minute, and after five minutes, ramp up the speed to ten meters per minute.On the third day, run the mice for a full 90 minutes. Start the treadmill at ten meters per minute and encourage the mice to run using gentle nudges to avoid repeated foot shocks. It is critical to observe and manipulate the mice so they don't receive too many electric shocks during the 90 minute run.Throughout the run, use your fingers to encourage the mice to stay on the treadmill. After 40 minutes, 6increase the speed by one meter per minute. After 50 minutes, increase the speed by one meter per minute again.After 60 minutes, increase the speed by another meter per minute. After 70 minutes, start increasing the speed every five minutes so that the last five minutes of the 90 minute run are 17 meters per minute. After the end of the trial, immediately euthanize the mice for tissue collection if desired.For this test, house the mice individually. Use the same strain as previously described. Prepare an 11.4 centimeter diameter mouse running wheel with an attached bike odometer.Each rotation should measure 358 millimeters of travel. Move the mouse into the cage containing the wheel and let the mouse use it freely for two weeks. Every 24 hours, record the distance run by the mouse from the odometer.After the two weeks, harvest tissues for analysis as needed. To measure the autophagic flux, treat the mice with the autophagy inhibitor, chloroquine, for three days. Dissolve the chloroquine in PBS at five milligrams per milliliter and inject it into mice intraperitoneally at the dose of 50 micrograms per gram.Give the mice one injection daily for three consecutive days and harvest tissues three hours after the last injection. For the treadmill exercised mice, start the 90 minute run 90 minutes after the injection on the third day so the run ends three hours after the injection. Then euthanize the mice and immediately harvest their tissues.Prepare to profuse an animal within 90 minutes of being exercised by preparing the fixative. Load a 30 milliliter syringe with 15 to 20 milliliters of freshly made four percent paraformaldehyde and connect the syringe with a 20 gauge catheter needle. Attach the loaded syringe pump and set it to 90 milliliters per hour of continuous flow.With the animal on a clean work surface, in a supine position, cut open the thoracic cavity through the diaphragm to expose the heart. Now, insert the catheter needle into the right ventricle. Next, make a small incision on the liver to drain the blood and start the pump.Inject the fixative until the lung and liver turn completely pale. Usually, 15 milliliters of fixative will suffice. After the profusion, remove the needle and collect tissues of interest.To collect skeletal muscle, dissect the vastus lateralis. Briefly pull the skin of the leg backwards to expose the muscles and localize the quadriceps femoris. Then, dissect out the vastus lateralis, which is the external muscle attached to the upper portion of the femoral bone.For further fixation and dehydration, place the harvested tissues in four percent paraformaldehyde for 24 hours at four degrees celsius. The next day, transfer the tissues to 15 percent sucrose PBS and let them soak at four degrees celsius overnight or until they have settled in the solution. Then, transfer the tissues to 30 percent sucrose PBS at four degrees celsius for overnight or longer.Keep the tissues in the dark as much as possible during these baths. Next, place the tissues in an embedding medium such as OCT and cut them into smaller pieces if necessary. Then, allow them to acclimate for a few minutes in a small petri dish.Afterwards, transfer them to cryomolds with enough OCT to cover. In the molds, orient the sectioning surfaces toward the bottom and avoid forming bubbles. Then, freeze the samples in a covered foam cooler filled with dry ice until the OCT turns white.Now, wrap individual samples in labeled foils, seal them in a plastic bag and store them at negative 80 degrees celsius until they can be processed. Using GFP tagged LC3 as a reporter system, autophagy induction was observed in mice exercised as described. After autophagy stimulation, LC3 translocated from the cytosol to the autophagosome in punctuate structures.Exercised mice had many more GFP-LC3 puncta in both skeletal muscle and the cerebral cortex compared to resting condition mice. Western blot analysis using an LC3 antibody showed that exercise significantly induced the generation of lipid conjugative LC3-II, suggesting that there was increased conversion of cytosolic LC3-I into LC3-II. Next, the degradation of the autophagic cargo receptor, p62, was measured.90 minutes of treadmill activity caused a higher degradation of p62 in skeletal muscle than the resting condition. This effect was rescued by an injection with chloroquine prior to exercise. As chloroquine is an inhibitor of lysosomal degradation, these data indicate that the observed phenomena is due to an elevated autophogagic flux rather than a block in autophagosome degradation.After its development, this technique had precursors in the field of autophagy to explore the effects on the mechanism of exercise in the degradation of metabolism and animal behaviors. While attempting this procedure, it's important to monitor the mice while they are running. Following this procedure, are the methods like electric microscopy.Animal microscopy can be performed in order to answer additional questions like exercise in select autophagy pathways, such as mitophagy and lipophagy.

Research

JoVE Journal

Biology

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

Агар-Block Микрокосмы для управляемого разложения тканей завод по Аэробные Грибы

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

Determining the Contribution of the Energy Systems During Exercise

Определение вклада Energy Systems во время тренировки

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Кратковременная экспрессия белков гидродинамическими доставки генов у мышей

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

В Vivo 4-мерном Tracking гемопоэтических стволовых и клеток-предшественников в взрослой мыши свода черепа костного мозга

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

Анализ аутофагии в<em&gt; Пеницилл золотистый</em&gt; С помощью голодания колодки в сочетании с флуоресцентной микроскопии

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Фекальные глюкокортикоиды Анализ: безоперационная Надпочечная Мониторинг в Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Study of Protein-protein Interactions in Autophagy Research

Исследование взаимодействий протеин протеина в Autophagy исследований

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

In Situ Иммунофлуоресцентное окрашивание аутофагии в мышечных клетках

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Лактатдегидрогеназа Assay поглощение — Простой и надежный метод для определения активности сыпучих Autophagic поглощения в клетках млекопитающих

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy

Micropuncture боуменова пространства в мышей, облегчается 2 фотона микроскопия

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional ...