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).

hayvanları da içeren tüm işlemleri Northwestern University Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından onaylanan kurallara göre gerçekleştirilmiştir. 1. Fare Modelleri <ol><li> egzersiz 8-12 haftalık fareler kullanın. In vivo olarak, egzersiz kaynaklı Otofaji tespit etmek için, görüntüleme çalışmaları ve biyokimyasal analizler için C57BL / 6 fareleri için GFP LC3 transgenik fareler (C57BL / 6 plan) kullanın. </li></ol> 2. Egzersiz kaynak...

<ol>
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Otofaji enerji sağlar ve sitoplazma bileşenleri veya zarar görmüş organellerin lizozomal degradasyonu ile sitotoksisitesini azaltır katabolik işlemdir. Otofaji incelenmesi hücresel homeostazının düzenlenmesi ve stres yanıtı mekanizmalarını anlamak için önemlidir. Yeni modeller ve yöntemler çok sayıda patolojik süreçler 16, 17 nasıl katkı bozulmuş otofaji incelemek için, araştırma alanında 15 ortaya çıkıyor....

Otofaji örneğin açlık ve hipoksi, 1, 2 gibi çeşitli stres koşullarına karşılık olarak indüklenir evrimsel olarak korunmuş bir parçalanma yolu vardır. Otofaji sırasında, çift zar kabarcıklar, gereksiz veya hasarlı hücre içi bileşenleri birleştirmek ve bozulması 3 lizozomlar içine taşımak, autophagosomes çağırdı. Bazal Otofaji nörodejenerasyon, tümör oluşumu ve tip 2 diyabet 4, 5, 6 dahil olmak üzere birçok hastalıkta implike olduğu hücresel fonksiyon ve organizmanın gelişimi, ve bozulmuş bazal Otofaji için gereklidir. En iyi bilinen fizyolojik otofaji indükleyici açlık olduğunu. Bununla birlikte, iki önemli kısıtlamalara sahiptir. İlk olarak, açlık, örneğin, etkili hayvanlarda Otofaji uyarılması için farelerde besin yasaklaması 48 saat uzun sürdüğüEn organlarda. İkincisi, açlık zorlukla nedeniyle beyinde göreli olarak istikrarlı bir besin kaynağı, beyin Otofaji neden olur. Aslında, birçok ilaç, kan beyin bariyerini geçemez olarak, küçük moleküllü indüklecilerle otofaji indüksiyon tespit etmek de zordur. Bu nedenle, daha iyi bir hastalığın patogenezinde Otofaji aktivasyon fonksiyonunu analiz etmek için, en son egzersiz süresi 7, 8, 9, kısa bir süre içinde Otofaji uyarılması için daha güçlü bir fizyolojik bir yöntem olduğu keşfedilmiştir. açlık ile karşılaştırıldığında, otofaji etkili bir koşu bandı hızlı 30 olarak dakika çalıştırarak indüklenir. Böylece, egzersiz sağlık yararları aracılık ve hastalıkların önlenmesinde otofaji mekanizmasını incelemek için kullanışlı ve güçlü fizyolojik bir yaklaşımdır. LC3 ve P62 dahil olmak üzere, Otofaji aktivitesinin tespit edilmesi için, bir çok protein işaretleri vardır. LC3 (mikrotübül ilişkili protein 1A / 1B-ışık cHain 3) Bir hücre sıvısı protein (IS LC3 I Otofaji indüksiyondan sonra PE (fosfatidiletanolamin) konjüge edilmiş olduğu) oluşturur. PE-lipide LC3 (LC3 II formu) autophagosomal zarları üzerine işe ve GFP ile işaretlenerek autophagosomes görselleştirmek için kullanılabilmektedir. mikroskopta autophagosomes yapılarını noktalı yanına sitosolden translokasyonu Otofaji indüksiyon gösterir. P62 (örneğin, ubikitinasyon proteinleri gibi) Otofaji yüzeyler için bir kargo reseptördür ve de autophagosomes dahil edilir. Protein autophagosomes birlikte lizozomların parçalanır yana, seviyeleri otofaji akının ölçülmesi için kullanılabilir. Burada zorunlu egzersiz (koşu bandı) ve gönüllü egzersiz (tekerlekler çalıştıran) de dahil olmak üzere aerobik egzersiz, tarafından uyarılan farklı fare dokularda Otofaji ölçmek için bu işaretçileri nasıl kullanılacağını gösterir. Aynı prosedürler, diğer indükleyicilerin tedaviden sonra Otofaji in vivo ölçümü uygulanabilir.

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			<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>
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		<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>
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			<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>
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			<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>
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		<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>
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			<td height="21" style="height:21px;width:247px;">ImageJ</td>
			<td style="width:204px;">NIH</td>
			<td style="width:195px;"></td>
			<td></td>
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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.

Bu protokol aerobik egzersiz fare dokularda Otofaji ikna etmek için iki farklı yöntem açıklanır: aklimasyonundan iki gün ilerledi çok şeritli koşu bandı üzerinde zorunlu egzersiz 90 dakika olmak üzere toplam; veya tek ev sahipliği fareler tarafından kullanılan bir koşu tekerlek üzerinde gönüllü egzersiz iki hafta. Her seri protokol, çeşitli organlarda, flüoresans mikroskopisi ve Western blot analizi ile Otofaji akı ölçebilir. <p class="jove_content" fo:keep...

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

Activating Autophagy by Aerobic Exercise in Mice

Otofaji aktivasyonu çeşitli hastalıkların önlenmesinde yararlıdır. Fizyolojik yaklaşımlardan biri in vivo otofaji fiziksel egzersiz teşvik etmek. Burada aerobik egzersiz ile Otofaji etkinleştirmek ve farelerde otofaji düzeylerini ölçmek için nasıl gösterir.

Farelerde Aerobik Egzersiz ile Autophagy etkinleştirme

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

Aerobik Mantarlar Kontrollü Bitki Doku Ayrışma Agar-Blok Microcosms

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

Biyoloji

Determining the Contribution of the Energy Systems During Exercise

Egzersiz sırasında Enerji Sistemlerinin Katkısı Belirlenmesi

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

Farelerde Hidrodinamik Gen Taşıyıcı Proteinlerin Geçici İfadesi

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

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

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

Otofaji Analiz<em&gt; Penicillium chrysogenum</em&gt; Floresan Mikroskopi ile Kombinasyon Açlık Pedleri kullanarak

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

Fekal Glukokortikoid Analizi: Tek tırnaklılarda in Non-invaziv Adrenal İzleme

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

Protein-protein etkileşimlerinin Autophagy araştırma çalışma

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

Otofajinin Kas Hücrelerinde Yerinde İmmunofloresan Boyanması

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

Laktat dehidrogenaz haciz tahlil — Toplu Autophagic haciz etkinliğini memeli hücreleri belirlemek için basit ve güvenilir Yöntem

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 tarafından 2 foton mikroskobu kolaylaştırdı farelerde Bowman'ın alanı

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