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).

Alle Verfahren mit Tieren wurden von der Northwestern University Institutional Animal Care und Use Committee (IACUC) zugelassen nach Richtlinien durchgeführt. 1. Mausmodelle <ol><li> Verwenden Sie 8-12 Wochen alten Mäusen in der Ausübung Ausbildung. Zur Erkennung ausgeübt induzierten Autophagie in vivo, verwenden Sie das GFP-LC3 - transgenen Mäusen (C57BL / 6 Hintergrund) für die Bildgebung Studien und C57BL / 6 - Mäuse für biochemische Analysen. </li></ol> 2. Übung induzierten Autophagie <ol><li> Laufband - Setup - gezwungen Übung <ol><li> Akklimatisieren und trainieren, um die Mäuse auf einem 10 ° bergauf offen Tretmühle für 2 Tage. Am Tag 1 üben die Mäuse für 5 min bei 8 m / min, und am Tag 2, üben die Mäuse für 5 min bei 8 m / min um weitere 5 min , gefolgt von 10 m / min 10. Ermutigen Sie die Mäuse durch sanfte Hand Schubs zu laufen. </li><li> Am 3. Tag, ließ die Mäuse einen einzigen Anfall von Laufen für 90 Minuten auf 10 ° bergauf Laufband, starti unterziehenng mit der Geschwindigkeit von 10 m / min. Nach 40 min bei einer Geschwindigkeit von 1 m / min alle 10 min für insgesamt 30 Minuten, um die Laufbandgeschwindigkeit zu erhöhen, und erhöhen Sie die Geschwindigkeit mit der Geschwindigkeit von 1 m / min alle 5 min bis 17 m / min erreicht, für insgesamt 90 min von Trainingszeit und 1.070 Meter Distanz. </li><li> Stellen Sie den elektrischen Reiz auf einem niedrigen Intensitätsbereich (1,2 Hz), und fördern die Mäuse zu laufen wiederholt Fuß-Schock zu vermeiden. </li><li> Für Gewebesammlung, einschläfern die Mäuse durch Isofluran Inhalation, durch Genickbruch folgte unmittelbar nach dem Training. Anderen zugelassenen Euthanasie Methoden können auch verwendet werden, wie beispielsweise CO2 oder IP-Injektion von anderen Anästhetika. Die Höhe der Autophagie scheint nicht durch Methoden der Euthanasie betroffen sein. </li></ol></li><li> Laufen Rad Setup - Kür <ol><li> Platzieren einer Maus-Laufrad (11,4 cm Durchmesser) in einem Käfig mit einem einzigen untergebracht Maus. </li><li> Überprüfen Sie die Lauffähigkeit ein Fahrrad Kilometerzähler verwenden.Richten Sie das Rad Parameter [Abstand (in mm) pro volle Umdrehung des Rades] bei 358 (11,4 * π). Messen Sie die Laufstrecke nach 24 Stunden. </li><li> Lassen Sie die Maus freiwillig für 2 Wochen mit dem Laufrad laufen. Für Gewebesammlung, opfern, um die Maus am Morgen nach der Laufzeit. </li></ol></li></ol> 3. Autophagy Flux Bewertung <ol><li> Um die autophagischen Fluss unter Ruhe- und Belastungs-Bedingungen behandeln Mäuse mit dem Autophagie Inhibitor Chloroquin für drei Tage messen und vergleichen sie mit Mäusen, die mit PBS behandelt. <ol><li> Auflösen Chloroquin in PBS (5 mg / ml) und Injizieren Chloroquin in Mäuse intraperitoneal in einer Dosis von 50 mg / kg / Tag für drei aufeinanderfolgende Tage. Sacrifice Mäuse und sammeln Gewebe 3 Stunden nach der letzten Injektion. </li><li> Bei ausgeübten Mäusen vorzubehandeln sie mit Chloroquin für zwei aufeinanderfolgende Tage in einer Dosis von 50 mg / kg / Tag. Am dritten Tag spritzen die Mäuse mit der gleichen Konzentration von Chloroquin und nach90 Minuten ließ sie für weitere 90 Minuten auf dem Laufband laufen. </li><li> Euthanize die Mäuse durch Inhalation von Isofluran, durch zervikale Dislokation gefolgt. Sammeln Gewebe unmittelbar nach der Ausführung, so daß die gesamte Inkubationszeit von Chloroquin die gleiche wie Kontrolle (Ruhe-) -Mäuse (3 h). HINWEIS: Alternativ kann eine einzelne Injektion von Chloroquin verwenden, um die Autophagie Fluss in Mausgeweben zu bestimmen. </li></ol></li></ol> 4. Gewebe Ernte und Fixierung <ol><li> Vor Gewebe Ernte vorbereiten phosphatgepufferter Salzlösung (PBS) und frischem 4% Paraformaldehyd (PFA, Vorsicht: Persönliche Schutzausrüstung erforderlich) in PBS bei einem endgültigen pH-Wert von 7,4. Bewahren Sie beide Lösungen bei 4 ° C. </li><li> Füllen 30 ml Spritze, die mit 15 bis 20 ml von 4% PFA (für GFP-LC3-Mäuse). Verbinden Sie die Spritze mit einer 20-G-Katheter Nadel. </li><li> Nach der Übung, einschläfern die Mäuse durch Isofluran Inhalation, durch Genickbruch folgte. </li><li> Legen Sie das Tier auf eine saubere Arbeitsfläche auf seiner back. Schneiden Sie und öffnen Sie die Brusthöhle durch das Zwerchfell schneiden das Herz aussetzen. </li><li> Machen Sie einen kleinen Schnitt an der Leber das Blut zu lassen kommen, und die Injektion sollte langsam in den rechten Ventrikel des Herzens über den Katheter insgesamt 15 ml 4% PFA, bis die Lunge und Leber völlig blaß. Perfundieren die Maus unmittelbar nach der Trainingseinheit, eine Spritzenpumpe mit einer konstanten Strömungsgeschwindigkeit von 90 ml / h verwendet wird. </li><li> Die Nadel nach der Perfusion, zu entfernen und Gewebe von Interesse (zB Gehirn 11 und Skelettmuskel) sammeln. Für Skelettmuskel, sezieren die vastus lateralis. Kurz gesagt, nach hinten in die Haut des Beins ziehen die Muskeln zu entlarven, die Quadrizeps femoris 12 zu lokalisieren, und vastus lateralis sezieren aus, die der externe Muskel mit dem oberen Teil des Oberschenkelknochens angebracht ist. </li><li> Für weitere Fixierung und Dehydrierung, legen Sie die Gewebe von GFP-LC3 Mäuse in 4% PFA für 24 Stunden bei 4 ° C, und sie dann auf 15% übertragenSucrose-PBS bei 4ºC über Nacht oder bis das Gewebe angesiedelt haben, und dann auf 30% Saccharose-PBS bei 4ºC über Nacht oder für längere Lagerung. Halten Sie die Proben im Dunkeln so viel wie möglich, da sie zu stark lichtempfindlich sein. </li><li> Für Western-Blot-Analyse lassen Sie die Gewebe von C57BL / 6-Mäuse direkt in flüssigem Stickstoff, und lagern Sie sie bei -80 ° C einfrieren. </li></ol> 5. Imaging Analyse von GFP-LC3 Puncta <ol><li> Tissue - Prozess <ol><li> Pre-Behandlung das Gewebe (vorher in PFA fixiert und in 30% Saccharose gespeichert) in Einbettungsmedium, wie beispielsweise &quot;Optimal Cutting Temperatur compound&quot; (OCT) für ein paar Minuten in einem markierten kleine Petrischale. </li><li> Übertragen Sie das Gewebe in einem markierten cryomold genügend frisches Oktober mit dem Gewebe zu decken. Richten Sie die Schnittfläche in Richtung der Unterseite des cryomold (Querschnitt für den Musculus vastus lateralis und Sagittalschnitt für den Hemi-Gehirn). Vermeiden Sie die Bildung von Blasen. </li><li> freeze die Proben durch die cryomold für ein paar Minuten in einem bedeckten Schaum-Kühler mit Trockeneis gefüllt platzieren, bis die OCT-weiß wird. Wickeln Sie einzelne Proben in markierten Folien, versiegeln sie in eine Plastiktüte, und lagern Sie sie bei -80 ° C über Nacht oder länger. </li><li> Verwenden Sie einen Kryostaten Probenabschnitte mit einer Dicke von 10 um, und montieren Sie die Abschnitte auf den Folien zu schneiden. Lagern Sie die Objektträger bei -20 ° C, und halten Sie immer die Dias weg vom Licht, wann immer möglich. </li></ol></li><li> Slide Vorbereitung <ol><li> Die Objektträger aus dem Gefrierschrank und tauen sie bei Raumtemperatur. Decken Sie den Gewebeschnitt mit einem Tropfen Eindeckmedium mit DAPI. Legen Sie ein Deckglas geeigneter Größe auf und Blasenbildung zu vermeiden. </li><li> Verwenden Sie Nagellack die Ränder des Deckglases zu versiegeln, damit Nagellack im Dunkeln bei Raumtemperatur trocknen, und dann speichern Sie die Folien in einem lichtdichten Behälter bei 4 ° C, oder gehen Sie mit Imaging-Erfassung von GFP-LC3 puncta. </li></ol></li><li> &lt;strong&gt; Die Quantifizierung von GFP - LC3 puncta durch Fluoreszenzmikroskopie <ol><li> Führen Sie Epifluoreszenzmikroskopie, Bilder im gleichen Zustand erfassen (Belichtung und Einstellungen) für alle Proben. Für GFP, verwenden Sie eine Emissionswellenlänge von 525 nm; für DAPI, verwenden 490 nm. mindestens zehn Bilder pro Probe Erwerben Sie ein 60X Ziel für vastus lateralis und den frontalen Kortex Region des Gehirns verwendet wird. </li><li> Quantifizierung die Anzahl der GFP-LC3 puncta in einem Gewebebereich von 2,500 &amp; mgr; m 2 in jedem Bild für die statistische Analyse, für die experimentellen Gruppen verblindet. Berechnen der durchschnittlichen Zahl von 10 Bilder von jeder Probe, wobei die automatische Messfunktion von &quot;Object Count&quot; verwendet wird. Einstellungen für den Musculus vastus lateralis sind: Schwellenwert L = 10, H = 200; 2X glätten; 1X sauber, und die Einstellungen für das Gehirn frontalen Kortex sind: Schwellenwert L = 11, H = 200; 2X glätten; 1X sauber. </li></ol></li></ol> 6. Western-Blot-Analyse auf Autophagy Marker <ol><li> &lt;strong&gt; Tissue Prozess <ol><li> Bereiten Sie die Lysepuffer: 50 mM Tris-HCl; 150 mM NaCl; 1 mM EDTA; 1% Triton X-100; Protease-Inhibitor und Phosphatase-Inhibitor (frisch hinzugefügt). </li><li> Entfernen des Gewebes von C57BL / 6-Mäuse von -80 ° C der Lagerung. Schneiden Sie das Muskelgewebe in kleine Stücke mit einer Rasierklinge vor dem folgenden Prozess. Hinzufügen, 800 &amp; mgr; l (für hemi-Hirn) oder 500 &amp; mgr; l (für vastus lateralis) kaltem Lysepuffer zur Probe. Homogenisieren der Probe bei 4 ° C mit einem Homogenisator bei einer mittleren Geschwindigkeit. </li><li> Vollständig lysieren die homogenisierte Mischung für weitere 30 min bis 1 h bei 4 ° C unter Rotation. </li><li> Spin down das Homogenat bei 12.000 × g für 10 min bei 4 ° C, verwerfen das Pellet, und sammeln Sie den Überstand in ein neues Röhrchen. </li></ol></li><li> autophagy Analyse <ol><li> Bestimmung der Proteinkonzentration von Gewebelysaten des BCA-Protein-Assay-Kit nach den Angaben des Herstellers. Normalisieren jede Probeauf die gleiche Konzentration. </li><li> Kombinieren Sie die Probe mit dem 2x Laemmli - Probenpuffer bei einem 1: 1 - Verhältnis, kochen sie bei 95 ° C für 5 - 10 min, und fahren Sie mit Western - Blot - Analyse auf Autophagie - Marker 13, 14, wie p62 (anti-p62 - Antikörper, 1: 500 Verdünnung) und LC3 (anti-LC3-Antikörper, 1: 500 Verdünnung). Koche die Proben unmittelbar nach der Zugabe des Probenpuffers, da längere Inkubation auf Eis mehrere abgebauten Banden LC3 erzeugen. </li><li> Quantifizieren p62 und LC3 Banden durch Densitometrieanalyse mit ImageJ und normalisieren den Wert auf den entsprechenden Aktin-Band. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Autophagie ist ein katabolischen Prozess, der Energie liefert, und reduziert die Zytotoxizität von lysosomalen Abbau von Cytoplasma-Bestandteile oder beschädigte Organellen. Autophagie Studium ist wichtig, dass die Regulation der zellulären Homöostase und die Mechanismen der Stressreaktion zu verstehen. Neue Modelle und Methoden entstehen 15, im Bereich der Forschung zu untersuchen , wie beeinträchtigte Autophagie zu zahlreichen pathologischen Prozessen beiträgt 16,</...

Autophagy ist ein evolutionär konservierten Abbauweg, die in Reaktion auf verschiedene Stressbedingungen, wie Hunger und Hypoxie 1, 2 induziert wird. Während der Autophagie, Doppelmembranbläschen, die so genannte Autophagosomen, integrieren unnötige oder subzellulären Komponenten beschädigt und transportieren sie in die Lysosomen zum Abbau 3. Basal autophagy ist wesentlich für die zelluläre Funktion und Entwicklung des Organismus und die Beeinträchtigung der basalen autophagy wird in vielen Störungen in Verbindung gebracht, einschließlich Neurodegeneration, Tumorgenese und Typ 2 Diabetes 4, 5, 6. Die bekannteste physiologischen Autophagie Induktor Hunger. Es hat jedoch zwei wesentliche Beschränkungen. Zuerst nimmt Hunger eine lange Zeit , um effektiv Autophagie bei Tieren, zum Beispiel 48 Stunden von Lebensmitteln Einschränkung bei Mäusen induzierenin den meisten Organen. Zweitens induziert Verhungern kaum Gehirn autophagy, aufgrund einer relativ stabilen Nährstoffversorgung im Gehirn. In der Tat ist es auch autophagy Induktion von kleinen Molekül-Induktoren zu detektieren schwierig, da viele Medikamente die Blut-Hirn-Schranke passieren können. So um besser auf die Funktion der Autophagie Aktivierung in der Pathogenese der Erkrankung zu analysieren, haben wir entdeckt , vor kurzem , dass die Übung ein potenter physiologische Methode ist die Autophagie in einem kurzen Zeitraum 7, 8, 9 zu induzieren. Verglichen mit Hunger wird Autophagie effektiv induziert durch Laufband so schnell wie 30 Minuten läuft. Somit Übung ist eine bequeme und potente physiologische Ansatz, den Mechanismus der Autophagie zu studieren Nutzen für die Gesundheit bei der Vermittlung und Prävention von Krankheiten. Es gibt mehrere Proteinmarker zum Nachweis von autophagy Aktivität, einschließlich LC3 und p62. LC3 (Mikrotubuli-assoziierten Protein 1A / 1B-Licht chain 3) eine cytosolische Protein (LC3-I bilden), die an PE (Phosphatidylethanolamin), auf autophagy Induktions konjugiert ist. PE-lipidierter LC3 (LC3-II-Form) auf autophagosomalen Membranen rekrutiert und kann verwendet werden, Autophagosomen sichtbar zu machen, wenn sie mit GFP-markierten. Seine Translokation aus dem Cytosol Strukturen Autophagosomen unter Mikroskopie punktiert ist ein Hinweis auf autophagy Induktion. p62 ist ein Fracht Rezeptor für autophagy Substrate (wie Ubiquitinierung Proteine), und wird in Autophagosomen als auch eingearbeitet. Da das Protein in Lysosomen mit Autophagosomen abgebaut wird, kann dessen Ebenen die autophagy Fluss zu messen, verwendet werden. Hier zeigen wir, wie diese Marker zu verwenden, die Autophagie in verschiedenen Geweben der Maus durch Aerobic-Übungen induziert zu quantifizieren, einschließlich Zwangs Bewegung (Laufband) und Kür (Laufräder). Die gleichen Verfahren können auch zur in - vivo - Messung von autophagy nach der Behandlung von anderen Induktoren angewendet werden.

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			<td style="width:204px;">Columbus Instruments</td>
			<td style="width:195px;">150-RM Exer 3/6</td>
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			<td height="21" style="height:21px;width:247px;">Mouse running wheel</td>
			<td style="width:204px;">Super Pet</td>
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			<td>diameter 11.4 cm</td>
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			<td height="42" style="height:42px;width:247px;">Fluorescence microscope</td>
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			<td style="width:195px;">Model: inverted microscope ECLIPSE</td>
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			<td height="21" style="height:21px;width:247px;">Cryostat</td>
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			<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>
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			<td height="42" style="height:42px;width:247px;">Chloroquine</td>
			<td style="width:204px;">CAYMAN CHEMICAL COMPANY</td>
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			<td height="21" style="height:21px;width:247px;">Parafolmaldehye</td>
			<td style="width:204px;">SIGMA-ALDRICH</td>
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			<td>Personal protection equipment required. This product may release formaldehyde gas, a chemical known to cause cancer</td>
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			<td style="width:204px;">Vector Laboratories</td>
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			<td height="21" style="height:21px;width:247px;">p62 antibody</td>
			<td style="width:204px;">BD Biosciences</td>
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			<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>
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			<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>
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			<td style="width:204px;">NIH</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.

Dieses Protokoll beschreibt zwei verschiedene Methoden Autophagie in Geweben der Maus durch Aerobic-Übungen zu induzieren: insgesamt 90 min von Zwangs Übung auf einer mehrspurigen Tretmühle von zwei Tagen der Eingewöhnung verlief; oder zwei Wochen nach der Kür auf einem Laufrad durch einzeln untergebracht Mäuse verwendet. In jeder Trainingsprotokoll, können wir die autophagy Fluss durch Fluoreszenzmikroskopie und Western Blot-Analyse in verschiedenen Organen zu messen. <p clas...

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

Activating Autophagy by Aerobic Exercise in Mice

Autophagy Aktivierung ist bei der Prävention von einer Reihe von Krankheiten nützlich. Eine der physiologischen Ansätze Autophagie in vivo zu induzieren körperliche Bewegung ist. Hier zeigen wir, wie die Autophagie von Aerobic-Übungen zu aktivieren und die Autophagie Ebenen bei Mäusen messen.

Die Aktivierung Autophagy von Aerobic-Übung bei Mäusen

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

activating-autophagy-by-aerobic-exercise-in-mice

Research

JoVE Journal

Biology

11.7K Aufrufe.  Northwestern University.  Autophagy Aktivierung ist bei der Prävention von einer Reihe von Krankheiten nützlich. Eine der physiologischen Ansätze Autophagie in vivo zu induzieren körperliche Bewegung ist. Hier zeigen wir, wie die Autophagie von Aerobic-Übungen zu aktivieren und die Autophagie Ebenen bei Mäusen messen. 

Serie: Activating Autophagy by Aerobic Exercise in Mice

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

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; agar-block). It is well-accepted that soil-block microcosms yield higher decay rates, fewer moisture issues, lower variability among studies, and higher thresholds of preservative toxicity. Soil-block testing is thus the more utilized technique and has been standardized by American Society for Testing and Materials (ASTM) (method D 1413-07). The soil-block design has drawbacks, however, using locally-variable soil sources and in limiting the control of nutrients external (exogenous) to the decaying tissues. These drawbacks have emerged as a problem in applying this method to other, increasingly popular research aims. These modern aims include degrading lignocellulosics for bioenergy research, testing bioremediation of co-metabolized toxics, evaluating oxidative mechanisms, and tracking translocated elements along hyphal networks. Soil-blocks do not lend enough control in these applications. A refined agar-block approach is necessary.
Here, we use the brown rot wood-degrading fungus Serpula lacrymans to degrade wood in agar-block microcosms, using deep Petri dishes with low-calcium agar. We test the role of exogenous gypsum on decay in a time-series, to demonstrate the utility and expected variability. Blocks from a single board rip (longitudinal cut) are conditioned, weighed, autoclaved, and introduced aseptically atop plastic mesh. Fungal inoculations are at each block face, with exogenous gypsum added at interfaces. Harvests are aseptic until the final destructive harvest. These microcosms are designed to avoid block contact with agar or Petri dish walls. Condensation is minimized during plate pours and during incubation. Finally, inoculum/gypsum/wood spacing is minimized but without allowing contact. These less technical aspects of agar-block design are also the most common causes of failure and the key source of variability among studies. Video publication is therefore useful in this case, and we demonstrate low-variability, high-quality results.

This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.

Agar-Block Mikrokosmen für Controlled Pflanzengewebekultur Zersetzung durch aerobe Pilze

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

Forschung

Biologie

Determining the Contribution of the Energy Systems During Exercise

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 physical activity. Although some sports are relatively easy to be reproduced in a laboratory (e.g., running and cycling), a number of sports are much more difficult to be reproduced and studied in controlled situations. This method presents how to assess the differential contribution of the energy systems in sports that are difficult to mimic in controlled laboratory conditions. The concepts shown here can be adapted to virtually any sport.
The following physiologic variables will be needed: rest oxygen consumption, exercise oxygen consumption, post-exercise oxygen consumption, rest plasma lactate concentration and post-exercise plasma peak lactate. To calculate the contribution of the aerobic metabolism, you will need the oxygen consumption at rest and during the exercise. By using the trapezoidal method, calculate the area under the curve of oxygen consumption during exercise, subtracting the area corresponding to the rest oxygen consumption. To calculate the contribution of the alactic anaerobic metabolism, the post-exercise oxygen consumption curve has to be adjusted to a mono or a bi-exponential model (chosen by the one that best fits). Then, use the terms of the fitted equation to calculate anaerobic alactic metabolism, as follows: ATP-CP metabolism = A1 (mL . s-1) x t1 (s). Finally, to calculate the contribution of the lactic anaerobic system, multiply peak plasma lactate by 3 and by the athlete&#x2019;s body mass (the result in mL is then converted to L and into kJ).
The method can be used for both continuous and intermittent exercise. This is a very interesting approach as it can be adapted to exercises and sports that are difficult to be mimicked in controlled environments. Also, this is the only available method capable of distinguishing the contribution of three different energy systems. Thus, the method allows the study of sports with great similarity to real situations, providing desirable ecological validity to the study.

This protocol allows researchers focused on exercise and sports sciences to determine the relative contribution of three different energy systems to the total energy expenditure during a large variety of exercises.

Bestimmung des Beitrages der Energy Systems während des Trainings

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

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

Transiente Expression von Proteinen durch Hydrodynamische Gentransfer in Mäusen

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

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

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

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In-vivo-4-Dimensional-Tracking von hämatopoetischen Stamm-und Vorläuferzellen im erwachsenen Maus Schädeldachknochenmark

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

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 possess several lines of defense against damage to biologically relevant molecules like nucleic acids, lipids and proteins. In addition to organelle dynamics (fusion/fission/motility/inheritance) and tightly controlled protease activity, the degradation of surplus, damaged or compromised organelles by autophagy (cellular &#8216;self-eating&#8217;) has received much attention from the scientific community. The regulation of autophagy is quite complex and depends on genetic and environmental factors, many of which have so far not been elucidated. Here a novel method is presented that allows the convenient study of autophagy in the filamentous fungus Penicillium chrysogenum. It is based on growth of the fungus on so-called &#8216;starvation pads&#8217; for stimulation of autophagy in a reproducible manner. Samples are directly assayed by microscopy and evaluated for autophagy induction / progress. The protocol presented here is not limited for use with P. chrysogenum and can be easily adapted for use in other filamentous fungi.

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

A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

Die Analyse der Autophagie in<em&gt; Penicillium chrysogenum</em&gt; Mithilfe von Starvation Pads in Kombination mit Fluoreszenzmikroskopie

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

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecal Glucocorticoid-Analyse: Nicht-invasive Monitoring-Adrenal in 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

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

Untersuchung von Protein-Protein-Interaktionen in der Autophagie-Forschung

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

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

In situ Immunfluoreszenz Färbung der Autophagie in Muskelstammzellen

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

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

Laktat-Dehydrogenase Sequestrierung Assay – Eine einfache und zuverlässige Methode zur Bestimmung Bulk Selbstverdauende Sequestration Aktivität in Säugerzellen

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

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional microscopy to surface nephron features, and 2-photon studies are limited in that interventions can only be assessed at the organ, rather than the nephron level. In particular, micropuncture studies of the glomeruli of mice have been challenged by the paucity of surface glomeruli in mice. To address this limitation in order to pursue studies of aspirate from Bowman&#39;s space in mouse physiologic models, we developed 2-photon glomerular micropuncture. We present a novel surgical preparation that allows lateral access to the kidney while preserving the required vertical imaging column for 2-photon microscopy. Administration of high molecular weight fluorescein isothiocyanate (FITC)-dextran is used to render the renal vasculature and therefore glomeruli visible for 2-photon imaging. A quantum dot-coated pipette is then introduced under stereotactic guidance to a glomerulus selected from the several to many which may be visualized within the imaging window. In this protocol, we provide details of the preparation, materials, and methods necessary to carry out the procedure. This technique facilitates previously-impossible physiologic study of the kidney, including recovery of filtrate from Bowman&#39;s space and all segments of the nephron within the imaging depth limit, about 100 &#181;m below the renal capsule. Pressure, charge and flow may all be measured using the introduced pipette. Here, we provide representative data from liquid chromatography/mass spectrometry performed on aspirate from Bowman&#39;s space. We expect this technique to have wide applicability in renal physiologic investigation.

We present use of 2-photon microscopy to place a micropipette within Bowman&#39;s urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology studies.

Mikropunktion Bowmans Platz bei Mäusen durch die 2-Photonen-Mikroskopie

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