Name: an anoxia-starvation model for ischemia/reperfusion in c. elegans
Uploaded: 2014-03-11T18:00:00
Duration: 9 min 24 s
Description: Watch this Scientific Journal Video about An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans at JoVE.com

Figures 1 and 2 were previously published in Free Radical Biology &#38; Medicine (Queliconi&#160;et al.5) and have copyright held by Elsevier. This work was supported by the Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de S&#227;o Paulo (FAPESP), the Instituto Nacional de Ci&#234;ncia e Tecnologia de Processos Redox em Biomedicina, the N&#250;cleo de Apoio &#224; Pesquisa de Processos Redox em Biomedicina, USPHS NS064945 (K.N.), and USPHS GM087483 (K.N.). B.B.Q. is a doctoral student supported by a FAPESP fellowship.

1. C. elegans&#160;Growth
<ol>
	<li>Prepare 35&#160;mm Nematode Growth Media (NGM) agar plates seeded with OP50 bacteria, as per standard cultivation methods1.</li>
	<li>Synchronize C. elegans by placing 6 gravid adults onto a seeded NGM plate. Remove the adults after ~100 eggs have been laid (~3 hr).</li>
	<li>Incubate the plates for 3 days at 20 &#176;C, at which point the worms will have developed to young adult stage and are optimal for this experiment. Alternative protocols to synchronize C. elegans are described elsewhere10.</li>
</ol>
2. Materials for AS
<ol>
	<li>M9 buffer (22 mM KH2PO4, 42 mM Na2HPO4, 86 mM NaCl, 1 mM MgSO4) should be purged of oxygen by equilibrating with N2 (argon can also be used) during 30 min prior to the experiment and kept surrounded by ice.</li>
	<li>Make a small hole (3&#160;mm wide) in the lid of 1.5&#160;ml&#160;microcentrifuge tubes in which the worms will be placed during AS. The hole allows for gas exchange without keeping the lid open, as this can lead to excessive media evaporation.</li>
</ol>
3. Anoxia/Starvation
<ol>
	<li>Conduct all experiments at least in triplicate.</li>
	<li>Add 1 ml&#160;of RT, oxygenated M9 buffer to each 35&#160;mm plate containing young adult worms grown as described above. Be careful to avoid removing bacteria from the plate (add M9 to the edges where bacteria weren&#39;t seeded). After ~1 min the worms should be swimming in the M9.</li>
	<li>Coat a 1&#160;ml&#160;pipette tip with BSA by pipetting and expelling a sterile 1% BSA solution.</li>
	<li>Carefully incline the plate containing the worms in M9 buffer, remove the M9 from the same place where it was added, and place the suspension in the prepared microcentrifuge tubes. Let rest in ice until the worms have dropped to the bottom of the tubes (~1-2 min).</li>
	<li>Remove the maximum amount of M9 possible without disturbing the worms from the tube (leaving around 100 &#956;l), add 1 ml&#160;of the oxygen-purged and iced M9 and place the tube over ice until the worms settle to the bottom again. Repeat the last step 3x. In the last wash, remove the M9 until ~100 &#956;l&#160;remains in the tube.</li>
	<li>Place the tubes with the worms in deoxygenated M9 inside an anoxic/hypoxic chamber (see instructions below, Section&#160;3.6.1) or, alternatively, into a custom sealed container (see instructions below, Section 3.6.2).
	<ol>
		<li>If a commercial anoxic/hypoxic chamber is to be used, leave some purged M9 inside the chamber before starting step 3.5 and repeat step 3.5 once the worms are inside the chamber.</li>
		<li>If a custom chamber is to be used, a detailed description of how to create a low-cost apparatus is presented at the end of this protocol and has been described by another group previously11. Briefly:
		<ol>
			<li>Make two holes in a 250&#160;ml&#160;Tupperware-type container lid and add tube connectors to them. One will be used to inject the gas mixture while the other will be the gas exit into a water bath.</li>
			<li>Place the worms into the chamber and seal the chamber using the lid.</li>
			<li>Gas with a constant N2 flux (100 ml/min).</li>
			<li>Place the container inside a water bath at 26 &#176;C. Ensure that the exit tube is well immersed in water so no air returns and that there are no leaks in the system (apparent by bubbles arising from the chamber itself).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Incubate the worms at 26 &#176;C for 20 hr.</li>
</ol>
4. Simulated Reperfusion
<ol>
	<li>After 20 hr under AS conditions, remove the tubes from the incubation chamber into room air.</li>
	<li>Pipette C. elegans with a BSA-coated tip (as in step 3.3) onto an OP50 seeded 35&#160;mm NGM plate. Incubate the plates at 20 &#176;C for 24 hr. After this, the worms will be ready to be scored.</li>
</ol>
5. Identifying Dead Versus Live Worms
<ol>
	<li>Using a platinum pick, lightly touch the top of the head of the worm. Live worms will move backwards after being touched. If the worms are nonresponsive, score as dead. Occasionally, a worm will not move backwards, but may exhibit a slight side-to-side head movement. Although not technically dead, these worms are almost certain to expire within hours, and should be scored as dead. If one waits too long to score the worms, internal hatching of progeny can occur and these &#8220;bags-of-worms&#8221; can rupture, making detecting the carcasses very difficult.</li>
	<li>Remove both live and dead worms from the plate as they are counted to avoid duplicate counts. The live worms can be moved to a separate plate to perform behavioral assays. The amount of living worms relative to the total is represented as the % of surviving worms (Figure&#160;1A).</li>
</ol>
6. Touch Response Assay
<ol>
	<li>A detailed description of this protocol has been presented in Hart, Anne C., ed. Behavior (July 3, 2006), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.87.1, http://www.wormbook.org.
	<ol>
		<li>To score for touch response, identify worms that are moving and lightly touch the side of the worm&#39;s head (near the middle part of the pharynx) with an eyelash pick (Figure&#160;3).</li>
		<li>If the worm moves backward, score as responsive, if not, score as nonresponsive (Figure&#160;1B). Repeat this step 10-15x for each worm with 10 sec intervals. Probe at least 10 worms in each group to have accurate data. The same assay can be used to assess forward locomotory changes following light touch to the posterior body wall. Be sure not to touch the head or tail of the worm, since this stimulates a distinct behavior.</li>
		<li>Finally, incorporate genetic control strains [N2 Bristol wild-type for positive, mec (mechanosensory) mutant for negative -&#160;e.g. CB1338 mec-3 (e1338)IV] to calibrate the force with which to touch the worms. Too harsh of a stimulation will elicit a behavioral response in mec mutants, too little will not elicit an effect in wildtype N2 controls.</li>
	</ol>
	</li>
</ol>
7. Neuronal Modifications
<ol>
	<li>To visualize post AS neuronal modifications, use a strain which expresses GFP in the touch-responding mec neurons4,5. These neurons have long processes that run along the body wall and are easily scored for morphological abnormalities. In addition, the morphologic information can be integrated with behavioral measures of neuronal function, as described above. One strain that can be used for this purpose is TU2583, which is available from the C. elegans Genetics Center and contains the uIs25 integrated transgene expressing GFP from the mec-18 promoter, or alternatively TU2562, which contains an integrated mec-3::GFP fusion.</li>
	<li>Prepare an agarose pad.
	<ol>
		<li>Melt 2% agarose in water, bring to 1x M9 with a 10x stock, and maintain the solution at 70 &#176;C.</li>
		<li>Place a drop (~20 &#956;l) of this solution on a glass slide (25 mm x 75 mm) that has been placed between two other slides, each of which have a single piece of tape on their bottom side.</li>
		<li>Place a final glass slide perpendicular to the first on top of the 2% agarose solution, and let it cool at RT. This should form a pad that is the thickness of the tape.</li>
		<li>Remove the top slide and add 10 &#956;l&#160;of M9 containing 0.1% tetramisole, which will immobilize the worms.</li>
	</ol>
	</li>
	<li>Move some (~10) live worms onto the 2% agarose slide. Place a coverslip over them and visualize GFP using a fluorescence microscope under a 100X&#160;objective with the appropriate illumination.</li>
	<li>Damaged neurons will show a cytosolic membrane string of pearls pattern in the processes, comprised of multiple punctum (Figure&#160;2A), and/or abnormalities where the processes appear to be broken (Figure&#160;2B). Count punctum and abnormalities in both neurons for each worm, using a total of at least 10 worms.</li>
</ol>
8. Lab-made Hypoxic Chamber
<ol>
	<li>A Tupperware-style, sealable, airtight container can be retrofit as an anoxic chamber. Take care to select a container that is as small as feasible. A small container creates a hypoxic environment faster and is easier to fit in the water bath (Figure&#160;4).</li>
	<li>Make two holes on the lid (can be done with a screwdriver or by heating forceps and forcing into the lid) and insert a plastic or metal connector that fits the tubing (Figure&#160;5A). Use glue that can be placed under water, and also gets hard after drying, avoiding movement of the connector and hence reducing the possibility of leakage (Figure&#160;5B).</li>
	<li>The container should be placed into the water bath so that it is submerged completely (Figure&#160;4). To immerse the container, use a bottle weight or a diving weight.</li>
	<li>One of the tube connections will be used to introduce a gas mixture and the other tube will act as a pressure relief, hence it should be left under the water so that the container is sealed off from the external environment (Figure&#160;4).</li>
	<li>The air composition can be modulated by using custom made gas, mass flow controllers or any apparatus that mixes the desired gases. The gas flow should be controlled, as high flow can create excessive evaporation. For example, in a 250 ml&#160;container, 100 ml/min of gas will provide good space for the worms and a suitable flux.</li>
	<li>The length of the tubing should be kept to a minimum, as they can be permeable to gas, allowing the entrance of O2. It is important to avoid using silicone, Tygon tubes as these materials are permeable to gas exchange. We prefer polypropylene or nylon tubes. Glass or metal tubing maintain the gasses well, but are also harder to work with.</li>
</ol>

<ol>
	<li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77 (1), 71-94 (1974).</li><li>Maine, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+gene+function+in+Caenorhabditis+elegans+using+RNA-mediated+interference.">Studying gene function in Caenorhabditis elegans using RNA-mediated interference.</a> Briefings Funct. Genom. Proteom. 7 (3), 184-194 (2008).</li><li>Vanden Hoek, ., L, T., Shao, Z., Li, C., Zak, R., Schumacker, P. T., Becker, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reperfusion+injury+on+cardiac+myocytes+after+simulated+ischemia.">Reperfusion injury on cardiac myocytes after simulated ischemia.</a> Am. J. Physiol. 270 (4), 1334-1341 (1996).</li><li>Ba Scott, ., Avidan, M. S., Crowder, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+hypoxic+death+in+C.+elegans+by+the+insulin/IGF+receptor+homolog+DAF-2.">Regulation of hypoxic death in C. elegans by the insulin/IGF receptor homolog DAF-2.</a> Science. 296 (5577), 2388-2391 (2002).</li><li>Queliconi, B. B., Marazzi, T. B. 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=Bicarbonate+modulates+oxidative+and+functional+damage+in+ischemia-reperfusion.">Bicarbonate modulates oxidative and functional damage in ischemia-reperfusion.</a> Free Rad. Biol. Med. 55, 46-53 (2013).</li><li>Wojtovich, A. P., DiStefano, P., Sherman, T., Brookes, P. S., Nehrke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+ATP-sensitive+potassium+channel+activity+and+hypoxic+preconditioning+are+independent+of+an+inwardly+rectifying+potassium+channel+subunit+in+Caenorhabditis+elegans.">Mitochondrial ATP-sensitive potassium channel activity and hypoxic preconditioning are independent of an inwardly rectifying potassium channel subunit in Caenorhabditis elegans.</a> FEBS Lett. 586 (4), 428-434 (2012).</li><li>Dasgupta, N., Patel, A. M., Scott, B. A., Crowder, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxic+Preconditioning+Requires+the+Apoptosis+Protein+CED-4+in+C.+elegans.">Hypoxic Preconditioning Requires the Apoptosis Protein CED-4 in C. elegans.</a> Curr. Biol. 17 (22), 1954-1959 (2007).</li><li>Jia, B., Crowder, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volatile+anesthetic+preconditioning+present+in+the+invertebrate+Caenorhabditis+elegans.">Volatile anesthetic preconditioning present in the invertebrate Caenorhabditis elegans.</a> Anesthesiology. 108 (3), 426-433 (2008).</li><li>Wojtovich, A. P., Sherman, T. A., Nadtochiy, S. M., Urciuoli, W. R., Brookes, P. S., Nehrke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLO-2+is+cytoprotective+and+contributes+to+mitochondrial+potassium+transport.">SLO-2 is cytoprotective and contributes to mitochondrial potassium transport.</a> PloS One. 6 (12), e28287 (2011).</li><li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Cer&#243;n, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Caenorhabditis+elegans+methods:+synchronization+and+observation.">Basic Caenorhabditis elegans methods: synchronization and observation.</a> J. Vis. Exp. (64), e4019 (2012).</li><li>Fawcett, E. M., Horsman, J. W., Miller, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+defined+gaseous+environments+to+study+the+effects+of+hypoxia+on+C.+elegans.">Creating defined gaseous environments to study the effects of hypoxia on C. elegans.</a> J. Vis. Exp. (65), e4088 (2012).</li><li>Butler, J. A., Mishur, R. J., Bokov, A. F., Hakala, K. W., Weintraub, S. T., Rea, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+the+anaerobic+response+of+C.+elegans+using+GC-MS.">Profiling the anaerobic response of C. elegans using GC-MS.</a> PLoS One. 7 (9), 10-1371 (2012).</li><li>Padilla, P. A., Nystul, T. G., Zager, R. A., Johnson, A. C., Roth, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dephosphorylation+of+cell+cycle-regulated+proteins+correlates+with+anoxia-induced+suspended+animation+in+Caenorhabditis+elegans.">Dephosphorylation of cell cycle-regulated proteins correlates with anoxia-induced suspended animation in Caenorhabditis elegans.</a> Mol. Biol. Cell. 13 (5), 1473-1483 (2002).</li><li>Jiang, H., Guo, R., Powell-Coffman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+hif-1+gene+encodes+a+bHLH-PAS+protein+that+is+required+for+adaptation+to+hypoxia.">The Caenorhabditis elegans hif-1 gene encodes a bHLH-PAS protein that is required for adaptation to hypoxia.</a> Proc. Natl. Acad. Sci. U.S.A. 98 (14), 7916-7921 (2001).</li><li>Twumasi-Boateng, K., Wang, T. 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=An+age-dependent+reversal+in+the+protective+capacities+of+JNK+signaling+shortens+Caenorhabditis+elegans+lifespan.">An age-dependent reversal in the protective capacities of JNK signaling shortens Caenorhabditis elegans lifespan.</a> Aging Cell. 11 (4), 659-667 (2012).</li><li>Wang, Y., Chen, J., Wei, G., He, H., Zhu, X., Xiao, T., Yuan, J., Dong, B., He, S. S. k. o. g. e. r. b. &. #. 2. 4. 8. ;. G., Chen, 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+Caenorhabditis+elegans+intermediate-size+transcriptome+shows+high+degree+of+stage-specific+expression.">The Caenorhabditis elegans intermediate-size transcriptome shows high degree of stage-specific expression.</a> Nucleic Acids Res. 39 (12), 5203-5214 (2011).</li><li>Perry, C. N., Huang, C., Liu, W., Magee, N., Carreira, R. S., Gottlieb, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenotransplantation+of+mitochondrial+electron+transfer+enzyme,+Ndi1,+in+myocardial+reperfusion+injury.">Xenotransplantation of mitochondrial electron transfer enzyme, Ndi1, in myocardial reperfusion injury.</a> PloS One. 6 (2), e16288 (2011).</li><li>Chai, Y., Li, W., Feng, G., Yang, Y., Wang, X., Ou, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+of+Cellular+Dynamics+During+Caenorhabditis+elegans+Postembryonic+Development.">Live Imaging of Cellular Dynamics During Caenorhabditis elegans Postembryonic Development.</a> Nature Protoc. 7 (12), 2090-2102 (2012).</li><li>Nehrke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reduction+in+intestinal+cell+pHi+due+to+loss+of+the+Caenorhabditis+elegans+Na+/H++exchanger+NHX-2+increases+life+span.">A reduction in intestinal cell pHi due to loss of the Caenorhabditis elegans Na+/H+ exchanger NHX-2 increases life span.</a> J. Biol. Chem. 278 (45), 44657-44666 (1074).</li></ol>

The authors have nothing to disclose.

AS has been widely used in C. elegans to model IR injury. Some key points should be highlighted for this protocol: C. elegans are resistant to a wide array of injuries, justifying the need for 3 concomitant insults (heat, starvation and anoxia) to achieve death using this system. Anoxia alone does not kill the worms in this window of time14. Furthermore, temperature increase is an additional stress, so it is important to monitor closely. Strictly speaking, starvation does not contribute significantly to the degree of mortality observed7, per se, but it appears to reduce variability among experimental replicates. Given that there can be significant variability from day-to-day, it is extremely important to compare samples run directly in parallel, and to repeat experiments over multiple days. In general, outcomes are measured for three separate plates of 50-100 worms/experimental condition and these are then averaged and considered as a single experimental replicate. Generally, between seven and nine replicates appear to be sufficient to achieve or rule out statistical significance.
The developmental stage of the worms used in the experiment also needs to be carefully monitored as the susceptibility of different stages to AS damage varies significantly15,16. The use of young adults is standard, and larval stage (L3 and L4) worms appear to be more resistant to the damaging effects of AS (unpublished data).
This protocol presents two ways to perform the experiments, one using a lab-made apparatus (using Tupperware-type containers and gas input11) and other using a commercial hypoxic chamber4,6. Anoxia can be achieved by other means that consume the oxygen, as described elsewhere13,17. The use of alternate techniques to create a hypoxic environment may change the AS incubation time necessary to create the desired amount of death. Targeting ~20% survival is an ideal starting point for studying protective interventions, while 80% is similarly ideal for interventions that exacerbate the detrimental effects of AS. Another important caveat is the time at which the observer scores dead/alive worms. If the time for analysis is extended beyond 24 hr, the data may be misleading since dead worms become increasingly difficult to identify. This may be due to worm carcasses becoming relatively transparent over time, but also to the fact that fertilized embryos can develop into progeny post-mortem inside of the carcasses and disrupt them as they emerge.
The analysis of neuronal morphology can be modified to look at protein expression patterns18, nuclear fragmentation4 and other parameters19 by substituting a worm strain that expresses the appropriate genetically encoded marker. One final caveat is that the visualization of the neuronal processes should be done less than 30 min after placing the worms on the slide. Animals kept under anesthesia on slides for longer periods can exhibit AS-independent damage. Adjust the amount of animals per slide according to the time needed to track and analyze them.

C. elegans is a nematode that has been widely adopted as a multicellular eukaryotic model organism since its introduction by Brenner1. It is a cheap, simple, and versatile model, which allows easy links between genetic alterations and phenotypic changes2.
Ischemia is characterized by a lack of nutrients and oxygen supply to a tissue, followed by reperfusion, when a burst of reactive oxygen species is produced3 and most of the damage occurs. In 2002, a model of ischemia/reperfusion (IR) in C. elegans was developed4 involving submitting the whole worm to anoxia, nutrient deprivation and heat stress for approximately 20 hr&#160;followed by 24 hr&#160;under normal conditions. Although this model is technically an anoxia-starvation (AS) condition, cell death occurs through mechanisms that are conserved in mammals, including damage induced by oxidants during reperfusion5. Furthermore, similar to mammalian IR, damage induced by AS in C. elegans can be prevented by ischemic preconditioning6,7 or anesthetic preconditioning8,9.
The protocols below demonstrate how to mimic IR in C. elegans using the AS model, how to score morphological and behavioral abnormalities that result from AS, and how to adapt the protocol in a way that allows the experiment to be conducted with a lower initial investment using a custom-made, easily-constructed chamber alternative.

<table border="0" cellpadding="0" cellspacing="0" width="1146">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">N2&#160; strain</td>
			<td style="width:269px;">CGC (http://www.cbs.umn.edu/CGC/)</td>
			<td style="width:208px;"></td>
			<td style="width:453px;">Wild type strain</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">TU2583 uIs25 (Pmec-18::GFP)</td>
			<td style="width:269px;">CGC (http://www.cbs.umn.edu/CGC/)</td>
			<td style="width:208px;">TU2583</td>
			<td style="width:453px;">integrated fluorescent transgene used to label touch neurons</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CB1338 mec-3 (e1338)IV</td>
			<td style="width:269px;">CGC (http://www.cbs.umn.edu/CGC/)</td>
			<td style="width:208px;">CB1338</td>
			<td style="width:453px;">canonical mec-3 mutant that is touch insensitive</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Microcentrifuge Tube</td>
			<td style="width:269px;">Eppendorf</td>
			<td style="width:208px;">0030 120.086</td>
			<td style="width:453px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Nikon Eclipse TE2000-U Microscope&#160;</td>
			<td style="width:269px;">Nikon USA</td>
			<td style="width:208px;">TE2000-U</td>
			<td style="width:453px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Low Temperature Incubator</td>
			<td style="width:269px;">Sheldon Manufacturing Inc.</td>
			<td style="width:208px;">Model 2005</td>
			<td style="width:453px;"></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Eyelash Pick</td>
			<td style="width:269px;"></td>
			<td style="width:208px;"></td>
			<td style="width:453px;">An eyelash pick can be prepared by attaching&#160; an eyelash onto a wooden toothpick, then attaching the toothpick in a glass Pasteur pipette (Figure 3)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Hypoxic Chamber</td>
			<td style="width:269px;">Coy</td>
			<td style="width:208px;">8307030</td>
			<td>Hypoxic Glove box equipped with paladium catalyst and CO2 controller.</td>
		</tr>
	</tbody>
</table>

an anoxia-starvation model for ischemia/reperfusion in c. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

Subjecting C. elegans to 20 hr&#160;of AS at 26 &#176;C in a custom lab-made incubation chamber as described (Section 3.6.2) resulted in significant mortality (Figure 1A)5. Subsequent fluorescent imaging of punctum and breaks in the GFP labeled neuronal processes of survivors confirmed the presence of morphological abnormalities (Figure&#160;2). The survivors also responded poorly to light body wall touch (Figure&#160;1B). This model has been used by multiple groups to study how genetic predisposition, pharmaceutical intervention, and metabolic plasticity affects AS dependent outcomes4-6,8,9,12,13.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51231/51231fig1highres.jpg" src="/files/ftp_upload/51231/51231fig1.jpg" /> 
Figure 1. C. elegans survival and touch response after AS.&#160;A)&#160;Percentage of worms that exhibit 24 hr post-AS survival. The N2 strain is the wild type genetic background, and KWN85 contains an integrated transgene that labels mec neurons with GFP.&#160;B)&#160;Response to touch stimuli of living C. elegans (N2 Strain) after AS. <a href="https://www.jove.com/files/ftp_upload/51231/51231fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51231/51231fig2highres.jpg" src="/files/ftp_upload/51231/51231fig2.jpg" /> 
Figure 2. Touch neuron modifications after AS. Touch neuron (PLML and PLMR) abnormalities such as tortuous processes and breaks (A) or the accumulation of GFP aggregates in the processes (B) were monitored in surviving anesthetized C. elegans after AS.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51231/51231fig3highres.jpg" src="/files/ftp_upload/51231/51231fig3.jpg" /> 
Figure 3. Eyelash pick. An assembled eyelash pick. In the inset, a detail of the eyelash glued to the toothpick wood.
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51231/51231fig4highres.jpg" src="/files/ftp_upload/51231/51231fig4.jpg" /> 
Figure 4. Lab-made hypoxic chamber inside the water bath. A closed container ready to start the experiment. Arrows indicate the gas entrance and exit and the bottle weight used to keep it from floating.
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/51231/51231fig5highres.jpg" src="/files/ftp_upload/51231/51231fig5.jpg" /> 
Figure 5. Details of the internal and external side of the container lid. A) External side of the container lid, indicating the tube and its connector attached to the previously made hole. B)&#160;External side of the container lid, indicating the tube connector attached to the previously made hole.

Watch this Scientific Journal Video about An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans at JoVE.com

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

an-anoxia-starvation-model-for-ischemiareperfusion-in-c-elegans

Research

JoVE Journal

Biology

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...