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

1. C. elegans Growth

1. Prepare 35 mm Nematode Growth Media (NGM) agar plates seeded with OP50 bacteria, as per standard cultivation methods¹.
2. Synchronize C. elegans by placing 6 gravid adults onto a seeded NGM plate. Remove the adults after ~100 eggs have been laid (~3 hr).
3. Incubate the plates for 3 days at 20 °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.

2. Materials for AS

1. 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.
2. Make a small hole (3 mm wide) in the lid of 1.5 ml 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.

3. Anoxia/Starvation

1. Conduct all experiments at least in triplicate.
2. Add 1 ml of RT, oxygenated M9 buffer to each 35 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't seeded). After ~1 min the worms should be swimming in the M9.
3. Coat a 1 ml pipette tip with BSA by pipetting and expelling a sterile 1% BSA solution.
4. 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).
5. Remove the maximum amount of M9 possible without disturbing the worms from the tube (leaving around 100 μl), add 1 ml 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 μl remains in the tube.
6. Place the tubes with the worms in deoxygenated M9 inside an anoxic/hypoxic chamber (see instructions below, Section 3.6.1) or, alternatively, into a custom sealed container (see instructions below, Section 3.6.2).
   1. 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.
   2. 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:
      1. Make two holes in a 250 ml 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.
      2. Place the worms into the chamber and seal the chamber using the lid.
      3. Gas with a constant N₂ flux (100 ml/min).
      4. Place the container inside a water bath at 26 °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).
7. Incubate the worms at 26 °C for 20 hr.

4. Simulated Reperfusion

1. After 20 hr under AS conditions, remove the tubes from the incubation chamber into room air.
2. Pipette C. elegans with a BSA-coated tip (as in step 3.3) onto an OP50 seeded 35 mm NGM plate. Incubate the plates at 20 °C for 24 hr. After this, the worms will be ready to be scored.

5. Identifying Dead Versus Live Worms

1. 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 “bags-of-worms” can rupture, making detecting the carcasses very difficult.
2. 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 1A).

6. Touch Response Assay

1. 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.
   1. To score for touch response, identify worms that are moving and lightly touch the side of the worm's head (near the middle part of the pharynx) with an eyelash pick (Figure 3).
   2. If the worm moves backward, score as responsive, if not, score as nonresponsive (Figure 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.
   3. Finally, incorporate genetic control strains [N2 Bristol wild-type for positive, mec (mechanosensory) mutant for negative - 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.

7. Neuronal Modifications

1. To visualize post AS neuronal modifications, use a strain which expresses GFP in the touch-responding mec neurons^4,5. 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.
2. Prepare an agarose pad.
   1. Melt 2% agarose in water, bring to 1x M9 with a 10x stock, and maintain the solution at 70 °C.
   2. Place a drop (~20 μl) 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.
   3. 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.
   4. Remove the top slide and add 10 μl of M9 containing 0.1% tetramisole, which will immobilize the worms.
3. 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 objective with the appropriate illumination.
4. Damaged neurons will show a cytosolic membrane string of pearls pattern in the processes, comprised of multiple punctum (Figure 2A), and/or abnormalities where the processes appear to be broken (Figure 2B). Count punctum and abnormalities in both neurons for each worm, using a total of at least 10 worms.

8. Lab-made Hypoxic Chamber

1. 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 4).
2. 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 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 5B).
3. The container should be placed into the water bath so that it is submerged completely (Figure 4). To immerse the container, use a bottle weight or a diving weight.
4. 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 4).
5. 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 container, 100 ml/min of gas will provide good space for the worms and a suitable flux.
6. 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.