1Department of Biochemistry, University of Washington, 2Molecular and Cellular Biology Program, University of Washington
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Fawcett, E. M., Horsman, J. W., Miller, D. L. Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans. J. Vis. Exp. (65), e4088, doi:10.3791/4088 (2012).
Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition <10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.
1. Construction of Environmental Chambers
2. Connecting the Gas to the Environmental Chamber
3. Selecting Flow Rate
4. Preparation of Samples for Viability Assay
5. Rapid Harvest of Hypoxia-exposed Worms (Example: Preparation of Samples for HIF-1 Western Analysis)
Many hypoxia-induced effects are quickly reversed upon return to room air, including the resumption of egg production12, phosphorylation of mitotic epitopes in embryogenesis13 and degradation of the HIF-1 protein9,19. Rapid isolation of animals exposed to hypoxia is required to obtain reproducible effects in these conditions. With this setup, animals can be harvested and frozen in liquid nitrogen in less than two minutes. While glove box hypoxia chambers allow for manipulation of samples in anoxic conditions, their cost and practicality for conditions other than anoxia limit their usefulness.
6. Representative Results
Organismal effects of hypoxia can be seen by examining the viability to adulthood of C. elegans (Figure 2). Embryos laid by wild-type Bristol (N2) and hif-1(ia04) deletion mutants are all survive in house air O2 concentrations (210,000 ppm O2). N2 worms are able to adapt and survive to adulthood in 5,000 ppm O2, while hif-1 embryos are not viable. This shows that HIF-1 is essential for adapting to the changing levels of oxygen available in the environment10. Neither N2 nor hif-1 animals can survive exposure to 1,000 ppm O2.
Visualizing worm directly in hypoxia is feasible with the use of a dissecting scope and clear container (Figure 3). By directly placing the hypoxia chamber on the dissecting scope, there is no need to remove the worms from hypoxia to observe organismal reactions. The scope can be fitted with fluorescence illumation (as in Figure 3), further extending the types of observations in hypoxia that are possible.
Figure 1. Example of Hypoxia chamber. Direction of gas flow is indicated by arrows. Gas is stored in compressed gas tanks with defined O2 concentrations (1) and a two stage regulator is attached (2). Gas enters the bottom of the flow tube (3), exiting the top at the correct flow rate. Gas then flows into the bubble flask (4), hydrating the gas (ensure correct connection of bubble flask by observing bubbles). Hydrated gas then passes into the hypoxia chamber at the inflow valve (5), exposing the samples to hypoxia. The gas finally vents into the room through an exhaust hole drilled in the chamber.
Figure 2. Viability of embryos exposed to 1,000 ppm O2, 5,000 ppm O2 and normoxia (~210,000 ppm O2). Embryos were exposed to each oxygen conditions as embryos for 24 hours in continuous flow oxygen chambers. Worms were moved to normoxic conditions, allowed to develop to adulthood for 48 hours, and then scored for viability to adulthood. n>50, N=5.
Figure 3. Visualization of C. elegans in hypoxia with microscopy. Worms are exposed to hypoxia using the methods outlined. The transparent environmental chamber (constructed with a pyrex crystallization dish and glass plate) is placed directly on the stage of a dissecting scope. Two views are shown, one including the entire gas flow set up, the other with just the chamber on the microscope stage.
This method presents a strategy for constructing a hypoxic environment that allows for environments with precise concentrations of oxygen to be maintained in the laboratory. These chambers provide a simple method for exposing organisms to specific low concentrations of O2 and monitoring the molecular and physiological outputs. The environmental chamber described is assembled by the lab instead of commercially purchased and can thus be modified to fit the needs of the experiment.
One distinct advantage of this method is the continuous flow design. This eliminates the difficulties normally encountered with maintaining low concentrations of O2 in chambers when the external O2 concentration is much higher (210,000 ppm O2 in room air). The alternative is a stopped-flow method, in which a hypoxic environment is maintained in a sealed chamber. Even small leaks, which can be difficult to detect, prevent the maintenance of hypoxic conditions using stopped-flow methods. The continuous flow method continually exchanges the air in the chamber with the defined oxygen concentration in the compressed air tank and maintains a positive pressure that prevents leaks from disrupting the hypoxic conditions.
Obtaining exact, pre-mixed oxygen concentrations from the gas supplier solves another difficult problem with hypoxia. It is quite difficult to measure extremely low concentrations of O2. Most O2 sensors are diffusion limited and quite expensive. Because O2 diffuses slowly, measuring low O2 concentrations can be slow or inaccurate20. In contrast, it is quite easy to generate gas mixtures by measuring the weight of gasses. The mixtures we regularly purchase are certified standard to be within 2% O2 content of the desired mix.
This method can be used to elicit observable hypoxia-induced changes both at the organismal and molecular level. While this method outlines survival assays and rapid whole worm isolation for molecular experiments, there are myriad downstream readouts that could be used. For example, this design allows for direction visualization of worms in hypoxia for study of real time behavior and changes to reporter constructs. To visualize worms with a dissecting scope, assemble the chamber using transparent boxes with small volume and minimal height. The entire chamber can be placed on the dissecting scope and is easily maneuverable for optimal visualization (see Figure 3). It would also be possible to observe samples at higher magnification by using perfusion chambers with an inverted microscope. This requires some adaptation of the chambers to interface it with tubing that is normally used for gas flow, and determine an appropriate flow rate. The representative results shown only scratch the surface of experimental possibilities, as hypoxia has been shown to affect cellular systems from DNA synthesis to protein degradation21,22.
The practical nature of this method is not limited to C. elegans. As long as appropriate-sized chambers are used, this method is readily adaptable to almost any model system. For adaptation to liquid media or cell culture, oxygen diffusion constants in solution, outgassing from plastic and time to equilibrate in culture must be taken into account, and it may be most appropriate to use O2 permeable culture plates23,24.
It is possible to modify the chambers presented in this protocol for use with other gasses. For instance, chambers can be adapted to provide an anoxic environment merely by omitting the O2 in the compressed gas tanks used to create a hypoxia chamber (with the balance being filled with nitrogen). This has allowed for observation of C. elegans in suspended animation (data not shown)10,13,25. Slight modifications must be made based on the properties of the gas mixture used. The composition of the tubing used to pipe gas into and out of the chamber may have to be varied. Some plastics are permeable to CO2, while others are not compatible with corrosive gasses such has hydrogen sulfide (H2S)16,25. A list of compatible plastics can be found on the Cole-Parmer website.
For toxic gasses the gas outlet from the chamber must be vented into a certified fume hood and appropriate personal protection, such as detectors, must be employed. Additionally, EH&S officers should be consulted before beginning any experiment using potentially hazardous gasses. Corrosive gasses may also require special attention. For example, H2S can corrode many of the plastics used in standard tubing material as well as brass fitting will corrode. We generally make sure that any wetted plastic is Kalrez or equivalent in instruments used with H2S. Certain gasses may interact with impurities in tap water, so DiH20 should be used in the bubble flask. Special considerations concerning glassware may also be required; for example, H2S necessitates equipment with wetted O-rings.
Both organismal and molecular changes are observed utilizing experiments which can be completed in a day. This ability to rapidly introduce samples to hypoxia provides a valuable tool in fields from aging and cancer to development.
No conflicts of interest declared.
We thank members of the Miller lab for discussion and critical reading of the manuscript. This work was supported by a new investigator award from the Nathan Shock Center of Excellence in the Basic Biology of Aging to DLM and the National Institutes of Health award R00 AG030550 to DLM.
|Tubing (FEP and PTFE)||Cole Parmer Tygon||YO-95821-00 (1/8" FEP) 06605-27 (1/16 x 1/8" PTFE)’ R-3603|
|Compression fittings||Seattle Fluid Systems||06363-58 (M. coupler 1/16") 06363-62 (F. coupler 1/16") 06363-60 (M. coupler 1/8") 06363-61 (F. coupler 1/8")|
|Flow tube||Aalborg||PMR3-010073 (3 output) PMR1-013520 (1 output)|
|Mass flow controller||Sierra Instruments||810S-L-DR-2-OV1-SK1-V1-S1 (Mass Trak) C100L-DD-2-OV1-SV1-PV2-V1-SO-C10 (Smart Trak 2)|
|Compressed gas tank||AirGas||Made to order|
|Plastic male Luer to hose barb fittings||Cole Parmer||45505-41 (500 series 1/16")|
|Cast acrylic boxes||Ellard Instrumentation||Made to order|
|Pipe fittings (Brass or stainless steel)||Seattle Fluid Systems||B-402-1 (1/4" nut) B-200-3 (1/8" union tee) B-400-set (1/4" ferrules) B-QM2-B1-200 (QM Body QC) B-200-1-2 (1/8 x 1/8" male conn)|
|Dow Corning Vacuum Grease||Sigma-Aldrich||Z273554|
|AnaeroPack box||Misubishi Gas Chemical Company||R684004 (0.4 liter) R685025 (2.5 liter) R685070 (7.0 liter)|
|Pyrex gas wash bottle||Sigma-Aldrich||CLS31770500C (500 mL) CLS31770250C (250 mL) CLS31770125C (125 mL)|
|Goat anti-mouse IgG-horseradish peroxidase||Southern Biotechnology Associates||1032-05|
|SuperSignal West Pico Chemiluminsecent Substrate||Pierce Chemical||34077|
|100 x 50 glass crystallization dishes||Kimax Kimble||23000|