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

The overall goal of the following experiment is to demonstrate methods used to wound see elegance, and to visualize the epidermal responses to wounding. This can be achieved by wounding the epidermis with small punctures caused by pricks from a microinjection needle. A second wounding method uses laser irradiation.

This can be performed on animals mounted for microscopy and allows for immediate imaging of the wounding response. The results show that wounding triggers a rapid elevation of epidermal calcium, followed by the accumulation of an actin ring around the wound site that closes in a few hours, thus allowing the wound to heal The needle. And laser wounding methods shown here are currently the most widely used methods of skin wounding in the nematode C Elgan.

These methods are used to address key questions in the wound kidney field as how skin cells detect and respond to injury. Put a plate containing young adult worms on ice for about half an hour To slow the animals down. This chilling induced torpor is important for precise needle wounding.

As worms normally move around too quickly for wounding under the stereo microscope, view the worms and using a pick, move them all to the center of the plate. Then grip a pulled needle like that used for micro injection with a 100 microliter pipette tip. As a needle holder, bring the needle point to either end of a worm, avoiding the gonads, either between the posterior pH pharynx and interior gonad, or between the posterior gonad and the rectum.

Then add an approximately right angle to the cuticle. Make a brief gentle prick that penetrates about five to 10 microns into the worm. These needle wounds may also damage internal organs such as the intestine.

Wait a moment to observe cellular contents leaking from the wound. A few seconds of leakage is okay, but more than that suggests that the wound will be lethal. Imaging the wound responses will be easiest if the wound is made in the lateral epidermal syncytium or the lateral seam.

In needle wounding, it is critical to inflict a small puncture rather than a large prick. As the ladder can rapidly kill the worms practice is needed to reproducably cause the minimal damage necessary to induce a wound response Proceed with wounding each animal using the same needle until it breaks or becomes dirty. With experience wounding all 25 animals on the plate in five minutes is quite feasible To precisely on the nematodes.

This protocol uses a femto segment laser attached to a spinning disc focal microscope line or point scans using femto femtosecond liters such as those in two photo microscopes. Should also suffice. Begin by collecting 10 young adults on an auger pad.

Paralyze them in a two microliter drop of 12 millimolar lail solution. Then cover them with a cover slip and wait a few minutes while they paralyze. It is essential that animal be completely paralyzed.

12 milli more will not affect only response. However, other methods of immobilization can also be used. Ensure that the femtosecond laser power is set to 140 milliwatts as measured before the objective and the laser repetition rate is at 80 megahertz.

Now locate the worms under a spinning disc confocal microscope using a 100 x objective with a high numerical aperture focus on the lateral, anterior or posterior syncytial epidermis of the target worm, focusing on the apical surface of the epidermal cell. Shine the laser for two 200 millisecond pulses separated by 20 milliseconds. This should be sufficient to wound the epidermis.

Wait a moment and observe the local disruption of cytoplasm, which will appear as bubbling. If a fluorescent marker is present, localized bleaching can be observed. This protocol makes use of C elegance that express calcium sensors in the epidermis such as G Camp three and also express a red fluorescent protein such as TD tomato as an internal control for the transgenes expression level.

Although both wounding methods trigger calcium elevations to image this by spinning disc confocal microscopy, laser wounding must be used after the wounding acquire time lapse images with an interval time of two seconds and an excitation and a laser exposure of 114 milliseconds. The same magnification as used for laser wounding is appropriate over longer time courses, such as two hours images can be taken every 30 seconds or longer. The text protocol suggests analysis routines.

This protocol uses worms that express an F actin marker GFP mosin under the control of an epidermal specific promoter, such as call 19 after needle wounding culture. The wounded worms at 20 degrees Celsius incubator for an hour and then take Zacks to image formation of the actin ring to examine actin dynamics immediately after needle wounding, transfer the wounded animals to an auger pad on a slide and anesthetize them with lail. Then image the animals using conventional confocal microscopy.

Acquire Zacks with a 0.5 micrometer interval for one to two hours to measure the actin ring to quantitate ring diameter. First, make a maximum intensity projection of the Z stack and draw four line scans at 45 degrees from each other over the GFP MOIs and ring. Then calculate the diameter of the ring as the average peak tope distance of the four scans for rings that have closed.

The diameter is defined as zero O laser or needle wounding triggers a rapid and sustained rise in epidermal calcium levels as visualized by the calcium sensor G camp. A lateral view of the epidermis in the anterior body shows the laser wound marked by an X and N DeMars the epidermal nucleus. The calcium rise quickly spreads out from the wound site Quantification of the calcium levels at 2040 and 60 microns from one wound site helps illustrate to these early calcium response kinetics.

Calcium elevation remains for tens of minutes and needle wounding reliably. Results in the formation of f actin rings that gradually close at wound sites within two hours. This was visualized using P call 19 GFP MOIs in animals.

These rings are less often produced by precise laser wounding. The acton ring's diameter was quantified using a four line scan. Quantification of the line scans illustrates how the actin ring has different intensities at various distances from the wound.Site.

Development of these wound healing methods has allowed researchers to exploit sea elegance as a model for many aspects of wound repair, including innate immune responses and wound closure. Don't forget that femto segment lasers are are hazard. Anyone working with the Femto segment laser should have completed laser safety training.