As a powerful animal model in neurodegenerative and the behavioral studies, C.elegans can be used to efficiently screen and evaluate toxic compounds against polyglutamine neurotoxicity using Huntington's disease-like models. The main advantage of this technique is profiling and integration of multiple phenotypes in different C.elegans models. Also actually like C.elegans models are used in this method.
It does providing size into screening and the investigation of therapeutic candidates for other neurodegenerative delays and indeed other delays. Please pay attention to temperatures used in C.elegans growth and perform the test at the right stages of namical development. Demonstrating the procedure will be Jiang Yiyi, Xioa Yue, and Wang Qiangqiang, three graduate students.
Begin by transferring 300 to 500 synchronized L1 larvae of AM141 nematodes to each well of a 48 well plate with 500 microliters of S medium containing OP50 strain of E.coli and five milligrams per milliliter of astragalan. Seal the plate with parafilm and incubate at 20 degrees Celsius and 120 rotations per minute for desired time intervals. Transfer the nematodes in a sterile 1.5 milliliter microcentrifuge tube and wash with M9 buffer three times by centrifugation to remove the remaining OP50 cells.
Then, resuspend the AM141 nematodes in M9 buffer. Now, transfer 10 to 15 nematodes into each well in a 384 well plate, setting 10 replicate wells for each treatment and add 10 microliters of 200 millimolar sodium azide to each well to paralyze the nematodes by allowing them to settle down to the bottom. Place the plate in a high content imaging system to acquire fluorescent images.
Open the image acquisition software and set up the parameters mentioned in the text manuscript. Analyze the image data by opening the review plate data window and selecting the test plate for image analysis. Double click on a test well to display its image.
Then select the count nuclei as the analysis method and click on the configure settings button. Define the source image from the FITC channel and select the standard algorithm. Set the image analysis parameters as described in the text manuscript and test them to optimize the method of analysis.
Save the settings and run the analysis on all the wells. Export the total nuclei as the total number of Q40:YFP aggregates in each well. Count the number of nematodes in each well and calculate the average number of Q40:YFP aggregates per nematode in each group.
Then, calculate the inhibition index. To prepare nematodes for the polyQ neurotoxicity assay, transfer 300 to 500 synchronized L1 larvae of HA759 nematodes to each well of a 48 well plate with 500 microliters of S medium containing OP50 strain of E.coli and five milligrams per milliliter of astragalan. After sealing the plates, incubate, harvest, and resuspend the nematodes in M9 buffer as demonstrated previously.
Now, prepare in agarose pad by adding two grams of agarose to 100 milliliters of M9 buffer and heat the solution in a microwave to near boiling. Dispense 0.5 milliliters of melted agarose onto the center of a one millimeter thick microscopy glass slide placed between two pieces of two millimeter thick glass plates, covering with another slide vertically. Gently remove the top slide once agarose cools down and is solidified.
Begin the ASH neuronal survival assay by adding a drop of 20 millimolar sodium azide onto the agros pad and then transferring 15 to 20 HA759 nematodes into the drop to immobilize them. Place a cover slip gently over the nematodes. Now, keep the slide under a fluorescence microscope fitted with a digital camera.
Select a 40x objective lens and FITC filter to detect GFP positive ASH neurons in the head region of the nematodes. Select more than 50 nematodes in each group randomly to count the number of nematodes with GFP-labeled bilateral ASH neurons in their head region, and then calculate the survival rate of ASH neurons. For the osmotic avoidance assay, divide a food-free NGM plate into normal and trap zones by creating an eight molar glycerol line in the middle.
Then, spread a 200 millimolar sodium azide line at around one centimeter away from the glycerol line to paralyze the nematodes crossing through the glycerol barrier into the trap zone. Transfer around 200 nematodes each onto the normal zone of three replicate plates for each group. Then, add a drop of 1%butanedione onto the trap zone to attract the nematodes.
Cover the lid of the Petri dish immediately and incubate at 23 degrees Celsius for 90 minutes. Score the number of nematodes on both the zones under a microscope and calculate the avoidance index. The transgenic polyQ strain AM141 strongly expresses Q40:YFP fusion proteins in its body wall muscle cells, identified by this automated imaging and analysis protocol, whose increasing amount was inhibited by astragalan treatment demonstrating its protective potential.
A loss of GFP fluorescence and bilateral ASH neurons in the head regions of nematodes indicates the ASH neuronal death. This ASH neuronal survival assay can be used to visually evaluate the neuroprotective effect of test compounds on Caenorhabditis elegance neurons. The chemosensory avoidance assay was used to examine the effective test samples on the functional loss of ASH neurons mediated by polyQ aggregation.
The avoidance index of HA759 nematodes treated with astragalan at 15 degrees Celsius for three days increased to more than 0.6, demonstrating a neuroprotective effect of the polysaccharide against behavioral impairments. For evaluating the overall neuroprotective capacity of test compounds, the data from the individual assays were integrated and presented as a radar chart showing the area of the triangle in the astragalan treatment group greater than that of the control group, indicating the anti-polyQ effects of the polysaccharide. Please keep in mind that food, temperature, and moisture may affect their behavior of C.elegans.
