Out laboratory is focusing on basics and translational neuroscience. It's combining strong foundation in epilepsy research with innovative approaches in neuroprotection, neuroinflammation, and neuroregeneration. Combining AI, advanced E machine, and genetics tool with innovative models, like a Planaria neuromodulation devices hold promise for transforming the landscape to study basic seizure mechanisms.
Experimental seizure models are very valuable for understanding the mechanisms of epilepsy and testing for potential therapies. However, several challenges persist restraining their translational impact because of the limited understanding of the biology of seizures, the complexity of human and minimal seizure mechanisms in invertebrate models, the limit of standardized protocols for behavior and neurophysiological responses and drug efficacy across different models. A gap we're addressing is the relationship between dose-dependent, pilocarpine-induced planaria behavior and subsequent changes in their nervous system visualize histologically with immunofluorescent and golgi-staining techniques.
Planarians serve as cost-efficient and ethically favorable models for experimental seizures. Unlike other invertebrates or zebra fish, planarians regenerate neurons and exhibit different behavior phenotypes enhancing their value for anti-seizure drug testing. Their simplicity, and conservation of neuromechanisms bridge basic research and translational epilepsy studies.
Planarians serve as seizure models because of their bilateral symmetry encephalization, conserve neurotransmitter systems, and sensitivity to neurotoxic and proconvulsant agents. This versatile model connects the invertebrates and mammalian epilepsy models advancing translational research and anti-seizure drug discovery efforts. The findings inspire questions investigating seizure-induced neuronal regenerative mechanisms, neuronal connectivity changes, broader neurotoxicity research, genetic preservation and medication after seizures, and the social behavior planaria to study the comorbidity of epilepsy.
To begin, prepare two solutions of pilocarpine at concentrations of one and two millimolars dissolved in spring water. Pipette three milliliters of each solution into separate rows of a 4 X 3 well plate. Cut a pipette tip to make the opening large enough to fit the planaria without causing damage.
Using a transfer pipette with the cut tip, place one lab-reared, two-week-old Dugesia dorotocephala into each of the 12 wells. Position a camera over the plate under normal indoor lighting conditions and record the planaria's behavior for one hour. For golgi staining, place euthanized planaria in a beaker or petri dish filled with a one-to-one mixture of solutions A and B from the golgi stain kit.
The following day, transfer the planaria into a new beaker filled with a fresh mixture of solutions A and B.Remove planaria from the solution and place them in solution C in another beaker. After replacing solution C the following day, let the planaria sit for a minimum of 72 hours and up to one week in solution C.Using a transfer pipette, remove planaria from solution C and embed in a small amount of OCT compound on a chuck. Surround them carefully with the OCT compound, and transversely cut the embedded planaria into five-micrometer sections using a cryostat machine set to minus 24 degrees Celsius.
Using a transfer pipette, mount the planaria sections onto gelatin-coated slides with solution C.Let the slides dry in the dark at room temperature for up to three days. To stain the slides, submerge them sequentially in staining and washing solutions. Cover the slides using the histological mounting media and a cover slip before observing it under a brightfield microscope.
Obtain Dugesia dorotocephala treated with one to six millimolar pilocarpine. After the planaria are euthanized, place them into 4%paraformaldehyde for at least 24 hours. Next, transfer the planaria to 20%sucrose and leave them in the solution for one day.
Rinse them three to four times in PBS for five minutes each. After washing, transfer them to fresh 20%sucrose for storage until ready to stain. Now, place the planaria sequentially in appropriate solutions for a specific time.
Next, block the planaria for one hour with powdered milk and incubate the tube overnight at four degrees Celsius in the primary antibody solution. The following day, rinse the planaria in PBS for 10 minutes. Dilute the goat anti-mouse IgG secondary antibody to one microgram per milliliter in PBS and incubate the planaria tube with the antibody solution overnight at room temperature.
After washing, mount the whole planaria on the slide. Cover the worms with a cover slip. Seal them with aqueous mounting media and observe the slide under a fluorescent microscope.
To begin, obtain images of planaria after golgi and immunostaining. Launch the open source image analysis software application and install the color deconvolution2 jar plugin in the application. Select the image to analyze and drag it to the open source image analysis software application.
Go to the image tab, click color, and select color deconvolution2 version 2.1. In the vectors option, click H DAB and in the output option, click 8bit Transmittance. Ensure that simulated LUTs cross product for color 3, show matrices, and hide legend, are all selected, then click okay.
Select the black and white image to confirm that the open source image analysis software application can effectively analyze it. Now, go to the image tab. Click adjust and select threshold.
Adjust the threshold so the DAB staining, or foreground is read with a white background. Click analyze, then set measurements. Select area, min and max gray value, limit to threshold, display label, median, and mean gray value, and click okay.
Then click analyze followed by measure to obtain the results. Finally, click the results image, then click file and select save as to save the results. The oscillations and flicks were increased in six-millimolar, pilocarpine-treated planaria as compared to the control.
Planaria exposed to three-millimolar pilocarpine spent significantly more time in the center of the wells compared to the control group. Planaria treated with six-millimolar pilocarpine spent more time in the perimeter than both the control and three-millimolar groups. Six-millimolar pilocarpine treatment significantly reduced the number of neural structures compared to the control.
