Methylnitrosourea (MNU)-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

The overall goal of this procedure is to induce retinal degeneration and ensuing regeneration in the adult zebrafish using n methyl and nitro ueaa. This is accomplished by first incubating wild type zebrafish in water containing N methyl N nitrous urea, or MNU. Next visual function is quantified by measuring the optokinetic response or OKR and calculating the visual acuity at different time points.

Post-treatment, then the zebrafish are euthanized and the eyes in nucleated for histological examinations using h and e staining to observe morphological changes in the retinal tissue. Finally, cellular changes are visualized in the retina with specific staining for cell death and proliferation. Ultimately, measurement of visual acuity and quantification of morphological and cellular changes are used to visualize retinal D and regeneration.

The main advantage of this pharmacological model is that it is simple as well as fast to use and induces reliable regional degeneration and regeneration in adult zebra fish To carry out NM methyl n nitroso urea or MNU treatment begin by preparing water containing 150 milligrams per liter of dry MNU. Proceed with caution while working with nm methyl and nitrous urea as it is toxic and may cause cancer heritable genetic damage or harm. An unborn child maintain while type AB strain, zebrafish six to 12 months old under standard conditions.

Transfer zebrafish into the MNU water and incubate them for 60 minutes at room temperature. Then use fresh water to quickly rinse the fish before transferring them into a new fish tank without MNU. Maintain the fish under standard conditions as long as desired for experiments.

Prepare to take visual acuity measurements by starting the motor system. Then from the menu, choose testing and set the options to simple staircase. Install an infusion bottle filled with 500 milliliters of water about one meter above the motor system.

Place one zebrafish in the examination chamber and connect it to the infusion bottle. Then place the examination chamber in the automotive system. Next, start the measurement and observe the eye movement in real time on the computer screen.

A positive response or yes, is defined as consecutive secs in the correct direction, whereas a negative response or no represents random eye movements similar to the ones observed with stationary gratings. If the eyes of the zebrafish exhibit three or more subsequent optokinetic responses or OKRs, press yes. If not, press no.

Under results in the menu, extract the visual acuity, which has been calculated by the software by determining the threshold of the spatial frequency of the optic kinetic stimulus to carry out histological analysis. After euthanizing the zebrafish according to the text protocol and in nucleating, the eyes use 4%power of formaldehyde in PBS to fix the intact eyes at four degrees Celsius for 12 hours before using a graded alcohol series to dehydrate the samples, embed the samples in paraffin, then cut five micrometer sections through the optic nerve head or ONH and mount them on glass slides. Next, use Alam to stain the decarbonized sections for five minutes before dipping the slides into distilled water twice, followed by 0.2%hydrochloric acid and 0.8%ammonia.

Move the slides into tap water for at least 10 minutes. Transfer the sections into eosin for three minutes after washing and dehydrating the slides with an ethanol series. Use mounting medium to embed them and a light microscope to observe the samples for terminal, the oxy nucleotide transferase, DUPT Nick n labeling or tunnel.

Add 20 micrograms per milliliter pronase K to de parize sections, and incubate them at room temperature for 20 minutes. After using tris buffered saline to wash the sections three times for five minutes each add 50 microliters of the tunnel reaction mixture to the sections and incubate them in a humidified chamber at 37 degrees Celsius for 60 minutes. Once the samples have been washed and triss three times for five minutes each, use mounting medium containing dappy to mount the slides before them under a fluorescent microscope to carry out proliferating cell nuclear antigen or PCNA staining.

Place the samples in antigen retrieval, buffer and boil for three minutes before washing in TB S3 times for five minutes each h using blocking buffer, incubate the samples for one hour at room temperature. Then with a one to 200 dilution of anti-PC NA.Primary antibodies, incubate the samples in a humidified chamber overnight at four degrees Celsius after washing the samples with TBS plus tween 23 times for five minutes each. Use a one to 500 dilution of secondary antibodies to incubate the samples for one hour at room temperature.

Once the slides have been mounted with mounting medium containing dpi, observe them under the fluorescence microscope. This figure shows the visual acuity course for adult zebrafish after the application of 150 micrograms per liter of MNU. Beginning with day one, the measurements revealed a mark decrease in visual acuity reaching minimum values at day three before an increase in values.

Proceeded at day eight and reached a full recovery at day 30 as seen through histological analysis of the zebrafish eye retinal degeneration, which was followed for 30 days after MNU treatment included disruption of the INL with cavity formation of the ONL beginning at day three, and with maximal loss of Rod ONL observed on day eight through positive tunnel staining in various layers of the sensory retina. It was observed that retinal degeneration after MNU treatment was caused by apoptosis with the majority of tunnel positive cells found in the ONL at day three. However, dying cells were also seen in the INL at the same time point.

After watching this video, you should have a good understanding of how to induce retina degeneration in adult zebrafish by using m and u treatment, as well as how to quantify functional and morphological changes by using OKR measurement and immunohistochemical procedures.