The overall goal of this procedure is to develop a quantitative lateral line regeneration assay that can be applied to an adult zebrafish disease model. This is accomplished by first ablating the lateral line of adult zebra fish by treating them for 24 hours with Gentamycin. Next, the Gentamycin is washed out and the fish are allowed to regenerate neuro masts for various times in fresh water.
The fish are then stained with a vital dye to visualize lateral line recovery at designated time points. To confirm that the drug was effective in ablating neuro masts, some fish should be stained immediately after washout of Gentamycin. Then the neuro masts of the lateral lines on fish or skin preparations are counted using images obtained with a fluorescent or confocal microscope.
Finally, the experimental groups are quantitatively compared to the control group using a regenerative neuro mast standard curve. Ultimately, fluorescent and confocal microscopy is used to monitor the regeneration of the lateral line in adult zebrafish disease models. We developed this technique to provide a quantitative assay for the examination of neuronal regeneration in adult zebrafish disease models To induce hair cell necrosis begin by adding a solution of 50 milligrams per milliliter of Gentamycin sulfate in fish water to a final concentration of 0.004%Using a container large enough to house four to six month old zebra fish without aeration, fill it with Gentamycin solution.
Place the fish inside and incubate them at 28 degrees Celsius for 24 hours. Prepare a fish tank of normal fish water to wash out the gentamycin. After washing out the Gentamycin, place the fish in normal fish water and return them to the 28 degrees Celsius incubator for eight to 16 hours from a 15 milligram per milliliter working stock solution of the fluorescent vital dye.
Four four dathyl amino styro and methyl peridium iodide or four dye two A.Prepare a 0.08%concentration in fish water After the designated recovery time from Gentamycin treatment, place each fish in the well of a six well culture plate containing the vital dye. Cover the plates and place them in a bench drawer by the fluorescent microscope for examination of stained neuro masts. Turn off the lights to prevent quenching of the vital dye and incubate them for one hour at the selected hour, post washout of the gentamycin.
Anesthetize each fish by placing them in anesthetic water and wait until their swimming motion ceases approximately one to two minutes. Next, blot each fish on a paper towel and place them on a piece of dampened filter paper centered on the lid of a plastic petri dish. Then place the lid on the stage of a fluorescent stereo microscope and use two x magnification to capture images for quantitative analysis To determine the amount of regeneration, count the number of visible neuro masks within the four designated stitches on the bottom most ventral side of the fish just proximal to the right pectoral fin.
Use the student's T-test or ANOVA for statistical analysis and repeat all experiments three times with a minimum of five fish per experiment. If the quantitative analysis at the level of neuro mast is not significant analysis at the level of the individual hair cell can also be utilized to obtain a higher degree of resolution. After staining and washing out the fish, use a one to 500 dilution of two phenoxyethanol to euthanize them under subdued light.
Make an incision across the belly and make two vertical incisions on each side of the first two incisions. Then make a square skin flap by making an incision along the upper ribs of the fish until it is aligned with the anal fins. Place the skin specimen on a glass slide, place aros on top, and then place a circular glass cover slip over it to help anchor and flatten the tissue.
For subsequent digital imaging. Use a 60 x objective to take digital images of the hair cells within each neuro mast of the mid-body stitches. Then count the hair cells within individual neuro masks for comparative and quantitative analysis of the control and experimental groups.
As seen here, the zebra fish head has a significantly higher number of neuro masts compared to either the midsection or tail with the tail region having the least number of neuro masks as shown in this panel. Because the pattern of stitches in the head is complicated and significantly greater in the number of neuro masks, it did not lend itself as a region for quantitative analysis. In addition, regardless of the Gentamycin concentration tested, complete ablation of neuro masks throughout the head was rarely attainable, leaving spots of neuro masks observed after Gentamycin treatment as previously reported.
In contrast, the tail has two few neuro masks, and as such, we selected the mid-body region to quantitatively analyze neuro mast regeneration. In the adult in this region, we identified four stitches just posterior to the lateral pectoral fin that were consistent in neuro mast number among all adults. Importantly, we were able to consistently and completely ablate the neuro masts of this region by a 24 hour 0.004%Gentamycin treatment as previously reported, allowing for a subsequent accurate determination of neuro mast regeneration as shown in this figure, regeneration was monitored after all neuro masks within the four mid-body stitches were ablated following 24 hour Gentamycin treatment and positive regeneration was determined by the appearance of a minimum of three neuro mast within a stitch by eight hours post gentamycin.
Approximately one third of fish had some sign of recovery. Although as seen here, the intensity of neuromas was faint in the regenerating stitches. The number of neuro masts and their intensity continued to increase in a linear fashion until regeneration reached a plateau at 16 hours post Gentamycin.
If the results obtained from the neuromas analysis are not statistically significant between the control and experimental groups, one may extend these studies to the level of the individual hair cells to obtain a higher degree of resolution for quantitative comparisons at eight hours, 10 hours, and 12 hours of regeneration time. We found that control groups had a range of zero to four hair cells per neuro mast in the mid-body region, as expected for control groups when quantitatively analyzed. No statistical difference between neuromas was detected in terms of the of hair cells per neuro mast at these time points.
While attempting this procedure, it's important to always be conscious of time. Do not leave fish in genamicin for more than 24 hours or vital dye for more than 75 minutes after its development. This technique paved the way for researchers in the field of regenerative medicine to explore the lateral line regeneration in a disease state compared to the normal state and the zebrafish model.
