Effects of Ivermectin on Planaria Regeneration

By: INT5 Group 3, Branden Damus, Sophie Knifong, Charles Lee, Lucy Miller, Adam Moustapha, Nicolas Vargas

​

Background

• Original project by Nina Ferenc and Michael Levin.
• Planaria are one of many different species of flatworms and are known for their ability regenerate stem cells and damaged tissue.
• Ivermectin is typically used as a drug to treat tropical parasitic-infections caused by Nematodes.
• Effects voltage gated ion channels found in Nematodes(a sub-species of worms, roundworms) causing death by paralysis or starvation.
• This study focused on the changes in survival rate and regeneration rate.
• Also recorded changes and abnormalities in the planaria.

​

Figure 14. Chemical structure of Ivermectin B1A, C₄₈H₇₄O₁₄. The compound we used in the project. Image obtained from Pubchem.

Figure 15. Enhanced image of many planaria. The subjects of the project. Image obtained from HHMI.

Hypothesis

Null hypothesis A: If Planaria are treated with a .5μM Ivermectin and .01% DMSO (Dimethyl Sulfoxide) and spring water solution, and then dissected into the tail, trunk and head, the average rate of growth would not show a statistically significant difference between the average rate of growth of Planaria undergoing the same conditions except with only a .01%DMSO(Dimethyl Sulfoxide) and spring water solution.

Null hypothesis B: If Planaria are treated with a .5μM Ivermectin and .01% DMSO (Dimethyl Sulfoxide) and spring water solution for 48 hours, and then dissected into the tail, trunk and head and observed for 14 days, the percent probability that a randomly selected fragment would have physical abnormalities, would not show a statistically significant difference between the the percent probability that a randomly selected fragment would have physical abnormalities of Planaria undergoing the same conditions except with only a .01%DMSO(Dimethyl Sulfoxide) and spring water solution.

Methods

1. Create 10 mL of 10mM Ivermectin DMSO solution by combining 87.5mg of Ivermectin and 10 mL of DMSO.
2. Prepare Treatment 2,(A 0.5μM Ivermectin, .01% DMSO spring water solution) the Independent Variable of our project, in a series of 3 serial dilutions.
3. Prepare Treatment 1,(A .01% DMSO spring water solution),the Constant of the experiment.
4. Soak 12 planaria for 48 hours pre-amputation in Treatment 1,(The Control Group).
5. soak 12 planaria for 48 hours pre-amputation in Treatment 2(The Experimental Group).\
6. Trisect the 12 planaria in the Control group into a head, trunk and tail separating 3 homologous fragments into each well of your 6 well plate. Mark the wells that contain heads, trunks and tails with their respective names and name the well, Well 1.
7. Repeat with 12 planaria in the Experimental group in a separate well, but mark Well 2 instead.
8. Record measurements on the length, % of surviving fragments, and any physical abnormalities noticed on the day of amputation and 2,7,9, and 14 days post-amputation.

​

​

Analysis

Administration of Ivermectin

1. Drug approved for human consumption, effective in combating Nematodes
2. Ivermectin was diluted in a DMSO solution
3. Not all effects on the activation of voltage gated chloride channels on stem cells are known, but their effects on other traits are.
4. Traits reflecting Ivermectin successful administration of Ivermectin were found in the subjects

Figure 1: Video showcasing changes in neuromuscular signalling in Planaria in both the control and experimental groups. Control = Planaria fragments following treatment in .01%dimethyl sulfoxide and amputation. Experimental = Planaria fragments after the .05uM Ivermectin and .01% dimethyl sulfoxide treatment.

Survival data

1. Challenge that researchers before us faced was the lethality of drugs that alter voltage gated channels in worms.
2. Dealing in such small concentrations makes it difficult to dilute to other concentrations once past a certain point.
3. Forced us to use only one concentration as in other studies anything higher than .05μM led to nearly 100% mortality rates and we were unable to dilute past .05μM without precipitation occurring

Figure 2: Graph of Control and Experimental Groups’ percent survival after treatment on observation days. Control = Percentage of Planaria Fragments alive following treatment in .01% dimethyl sulfoxide and amputation on the recorded date. Exp. = Percentage of planaria fragments still alive after the .05uM IVM and .01% dimethyl sulfoxide treatment. Table created using rapidtables.com.

Regeneration data

Figure 3. Average Length of control and experimental planaria head fragments on the given date. Control = Planaria fragments following treatment in .01%dimethyl sulfoxide and amputation. Experimental = Planaria fragments after the .05uM Ivermectin and .01% dimethyl sulfoxide treatment.Bars represent 2SEM’s. Reveals there is no statistically significant difference the rate of regeneration in head fragments due to Ivermectin. Table created using Google Sheets.

Figure 5. Average Length of control and experimental planaria tail fragments on the given date. Control = Planaria fragments following treatment in .01%dimethyl sulfoxide and amputation. Experimental = Planaria fragments after the .05uM Ivermectin and .01% dimethyl sulfoxide treatment.Bars represent 2SEM’s. Reveals there is no statistically significant difference the rate of regeneration in tail fragments due to Ivermectin. Table created using Google Sheets.

​

Figure 4. Average Length of control and experimental planaria tail fragments on the given date. Control = Planaria fragments following treatment in .01%dimethyl sulfoxide and amputation. Experimental = Planaria fragments after the .05uM Ivermectin and .01% dimethyl sulfoxide treatment.Bars represent 2SEM’s. Reveals there is no statistically significant difference the rate of regeneration in trunk fragments due to Ivermectin.​ Table created using Google Sheets.

​

​

Recording

Recording

Physical

Abnormality Data

• Did not discover drastic changes in the regenerated fragments, ie: no fragments with two heads or two tails.
• Discovered other abnormalities that weren’t present in the control worms.
• All worms, in both the control and experimental groups, recorded on 10/25/2022 were fully regenerated(meaning they all had a head tail or trunk).

Figure 6: Image of a fully regenerated Control Group Planaria tail fragment. Control = Planaria Fragments following treatment in .01% dimethyl sulfoxide and amputation on the recorded date. Image captured 10/25/2022(14 days post-amputation)

Images of Physical Abnormalities

Figure 7. Image of Planaria trunk fragment from experimental group with improperly developed eyes. Experimental = Planaria fragments after the .05uM Ivermectin and .01% dimethyl sulfoxide treatment.Image captured 10/25/2022 (14 days post amputation)

Figure 9. Image of Planaria tail fragment from experimental group with uncommon pigmentation. Experimental = Planaria fragments after the .05uM Ivermectin and .01% dimethyl sulfoxide treatment.Image captured 10/13/2022 (2 days post amputation)

Figure 8. Image of Planaria head fragment from experimental group with a bifurcated tail. Experimental = Planaria fragments after the .05uM Ivermectin and .01% dimethyl sulfoxide treatment. Image captured 10/25/2022 (14 days post amputation)

Figure 10. Image of Planaria trunk fragment from experimental group with ridges visible on body. Experimental = Planaria fragments after the .05uM Ivermectin and .01% dimethyl sulfoxide treatment.Image captured 10/18/2022 (7 days post amputation)

Physical

Abnormality Data Cont.

1. Counted any physical change as an abnormality.
2. There could be many changes due to the alteration of gated voltage gated channels but we are unable to observe them.
3. Did not include changes in movement pattern due to possibility that it may be a direct effect of the voltage gated channels and not necessarily due to changes in the cell differentiation process.
4. Figure 11 represents the % probability that any given worm in either the control group or experimental group has an abnormality.

​

Figure 11. Bar graph depicting the percentage of planaria fragments that were observed to have a physical abnormality(difference in body structure, difference in color, difference in development, difference in appearance) 14 days post-amputation.Control = Percentage of planaria fragments showcasing abnormalities following treatment in .01% dimethyl sulfoxide, also referred to as solution 1, and amputation. Experimental = Percentage of planaria fragments showcasing abnormalities after the .05uM IVM and .01% dimethyl sulfoxide treatment, also referred to as solution 2, and amputation. Table provided by rapidtables.com.

Physical

Abnormality Data Cont.

• Utilized a one way ANOVA, analysis of variance, test to statistically support that there was a significant difference in cell differentiation due to the opening of glutamate gated chloride channels.
• ANOVA tests are used to measure if there is a difference between the means of 2 or more groups.
• At a significance value of .x, our data would support the idea that we are (100-x)% certain the difference in physical abnormalities is due to the difference in treatments

Figure 12. Explanation of a One-way ANOVA, Analysis of Variance Table provided by Analytics Buddha.

Physical

Abnormality Data Cont.

• Means used in One-way ANOVA test represented the percentage probability that a randomly selected.
• Able to compare treatment 1 and treatment 2 and see whether the data supports or rejects the hypothesis.
• The critical value, 7.085, highlighted in red, can be found in the F-Distribution table which tells us the corresponding p-value is .00983.

​

One way ANOVA test of Treatment 1 and Treatment 2

Figure 13. Completed One way ANOVA test, a statistical test used to measure and compare the difference of means of 2 or more groups. The source, Between-Treatments is representative of the two groups receiving treatments. The source, Within-Treatments is representative of the expected error. The symbol SS is representative of the square sum. The symbol df represents the degrees of freedom. The symbol MS is representative of the mean sum. The symbol F, highlighted in red, is representative of the mean sum of Between-Treatments divided by the mean sum of Within-Treatments, leaving you with your critical value. Critical values derived from a One-way ANOVA test can be used in the F-Distribution table to find a corresponding p-value. Values in table gained by following equations in Figure 12.

Conclusions

• Change in movement patterns suggest we were successful in diffusing Ivermectin into the Planaria.
• Lack of polyemia indicates glutamate gated chloride channels are not involved in the beta-catenin and WNT pathway.
• P-value of .00983, from ANOVA test, suggests that at a .01 significance rate, the data we collected from treatments 1 and 2 reject Null Hypothesis B with more than 99% certainty.
• Don’t have the tools to see if the reason behind the mutations is cell differentiation.
• Improperly analyzed the rate of growth for planaria fragments so we are unable to reject Null Hypothesis A.
• Given more time and more tools, could expand the treatment.

​

Acknowledgments

We would like to thank the Howard Hughes Medical Institute for allowing us to conduct this research. We would also like to thank Dr. DiResta and Dr. St. Hilaire for providing us with their guidance and support throughout the experimental process. We thoroughly enjoyed our time in this lab and we hope we are able to work together in the near-future.

References

Figures and Origins

Figure 1, Figure 6, Figure 7, Figure 8, Figure 9, Figure 10 and Figure 13: Captured by INT 5 Lab Group 3

Figure 2 and Figure 11: https://www.rapidtables.com/, Created by INT5 Lab Group 3

Figure 3, Figure 4, Figure 5: Google Sheets, Created by INT 5 Lab Group 3

Figure 12: https://analyticsbuddhu.wordpress.com/

Figure 14: https://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?cid=6321424&t=l

Figure 15: https://www.youtube.com/watch?v=refo1mi9xDM, Captured at time 1:10 in video.

​