4. Neurite Growth Research Project-JanPlan 2009

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N,N-Diethyl-meta-toulamide. The active chemical in DEET: 

Flavonoids and Terpenoids are the antioxidant elements in Ginkgo Biloba Extract:

 For many years the Tilden research lab has  focused on crustacean nervous systems, specifically the neural tissue of the fiddler crab, Uca Pugilator. Most of the resarch has focused on the effects of that melatonin has on neurite growth in neurons cultured from the x-Organ/Sinus gland complex of the fiddler crab.  This study was designed with the question of how we could prolong and/or promote the neurite growth in mind.

The fiddler crab was a great model organism for us in that the extraction of specific tissues from its eyestalk proved feasible in our undergraduate lab setting. Before delving into our project, Andrea briefed us on the structures we were about to examine.

Within the eyestalk sits an organ collectively known as the X-organ/ sinus gland, analogous to the hypothalamus and pituitary gland in humans. The somata comprise the x-organ portion of the complex, while the synaptic endings (axons) constitute the sinus gland. The X-organ/ sinus gland regulates and secretes a number of hormones directly into the hemolymph, where it reaches target organs marked by hormone-specific receptors. Cardioactive hormone, pigment dispersing and concentrating hormones, crustacean hyperglycemic hormone, and molt inhibiting hormone, are some of the many integral hormones under the control of the X-organ/ sinus gland.

The X-organ/ sinus gland has three lobes, but we focused primarily on the tissue directly opposite the blue sinus gland, which served as a visual landmark during dissection. We harvested only the X-organ cells because they most closely resemble human hippocampus cells, those cells most important for making synaptic connections during learning and memory,

In researching common dietary and herbal supplements used in Alzheimer's patients, we came across the antioxidant Ginkgo Biloba Extract. Ginkgo Biloba Extract has been used in Chinese medicine for centuries to treat nearly every type of ailment, and has relatively recently made it to the United States as a treatment/preventative measure for Alzheimer's and other neurodegenerative conditions. The flavonoid compounds found in the leaves of Ginkgo Biloba are antioxidants, which bind to free radicals in the cells. By binding to the free radicals, the flavonoids prevent the free radicals from "stealing" electrons from the growing neurite membranes. We hypothesized that this antioxidant property would result in enhanced neurite growth. Furthermore, Ginkgo Biloba extract contains terpenoid compounds, which are less studied, but are thought to enhance neural connections.

We next needed a way to induce oxidative stress within the growing neurites. We decided to treat the neurons with a commonly used insect repellant, DEET. We chose Repel 100, which is 100% DEET (N,N-Diethyl-meta-toulamide). We hypothesized that the free radicals found in the highly toxic substance would inhibit neurite growth and therefore decrease the overall amount of neurite growth in the cells treated with this compound.
The purpose of our project was to determine the impact of oxidative stress and the effects of exogenous antioxidants on neurite growth in neurons extracted from the X-organ/sinus gland of the fiddler crab Uca pugilator.

After removing the eyestalks from anesthetized crabs, we microdissected the eyestalks and removed neural tissue from the X-organ, located directly opposite the opalescent-blue sinus gland. We enzymatically digested the tissue fragments using trypsin, separated the tissue to break loose individual neurons, and cultured the cells for a period of forty-eight hours.

Neural cells grown in a normal culture medium were used as controls. Our experimental cells were cultured in a medium treated with either 100% DEET insect repellent (at a 50% lethal dose), or with 24% standardized Ginkgo Biloba extract (10X the human dose).

After forty-eight hours, the cell cultures were examined under a light microscope at 640X magnification and photographed using AxioVision software. Neurite areas and cell body areas were calculated using Image J, and a ratio of neurite to cell body was calculated.

The results from this experiment showed that both additives resulted in significantly greater neurite growth than the control subject pool, with DEET resulting in the highest growth, followed by Gingko biloba. Our data was compiled over a two-week span, and our sample sizes ranged from n=79 for DEET to n=158 for gingko. Our results do not support our hypothesis, in that the oxidative stress, DEET, proved to aid the neurite growth greater than our antioxidant, Gingko biloba and the control. Some reasons for these results are: 1. DEET sample size is much smaller than the Gingko sample size, 2. Gingko cultures accumulated bacteria from various unknown sources, possibly diminishing/aiding neurite growth in cultures, 3. Possible contaminants entering culture dishes during an allotted waiting period of 48 hours could have altered growth. 

Figure 1. Neurite growth as a function of cell culture additive 

If we had had more time for this experiemnt we would have liked to examine the effects of treating neurons previosuly exposed to the DEET compound, and therefore already oxidatively stressed, with the Ginkgo Biloba extract. Further research could be done on not only the enhancement properties, but also the repairative properties of Ginkgo Biloba Extract. We also would have liked to examined a larger sample size of cells treated with DEET, as this was our lowest smple size. We also encountered some problem with bacterial infections, most likely stemming from an infected culture medium or trypsin solution. We still got results despite the bacteria growth in some of our cultures, but with more time we would have eliminated the infections and hopefully examined more cell growth.

Deet technical fact sheet from the National Pesticide Information Center

 We would like to thank the Colby College Biology Department for their lovely facilities. We also thank Andrea for her wonderful guidance, as well as Escar Kusema and Max Mutter for their incredible Teaching Assistant Abilities, aand Ruth Langton and Jen Myers for their helpful insight.

This experiment was conducted by Ben Rooney, Victor Gagne, Amy Campbell, Christina Mok and Kimberly Parker.