Over the past four years, the rate of autism diagnosed has increased over 30%. Autism spectrum disorder is a neurodevelopmental disorder in which the diagnosed exhibit social and behavioral deficits. Since autism is a life-long impairment, researchers are attempting to rapidly find the causes in the hope to find a cure. It is theorized that prenatal hormone exposure could potentially result in ASD. Therefore, the prenatal hormones estradiol and dihydrotestosterone were subcutaneously injected into mother rats from the CD stand.
Once the female and male rats reached post-natal day 90 or older, they were euthanized, and their brains were extracted, sterilized, and stained in order to count the neurons in the left hemisphere of the cerebellum. Introduction: Over the past three decades, autism spectrum disorder (ASD) rates in children have culminated exponentially. Compared to the 1980`s in which 1 in every 10,000 children was diagnosed with (ASD), today 1 in every 68 children is recognized as having ASD, according to the Center for Disease Control and Prevention (“Facts about Autism”).
ASD is a developmental impairment in which the effected often have difficulty socializing and communicating with others. The disorder which is thought to be caused by a gene mutation can provoke a spectrum of symptoms ranging from lack of empathy and difficulty speaking to repetitive and eerie actions such as flailing one`s arms. As the symptoms may seem like normal, childlike behavior, autism is difficult to detect until the age of two since autism is diagnosed through behavior and social development (Filipek, et. ).
After diagnosis, the individual is able to take medication to help minimize the symptoms, but ASD is a life-long ailment as there is no cure. In order to better understand ASD in humans, researchers are attempting to mimic autistic-like behaviors in rats. Snow, Hartle, and Ivanco theorized that ASD may cause abnormal brain development in both humans and rats. In tandem, Cheng discovered that humans, autistic brains tend to have little differentiation in the genes of the temporal and frontal lobe.
In the temporal lobe, this leads to reduced cortical functional connectivity which affects the ability to create facial expressions due to reduced cortical functional connectivity. Similarly, the frontal lobe lacks the ability to control spatial functions due to reduced cortical functional connectivity (Cheng, et al. ). Researchers have found that when rats exemplify autistic-like tendencies their brain morphology differs from those of unaffected rats. For example, morphological analysis has revealed complex dendritic arborization in the apical dendrites of pyramidal cells in the motor cortex (Snow, Hartle, and Ivanco).
In addition, studied rats had lower neuronal counts in the dorsal hippocampus, which affects cognitive behavior, but greater counts in the ventral hippocampus, which affects temporal association (Bringas, et al. ). Furthermore, Murakami and Courchesne discovered that rats whose cerebellum was studied had significantly smaller cerebellar hemispheres than the control (Murakami, Courchesne, et al. ). Scientists are testing prenatal exposure utilizing an array of hormones such as estradiol and dihydrotestosterone which are bothv often used daily in an attempt to isolate what could cause ASD.
Estradiol is an estrogen sex hormone and is commonly consumed by women experiencing menopause or osteoporosis (Scutti). Additionally, dihydrotestosterone (5? -DHT) is an androgen sex hormone that is responsible for the development of the penis and the prostate in fetuses (Newman). Both sex hormones are believed to perpetuate ASD which is thought to be linked to sex hormones. In order to test this, pregnant rats were injected subcutaneously during days 15-19 – which coincides with the second human trimester – with estradiol, dihydrotestosterone, or corn oil (which acts as a control).
After the pups were born, their associative learning and cognitive mapping was tested through eye blink classical conditioning, Morris water maze, and neuronal changes in the cerebellum. It is hypothesized that the mother rats who are subcutaneously injected with the prenatal hormones estradiol and dihydrotestosterone will produce offspring that may exhibit autistic-like behavior caused by a difference in sexually dimorphic brain structure such as cognitive learning and associative learning. Methods: First, after the test rats from strain CD were euthanized, their brains were extracted and stored in a solution of 3% paraformaldehyde and 1. % glutaraldehyde.
All rat brains studied were postnatal day 90 or older (adults), but both female and male rats were studied. The cerebellum was then equally cleaved with a sterile razorblade to separate the right and left hemispheres. The left hemispheres were used in order to test the neuronal count. To sterilize and prepare the brain for mounting, the left cerebellum was soaked in various solutions of ethyl alcohol and infiltration solution and saturated by placing the brain and it`s solution on a digital shaker.
Immediately after cutting, each brain is placed in an amber vile and soaked with 2 mL 70% ethyl alcohol for two hours. Next, they are placed in 2 mL of 95% ethyl alcohol solution for two hours on the digital shaker. This process is repeated a second time. After four hours have elapsed, each left hemisphere is saturated in 2 mL of 100% ethyl alcohol for one hour. Then, a solution made of 10 mL of infiltration solution and 30 mL of 100% ethyl alcohol. 2 mL of this solution is then placed in each amber vile containing a brain; the samples are placed on the digital shaker for twenty-four hours.
Following, a new solution containing 20 mL of infiltration solution and 20 mL of ethyl alcohol is made, and each left hemisphere is soaked in the solution for twenty-four hours. A new solution containing 30 mL of infiltration solution and 10 mL of ethyl alcohol is made and used to soak the brains for an additional twenty-hour hours. Finally, the left hemispheres are saturated in a 100% solution of infiltration solution. Following this saturation process, the left hemispheres are placed inside the cerebellum molding plates for embedding.
Each sample must be oriented so that the left hemisphere of the cerebellum is anatomically correct as if it were in the rat`s body. A solution is then mixed with activator and infiltration solution mixed in a 1:15 ratio. 5 mL of the solution is then quickly pipetted into the cerebellum mold so that it surrounds and covers the rats` left cerebellum in the mold. The soaked cerebellums are left to sit at room temperature for an hour, and then placed in an oven to assure optimal hardening. Next, the left hemispheres of the rats are cut.
A glass knife is made by cutting a microtome glass with a Ralph glass knife maker. Prior to cleaving, ¬¬¬¬¬¬the glass knife maker must be thoroughly cleaned with pressurized air in order to dispose of previous glass particles. The knife is observed under a light microscope to guarantee there are no abrasions or abnormalities on the glass knife. Continuing, the glass knife and the specimen are both placed on the microtone so that the sample is parallel to the glass knife. Before cutting the brain, the knife and specimen must both be moist ensuring smooth and precise cuts.
The lever is then turned, and the cut is made. Using a #3 brush as a transfer brush, every sixth cut specimen is transferred to a microplate in which each well is filled halfway with distilled water while the other five cerebellum cuts are discarded, so therefore the cutting ratio is 1/6. Before making the next cut, the sample and knife are both remoistened. This process is repeated until the brain specimen is completely cut. Next, the cuts are placed on a slide. First, a petri dish is filled halfway with cold 20% ethyl alcohol.
A slide is placed into the alcohol, and samples, one by one, are carefully placed on the part of the slide submerged in alcohol. After placing, the area of the slide with the specimen is removed from the alcohol in order to dry. Once a slide has four separate brain samples, the slide is dried with kimwipes. Using the edge of each wipe and looking through a light microscope, each sample is dabbed so that any excess alcohol is removed. After a slide has four cerebellums, it is placed on the slide warmer for twenty-four hours.
This process is repeated until the entire left hemisphere is placed on a slide. In order to create a stain for counting the nuclei of the neurons, a working solution of cresyl violet stock is manufactured through mixing three separate stocks- A, B, and C. Solution A, 1% cresyl violet stock, is created through infusing 2 grams of cresyl violet and 200 mL of distilled water, and the solution is stirred for twenty-four hours with a magnetic stir bar. Filtration Solution B, a . 2 M stock, is formed through blending 12 mL of glacial acetic acid and 1 L of distilled water with a stir bar.
Solution C, a . 2 molar stock, is created by mixing 27. 2 sodium acetate and 1 L of distilled water with a stir rod. The final stock solution is made by mixing 83 mL of stock A, 35 mL of stock B, 48 mL of stock C, ad 83 mL of distilled water. To stain the samples, a staining system is created which contains different solutions with 250 mL in each well. In order, the solutions are 100% ethyl alcohol, 95% ethyl alcohol, 70% ethyl alcohol, distilled water, the cresyl violet stock, distilled water, differentiation, 95% ethyl alcohol, 100% ethyl alcohol, and a 100% ethyl alcohol.