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That Looks Fishy to Me

By Shirin Karimi

Ruth Burley

Jeff Watts

To the eyes of a human, small fish may seem trivial at first glance, often cast aside as county fair prizes and expendable pets. Yet for Ruth Burley, a master’s degree candidate in biology, fish may be a crucial tool in understanding diabetic retinopathy, the leading cause of blindness from diabetes. This progressive condition affects the blood vessels in the eye, leading to symptoms of retinal swelling or retinal scarring, resulting in retinal detachment and, ultimately, blindness. Diabetic retinopathy takes approximately 20 years for symptoms to develop, and since no cure has yet been found the only way to treat the condition is by regulating blood sugar, using lasers to blunt the effects of new vessel growth, and using certain drugs that combat capillary growth. Yet research like Burley’s may change the outlook for diabetic retinopathy.

Burley is continuing her studies at the same institution that helped fuel her passion for biology. Interestingly enough, her foray into research was propelled by a feeling that many college students feel at the conclusion of the spring semester: boredom. She wanted to stay on campus somehow and the biology department provided a solution. “The summer after sophomore year, I applied for the Dean’s Undergraduate Research Award,” Burley recounted. After she won the grant, Burley started her research in Professor Lynne Arneson’s lab, which involved inducing hyperglycemia (high blood sugar) in zebrafish and looking for the expression of the protein vascular enodothelial growth factor (VEGF), which is involved in new capillary growth.

In Arneson’s lab, Burley extracted samples of messenger RNA (mRNA) from the zebrafish eyes using an RNA isolation kit. With the mRNA isolated , she used reverse transcription to convert the mRNA product to a cDNA product and then amplified the quantity available for testing with a process called polymerase chain reaction. Next, using a process called gel electrophoresis, a procedure which separates DNA or RNA product based on sequence length, Burley ran her cDNA product to verify VEGF expression. “If I have VEGF protein expressed, it will appear as a band on the gel at the maker length which corresponds to the VEGF sequence length,” Burley explains.

Ever since that formative summer, diabetic retinopathy has guided Burley’s research career. Since hyperglycemia is a characteristic symptom of diabetes, Burley would observe the changes seen in the hyperglycemic zebrafish over a month and analyze these changes to see if the effects resemble those experienced by humans with diabetes.

Burley hopes zebrafish can be used as a model for humans in understanding the cellular basis of diabetic retinopathy since this specific species of fish is inexpensive, easy to take care of, and undergoes stages of development from the embryonic stage that are very similar to that of humans.

As a senior, she concentrated on another protein called glial fibrillary acidic protein (GFAP), a protein located in the retinal Müller glial cells that provides support and strength to glial cells. Since Burley knew from her literature research that GFAP expression increases within glial cells due to trauma, she wanted to see “if GFAP expression increases as a result of diabetic retinopathy.” The results of this experiment eventually culminated in Burley’s senior honors capstone project.

Burley graduated last May with her undergraduate degree in biology and wasted no time in continuing her research on diabetic retinopathy in the zebrafish model in the five-year combined BS/MS program. With a $500 grant from the biology department, she began researching in Professor Victoria Connaughton’s lab for her master’s thesis. Continuing her work concerning GFAP, Burley uses a procedure known as immunocytochemistry, which identifies protein expression in types of tissue. Using both hyperglycemia-induced zebrafish and untreated (control group) zebrafish, Burley cut the eye into slices, then stained and incubated the slices with antibodies so that they fluoresce in the presence of GFAP. Once the fluorescing antibodies are bound to the protein of interest in the retina of the zebrafish, Burley can examine the changes in the retinal tissue and compare the hyperglycemic fish to the control group. Her previous test for GFAP allowed her to distinguish if GFAP expression was occurring. With Burley’s new work she hopes to identify where the expression of GFAP is occurring within the retina.

Burley, however, does not spend all of her time conducting research. She is also busy working as a receptionist in an ophthalmologist’s office. Luckily, her employer provides very useful information on the condition that Burley has spent the past three years studying. Currently, she has been spending time researching pericytes, cells that surround the endothelial cells, preventing any permeability of the retina vessels. The ophthalmologist informed Burley that diabetic retinopathy causes an immediate decrease in pericytes, resulting in a leakage of the albumin, a protein in your blood, into the retina. “What we are hoping to do is use trypsin digest (the enzyme trypsin is a type of digestive enzyme found in the intestines) to expose the vasculature of the zebrafish eye,” Burley explains.

“In healthy individuals, you have a 1:1 ratio of pericytes to endothelial cells.” By examining the vasculature of the zebrafish eye, the ratio of the pericytes to endothelial cells can be calculated and compared in hyperglycemic and control animals. Burley will begin these experiments in the spring.

Since her research projects will conclude this year, the next step for Burley is a career in medicine. This spring, she plans to apply to medical schools and is contemplating specializing as a general practitioner. In the words of her master’s thesis advisor, Professor Connaughton: “Ruth has great potential as a scientific researcher. She is very comfortable in the lab and she is a thorough and careful scientist.” Whatever scientific career she decides to pursue, Burley certainly knows no bounds.