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Introduction The cornea is considered the clear outer “window” of the eye, it is an important integument. The cornea contributes ~70% of the eye’s optical power. There is no vascular supply in the cornea; nutrient, waste and gas exchange are between cornea, tears and the aqueous humor. The corneal stroma is surrounded on both of its sides by an epithelium and an endothelium that when damaged causes swelling. The epithelium and endothelium are what keeps the stroma thin and transparent. In eye banks the epithelium and endothelium can be damaged causing corneas to be untransplantable. The corneal stroma is composed of numerous sheets or layers stacked on top of each other into lamellae, which resembles the layers in plywood. It is also hydrophilic, and it is almost hygroscopic, meaning that it takes in most of its moisture from the surrounding air. These collagen fibrils are surrounded by proteoglycans that are hydrophilic also. These proteoglycans are composed of GAG-s (glycosaminoglycans) that are very polar and form gels in water. The negative charges of the GAG-s are located on the SO3-and COO-. Corneal edema is the swelling of the cornea. MgSO4 has already been proven to reduce the rate of edema in the corneal stroma. NaNO2 is collagen cross-linker that should reduce edema of the cornea even more when paired with MgSO4. Effects of MgSO4 and NaNO2 on Bovine Corneal Stromas • PBS v.MgSO4 Calf Eyes: Three vials are labeled with the numbers 1, 2, and 3. 1, and 2 were filled with PBS while vial 3 was filled with 148mM MgSO4. The corneas were • removed from the beaker and wet weights were measured at 0-180 • mins as before. Then the weight of the dry cornea was measured. • PBS v. MgSO4+NaNO2+NaH2PO4 v. NaNO2+NaCl+NaH2PO4: 6 • vials were labeled as group 1A-6A these jars contained PBS. 7 • vials were labeled as group 1B-7B these jars contained the • 48mM MgSO4+100mM NaNO2 mixture. 7 more vials were • labeled as group 1C-7C these vials contained 100mM NaNO2+48mM • NaCl mixture. The corneas were removed from the beaker and wet • weights were measured at 0-180 mins as before. Then the weight of • the dry cornea was measured. • 24 hr PBS v. MgSO4+NaNO2+NaH2PO4 v. NaNO2+NaCl+NaH2PO4 Pretreated corneas: After corneal buttons were punched they were bathed for 30 min in PBS (6 stromas), 48mM MgSO4+100mM NaNO2 mixture (7 stromas), 100mM NaNO2+48mM NaCl mixture (7 stromas). They were placed back into the humid beaker overnight. The corneas were removed from the beaker and wet weights were measured at 0-180 mins as before. Then the weight of the dry cornea was measured. • Fig.5 Pretreatment results of • Hydration v. Time • Fig.1 corneal • illustration • All of the corneas that were bathed in NaNO2 turned • Red to orange in color and those bathed in NaNO2+MgSO4 • Turned to a deep-dark red orange. Results The results were discovered by finding our Hydration v time during the experiment. Hydration was found by first entering the recorded data on to Excel, running the data through functions and equations, and by finally graphing it in Excel. The equation that was used to determine Hydration of the corneal stroma was: H=(wet weight x dry weight) / dry weight. For our last two sets of graphs we went with the average and took standard deviations of the groups since there were so many experimental groups (ranging from 1A-7A to 1C-7C). A T-test determined that corneas bathed in MgSO4 swelled slower than those bathed in PBS. Analysis of variance determined that pretreated corneas bathed in NaNO2 swelled slower than corneas bathed in PBS. • Fig.2 GAGs of • proteoglycans Cameron Byrd, and Dr. Joseph Huff Department of Biology, University of West Georgia, Carrollton, GA 30118 Conclusions The samples that were collected were bathed and then had their data run through Excel. The graphs to the left were the outcomes of each of the experiments. In Fig.3 it was seen that PBS swelled corneas faster than MgSO4. In Fig.4 it can be seen that the corneas soaked in MgSO4 and NaNO2 slightly reduced swelling rates of all the experimental groups. Regular NaNO2 with NaCl was not as high of a rate as PBS. Fig.5 shows results from the corneas that were bathed in the NaNO2 solution with and without MgSO4. From the data at the left it is shown that once more NaNO2 with MgSO4 was more effective at slowing the swelling rates of the corneal stroma than NaNO2 + NaCl and PBS. • Procedure • Bovine (beef) eyes were collected from a number of different slaughterhouses. Cattle eyes and a set of calf eyes were requested on the day of the kill. Upon the eyes arrival to the lab the corneal epithelium and endothelium were removed and a corneal button was punched out. • The corneal buttons were then placed on the inside of a humid beaker. • The beaker was covered and stored in a refrigerator overnight. All • experiments were started within a 24-48 hr. period after the death of the animal. • MgSO4 v. PBS (Phosphate Buffered Saline): • 4 vials were filled, two with PBS, two with MgSO4. • They were labeled groups A and B. Group A being • PBS, and group B being the MgSO4. The corneas • were removed from the beaker and placed on a • scale to take the 0 time wet weight. After the first • cornea was weighed and placed in the bathing • solution a timer was that was preset for 15 min. • was started. The remaining corneas were weighed and placed in their respective bathing solutions.When the 15 min. were up the corneas were weighed for wet weight and placed back in the bathing solution for another 15 min. The wet weight was taken at 30, 60, 90, 120, 150, and 180 time increments. After the last reading the corneas were placed inside of an oven overnight to dry. Then the weight of the dry cornea was measured. Hydration Analysis • Acknowledgements • Zach Hogan; assistance with the project. • Grant Funding National Science Foundation STEP grant # DUE-0336571 • Generating Enthusiasm in Math and Sciences (G.E.M.S) Groups: 1A=PBS 2A=PBS 1B=MgSO4 2B=MgSO4 • Fig.3 MgSO4 Results of Hydration v. Time • Fig.4 NaNO2 Results of Hydration v. Time
