Effects of MgSO4 and NaNO2 on Bovine Corneal Stroma

By: Cameron Byrd

Advisor: Dr. Joseph Huff

Biology Department

Abstract:

The objective of this research was to discover the effects that MgSO4, NaNO2, and bathed NaCl have on the corneal stroma. We already know that MgSO4 slows corneal edema a relative amount. What we wanted to find out is if NaNO2 cross-links the collagen of the stroma to reduce swelling even more than MgSO4. We wanted to explore this for their potential use in eye banks. Right now eye banks can only hold eyes for a limited amount of time; we hope to find a way to prolong the storage of corneas to insure that they are transplantable.

Introduction:

The cornea is considered the clear outer “window” of the eye; it is an important integument (protective layer). The cornea contributes ~70% of the eye’s optical power. There is no vascular supply in the cornea; nutrients, wastes and gasesare exchanged between cornea, tears and the aqueous humor. The corneal stroma is a connective tissue surrounded on both of its sides by an epithelium and an endothelium that when damaged causes swelling. The epithelium and endothelium are water transporting tissues that keep 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 can take in moisture from the surrounding air. Its 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 OSO3- and COO-. It is these negative charges on the GAG-s that produce corneal edema. Corneal edema is the swelling of the cornea.(1, 2) When the cornea swells it loses transparency. (3, 4)MgSO4 has already been proven to reduce the rate of edema in the corneal stroma.(5, 6) NaNO2 is collagen cross-linker that should reduce edema of the cornea even more when paired with MgSO4. Dr. Huff already ran the MgSO4 experiment before. First studied MgSO4 v. phosphate buffered saline (PBS) to get up to date with the lab and the procedures on how to conduct these experiments, and studied the effects of NaNO2 with NaCl or MgSO4 compared to PBS.

Materials and Methods:

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 was scraped off using a scalpel blade while applying pressure, but not enough to damage the cornea; just to remove epithelium. Once the epithelium was removed a scleral skirt was cut around the cornea about 2-4 mm from the outside edge of the cornea. The aqueous humour, irides, and lens were removed from the skirt. The skirt was then stretched on a Teflon block and a corneal button was punched from the skirt using a hammer and an arch leather punch. The corneal buttons were then placed on the side of a humid beaker. The beaker was covered with Parafilm and stored in a refrigerator overnight. All experiments were started within a 24-48 hr. period after the death of the animals.

  • MgSO4 v. PBS (Phosphate Buffered Saline): 4 vials were filled, two with 148mM PBS, two with 148mM MgSO4. They were labeled groups A and B. Group A beingPBS, 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 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 also taken at 30, 60, 90, 120, 150, and 180 time increments. In between readings empty vials were pre-weighed, and labeled so at the end of the 180 min the corneas were placed in an oven to dry over night. The corneas’ dry weightswere then measured.
  • PBS v. MgSO4 Calf Eyes: Three vials were 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 0-180mins as before. Then the weight of the dry corneas 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 corneas 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 corneas was measured.

Results:

The results were discovered by finding our Hydration v time duringthe 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 (H) 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 corneas per groups (ranging from 6-7). 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.

Conclusions:

The samples were collected were bathed and then had their data run through Excel. The graphs above were the outcomes of each of the experiments. In Fig.1 above it shows that the cow and bull eyes we received from the slaughterhouse had damages and were already kind of old; Even thought they were kind of old, the resutlts were the same, that MgSO4 inhibited the swelling of the corneas. In Fig. 2 it was seen that PBS also swelled calf corneas faster than MgSO4. The first experiment was to confirm results already found by Dr.Huff; I used experiments 1 and 2 to better understand the procedures in the lab and how to go through completing an experiment and its analysis. In Fig.3 it can be seen that the corneas soaked in MgSO4 and NaNO2 with NaCl, or NaNO2 with MgSO4 slightly reduced swelling rates of all experimental groups. Swelling in NaNO2 with NaCl or NaNO2 and MgSO4 was not as high of a rate as PBS. Fig.4 shows results from the corneas that were bathed in the NaNO2 solution with NaCl without MgSO4, or bathed in solution with NaNO2 with MgSO4. From the data above it is shown that once more NaNO2 was ore effective at slowing the swelling rates of the corneal stroma than NaNO2 + NaCl and PBS. The findings that NaNO2 pretreatment inhibited swelling are consistent with its ability to cross-link collagen in the stroma, reducing its hydration properties. These findings may be useful for inhibiting corneal swelling in the eye banks, or possibly for increasing or decreasing corneal refractive power in foresighted or nearsighted people.

References

1. Harris, J.E.; “Symposium on the Cornea Introduction: Factors influencing corneal hydration”; Investigative Ophthalmology; Vol. 1; Num. 2;pg.151-157; April 1962

2. Huff J.W; “Chapter 5: Corneal Stroma”; Bennet E.S, Weissman B.A; Clinical Contact

Lens Practice; Chp. 5; pg. 1-10.

3. Kostyuk O., Nalovina O., Mubard T.M., Regini J.W., Meek K.M., Quantock A.J.,

Elliot G.F., Hodson S.A.; “ Transparency of the bovine stroma at physiological hydration and its dependence on concentration of the ambient anion”; J. Physio.2002. 543; 633-642.Jul. 5.

4. Wiederholt M, Koch M; “Effect of intraocular irrigating solutions of intracellular membrane potentials and swelling rate of isolated human and rabbit cornea”; Investigative Ophthalmology and Visual Sci.; Vol 18. Num 3; p.313-317; March 1979.

5. Huff, J.W, Olanrewaju J., Uhkheme M., Sukhitshvili G.; “Ion-Dependent Hydration

and Transparency of the Corneal Stroma, a Polyelectrolyte Biogel”. Macromol. Symp. 2005, 227:311-320.

6. Paik D.C, Saito L.Y, Sugirtharaj D.D, Holmes J.W; ”Nitrite-Induced Cross-Linking

Alters Remodeling and Mechanical Properties of Collagenous Engineered Tissues”Connective Tissue Research, Volume 47, Issue 3 ;June 2006 , pages 163 – 176.