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How Place of Pressurization Effects Ocular Structures HOW PLACE OF PRESSURIZATION EFFECTS OCULAR STRUCTURES Mikayla Ferchaw, Ning-Jiun Jan, Ian Sigal, PhD. Laboratory of Ocular Biomechanics, Department of Ophthalmology, University of Pittsburgh School of Medicine INTRODUCTION loading could be observed and clearly indicated on the obtained Glaucoma is the second leading cause of irreversible images produced. Once the eyes were fixed, they were blindness worldwide [1]. The main risk factor for glaucoma is cryosectioned axially. Cryosectioning is a process where the elevated intraocular pressure (IOP), which is regulated by the eyes are frozen and then sliced into very thin sections, which in production and drainage of aqueous humor in the anterior this case is 30 microns. Next, the sections were imaged with chamber of the eye [2]. Whole eye pressurization experiments polarization light microscopy and then loaded into FIJI, an can be used to understand how increased IOP affects different image processing package, where the collagen fiber bundles structures in the eye and how that results in risk for were then marked in small increments. Simultaneously, the glaucomatous damage [3]. Current pressurization experiments, original images were processed to determine collagen fiber however, consider pressurization through the anterior and orientation. The final step to this experiment was performing vitreous chamber as interchangeable and equivalent. IOP is the statistical analysis in R, a statistical coding software, to find regulated by the dynamics of the anterior chamber, not the the difference in collagen waviness in the different regions of vitreous chamber [3]. The anterior chamber is continuously the eye when pressurized through the anterior chamber versus replenished with aqueous humor via the trabecular meshwork the vitreous chamber. There are 17 different regions of interest located at the base of the cornea, where the vitreous chamber where regions 1, 16, and 17 are classified as peripapillary sclera has a relatively constant amount of vitreous humor throughout a (PPS), regions 2-7 are classified as the nasal sclera, regions 8- person’s life [3]. It is with these details that the vitreous 10 are classified as cornea, and finally regions 11-15 are chamber of the eye is believed to be a closed vessel and act as classified as the temporal sclera. such when pressurization experiments are performed on eyes [3]. Many believe these two experimental conditions are RESULTS equivalent, but from what research has suggested, those Figure 1 below shows a boxplot of the graph of Waviness scientists seem to be mistaken. The local forces on the various vs. Region Number, where each region of interest is grouped parts of the eye may be different depending on the place of into PPS, nasal sclera, cornea and temporal sclera. It can be pressurization, which can be seen in Bellezza’s 2003 seen in this figure that the corneal regions (labeled in purple on experiment with monkey eyes, where the team saw differences the figure) have a higher amount of variance, or spread amongst in the size of ocular structures fixed at high IOP [4]. The term, the data, than the PPS, nasal sclera and temporal sclera. ‘waviness’, is used to describe the measure of the standard deviation of the fiber orientation in a small selection of tissue. High loading results in a straighter collagen arrangement and low waviness values. Conversely, low loading results in a more “wavy” arrangement, not as straight, with high waviness values. The objective of this research project is to determine whether cannulation of sheep eyes from the anterior chamber through the cornea or from the vitreous chamber through the sclera will result in different loading on different regions of the eye, namely the corneal and scleral regions under the two pressurization methods. The hypothesis for this research project is that pressurizing through the anterior chamber will have different effects on the local collagen microstructure than Figure 1. This boxplot graph shows waviness values for each pressurizing through the vitreous chamber. This experiment region number grouping within five eyes worth of data when will be considered successful if the various statistical analysis pressurized through the anterior chamber (shown in blue) and measures produce p values less than 0.003 for 17 regions and the vitreous chamber (shown in red). The corneal regions show 0.0125 for 4 regions via a Bonferroni correction, which would a higher variance than the PPS, nasal sclera and temporal sclera show that the waviness values for the anterior chamber and the overall. vitreous chamber are, in fact, significantly different. Linear mixed effect (LME) models created within R were then assessed to further affirm the claim that Figure 1 has METHODS exhibited. Figure 2, shown below, reports the results of the This study fixed five sheep eyes at a high IOP of 50 mmHg linear mixed effect models when testing the regions of interest, (normal sheep eye pressure is around 10 mmHg), in which two 1-17, standing alone (A) and when the regions are grouped eyes were pressurized through the anterior chamber and three together (B). A Bonferroni correction was used, set alpha to were pressurized through the vitreous chamber, where high 0.05, then we divided by the number of regions to get a 1 significance value of 0.003 for picture A and 0.0125 for picture assumes a skewed distribution. The cornea showed a B (divide by 17 regions for A and 4 regions for B). When the significant difference in waviness between the anterior and regions of interest were assessed separately (A), none of the vitreous regions at a value of 0.006. values of the means were deemed significantly different (less than 0.003). Thus, we decided to test if the means would be DISCUSSION significantly different when assessed in the respective We hypothesized that pressurizing through the anterior groupings (B). With this data, the cornea and temporal sclera chamber will have different effects on the local collagen resulted in a p value of less than 10e-15, which is a very small microstructure than pressurizing through the vitreous chamber. value close to 0, deeming these two regions to be significantly Based on the results of the study, this hypothesis was deemed different since the values were lower than 0.0125. to be true. Looking at the mean differences with the LME test, both temporal sclera and cornea were significant, but it should B be noted that the variability in the temporal sclera is a lot higher than the cornea and could contribute in a false positive for that test (Figure 1). Ansari-Bradley and F test would express the A same results if the data set had a normal distribution. Since the data was skewed, the Ansari-Bradley test was the most accurate test to use for our analysis. Only cornea showed differences in variability of waviness depending on pressurization location. Why? Since cornea was significant in Ansari-Bradley, it means that the cornea is more variable between anterior and vitreous pressurizations. Physically, this means that the vitreous chamber is a closed chamber. Because the temporal sclera was showing significance in the F test, but a large p value in the Ansari-Bradley, we deemed this a false positive as well. Figure 2. Linear mixed effect (LME) models used to assess Looking to the future, an experiment we can do is to measure significance of the regions of interest by way of a p value. In the pressure in the anterior chamber when pressurizing through figure A, the regions of interest were evaluated separately and the vitreous versus the anterior chamber. If the pressure in the none of them resulted in a value less than 0.003. In figure B, anterior does not match what we set it at when we pressurized the regions were evaluated in groups and both the cornea and through the vitreous chamber, then we know that the anterior temporal sclera were deemed significant since both of those chamber is leaking. regions reported a value lower than 0.0125. It was decided that since these tests did not produce ACKNOWLEDGMENTS consistent results when analyzing the data region by region vs I would like to thank my PI, Ian Sigal and my mentor, when the regions were grouped, we decided to test the Ning-Jiun Jan. Danielle Hu, my marking team, Zachary variability of the collagen fiber orientation using the F test and Adgate, Jennifer Panza, and Brienna Roys, and Ryan O’Malley the Ansari-Bradley test. Figure 3 shows these results below. deserve an acknowledgment as well, since my project focused The F test looks at the variability of one group and compares on obtaining data from their markings and analyzing that that to another group, assuming a normal distribution, where information. the Ansari-Bradley test numbers values from widest ranges into the middle and compares groups, assuming a skewed REFERENCES distribution. Since the Ansari-Bradley test takes away the [1] Sanchez, I. (2014). Measurement of Intraocular Pressure in power of the mean and focuses in on the median and quartiles, a Porcine Ex Vivo Model Eye. Ophthalmology we believe that it is the most correct test for our data set Research: An International Journal OR, 2(2), 55-64. because we know that all of the means and medians are far [2] Makoto Ishikawa, Takeshi Yoshitomi, Charles F. Zorumski, away from each other based off of our boxplot results, and Yukitoshi Izumi, “Experimentally Induced indicating a skewed distribution. Using the Ansari-Bradley test Mammalian Models of Glaucoma,” BioMed Research and Bonferroni correction making a p value of <0.0125 International, vol. 2015, Article ID 281214, 11 significant, the corneal region was the only region that fell pages, 2015. doi:10.1155/2015/281214 below the 0.0125 range with a value of 0.006.
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