Animal models play an important role in glaucoma research. With rodent models, for example, investigators can precisely control potentially confounding factors such as genetic background and diet, design invasive studies, and perform experiments on large populations. There are, however, significant limitations to animal research. With regard to the portions of the ocular anatomy affected by glaucoma, animal eyes are unable to completely duplicate the structures in the human eye. The optic nerve head (ONH) and trabecular meshwork (TM) in rats and mice come the closest, although these animals lack a true lamina cribrosa. Other structures also differ.
Moreover, animals do not spontaneously develop primary open-angle glaucoma, the most common form of the disease in humans. Researchers have worked around this obstacle by inducing glaucoma in rats and mice through the application of laser energy or by damaging the TM to occlude aqueous humor outflow. This effectively raises the IOP but does not mimic the mechanisms that lead to elevated IOP and glaucomatous damage in humans.
For the past 25 years, human tissue has been an invaluable resource in my efforts to identify the molecular and cellular pathways for damage to the TM in glaucoma. The Lions Eye Institute for Transplant and Research (LEITR) has supplied the majority of that tissue.
BROADENING ACCESS TO TISSUE
As the world's first combined eye bank and ocular research facility, not only does LEITR obtain large volumes of high-quality, fresh tissue, but the organization also recently enhanced its commitment to research. LEITR allocates about half of the donor tissue it collects annually to research. Additionally, the institute logs each donated eye into a comprehensive database with extensive information about the donor's clinical and familial medical history.
LEITR draws from a large population (mostly elderly) with high rates of glaucoma and other conditions of interest to ocular researchers. Investigators can dissect tissue, perform imaging or cell cultures, and begin experiments in LEITR's high-technology research facility, which even has on-site sleeping quarters for visiting researchers. This is a tremendous resource for those whose work is constrained by the need to obtain postmortem tissue very quickly.
IDENTIFYING PATHOGENIC PATHWAYS
A primary focus of my work has been identifying the molecular and cellular pathways for damage to the TM and ONH. My colleagues and I have used human tissue in a number of ways toward this goal. First, we have cultured cells from human TM and ONH tissue in both normal and glaucomatous eyes (Figure 1). We have also used an ex vivo perfusion organ culture model in which the anterior segment is specially mounted and perfused with media (Figure 2). This maintains the aqueous humor outflow pathway and allows the tissue to be cultured over a relatively long period of time (weeks instead of hours) to discover the molecular mechanisms that regulate IOP in the anterior chamber. This model has helped us to validate some of the signaling pathways initially identified in cultured cells.
We have also successfully harvested RNA from both cultured cells and the actual donor eye tissue to compare gene expression in normal versus glaucoma eyes. Once we identify the differential expression of a specific protein or gene in the cultured cells, my colleagues and I conduct immunohistochemical testing on tissues to make sure the differential expression is not just an artifact of a cell or organ culture (Figure 3).
The technology now exists with which to evaluate gene expression easily and rapidly using messenger RNA (mRNA), provided that the RNA can be obtained from fresh tissue. LEITR's ability to partially process this tissue and to preserve RNA for later analysis1 has been essential for conducting this research.
Through cell and organ cultures, immunohistochemistry, and RNA sequencing, my colleagues and I have discovered several pathogenic pathways in glaucoma. For example, transforming growth factor (TGF) β2 is a profibrotic factor that has been implicated in many disease processes and has been shown to be elevated in the aqueous humor of glaucoma patients.2 As we began to investigate the downstream consequences of elevated levels of TGFβ2, we found that this growth factor causes fibrosis of the TM.3 This fibrotic pathway is usually counterbalanced by bone morphogenetic proteins (BMPs), the signaling pathways of which modify TGFβ2 signaling. When something fails in this balance, the result is TM fibrosis and elevated IOP.
When we compared gene and protein expression in normal and glaucoma TM cells, we found a significant increase in the expression of the BMP inhibitor gremlin mRNA in glaucoma TM cells (Figure 4) and significantly elevated IOP in anterior segments perfused with gremlin. 4 There is similar pathogenic TGFβ2/BMP signaling and elevated expression of gremlin mRNA in the ONH.5
Another comparison of gene expression revealed significantly increased expression of sFRP-1 in glaucoma TM cells. sFRP-1 is a WNT antagonist, and when it is elevated, the normal WNT signaling that is necessary to maintain TM function is perturbed. This significantly decreases outflow facility and increases IOP.1 We have evidence that there is also interaction between the WNT signaling pathway and the TGFβ2 and BMP pathways.6
We have identified several other pathways, but the three I described herein are the most interesting because of the ways in which they seem to influence each other's signaling. None of these pathways would have been discovered without access to human donor tissue. With similar and coregulating pathogenic pathways occurring in two of the tissues central to glaucoma (the TM and ONH), it appears there are many ways to potentially perturb the system and get the same result. This suggests that there are several potential therapeutic targets by which to intervene in the glaucoma disease process.
WHAT THE FUTURE HOLDS
In the near future, we expect to be able to culture and study retinal ganglion cells from the retinas of eyes with glaucoma for comparison with normal eyes. Because the retina is sensitive to the loss of oxygen and trophic support, we will need donor eyes that are less than 6 hours postmortem for these studies. Identifying these pathogenic pathways is essential to understanding the early disease process of glaucoma, long before there is irrevocable damage to the retinal ganglion cells and the optic nerve.
The great news is that procuring the tissue used for this research is complementary to the quest for transplant tissue. Young, healthy corneas can be transplanted, and essential research can be conducted on tissue from older donors, eyes not qualified for transplantation, or even tissues typically discarded such as the posterior poles or corneoscleral rims.
I hope that the ophthalmic community can continue to educate prospective donors and their families about how valuable their generous gift is to understanding and preventing disease as well as to restoring sight. I also hope that more researchers will begin to think of human tissue as a resource with which to address questions that cannot be answered any other way.
Abbot F. Clark, PhD, is a professor of cell biology and anatomy at the University of North Texas Health Science Center, and he is director of the University of North Texas Eye Research Institute in Fort Worth, Texas. He receives research grant support from Alcon Research Ltd., the Department of Defense, LEITR, and the National Eye Institute. Dr. Clark may be reached at (817) 735-2094; abe.clark@unthsc.edu.
- Wang WH, McNatt LG, Pang IH, et al. Increased expression of the WNT antagonist sFRP-1 in glaucoma elevates intraocular pressure. J Clin Invest. 2008;118(3):1056-1064.
- Tripathi RC, Li J, Chen WF, Tripathi BJ. Aqueous humor in glaucoma eyes contains increased levels of TGF-beta2. Exp Eye Res. 1994;59:723-727.
- Fleenor DL, Shepard AR, Hellberg PE, et al. TGFbeta2-induced changes in human trabecular meshwork: implications for glaucoma. Invest Ophthalmol Vis Sci. 2006;47:226-234.
- Wordinger RJ, Fleenor DL, Hellberg PE, et al. Effects of TGF-beta2, BMP-4, and gremlin in the trabecular meshwork: implications for glaucoma. Invest Ophthalmol Vis Sci. 2007;48(3):1191-1200.
- Zode GS, Clark AF, Wordinger RJ. Bone morphogenetic protein 4 inhibits TGF-beta2 stimulation of extracellular matrix proteins in optic nerve head cells: role of gremlin in ECM modulation. Glia. 2009;57(7):755-66.
- Mao W, Sethi A, Wordinger RJ, Clark AF. Crosstalk between the Wnt and TGF-beta pathways in the trabecular meshwork. Paper presented at: ARVO Annual Meeting; May 8, 2012; Fort Lauderdale, FL.
