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H-7 in Live Monkey versus Cultured Human Eyes
Author Response: H-7 in Live Monkey versus Cultured Human Eyes
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Paul L. Kaufman
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kaufmanp{at}mhub.ophth.wisc.edu Paul L. Kaufman
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We read with interest the study "Pharmacologic disruption of Schlemm's canal cells and outflow facility in anterior segments of human eyes," by Bahler et al.1 and wish to comment on two points: 1. The loss of Schlemm's canal inner wall endothelial cells observed by the authors in post mortem organ cultured human eyes has not been seen in live monkey eyes carefully examined by light and electron microscopy.2,3 Among the possible explanations is that the inner wall endothelial cells in the post mortem human organ cultured preparations are already damaged, and their adhesions weakened, so that the addition of H-7 further weakens them sufficiently to slough in the presence of the perfusion pressure gradient. In essence, the tissue is already half dead and cannot readily withstand additional stress. Additionally, potential changes in the inner wall endothelial cells of the aged donor eyes (70±12 years) may also render the cell adhesions more sensitive to the drug and the pressure gradient. The relatively small facility increase caused by H-7 and cytochalasin D in this preparation, compared to the live monkey, are consistent with the interpretation that the entire trabecular meshwork system functions poorly. 2. The authors describe the extensive network of tendons and connecting fibrils in the human trabecular meshwork and the paucity of such tendons and fibrils in monkey and bovine eyes. They further suggest that the lack of tendons and fibrils might account for the resistance washout occurring during perfusion of the eyes in these latter species, compared to the absence of washout in human eyes perfused in organ culture. However, the monkey eye is rich in anterior ciliary muscle tendons which splay out within the trabecular meshwork, intermingle with the meshwork's elastic network and with connecting fibrils that insert into special surface modifications on the inner wall cells.4,5 Therefore, some other explanation for the apparent species differences in washout must be sought. Paul L. Kaufman and Baohe Tian Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison Medical School, Madison, WI References 1. Bahler CK, Hann CR, Fautsch MP, Johnson DH. Pharmacologic disruption of Schlemm's canal cells and outflow facility in anterior segments of human eyes. Invest Ophthalmol Vis Sci. 2004;45:2246-2254. |
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Douglas Johnson
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johnson.douglas{at}mayo.edu Douglas Johnson
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Dr. Kaufman's extensive and excellent work served as our inspiration for this project and many others over the years. Our findings do not refute his work, but rather were done to extend it from monkeys to humans. Drs. Kaufman and Tian's comments are "right on": the culture model is not perfect and not the final answer, but only one way to address the problem of glaucoma. We agree that our laboratory culture model, using post-mortem human eyes, does not replicate the living eye. Differentiating physiologic responses in the meshwork from changes due to "the entire trabecular meshwork system function[ing] poorly" can be challenging. In keeping with this, we have reported that 22% of cultures are not successful1 and have published three additional studies examining this question (Bahler CK, et al. IOVS 2003;44:ARVO E-Abstract 1156).2-4 Our approach to this has been to validate our results by 1) finding reproducibility in multiple eyes (n=25 pairs in this project), 2) use of fellow eye as control, and 3) light and microscopic assessment of the appearance of the meshworks. Both we and the nine other laboratories that use the technique feel that in the 78% of cultures that are successful (determined by histologic criteria), the cultures are functioning well enough that biochemical and molecular biological studies can be performed. Despite this, they are not living in a truly physiologic environment (no aqueous!), and caution is needed in interpreting such data, just as caution is required in interpreting data from monolayer cell culture studies. An unexpected benefit of this very problem of weakened cells in culture is the ability to study the effect of cell loss in a relatively "isolated" system without the confounding effect of platelets binding to cell breaks and without the influx of macrophages and leukocytes. In attempting to understand the site of aqueous outflow resistance, an isolated system can be an advantage: this was the rationale for our study. The second major point that Drs. Kaufman and Tian address is differences in experimental models: 1) age of subjects (younger monkeys versus older humans) and 2) species differences (monkey versus human). 1) The age of the human eyes we studied was indeed older than that of most monkeys studied, but in this regard actually more closely matches humans with POAG. Another species difference: the loss of extracellular material in the juxtacanalicular tissue in monkeys5 also appears to be a fundamental difference from human eyes: large empty spaces in this region were not seen in the human eyes in our experiment. This could also account for differences in facility change after H-7 between human and monkey. We agree that monkeys have connecting tendons and fibrils, but our unpublished work does not find them as commonly in monkeys as in humans. Our histologic findings after H-7 in the human eyes did not find the extension of the juxtacanalicular region, expansion of the inner wall, and loss of extracellular material shown in the monkey eye (see Figure 2 in Reference #5). This could be because of the anchoring effect of the tendons in the humans, because the focal adhesions and other connections between cell-ECM differ between monkeys and humans, or because the human JCT cells were unhealthy and did not relax after H-7. A future ARVO project? In summary, it is only through the work of pioneers such as Drs. Kaufman and Tian that advances are made. Repeating such work, with additional twists or changes in experimental models, serves to further our understanding of outflow physiology. Discussion of the potential reasons for differences in findings can serve as a springboard for new ideas and ultimately advance our knowledge. Douglas Johnson Department of Ophthalmology, Mayo Clinic, Rochester, MN References 1. Johnson DH, Tschumper RC. Human trabecular meshwork organ culture. A new method. Invest Ophthalmol Vis Sci. 1987;28:945-953. |
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