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Inappropriate Choice of Animal Age for Investigating Retinal Progenitor Cells
Author Response: Inappropriate Choice of Animal Age for Investigating Retinal Progenitor Cells
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Rajesh Sharma
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Rajesh.Sharma{at}jax.ufl.edu Rajesh Sharma
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We read the paper by Nishiguchi et al.1 with great interest. The authors have investigated whether or not VEGF is involved with the degenerative process in a model of retinal degeneration. This is a very relevant subject considering the use of anti-VEGF therapy in retinal disorders. Results are very interesting; however, a few points need clarification. In the Figure 2 legend, the authors state that BrdU-positive cells with VEGFR1/Flt1 expression represent vascular endothelial cells. However, the authors have not used any marker for the vascular endothelial cells, and it is not obvious from the histological details of the photomicrograph that these cells are vascular endothelial cells. On the other hand, it is also possible that the increased number of BrdU positive cells observed in the VEGF treated group could be vascular endothelial cells rather than retinal progenitor cells. For assessing the effect of VEGF in vitro, the authors cultured retinal explants from PN 9 eyes. At this stage, most of the retinal neurons are already postmitotic.2,3 Very few proliferating cells are likely to be in the extreme periphery, if any. It is possible authors may have assessed vascular endothelial cells. This question could have been resolved if the authors had used markers for vascular endothelial cells. The authors maintained their explants in vitro for 10 days. At this point, especially a relatively mature retina, as used in this study, is likely to lose morphology and therefore the accuracy of the count. Any proliferating cells in these experiments are likely to be from retinas at developmental stage PN 9 to PN 19 (developmental stage at the time of explant + days in vitro). At this developmental stage, the proliferating cells are not likely to be the retinal progenitor/stem cells as stated in the results section. Therefore, extrapolation of results from in vitro experiments for retinal stem cells is not accurate and can be misleading unless specific stem cell markers are demonstrated in these proliferating cells. In Figure 1, the authors demonstrate that when animals were injected with BrdU at postnatal day (PN) 6 and examined at postnatal day 18, retinal cells showed colocalization of Pax6 with BrdU. Pax6 is expressed very early in the evaginating optic vesicle and subsequently in all retinal progenitors before they differentiate.4,5 Therefore at PN 18, when all the neurons are postmitotic, if any retinal neurons are expressing Pax6, they should be identified. For in vivo experiments, the authors injected VEGF into the vitreous solution volume measuring 0.5 microL. This volume is so small that its delivery to the vitreous is very unreliable. Again, the authors labeled the retinas with BrdU at PN 12. At this point, there are likely to be no progenitor cells but rather only differentiated neurons. The authors measured proliferating cells at the site of injection which was often complicated by retinal detachment as stated by the authors. This is typically the site of Muller cell proliferation,6,7,8 retinal pigment epithelial,9,10 or inflammatory cell (due to blood retinal breakdown) proliferation. In Figure 5, the authors labeled the retinas at PN 12 and compared the number of labeled cells at the same day and at PN 30. The authors show that the number of labeled cells decreased. These results need explanation, since in both ages labeled cells are the cells that were proliferating at the time of injection. Since BrdU remained in the eye longer in PN 30 mice and the proliferating cells at the time of injection were allowed to proliferate for longer time (until all proliferation ceased), one would expect to see more labeled cells in the periphery (unless the labeled cells underwent cell death) of PN 30 retina (or at least the same number). Rajesh K. Sharma and Kakarla V. Chalam References 1. Nishiguchi KM, Nakamura M, Kaneko H, Kachi S, Terasaki H. The role of VEGF and VEGFR2/Flk1 in proliferation of retinal progenitor cells in murine retinal degeneration. Invest Ophthalmol Vis Sci. 2007;48:4315-4320. |
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Koji Nishiguchi
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kojinish{at}med.nagoya-u.ac.jp Koji Nishiguchi
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We would like to respond to the letter from Drs. Sharma and Chalam regarding our report on the role of VEGF on retinal progenitors in mice with retinal degeneration (rd1 mice).1 One of the main concerns pointed out was based on previous reports suggesting that proliferating retinal progenitors do not exist in the murine retina at the ages labeled with BrdU in the experiment. While this could be mostly true in wild-type animals, it may not be the case in mice with retinal degeneration. It is well known that animals with neurodegeneration have an increased number of neural stem/progenitor cells in the brain.2-4 Similar evidence has been reported in the retina.5 In addition, in rd1 mice, we observed increased numbers of BrdU positive cells in the peripheral retina of rd1 mice as compared to that of wild-type mice by roughly 2.5, 6.6, and 4.9 times at P12, P18, and P30, respectively (unpublished observation). This was accompanied by a prolonged period of retinal neurogenesis in these mice compared to wild-type mice (unpublished observation). Therefore, we believe that the choice of animals and their age is not entirely inappropriate to study retinal progenitors. Another concern brought up was the inadequate characterization of BrdU-positive cells and their progeny. We have recently shown that BrdU positive cells in the peripheral retina and pars plana may be remnants of neuroblast layer that participates in the histogenesis of the retina,6 which agrees with the result of fate analyses of BrdU-positive cells in this study. On the other hand, while we doubt that a significant proportion of the BrdU positive cells present mostly in the peripheral but not elsewhere in the retina is vascular endothelial cells, it is possible that some of them may be glial cells as suggested. However, even those glial cells may act as retinal progenitors.7,8 Regarding Figure 5 that showed the decrease in the number of BrdU-positive cells over time, we regret that the description of the method was insufficient. In this experiment, BrdU was injected 2 hours before sacrificing the mice at P12 and P30. The observed reduction of BrdU- positive cells over time may reflect the decreasing number of proliferating retinal progenitors after the termination of ocular histogenesis. We greatly thank Drs. Sharma and Chalam, the reviewers, and the editor for providing us a chance to promote the understanding of our study.1 Koji Nishiguchi Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan References 1. Nishiguchi KM, Nakamura M, Kaneko H, Kachi S, Terasaki H. The role of VEGF and VEGFR2/Flk1 in proliferation of retinal progenitor cells in murine retinal degeneration. Invest Ophthalmol Vis Sci. 2007;48:4315-4320. |
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