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Activation of Keratocyte Apoptosis in Response to Epithelial Scrape Injury Does Not Require Tears
Author Response: Activation of Keratocyte Apoptosis in Response to Epithelial Scrape Injury Does Not
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Steven E. Wilson, Professor and Chair Department of Ophthalmology, University of Washington
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sewilson{at}u.washington.edu Steven E. Wilson
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The careful work presented by Zhao et al.1 in their recent paper “Role of tears in keratocyte loss after epithelial removal in mouse cornea” was interesting and thought provoking. However, we would like to point out problems with the methodology and previous work that should be considered before accepting the hypotheses that tears alone induce keratocyte apoptosis after corneal epithelial injury and that the epithelium has no role in this process. First, the use of a simple fluorescent nuclear dye like DAPI is insufficient to determine the fate of cells in response to injury. This dye cannot distinguish living cells from dead cells. Our recent work has shown that cellular components, including the nucleus with fragmented DNA, can persist in tissues for up to a week in the stroma with apoptosis detected by TUNEL assay or apoptotic fragmentation of the cell detected by electron microscopy (Mohan and Wilson, unpublished data, 2001). Eventually these cellular fragments, that continue to stain with DAPI dye, undergo phagocytosis by inflammatory cells such as polymorphonuclear cells and monocytes. Thus, using the method in this study, many cells that were actually dead would likely have been detected. Methods that discriminate between viable and non-viable cells, and ideally between apoptosis and necrosis, should be used in these studies. Second, tears collected from the ocular surface are composed of lacrimal aqueous tear fluid and components such as interleukin-1 released from epithelial cells lining the ocular surface as they undergo the apoptosis that is a normal part of their physiology.2,3 Pure lacrimal tears cannot be collected from the ocular surface using the technique employed in this study. Collection of “pure” lacrimal gland tears would require cannulation of the lacrimal duct. It would not be surprising, however, if pure lacrimal tears still contain the components necessary to trigger apoptosis of keratocytes since lacrimal acinar cells also produce many of these apoptosis modulators. Thus, it is expected that the tears would contain the components from the epithelium that trigger keratocyte apoptosis. It is not clear, however, why the authors did not detect cell death after scraped epithelium was applied to bare stroma. This does not correspond to the results of similar experiments that we have performed using the TUNEL assay and electron microscopy, including placement of corneal epithelial debris beneath a lamellar flap (Mohan and Wilson, unpublished data, 2000). Likely, this relates to the staining methodology issue mentioned above. Finally, simpler experiments show there is no requirement for the presence of tears to trigger keratocyte apoptosis. If an eye is enucleated, rinsed several times in PBS to remove tears, scraped with a scalpel to injure the epithelium, and placed for one hour in a humidified chamber, apoptosis is detected in keratocytes with the TUNEL assay (Figure 1) or electron microscopy in either rabbit or mouse. Thus, no tears are required. We also previously reported that keratocyte apoptosis is triggered by firmly pressing a cloning cylinder or hard contact lens against the corneal epithelium without producing an epithelial defect.4 This maneuver probably injures basal epithelial cells and releases pro-apoptotic cytokines without signal sequences such as interleukin-1 into the anterior stroma. Again, tear contact with the stroma is unnecessary to produce apoptosis in the keratocytes.  Figure 1. Keratocyte apoptosis in rabbit and mouse corneas in response to corneal scrape in the absence of tears. Eyes were enucleated and rinsed several times in beakers of phosphate-buffered saline to remove tears. Experimental eyes had epithelial scrape performed with a scalpel blade. Experimental and control eyes were placed in a humidified chamber at 37°C for one hour. After one hour, eyes were cryofixed in OCT compound (Sakura Finetek, Torrance, CA). Seven-micrometer thick sections were assayed for fragmented DNA using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay (Intergen, Purchase, NY). There were no apoptotic keratocytes in control corneas that did not have epithelial scrape injury, but were rinsed in phosphate-buffered saline (not shown). (A) Wounded rabbit cornea with keratocytes (arrows) in the anterior stroma labeling for high levels of fragmented DNA (200X). (B) Wounded mouse cornea with keratocytes in the stroma (arrows) labeling for high levels of fragmented DNA. Some posterior keratocytes and endothelial cells also label, as has been reported previously in mouse corneas following epithelial scrape.5 In summary, the work of Zhao et al. is curious and seems to contradict conclusions drawn from previous publications of several investigators. We believe a closer examination of the methodologies employed in this paper is warranted. Steven E. Wilson Department of Ophthalmology, University of Washington, Seattle, WA Accepted for publication March 6, 2002 References 1. Zhao J, Nagasaki T, Maurice DM. Role of tears in keratocyte loss after epithelial removal in mouse cornea. Invest Ophthalmol Vis Sci. 2001;42:1743-1749. 2. Wilson SE, Schultz GS, Chegini N, Weng J, He Y-G. Epidermal growth factor, transforming growth factor a, transforming growth factor b, acidic fibroblast growth factor, basic fibroblast growth factor, and interleukin-1 proteins in the cornea. Exp Eye Res. 1994;59:63-72. 3. Solomon A, Rosenblatt M, Li DQ, Liu Z, Monroy D, Ji Z, Lokeshwar BL, Pflugfelder SC. Doxycycline inhibition of interleukin-1 in the corneal epithelium. Invest Ophthalmol Vis Sci. 2000;41:2544-2557. 4. Wilson SE. Keratocyte apoptosis in refractive surgery: Everett Kinsey Lecture. CLAO Journal. 1998;24:181-185. 5. Wilson SE, He Y-G, Weng J, Li Q, McDowall AW, Vital M, Chwang EL. Epithelial injury induces keratocyte apoptosis: hypothesized role for the interleukin-1 system in the modulation of corneal tissue organization and wound healing. Exp Eye Res. 1996;62:325-328. |
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David M. Maurice Department of Ophthalmology, Columbia University
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dmm35{at}columbia.edu David M. Maurice
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INTRODUCTION If an area of the corneal epithelium is scraped away, it is followed by a massive loss of keratocytes in the underlying stroma. Wilson1 attributed this to the influence of factors derived from the corneal epithelium. On the contrary, we recently published evidence2 using the mouse, that these cytotoxic factors were a normal component of tears and that the role of the epithelium was only to act as a barrier that prevented their access to the stroma. Wilson and Mohan now suggest that the discrepancy between their results and ours is due to faults in our methodology. Unfortunately, neither have they had the opportunity to examine our recent work3-5 nor we the two unpublished studies referred to in their letter. However, we welcome the opportunity to clarify the conflict between our views. DAPI STAINING Wilson and Mohan argue that DAPI staining is "insufficient to determine the fate of cells in response to injury" and that "many cells that were actually dead would likely have been detected." We would agree with these statements if the observation had been made only a short time after exposure to a cytotoxic agent. However, we observed healthy DAPI staining of the keratocyte nuclei 4 hours after the exposure of bare stroma to epithelial debris or cells, while staining was completely lost within 2 hours of tear treatment.2 Therefore, any cytotoxic effect that may be associated with our epithelial treatment was negligible, compared to that of the tears. Moreover, we used the whole mount cornea, which, unlike cross-sections, provided an en face view of the DAPI stained nuclei without histological artifact. With this technique, intact cell nuclei display a sharply demarcated, bright oval staining with several prominent nucleoli (see Figure 2 of our paper2), whereas apoptotic or necrotic nuclei exhibit deformation and/or dim staining. In fact, DAPI staining has been used to detect apoptosis and sometimes to distinguish apoptotic from necrotic cell death.6 Accordingly, we disagree with the assessment of Wilson and Mohan regarding the efficacy of our DAPI staining protocol in detecting keratocyte death. LACRIMAL VS. EPITHELIAL As we pointed out in the last paragraph of our paper,2 our experiments did not reveal the source of the tear cytotoxic factors, whether lacrimal fluid or surface epithelium, and Wilson and Mohan reiterate the same issue. Since then, we have carried out experiments that demonstrate that the lacrimal glands were the major source of the mouse tear cytotoxicity, rather than the corneal or conjunctival epithelium.4,5 Regarding our results on the effect of isolated epithelial debris or cells, Wilson and Mohan write "This does not correspond to the results of similar experiments that we have performed," but one of their experiments has not yet been published and we are unable to locate the other "similar experiments" in the literature, which makes us unable to respond. MECHANICAL Experiments of Wilson and Mohan represented in Letter Figure 1 show that keratocytes can be killed without the intervention of tears. They also present the keratocyte apoptosis that follows pressure by a hard object on the corneal surface1 as evidence that damaged epithelium is involved. It is possible, however, that in both sets of experiments and perhaps also those in their previous publications, apoptosis occurred as a result of mechanical injury to the keratocytes caused by the experimental procedures. In fact, some recent findings3 suggest that a physical impact on the stromal cells, such as that caused by epithelial scraping, can kill them. TUNEL assay would have detected both apoptotic and necrotic cell death if there was DNA fragmentation following physical injury. CONCLUSION We believe that all the points raised by the letter of Wilson and Mohan have been addressed, and we maintain our conclusion2 that factors in the tears are principally responsible for triggering the death of keratocytes, at least in the mouse, whereas the epithelium plays a minor role, if any. Our views remain opposed to those of Wilson and Mohan on these points, therefore. Finally, our current working hypothesis is that there are two distinct mechanisms active in killing keratocytes after injury; direct mechanical trauma and the access of tears to the stroma. Jin ZhaoTakayuki Nagasaki David M. Maurice Department of Ophthalmology, Columbia University, New York, NY References 1. Wilson SE. Everett Kinsey Lecture. Keratocyte apoptosis in refractive surgery. CLAO J. 1998;24:181-185. 2. Zhao J, Nagasaki T, Maurice DM. Role of tears in keratocyte loss after epithelial removal in mouse cornea. Invest Ophthalmol Vis Sci. 2001;42:1743-1749. 3. Maurice DM, Nagasaki T, Zhao J. Nuclear degeneration to the keratocytes near a cornea injury. 2002 ARVO Annual Meeting. Presentation Number 1711. 4. Zhao J, Nagasaki T, Maurice DM. Source of mouse tear factors that are cytotoxic to corneal keratocytes. 2002 ARVO Annual Meeting. Presentation Number 3132. 5. Zhao J, Nagasaki T. Lacrimal gland as the major source of mouse tear factors that are cytotoxic to corneal keratocytes. submitted. 2002. 6. Willingham MC. Methods for the detection of apoptosis. J Histochem Cytochem. 1999;47:1101-1110. |
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