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  Koji Kitazawa, Hidetoshi Tanioka, Chie Sotozono and Shigeru Kinoshita
  Published on: 1 December 2017
• eletter
  Philippe Gain, Zhiguo He, Thibaud Garcin and Gilles Thuret
  Published on: 23 November 2017

• Published on: 1 December 2017

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  • Koji Kitazawa, M.D., Ph.D. Kyoto Prefectural University of Medicine
  • Other Contributors:
    • Hidetoshi Tanioka, Ph.D.
    • Chie Sotozono, M.D., Ph.D.
    • Shigeru Kinoshita, M.D., Ph.D.

  We read with interest the insightful comments in the e-letter submitted by Dr. Gain, Dr. He, Dr. Garcin, and Dr. Thuret on our recently published article.1 As stated in their letter, they previously reported that by using a similar triple staining (i.e., Hoechst 33342, ethidium homodimere and calcein-AM) on the endothelium of whole donor corneas stored in long-term organ culture, the endothelial cell (EC) density in the whole pool of viable ECs in the cornea is routinely, and quite substantially, overestimated.2,3 We completely agree with the authors regarding the importance of assessing the whole pool of viable ECs in corneal grafts.

  We first reported at the 2009 Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO) that triple-staining a donor graft with propidium iodide, calcein-AM, and Hoechst 33342 allowed for a distinct discrimination between living cells and dead cells. In that report, we hypothesized that the existence of dead cells on the endothelium of the donor cornea suggests an association with the rapid loss of corneal ECs, at least at the early phase, post keratoplasty. However, Gauthier and associates reported3 that there was no difference between the density of viable ECs at day 0 and at day 5 postoperative, thus suggesting that very early EC loss in the host recipient is almost negligible. However, similar to Gain and associates, we believed that it was quite important to measure viable ECD, not to calculate just ECD by...

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  We read with interest the insightful comments in the e-letter submitted by Dr. Gain, Dr. He, Dr. Garcin, and Dr. Thuret on our recently published article.1 As stated in their letter, they previously reported that by using a similar triple staining (i.e., Hoechst 33342, ethidium homodimere and calcein-AM) on the endothelium of whole donor corneas stored in long-term organ culture, the endothelial cell (EC) density in the whole pool of viable ECs in the cornea is routinely, and quite substantially, overestimated.2,3 We completely agree with the authors regarding the importance of assessing the whole pool of viable ECs in corneal grafts.

  We first reported at the 2009 Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO) that triple-staining a donor graft with propidium iodide, calcein-AM, and Hoechst 33342 allowed for a distinct discrimination between living cells and dead cells. In that report, we hypothesized that the existence of dead cells on the endothelium of the donor cornea suggests an association with the rapid loss of corneal ECs, at least at the early phase, post keratoplasty. However, Gauthier and associates reported3 that there was no difference between the density of viable ECs at day 0 and at day 5 postoperative, thus suggesting that very early EC loss in the host recipient is almost negligible. However, similar to Gain and associates, we believed that it was quite important to measure viable ECD, not to calculate just ECD by using specular microscope.

  In our reported case series, we assessed the relationship between the number of dead cells and the EC density post surgery, in both the early phase and late phase post keratoplasty, as it possibly affects the longevity of the cells over the long-term follow-up. We are currently conducting further investigation on this matter.

  References
  1. Kitazawa K, Inatomi T, Tanioka H, et al. The existence of dead cells in donor corneal endothelium preserved with storage media. Br J Ophthalmol. 2017;101:1725-1730.
  2. Pipparelli A, Thuret G, Toubeau D, et al. Pan-corneal endothelial viability assessment: application to endothelial grafts predissected by eye banks. Invest Ophthalmol Vis Sci. 2011;52:6018-6025.
  3. Gauthier AS, Garcin T, Thuret G, et al. Very early endothelial cell loss after penetrating keratoplasty with organ-cultured corneas. Br J Ophthalmol. 2017;101:1113-1118.

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  Conflict of Interest:
  None declared.

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• Published on: 23 November 2017

  eletter

  • Philippe Gain, Ophthalmic surgeon and researcher, head of the ophthalmology dept Laboratory "Biology, engineering and imaging of the Corneal graft", EA2521, University of St-Etienne, France
  • Other Contributors:
    • Zhiguo He, Research Engineer
    • Thibaud Garcin, Resident in ophthalmology
    • Gilles Thuret, Ophthalmic surgeon and researcher

  We noticed the article entitled “The existence of dead cells in donor corneal endothelium preserved with storage media” by Kitazawa with interest (Br J Ophthalmol 2017. Oct 5).
  Authors clearly demonstrated, using a triple staining with Hoechst, Propidium Iodide and Calcein-AM, that corneas stored at 4°C in Optisol-GS for 3 to 7 days, the technic most used worldwide, bear a significant number of dead endothelial cells (ECs). They underlined that these non-viable ECs are not recognized by specular cell count done by the eye bank and that this could explain the “cell loss” inevitably noticed post graft. Our team applied, for the first time in 2011 [1] and again in 2016 [2], a similar triple staining on the endothelium of whole corneas stored in long-term organ culture at 31°C, the dominant technic in Europe. We made the same findings as Kitazawa et al. and defined the notion of viable ECD (vECD) as the number of viable ECs per surface unit. Areas without ECs (especially in Descemetic folds), dead and dying EC (that will not survive the storage) clearly explain the important discrepancy between the cell count done by the eye bank (unable to spot them) and the very early postoperative ECD. Our team also demonstrated long ago that vital Trypan blue staining used by certain eye bank is unable to spot all dying cells because its time window of positivity is very narrow, corresponding only to ECs near to desquamate [3]. Viable ECD determined by triple staining therefore appea...

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  We noticed the article entitled “The existence of dead cells in donor corneal endothelium preserved with storage media” by Kitazawa with interest (Br J Ophthalmol 2017. Oct 5).
  Authors clearly demonstrated, using a triple staining with Hoechst, Propidium Iodide and Calcein-AM, that corneas stored at 4°C in Optisol-GS for 3 to 7 days, the technic most used worldwide, bear a significant number of dead endothelial cells (ECs). They underlined that these non-viable ECs are not recognized by specular cell count done by the eye bank and that this could explain the “cell loss” inevitably noticed post graft. Our team applied, for the first time in 2011 [1] and again in 2016 [2], a similar triple staining on the endothelium of whole corneas stored in long-term organ culture at 31°C, the dominant technic in Europe. We made the same findings as Kitazawa et al. and defined the notion of viable ECD (vECD) as the number of viable ECs per surface unit. Areas without ECs (especially in Descemetic folds), dead and dying EC (that will not survive the storage) clearly explain the important discrepancy between the cell count done by the eye bank (unable to spot them) and the very early postoperative ECD. Our team also demonstrated long ago that vital Trypan blue staining used by certain eye bank is unable to spot all dying cells because its time window of positivity is very narrow, corresponding only to ECs near to desquamate [3]. Viable ECD determined by triple staining therefore appears as the only reliable measure of the “useful graft endothelial capital” that is, in our opinion, a major determinant of graft survival. As a reminder, fresh grafts done in the 80’s (that the first author of this e-letter aged over 55 years knows…), contained virtually only viable ECs and were known to have a prolonged survival, around 20-25 years, largely better than the corneas stored today.
  Authors conclude that the decreased EC viability may be due partly to the storage conditions. Without pleading for the return of fresh grafts, we are convinced that its improvement will require a profound change in storage process because the two current methods (4 and 31°C) are passive (simple immersion in storage medium). We recently developed the notion of active storage using a corneal bioreactor that restores the intraocular pressure, which is essential for corneal physiology. It naturally limits stromal oedema[4] and significantly improves EC viability[5].

  Reference List
  1. Pipparelli A, Thuret G, Toubeau D, et al. Pan-corneal endothelial viability assessment: application to endothelial grafts predissected by eye banks. Invest. Ophthalmol. Vis. Sci. 2011;52:6018-25.
  2. Gauthier AS, Garcin T, Thuret G, et al. Very early endothelial cell loss after penetrating keratoplasty with organ-cultured corneas. Br J Ophthalmol 2017;101:1113-18.
  3. Gain P, Thuret G, Chiquet C, et al. Value of two mortality assessment techniques for organ cultured corneal endothelium: trypan blue versus TUNEL technique. Br J Ophthalmol 2002;86:306-10.
  4. Guindolet D, Crouzet E, He Z, et al. Storage of porcine cornea in an innovative bioreactor. Invest. Ophthalmol. Vis. Sci. 2017;in press.
  5. Garcin T, Forest F, Verhoeven P, et al. Preclinical randomized controlled study comparing long-term stored human corneas in an innovative bioreactor versus standard organ-culture. Invest. Ophthalmol. Vis. Sci. 2017;58:ARVO E-Abstract 3793.

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  Conflict of Interest:
  None declared.

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