Seven-day storage of pneumatically dissected Descemet s endothelial grafts with and without Dua s layer

Seven-day storage of pneumatically dissected Descemet s endothelial grafts with and without Dua s layer Sherif A. GamalElDin, Marwa M. Salama and Malak I. ElShazly Ophthalmology, Cairo University, Cairo, Egypt ABSTRACT. Purpose: To evaluate the efficacy of big-bubble (BB) technique in separating Dua s layer together with the Descemet s membrane endothelial (DE) graft and the effect of 7-day storage of the prepared tissues on the endothelial cells. Method: This is an experimental study in which 21 human corneo-scleral tissues unsuitable for transplantation were used. Grafts were mounted on artificial chamber; epithelial side-up with BB technique was used to detach the DE graft. The resultant tissues were stored in tissue culture medium for 7 days. Dua s layer presence, endothelial cell density (ECD), endothelial cell loss and viability were assessed after the dissection and at 7 days after storage. Results: Complete detachment of DE grafts was achieved in 20 cases (95.24%). Histological analysis revealed Dua s layer presence in 14 cases (70%). The mean ECD for the corneas before dissection was 2375 338 cells/mm 2 with significant reduction to 2200 319 cells/mm 2 (p < 0.00001) with endothelial cell loss of 7.3 3.5% and non-viable cells of 6.5 2.5% at the end of the 7-day storage. On comparing the dissected DE grafts with Dua s layer versus those without at 1 day after dissection and at 7 days after storage, we found no statistically significant changes in endothelial cell loss (p = 0.387; p = 0.836 respectively) and non-viable cells percentage (p = 0.180; p = 0.260, respectively). Conclusion: Big-bubble technique is reproducible in dissecting DE grafts with minimal damage to the endothelial cells. The percentage of endothelial cell loss and non-viable cells is similar in the DE grafts with/without Dua s layer after 7-day storage. Key words: big bubble Descemet s membrane endothelial grafts Dua s layer endothelial cell loss Acta Ophthalmol. 2016: 94: e130 e134 ª 2015 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd doi: 10.1111/aos.12823 Introduction Lamellar corneal surgeries for replacement of corneal endothelium are nowadays becoming the standard technique for treatment of endothelial dysfunctions. The most widely used Descemet s stripping automated endothelial keratoplasty (DSAEK) has a disadvantage of interface abnormalities that might affect the final visual quality which is due to the included stromal tissue within the donor corneal graft (Sub et al. 2008). This drawback was avoided by the development of the Descemet s membrane endothelial keratoplasty (DMEK) procedure, in which no stroma-to-stroma interface existed, hence providing better visual quality and rapid recovery (Tourtas et al. 2012). However, DMEK is not widely adopted due to the requirement of high surgical skills for both harvesting and transplanting the donor endothelium (Boynton & Woodward 2014; Salvalaio et al. 2014). Dua et al. (2013) suggested that the collagenous pre-descemet s layer (Dua s layer (PDL) may act as a splint for the Descemet s membrane endothelial (DE) graft with better handling and less scrolling, hence the emergence of pre-descemet s endothelial keratoplasty (PDEK) (Agarwal et al. 2014). Pneumatic dissection had been used several years ago for DMEK graft preparation (Venzano et al. 2010; Zarei-Ghanavati et al. 2010; Busin et al. 2011) and recently used by Agarwal et al. (2014) for PDEK graft preparation. The aim of our study was to evaluate the feasibility of utilizing the big-bubble (BB) technique in the separation of Dua s layer together with the DE graft and to study the effect of 7-day tissue culture medium storage of the prepared tissues on the endothelial cells. Materials and Methods Twenty-one human corneo-scleral discs with a sclera rim of more than 2 mm, in Optisol-GS medium, were obtained from the Eye Bank of Kasr El Aini Hospital, Cairo University. The e130

average storage time before dissection was 7.29 1.9 days. These grafts were unsuitable for transplantation due to positive hepatitis C serology. Consent was obtained for these corneas to be used in research, and ethical approval was obtained from the local ethical committee. The average donor s age was 57.5 12.6 years. Before dissection, the grafts were evaluated for cell density and viability using Trypan blue 0.1% for 1 min. Viable cells expel the dye and the nuclei of the dead cells take the stain. The grafts were then washed in a balanced salt solution and placed on Petri dish containing sucrose solution. The endothelium was examined by inverted light microscope at 9100 magnification (Leitz/Leica, Wetzlar, Germany). Endothelial cell density (ECD) was estimated by a 10 9 10 calibrated reticule mounted in the eyepiece of the microscope (fixed-frame analysis technique). The ECD was expressed as the mean of 10 different counts; each performed in a different area of the central cornea. On the same day of the assessment, the dissection was performed, starting by staining the endothelial side of the graft using Trypan blue 0.1%. The graft was then mounted on an artificial anterior chamber (AAC) (Katena Inc., Denville, NJ, USA) with the epithelial side-up. The endothelium was protected by viscoelastic solution. The BB technique, described by Anwar with few modifications, was used for the dissection of the desired tissue (Anwar & Teichmann 2002). A paracentesis was performed using an MVR at 3 O clock position to control the anterior chamber pressure throughout the procedure (Fig. 1A). A superficial crescent incision 1 mm inside the limbus at 11 O clock was performed, through which a 30-gauge needle attached to a 3-ml air-filled syringe was introduced with the bevel down, and pushed forwards for 5 mm (Fig. 1B,C). The stained endothelium helped in monitoring the depth of the needle tip. The pressure was checked to be on the lower side before air injection. Air was injected to create a big bubble (Fig. 1D). The graft was everted to ensure the formation of the big bubble and separation of the DE graft then remounted on the AAC (Fig. 1E). Partial thickness trephination (8 mm diameter) of the cornea was performed to a depth of 300 lm (Fig. 1F). Anterior lamellar keratectomy using a crescent knife was performed (Fig. 1G). The residual stroma was incised at the centre using a crescent knife and removed till the edge of trephination using corneal scissors (Fig. 1H,I). The resulting separated tissues remained adherent at the periphery to the corneo-scleral rim and preserved in tissue culture medium. Re-examination of the separated tissues was performed as described above, the same day of the procedure and after 7-day storage, for ECD and viability. Histological examination using H&E staining and periodic acid Schiff (PAS) was performed 7 days after dissection for the presence of Dua s layer (Fig. 2). Statistical analysis was performed using MEDCALC biomedical statistical software version 14.8 (Medcalc software bvba, Ostend, Belgium). Descriptive statistics were calculated, the data was summarized as mean SD, and frequencies were reported with numbers and percentages. Comparisons between preprocedure data and postprocedure data at 1 and 7 days were carried out using Friedman s test. Comparisons between the two study subgroups (with and without Dua s layer) were carried out using Mann Whitney U-test. The results were considered statistically significant with a p- value 0.05. Results The average age of the donors was 57.5 12.6 years. They were 13 men and eight women. Complete detachment of DE graft was achieved in 20 of 21 cases (95.24%). Bubble burst occurred in one case. Seven days after storage, histological analysis of H&Eand PAS-stained transverse sections revealed the presence of Dua s layer in 14 of 20 dissected grafts (70%). It was of an average thickness of 15.4 4.1 lm, made of 5 6 of tightly packed bundles with no evidence of keratocytes. The mean ECD, non-viable cells and endothelial cell loss percentages before and after separation and after 7-day storage, for all 20 cases as well as separately for the 14 cases with Dua s and the six cases without, are shown in Table 1. For the 20 cases, there was a significant reduction in the ECD 7 days after storage compared to the predissection and postdissection values (Friedman test, p < 0.00001), with significant endothelial cell loss (Friedman s test, p < 0.00001), and increase in non-viable cells percentage (Friedman s test, p < 0.00001). In the 14 dissected DE grafts where Dua s layer was detected, there was a significant reduction in the ECD (Friedman s test, p < 0.00001), with significant endothelial cell loss (Friedman test, p = 0.00222), and increase in the non-viable cells percentage at 7 days after storage compared to the predissection and postdissection values (Friedman s test, p < 0.00001). Similarly, the remaining six dissected DE grafts showed a significant reduction in the ECD (Friedman s test, p < 0.00001), with significant endothelial cell loss (Friedman test, p = 0.00084), and increase in the nonviable cells percentage at 7 days after storage compared to the predissection and postdissection values (Friedman s test, p < 0.00001). On comparing the dissected DE grafts with Dua s layer versus those without at 1 day after dissection and at 7 days after storage, we found no statistically significant changes in endothelial cell loss (Mann Whitney U-test, p = 0.387, and p = 0.836 respectively) and non-viable cells percentage (Mann Whitney U-test, p = 0.180, and p = 0.260 respectively). Discussion Posterior lamellar Keratoplasty has become a good alternative to penetrating keratoplasty in treating cases with endothelial dysfunction. Descemet s membrane endothelial keratoplasty overcomes many drawbacks associated with DSAEK, hence its superiority regarding visual quality. However, DMEK requires higher surgical skills for harvesting and implanting the DE graft. Agarwal et al. (2014) presented a new technique in which the DE graft together with Dua s layer can be dissected and used for PDEK. The presence of this collagenous layer acts as a splint to the DE graft, allowing easier handling and implantation. Together with the advantage of no interface haze reported with DMEK, the PDEK is expected to gain more popularity in the coming few years. e131

(A) (B) (C) (D) (E) (F) (G) (H) (I) Fig. 1. Pneumatic dissection of Descemet s membrane endothelial (DE) graft. (A) Paracentesis at 3 o clock. (B) Superficial crescent incision at 11 o clock 1 mm inside the limbus. (C) Pushing a 30-gauge beveled down needle 5 mm towards the centre. (D) Air injection and creation of a big bubble. (E) Eversion of the graft to ensure big-bubble formation. (F) Partial thickness trephination of the cornea. (G) Lamellar keratectomy. (H) Central crescent knife incision of the residual stroma. (I) Excision of the residual stroma using scissors. Several techniques had been suggested for preparing DE grafts with or without Dua s layer from donor corneas (Boynton & Woodward 2014), of which pneumatic dissection was performed by different methods (Studeny et al. 2010; Venzano et al. 2010; Zarei-Ghanavati et al. 2010; Busin et al. 2011; McKee et al. 2011; Agarwal et al. 2014; Altaan et al. 2015). In our study, pneumatic dissection using BB technique with epithelial sideup was used to dissect the DE graft. We succeeded in separating 20 of 21 corneo-scleral grafts without requiring high surgical skills. Ensuring proper depth of the needle guided by Trypan blue staining of the endothelium and adequate hypotony by reducing the pressure in the AAC before air injection facilitates the formation of a big bubble and DE dissection. Mounting the graft with epithelial side-up and protecting the endothelium with viscoelastic material in the AAC provide a no-touch technique in which the sharp instruments used for dissection do not come in contact with the endothelium. Rapid deflation of the air bubble avoids prolonged mechanical stretch that may be associated with damage to the endothelial intercellular junctions. Keeping the dissected tissue attached to the remaining corneo-scleral rim helped in preventing the rolling of the tissues during preservation with easy handling and proper orientation for later transplantation. The above-mentioned tips applied in our study helped to minimize trauma to the endothelial cells with a resulted loss of 2.97% after dissection. This was comparable to the endothelial cell loss of 3% in the group C of a study performed by Venzano et al. (2010), in which rapid deflation of the air bubble was induced after dissection. The endothelial cell loss found in our study was lower than that reported by Lie et al. (2008) (4 7%) in which preparation of the DE grafts was performed by Descemet s membrane stripping. In our study, Dua s layer was detected in 14 cases of the 20 dissected grafts. There was an increase in its average thickness than the thickness described by Dua et al. (2013) (10.15 e132

Fig. 2. Histological appearance of a dissected graft with the presence of Dua s layer. (a) Dua s layer. (b) Descemet s membrane. (c) Stroma. (d) Strands of collagen bundles. Table 1. The mean ECD, non-viable cells and endothelial cell loss percentages before and after separation and after 7-day storage, for all cases, the 14 cases with Dua s and the six cases without. 3.6 lm) which could be related to the tissue oedema as a result of storage. The lamellae of bundles and the absence of keratocytes were barely detected using inverted light microscopy, yet similar to the criteria mentioned by Dua et al. (2013). The endothelial cell loss and the increase in the non-viable cells percentage in these dissected grafts were significant after separation compared to the predissection values. This was different from the results obtained by ECD (cells/mm 2 ) Non-viable cells (%) EC % loss/(before) All 20 cases Before dissection 2375 338 1.3 1.1 After dissection 2305 342 3.4 1.7 2.97 3.18 After 7-day storage 2200 319 6.5 2.5 7.33 3.43 DE graft with Dua s layer Before dissection 2521 275 1.3 1.3 After dissection 2442 295 3.0 1.6 3.2 2.75 After 7-day storage 2335 259 6.1 2.3 7.3 3.19 DE graft without Dua s layer Before dissection 2033 196 1.3 0.8 After dissection 1983 204 4.2 1.9 2.4 4.30 After 7-day storage 1883 204 7.3 2.8 7.4 4.28 DE = Descemet s membrane endothelial, ECD = endothelial cell density, EC = endothelial cells. Altaan et al. (2015), who showed no significant difference in endothelial cell loss for PDEK tissue before and after bubbling, which could be explained by the small sample size used in their study as they assumed. Despite the significant endothelial cell loss and the increase of the non-viable cells percentage after dissection in grafts with Dua s layer, we found similar significant results in grafts without Dua s layer. In our study, there was a noticeable difference in the baseline ECD in DE grafts with Dua s layer (2521 275/mm 2 ) and in DE grafts without Dua s layer (2033 196/mm 2 ). By referring to the corneo-scleral graft donor s age, we found that DE grafts with Dua s layer belonged to younger donors. This matches the suggestion of Agarwal et al. (2014) that the pre-pdl bubble can be easily created in young eyes. After 7 days of tissue culture storage, we reported a significant endothelial cell loss and an increase in the nonviable cells percentage in the DE graft with Dua s layer of 7.3% and 6.1%, respectively. These changes were similar to those found in the studied DE grafts without Dua s layer. To our knowledge, this study is the first to report the endothelial cell loss and nonviable cells percentage in DE grafts with Dua s layer after 7-days storage. In a study performed by Busin et al. (2010), an endothelial cell loss of 4.4% was reported in the 19 pneumatically dissected DE grafts without Dua s layer after storage in tissue culture medium. Although we noted higher percentage of endothelial cell loss, we found that the amount of loss after dissection till the end of the 7-day storage period was 4.1% in the DE grafts with Dua s layer and 5% in the DE grafts without. However, Busin et al. did not report the endothelial cell loss immediately after dissection. A study performed by Parekh et al. (2014), on 54 DE grafts without Dua s layer dissected by a submerged hydroseparation technique and preserved for 7 days in tissue culture medium reported an endothelial cell loss of 11.5%, which was higher than that found in our study. Also, Salvalaio et al. (2014) reported a loss of 27.7% in 30 DE grafts without Dua s layer stored in deturgescent medium for 7 days after SubHyS dissection technique. Although we studied the viability of the endothelial cells for both groups but did not study their morphology after 7-day storage, the ECD still remains the most important factor determining the suitability for using PDEK and DMEK tissue for transplantation. A limitation to our studied technique is that it only provides DE grafts with or without Dua s layer for PDEK and DMEK. As the stroma is sacrificed during dissection, DALK tissues can- e133

not be supplied. Also, although being reproducible and reliable in separating DE grafts, it does not ensure Dua s layer incorporation with the DE grafts after successful air dissection. In the study performed by Agarwal et al. (2014), a technique to ensure Dua s layer dissection together with DE grafts for PDEK transplantation was described. As the Dua s layer tissue is essential to perform PDEK technique, if DE tissue is only obtained, the surgeon will have to perform DMEK technique which is more challenging. We found no significant changes in the endothelial cell loss and in the increase of the non-viable cells percentage between the DE grafts with and without Dua s layer after 7-day storage; however, further studies on a larger number of preserved dissected grafts with Dua s layer are recommended. Big-bubble technique is reproducible in dissecting DE grafts with minimal damage to the endothelial cells. The percentage of endothelial cell loss and non-viable cells is similar in the DE grafts with Dua s layer and those without after 7-day tissue culture storage. References Agarwal A, Dua HS, Narang P et al. (2014): Pre-Descemet s endothelial keratoplasty (PDEK). Br J Ophthalmol 98: 1181 1185. Altaan SL, Gupta AE, Sidney LE et al. (2015): Endothelial cell loss following tissue harvesting by pneumodissection for endothelial keratoplasty: an ex vivo study. Br J Ophthalmol 99: 710 713. Anwar M & Teichmann KD (2002): Bigbubble technique to bare Descemet s membrane in anterior lamellar keratoplasty. J Cataract Refract Surg 28: 398 403. Boynton GE & Woodward MA (2014): Eyebank preparation of endothelial tissue. Curr Opin Ophthalmol 25: 319 324. Busin M, Scorcia V, Patel AK et al. (2010): pneumatic dissection and storage of donor endothelial tissue for Descemet s membrane endothelial keratoplasty. Ophthalmology 117: 1517 1520. Busin M, Scorcia V, Patel AK et al. (2011): Donor tissue preparation for Descemet membrane endothelial keratoplasty. Br J Ophthalmol 95: 1172 1173. Dua HS, Faraj LA, Said DG et al. (2013): A novel pre-descemet s layer (Dua s layer). Ophthalmology 120: 1778 1785. Lie JT, Birbal R, Ham L et al. (2008): Donor tissue preparation for Descemet membrane endothelial keratoplasty. J Cataract Refract Surg 34: 1578 1583. McKee H, Irion L, Carley F et al. (2011): Residual corneal stroma in big-bubble deep anterior lamellar keratoplasty: a histological study in eye-bank corneas. Br J Ophthalmol 95: 1463 1465. Parekh M, Ruzza A, Salvalaio G et al. (2014): Descemet membrane endothelial keratoplasty tissue preparation from donor corneas using a standardized submerged hydroseparation method. Am J Ophthalmol 158: 277 285. Salvalaio G, Parekh M, Ruzza A et al. (2014): DMEK lenticule preparation from donor corneas using a novel SubHyS technique followed by anterior corneal dissection. Br J Ophthalmol 98: 1120 1125. Studeny P, Farkas A, Vokrojova M et al. (2010): Descemet membrane endothelial keratoplasty with a stromal rim (DMEK- S). Br J Ophthalmol 94: 909 914. Sub LH, Yoo SH, Deobhakta A et al. (2008): Complications of Descemet s stripping with automated endothelial keratoplasty: survey of 118 eyes at one institute. Ophthalmology 115: 1517 1524. Tourtas T, Laaser K, Bachmann BO et al. (2012): Descemet membrane endothelial keratoplasty versus descemet stripping automated endothelial keratoplasty. Am J Ophthalmol 153: 1082 1090. Venzano D, Pagani P, Randazzo N et al. (2010): Descemet membrane air-bubble separation in donor corneas. J Cataract Refract Surg 36: 2022 2027. Zarei-Ghanavati S, Khakshoor H & Zarei- Ghanavati M (2010): Reverse big bubble: a new technique for preparing donor tissue of Descemet membrane endothelial keratoplasty. Br J Ophthalmol 94: 1110 1111. Received on April 1st, 2015. Accepted on July 6th, 2015. Correspondence: Malak I. ElShazly, MD 5 Ibn El Nabih Street Zamalek, Cairo 11211 Egypt Tel: +201222244843 Fax: +20223636504 Email: melshazly75@hotmail.com This article (the following original article) was not presented at any meeting. The authors are thankful to Dr. Hala Gabr, Professor of Clinical pathology, Cairo University, for providing technical assistance, equipments and facilities for the preparation of the manuscript. e134