August 2012
Volume 53, Issue 9
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Cornea  |   August 2012
Comparison of Different Methods of Glycerol Preservation for Deep Anterior Lamellar Keratoplasty Eligible Corneas
Author Affiliations & Notes
  • Jinyang Li
    From the School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, China;
  • Shuai Shi
    From the School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, China;
  • Xin Zhang
    From the School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, China;
  • Shouxiang Ni
    From the School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, China;
  • Yu Wang
    From the School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, China;
  • Christine A Curcio
    Global Sight Network, Alabama Eye Bank, Birmingham, Alabama; and
    Department of Ophthalmology, EyeSight Foundation of Alabama Vision Research Laboratories, University of Alabama School of Medicine, Birmingham, Alabama.
  • Wei Chen
    From the School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, China;
  • Corresponding author: Wei Chen, The School of Ophthalmology and Optometry, Wenzhou Medical College, 270 Xueyuan West Road, Wenzhou, Zhejiang, 325027, People's Republic of China; chenweimd@hotmail.com
Investigative Ophthalmology & Visual Science August 2012, Vol.53, 5675-5685. doi:10.1167/iovs.12-9936
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      Jinyang Li, Shuai Shi, Xin Zhang, Shouxiang Ni, Yu Wang, Christine A Curcio, Wei Chen; Comparison of Different Methods of Glycerol Preservation for Deep Anterior Lamellar Keratoplasty Eligible Corneas. Invest. Ophthalmol. Vis. Sci. 2012;53(9):5675-5685. doi: 10.1167/iovs.12-9936.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To compare different methods of glycerol-preserved corneas intended for deep anterior lamellar keratoplasty (DALK).

Methods.: We analyzed transparency, transmittance, thickness, biomechanics, morphology, and antigenicity of donor corneas preserved by four different glycerol-based methods (n = 6 per group) for 3 months, as follows: tissues in anhydrous glycerol without aluminosilicate molecular sieves at room temperature (GRT); tissues in anhydrous glycerol with aluminosilicate molecular sieves at room temperature (SRT); tissues in anhydrous glycerol without aluminosilicate molecular sieves at −78°C (G78); and tissues in anhydrous glycerol without aluminosilicate molecular sieves at −20°C (G20).

Results.: Slit lamp images and transmittance curves obtained by spectrophotometer show that the G78 cornea was the most transparent tissue. Stress-strain behavior indicated that corneas in the G78 group were the most pliable, and SRT corneas were the stiffest. Electron microscopy analysis indicated that corneal cytoarchitecture and keratocyte integrity was destroyed in all glycerol-preserved corneas. Disorganized stromal collagen fibers were evident in groups stored at RT. Especially in SRT corneas, parallelism was lost, fibrils were extremely tortuous and discontinuous, and widespread fibril degeneration could be found. Antigenicity of tissue, assessed via immunohistochemistry for CD45-positive cells, HLA-ABC and HLA-DR, was lowered after glycerol preservation relative to fresh cornea tissues, and immunoreactivity was located mainly on corneal epithelium and limbus rather than stroma.

Conclusions.: Anhydrous glycerol preservation without molecular sieves in a −78°C freezer was the best method to obtain DALK-eligible tissues that were both transparent and pliable.

Introduction
Corneal impairment is the third leading cause of blindness in the developing world and the prevalent corneal conditions are advanced infectious keratitis and industrial chemical burns. 14 Most corneal blindness is treatable with keratoplasty, but these procedures are currently available to only a small percentage of eligible patients. Access to sight-restoring keratoplasty in China is severely limited by the lack of developed eye-banking networks and a critical shortage of tissue for transplantation. Beyond the developed world, corneal transplantation using fresh corneal tissue is further hindered by unreliable storage and transportation facilities, unorganized distribution networks, the cost-prohibitive nature of imported tissue, unreliable compliance with medications and follow-up instructions, and inadequate health and education services. 
Deep anterior lamellar keratoplasty (DALK) is a surgical technique for recipients with healthy endothelium involving removing all host corneal layers except Descemet's membrane and leaving the anterior chamber intact. As surgical innovation in lamellar corneal surgery expands the potential use of acellular corneal tissue, long-term preservation techniques are being revisited as a way to increase availability of corneal tissue to corneal surgeons throughout the developing world. Constraints of availability, cost, storage, and transportation may be substantially alleviated by the improved availability of glycerol-preserved corneas (GPCs). 5 As a dehydrating agent, glycerol has antimicrobial and antiprotease properties and maintains corneal structure, making it suitable for long-term storage of corneas for purposes not requiring viable cell layers. 68 Our previous clinical studies have shown that GPCs can be used safely and effectively in patients with low-risk and high-risk corneal conditions. 9,10 In a prospective randomized clinical trial, Farias and associates 11 concluded DALK using lyophilized corneas seems to yield clinical results that are as good as and perhaps better than DALK using tissues preserved in Optisol (Bausch & Lomb, St. Louis, MO). 
Tissues preserved using different formulations of glycerol can have noticeably different properties. 12,13 To the best of our knowledge, no comparative studies have yet determined if different glycerol-based corneal preservation methods meet the demands of DALK-eligible tissue. The goal of this study was to compare and characterize the transparency, transmittance, thickness, biomechanics, morphology, and antigenicity of donor corneas preserved by four different glycerol-based methods intended for DALK. 
Methods
Experimental Groups
Corneas were obtained from medically eligible donors and recovered into intermediate term media (Optisol GS; Bausch & Lomb), then transferred and preserved by four different glycerol-based methods (n = 6 per group), as follows: tissues in anhydrous glycerol without aluminosilicate molecular sieves at room temperature (GRT); tissues in anhydrous glycerol with aluminosilicate molecular sieves at room temperature (SRT); tissues in anhydrous glycerol without aluminosilicate molecular sieves at −78°C (G78); tissues in anhydrous glycerol without aluminosilicate molecular sieves at −20°C (G20). Donor corneas used for GRT in this study came from two different sources: Global Sight Network (GSN), Birmingham, AL, and Wenzhou Eyebank, Wenzhou, Zhejiang, China. Donor corneas for SRT came from GSN only. All GPCs in the four groups were kept in storage for 3 months. Fresh donor corneas served as a comparison group for these GPCs. 
Experimental Methods
Frozen corneas were thawed before rehydration and further analysis. All glycerol-preserved tissues were rehydrated at room temperature for 30 minutes in balanced salt solution (BSS) before the following laboratory experiments were conducted. Assays were chosen to assess five properties important to the usability of glycerol-preserved tissue as an allograft material for DALK. All experiments were conducted under the pre-established flow in a time-locked manner. Thus, each GPC in different groups was tested individually from slit-lamp observation, light transmittance to thickness, then biomechanical behavior or immunohistochemical staining and electron microscope observation (one cornea cut in half for two testings). The experimental flow chart and the number of samples in each assays is shown in Figure 1
Figure 1. 
 
The experimental flow chart.
Figure 1. 
 
The experimental flow chart.
Transparency.
Using a spectrophotometer (UV-Visible-NIR Spectrophotometer U-4100; Hitachi High-Tech, Tokyo, Japan), total light transmittance (the sum of parallel and dispersed light transmittance) of the corneal tissues was measured from 280 to 800 nm. The percent transmission was determined with a resolution of 1 nm at a rate of 600 nm/min. In each donor sample, the area of central cornea was measured. The average percent transmission of each sample was calculated for every 1 nm of light wavelength. In this study, we compared only average percent transmission of visible light (wavelength range, 380–780 nm). The average percent transmission was calculated using the following equation: where T(λ) = percent transmission at the wavelength λ. The boundaries of range (in nanometers) were α = 380, β = 780. The mean ± standard error (SD) of the average percent transmission was calculated, and the differences were determined by one-way ANOVA combined with a Scheffe's test (SPSS 13.0; SPSS Inc., Chicago, IL), P < 0.05 considered statistically significant. Transparency of each corneal tissue was additionally assessed in slit-lamp images. For comparison, we also measured light transmittance and performed slit-lamp observation of fresh cornea. 
Thickness.
The center thickness of each corneal tissue was determined by taking the average of five measurements from an electronic thickness gauge specifically designed for measuring soft lenses (model ET-3; Rehder Development, Castro Valley, CA). Corneal tissue was centered on a steel ball carrier and a sensor automatically lowered to the anterior surface of the cornea by a motorized drive. This device lowers the sensor at a constant velocity and applies a constant amount of force, thereby increasing measurement precision while maintaining an accuracy of ±2 μm. Statistical comparisons of four groups were performed with ANOVA (SPSS 13.0), P < 0.05 considered statistically significant. 
Biomechanics.
Corneas were mounted onto a custom-built pressure chamber of an inflation test rig. Mechanical clamps and cyanoacrylate glue were used to provide a tight connection along a ring of scleral tissue for each specimen. The pressure chamber was filled with a BSS (Bausch & Lomb) and connected to a loading tank, which was moved up and down with a motor that controlled the pressure inside the chamber. The connection between the chamber and the tank went through a water tank equipped with a temperature controller (Julabo, EHSY, Beijing, China) to maintain the BSS in the pressure chamber at 37°C throughout the test. The motor attached to the tank operated at a speed equivalent to a pressure application rate of 5.8 mm Hg/min (Motion Assistant software; National Instruments Corporation, Austin, TX). All corneas were first subjected to an initial inflation pressure of 0.75 mm Hg, the minimum required to achieve a fully inflated and smooth corneal surface. This was followed by 3 cycles of loading up to a max pressure of 45 mm Hg and unloading to precondition the tissue and stabilize its behavior before considering results from the fourth cycle as representative of the cornea's biomechanical behavior. A CCD laser displacement sensor (LK series; Keyence, Milton Keynes, UK) was used to monitor the displacement of the corneal apex continually. The laser beam and pressure transducer were connected to a computer to record data automatically every 0.25 second during the test. 
Experimentally obtained relationships between the posterior pressure and apical rise were converted into stress-strain behavior using mathematical shell analysis, as described. 14  
Morphology of Collagen Fibrils.
Corneal tissues were first thoroughly rinsed with 0.1 mol/L sodium cacodylate buffer (pH 7.4) and then preserved by immersion in 2% glutaraldehyde in 80 mM sodium cacodylate (pH 7.4, 320–340 mOsm/kg) overnight at 4°C. All subsequent steps were performed at room temperature unless noted. After thorough fixation, 1 × 3-mm pieces were cut from the central region, rinsed in cacodylate buffer, and postfixed by immersion in freshly prepared 1% osmium tetroxide in 100 mM cacodylate buffer for 1 hour under subdued lighting. Samples were again washed in cacodylate buffer and dehydrated through a graded alcohol series (30% ∼100% in six steps). Samples were then infiltrated with propylene oxide (3 changes at 10-minute intervals) and a 3:1 vol/vol mixture of propylene oxide and Spurr's resin (product number 4300, Electron Microscopy Sciences, Fort Washington, PA) for 3 hours. This was followed by overnight immersion in a 1:1 vol/vol mixture of propylene oxide and Spurr's resin, followed by transfer to 100% Spurr's resin overnight. Tissue samples were oriented in embedding molds and left for overnight polymerization at 60°C. Thick transverse sections (0.5∼1 μm) were cut and stained with toluidine blue for light microscopic observations and to determine tissue orientation. Ultrathin sections were obtained and mounted on parallel-bar copper grids (115 μm bar spacing) (product number 2415C-XA, SPI Supplies, West Chester, PA). Sections were double-stained first in 3.5% aqueous uranyl acetate for 20 minutes at 60°C, followed by Reynold's lead citrate for 10 minutes. Grids were examined in a transmission electron microscope (H-7650; Hitachi, Tokyo, Japan). Micrographs were chosen at random for analysis. 
Antigenicity.
Sections of formalin-fixed, paraffin-embedded tissue blocks were used for immunohistochemical analysis. Sections were de-paraffinized, followed by antigen retrieval with epitope retrieval solution (10 mmol citrate buffer, pH 6.0; Dakocytomation Inc., Carpinteria, CA) in an autoclave (121°C, 20 minutes). Endogenous peroxidase was blocked by a peroxidase blocking solution (ChemMate; Dakocytomation). Primary antibodies (HLA Class I ABC antibody [ab70328]; 1:100; HLA Class II DR antibody [ab20181]; 1:100; CD45 antibody [ab10559], 1:1000; Abcam, Cambridge, UK) were applied to sections at 4°C overnight. Thereafter, sections were incubated with streptavidin-biotin complex (Simple Stain MAX-PO kit; Nichirei, Tokyo, Japan) for 30 minutes. Sections were treated with 3,3′-diaminobenzidine (Dakocytomation) for 5 minutes and counterstained with hematoxylin. Slides were examined by light microscopy, a positive reaction being indicated by a brown color. Sections for which primary antibodies were omitted were used as negative controls (not shown). Sections of fresh thymus tissue served as positive controls (not shown). 
Results
Transparency
As shown in Figure 2, both GRT and SRT corneas transmitted less light than the two frozen GPCs (G78 and G20) at all wavelengths. Transmission of the G78 corneas was closest to that of fresh cornea of all the groups, and it decreased gradually with shorter wavelengths. This result is consistent with slit-lamp observation (Fig. 3), which revealed that G78 corneas were the most transparent, and SRT corneas were extremely cloudy and edematous. As summarized in the Table, average percent transmission between visible light wavelengths differed significantly among the among the four groups (P < 0.05). The transmission value for one fresh cornea, which had been preserved in intermediate-term media for 12 days, was 66.6 % as control. 
Figure 2. 
 
The percent transmission curves for glycerol-based corneas in four groups and one fresh cornea. Each point on each curve represents an average of the five corneal tissues in each group. The entire light spectrum is shown on the horizontal axis. Red curve, G78 group; blue curve, G20 group; green curve, GRT group; magenta curve, SRT group; black curve, one fresh cornea for control.
Figure 2. 
 
The percent transmission curves for glycerol-based corneas in four groups and one fresh cornea. Each point on each curve represents an average of the five corneal tissues in each group. The entire light spectrum is shown on the horizontal axis. Red curve, G78 group; blue curve, G20 group; green curve, GRT group; magenta curve, SRT group; black curve, one fresh cornea for control.
Figure 3. 
 
Slit-lamp images of glycerol-based corneas in four preservation groups show the G78 cornea is transparent just as fresh tissue, but the SRT cornea looks cloudy and extremely edematous.
Figure 3. 
 
Slit-lamp images of glycerol-based corneas in four preservation groups show the G78 cornea is transparent just as fresh tissue, but the SRT cornea looks cloudy and extremely edematous.
Table. 
 
Average Percent Transmission of Glycerol-Based Corneas in Four Preservation Groups between Visible Light Wavelength
Table. 
 
Average Percent Transmission of Glycerol-Based Corneas in Four Preservation Groups between Visible Light Wavelength
Group (n = 6) Average Transmittance (%)
SRT 27.4 ± 1.80
GRT 31.9 ± 1.63
G20 51.5 ± 1.03
G78 57.0 ± 0.91
Thickness
Mean center thicknesses of glycerol-preserved corneas are shown in Figure 4. The mean corneal thickness in the SRT group was dramatically increased to 1651 ± 112.5 μm after 30 minutes of rehydration. There were statistically significant differences among groups SRT, GRT, and G78, but not between G20 and G78 groups (P = 0.469). Relative to the other three groups, the differences of GRT and SRT corneas were all significant (P < 0.05). There was no statistically significant difference between G20 and G78 groups (P = 0.469). 
Figure 4. 
 
The center thicknesses of glycerol-based corneas in four groups after rehydration. Values of corneal thickness is given as the mean ± SD, scale in μm. Black point, SRT group; red point, GRT group; blue point, G20 group; green point, G78 group.
Figure 4. 
 
The center thicknesses of glycerol-based corneas in four groups after rehydration. Values of corneal thickness is given as the mean ± SD, scale in μm. Black point, SRT group; red point, GRT group; blue point, G20 group; green point, G78 group.
Biomechanical Behavior
Stress-strain behavior of corneal specimens was derived using shell theory. In all cases, stress-strain behavior followed an exponential function that was determined to be best fit by the average value within each group. Mean stress at each strain level was obtained for each of the four groups. As shown in Figure 5A, results demonstrated clear stiffening associated with higher preservation temperature. At the same strain levels, including low and high, there were consistent increases in stress associated with the high-preservation temperature. This test indicated that G78 corneas were the most pliable, and SRT corneas were the stiffest. The stress-strain behavior of a fresh corneal specimen is shown in Figure 5B as a reference. 
Figure 5. 
 
( A) Comparison of average stress-strain behavior of the four specimen groups. The error bars represent the SDs of stress values. (B) The stress-strain behavior of one fresh corneal specimen. The range of strain levels is much larger than glycerol-based preserved corneas.
Figure 5. 
 
( A) Comparison of average stress-strain behavior of the four specimen groups. The error bars represent the SDs of stress values. (B) The stress-strain behavior of one fresh corneal specimen. The range of strain levels is much larger than glycerol-based preserved corneas.
Ultrastructure
Electron microscopy analysis indicated that keratocytes in a normal cornea usually lie between collagen lamellae. The cell body contains a large nucleus. After glycerol preservation, keratocytes were destroyed and collapsed to a certain extent in all four groups. As demonstrated, in GPC samples, organelles of keratocytes were absent, and the nucleus and cytoplasm were compact (Fig. 6). 
Figure 6. 
 
Transmission electron micrograph showing cytoarchitecture and organelles in a fresh cornea (A); the integrity of keratocytes was destroyed and condensed in glycerol-preserved corneas, and keratocytes exhibit loss of intracellular organelles and dissolution of cytoplasm (B, SRT; C, GRT; D, G78).
Figure 6. 
 
Transmission electron micrograph showing cytoarchitecture and organelles in a fresh cornea (A); the integrity of keratocytes was destroyed and condensed in glycerol-preserved corneas, and keratocytes exhibit loss of intracellular organelles and dissolution of cytoplasm (B, SRT; C, GRT; D, G78).
Electron microscopy of the stroma reveals collagen fibrils cut perpendicularly and tangentially. Fibrils in a fresh cornea have uniform diameters and form compact parallel arrays (Figs. 7A, 8A). In contrast, the regular arrangement of collagen fibrils exhibited vast differences among 4 kinds of glycerol preservation methods with the same rehydration time. In G78 corneas, regularly arranged collagen fibrils re-approximated their original spacing when the tissue was rehydrated. Collagen fibrils also maintained parallelism and organization (Figs. 7B, 8B). In G20 corneas, fibrils maintained parallelism, but the spaces between the lamellae varied. There were blank regions, often called lakes, where fibrils are missing (Fig. 7C). In GRT corneas, parallelism was partially maintained, spaces between the lamellae became larger, fibrils were tortuous, and numerous lakes were found. Moreover, threadlike high-density shadows around the fibrils, representing degenerating fibrils, were apparent (Fig. 8C). In SRT corneas, parallelism was lost, fibrils were extremely tortuous and discontinuous, and widespread fibril degeneration could be found (Figs. 7D, 8D). 
Figure 7. 
 
Transmission electron micrograph of collagen fibrils in the midcentral stroma illustrating the arrangement of individual collagen fibrils within various lamellae (A, fresh cornea; B, G78; C, G20; D, SRT) (bar, 500 nm).
Figure 7. 
 
Transmission electron micrograph of collagen fibrils in the midcentral stroma illustrating the arrangement of individual collagen fibrils within various lamellae (A, fresh cornea; B, G78; C, G20; D, SRT) (bar, 500 nm).
Figure 8. 
 
Transmission electron micrograph of collagen fibrils in the midcentral stroma illustrating the parallelism of the collagen fibers (A, fresh cornea; B, G78; C, GRT; D, SRT) (bar, 500 nm).
Figure 8. 
 
Transmission electron micrograph of collagen fibrils in the midcentral stroma illustrating the parallelism of the collagen fibers (A, fresh cornea; B, G78; C, GRT; D, SRT) (bar, 500 nm).
Antigenicity
After glycerol preservation, HLA-ABC was readily detected on corneal epithelium and limbus and rarely detected on endothelium and keratocytes in the anterior stroma beneath epithelium (Figs. 9A, 9B). HLA-DR was seen in corneal epithelium and was more abundant near the limbus. HLA-DR was not found in stromal keratocytes or in the endothelium (Figs. 9C, 9D). CD45-immunoreactivity was present in fewer cells than the other markers and was located dispersed on the limbus only (Fig. 10). Altogether, positive reactions for classes I and II, and CD45 were reduced in all glycerol-preserved corneas and were mainly located on corneal epithelium and limbus rather than stroma. For the two RT groups, immunoreactivity was further reduced. 
Figure 9. 
 
The glycerol-preserved cornea (G78) show positive reaction (brown) for HLA-ABC (A, limbus; B, epithelium and anterior stroma) and HLA-DR (C, limbus; D, epithelium) (light microscopy; bar, 20 μm).
Figure 9. 
 
The glycerol-preserved cornea (G78) show positive reaction (brown) for HLA-ABC (A, limbus; B, epithelium and anterior stroma) and HLA-DR (C, limbus; D, epithelium) (light microscopy; bar, 20 μm).
Figure 10. 
 
The fresh cornea and glycerol-preserved cornea show positive reaction (brown) for CD45 (A, fresh, limbus; B, fresh, epithelium and anterior stroma; C, G78, limbus; D, G78, epithelium) (light microscopy; bar, 20 μm).
Figure 10. 
 
The fresh cornea and glycerol-preserved cornea show positive reaction (brown) for CD45 (A, fresh, limbus; B, fresh, epithelium and anterior stroma; C, G78, limbus; D, G78, epithelium) (light microscopy; bar, 20 μm).
Discussion
A goal of the study was to determine how to best exploit properties of glycerol for preparing DALK-ready tissues. We showed organelles of keratocytes were absent, nucleus and cytoplasm were compact, and cellular antigens were lost through all four methods of glycerol preservation. Preservation in anhydrous glycerol without molecular sieves, stored at −78°C, was the best method to obtain DALK-eligible tissues. 
Glycerol is a chemical compound commonly called glycerin. This colorless, odorless, and viscous liquid has three hydrophilic hydroxyl groups that impart both its solubility in water and its hygroscopic nature. 6 Glycerol acts as a cryo-protectant for frozen tissue storage, because it binds strongly to water, prevents water molecules from joining a growing ice crystal, and forestalls the formation of damaging ice crystals. 15 An early goal of cryo-biology was survival of healthy corneal endothelial cells after glycerol cryo-protection and freezing. 16 Glycerol has excellent antibacterial, antifungal, and antiviral properties, well documented by literature on skin and bone banking. 12,13,1721 As a dehydrating agent, glycerol permits the use of GPC as scaffolding for procedures not requiring viable cell layers; however, tissues preserved using different formulations of glycerol have noticeably different properties. For example, skin preserved in 85% glycerol/15% water, a virucidal concentration, is also more supple than skin preserved in anhydrous glycerol. 12 Bone grafts kept in 98% glycerol at room temperature exhibited similar bone matrix preservation to cryopreserved bone; no bacteria or fungi were found in the samples and there was no difference between the distributions of histomorphologic variables and cell populations. 13 Thus, preservation protocols must be optimized for target tissue and intended surgical purpose. 
Our results show, after the same rehydration time, the extent of corneal edema was remarkably greater in RT groups than cryo-preserved groups. This led to a significant decrease of transparency and increase of thickness for RT corneas. Especially for SRT cornea, the mean corneal thickness was dramatically increased to 1651.0 ± 112.5 μm after 30 minutes of rehydration, almost triple that of normal fresh cornea. Excessive stromal edema also influenced the corneal biomechanical behavior, which demonstrated stiffening associated with higher preservation temperature. GPC stored at −78°C was the most pliable cornea as compared with other preservation temperatures. 
To investigate the effect of different preservation temperatures on stromal edema, we compared the ultrastructure of collagen lamellae using electron microscopy. The results show collagen fibers maintained parallelism and organization in G78 corneas. In SRT corneas, however, parallelism was lost and fibrils were extremely tortuous and discontinuous; moreover, widespread degeneration could be found around the fibrils. We attribute the extreme edema of SRT cornea to destruction of corneal collagen structure and degeneration of collagen fibers. However, one limitation of this study is that the original corneal thickness was unmeasured and we cannot comment on the relative hydration of each cornea. Ideally, DALK-ready tissues, which are not dependent on living cells, would exhibit the following characteristics: an adequate thickness of stromal lamellae conferring appropriate strength and rigidity similar to fresh cornea, and suitable for intra-operative suture to host bed; quick restoration of transparency postoperatively, to provide an acceptably early good visual acuity; and resistance of corneal stromal structure to postoperative melt. On the basis of our current data, we conclude that G78 cornea was the most eligible GPC for DALK. 
Lamellar keratoplasty using GPC was established by the pioneering experimental and clinical studies of J.H. King, 7,8,22,23 who first preserved corneas in 95% glycerol under vacuum to ensure an anhydrous state. Later, equivalent dehydration was achieved by including in the glycerol molecular sieves (sodium and calcium alumino-silicates) that, as physical adsorptive agents, removed water to an extremely low vapor pressure. With new surgical techniques and instrumentation innovation, corneal transplantation is now at a new watershed, with a major paradigm shift from traditional penetrating keratoplasty (PK) to newer and innovative forms of lamellar keratoplasty aimed at targeted replacement of only diseased tissue layers. 24 A recent editorial 25 concluded that DALK is rapidly gaining acceptance as the procedure of choice for such patients whose endothelium is not compromised. DALK does not require donor endothelial cells. Therefore, it is the leading application for the use of acellular long-term preserved tissue. Of importance to this application, DALK performed with acellular corneas using lyophilization 11 or glycerin-cryopreservation 9,10 yields results equal to or superior to fresh corneas. GPC could decrease the possibility of immune rejection in the setting of DALK, which is mainly ascribed to its de-cellularized nature, as demonstrated in our previous clinical studies. 9,10 Cells in the fresh corneal tissue, such as epithelium, keratocytes and bone marrow–derived cells, are important in corneal allograft response. De-cellularization has been successfully demonstrated to reduce tissue immunogenicity, and low antigenicity of the corneal tissue could reduce graft rejection and offer long-term graft survival. As demonstrated by Zhang et al., 26 anti-CD45 antibody plus complement-mediated targeting of ex vivo donor tissue was a highly efficient way to deplete corneal passenger leukocytes. Corneas de-cellularized by other methods such as lyophilization and treatment with detergents and proteases 11,27,28 are also being explored for their potential as lamellar grafts; however, none of these methods have been used clinically up to now. Recently, Utine et al. 29 reported that sterile gamma-irradiated corneas may be considered in lieu of fresh donor corneas for lamellar corneal patch grafts for lack of immunogenicity. However, these tissues required a new preoperative management for fresh corneas and relatively expensive equipment. In the present study, the results of immunohistochemistry showed that positive reaction for HLA-ABC, HLA-DR, and CD45 was reduced in all GPCs and was mainly located on corneal epithelium and limbus. The antigenicity of stroma was low or almost lost, which was consistent with our electron microscopy findings showing the keratocytes were collapsed and destroyed after glycerol preservation. This is more advantageous for DALK-ready tissues, whose epithelium and endothelium will be removed before surgical operation. Thus, among different long-term corneal-preservation techniques, the application of glycerol was a simple yet effective technique to achieve decellularization. 30  
The use of GPC in lamellar keratoplasty could increase the pool of corneas acceptable for transplantation. This potential advantage must not be overlooked in the emerging economies, where donor corneas and eye banks are limited. In 2010, US members of the Eye Bank Association of America, while distributing 59,271 corneas for transplantation, also recovered 9471 medically eligible tissues that were determined not usable for transplantation, because of low endothelial cell density, stromal scar, or small clear zone. 31 GPCs from this latter source, preserved by GSN using the SRT method tested in the current study, are also being used as patch grafts for glaucoma drainage device surgery. 32,33 For that purpose, GPC tissue clarity is adequate for laser lysis of sutures, and resistance to erosion is good up to 1 year postoperatively (Wigton E, Swanner J, Joiner DW, et al., manuscript submitted, 2012); however, in this study, the tissues with best potential for DALK were frozen quickly after immersion in glycerol and stored frozen. This preparation method would be most readily implemented in settings with a local eye bank, given the current regulations governing international tissue distribution. 
In conclusion, anhydrous glycerol preservation induced de-cellularization and reduced antigenicity. Anhydrous glycerol preservation without molecular sieves in a −78°C freezer was the best method to obtain DALK-eligible tissues that were both transparent and pliable. All methods of glycerol preservation exhibited loss of cellular antigens that may decrease rejection risk. 
References
Xie L Qi F Gao H Wang T Shi W Zhao J. Major shifts in corneal transplantation procedures in north China: 5316 eyes over 12 years. Br J Ophthalmol . 2009;93:1291–1295. [CrossRef] [PubMed]
Shi W Jin H Li S Liu M Xie L. Indications of paediatric keratoplasty in north China. Clin Experiment Ophthalmol . 2007;35:724–727. [CrossRef] [PubMed]
Xie L Song Z Zhao J Shi W Wang F. Indications for penetrating keratoplasty in north China. Cornea . 2007;26:1070–1073. [CrossRef] [PubMed]
Zhang C Xu J. Indications for penetrating keratoplasty in East China, 1994-2003. Graefes Arch Clin Exp Ophthalmol . 2005;243:1005–1009. [CrossRef] [PubMed]
Feilmeier MR Tabin GC Williams L Oliva M. The use of glycerol-preserved corneas in the developing world. Middle East Afr J Ophthalmol . 2010;17:38–43. [PubMed]
Pegg DE. The preservation of tissues for transplantation. Cell Tissue Bank . 2006;7:349–358. [CrossRef] [PubMed]
King JH Jr McTigue JW Meryman HT. Preservation of corneas for lamellar keratoplasty: a simple method of chemical glycerine-dehydration. Trans Am Ophthalmol Soc . 1961;59:194–201. [PubMed]
King JH Jr Townsend WM. The prolonged storage of donor corneas by glycerine dehydration. Trans Am Ophthalmol Soc . 1984;82:106–110. [PubMed]
Li J Yu L Deng Z Deep anterior lamellar keratoplasty using acellular corneal tissue for prevention of allograft rejection in high-risk corneas. Am J Ophthalmol . 2011;152:762–770. [CrossRef] [PubMed]
Chen W Lin Y Zhang X Comparison of fresh corneal tissue versus glycerin-cryopreserved corneal tissue in deep anterior lamellar keratoplasty. Invest Ophthalmol Vis Sci . 2010;51:775–781. [CrossRef] [PubMed]
Farias R Barbosa L Lima A Deep anterior lamellar transplant using lyophilized and Optisol corneas in patients with keratoconus. Cornea . 2008;27:1030–1036. [CrossRef] [PubMed]
van Baare J Buitenwerf J Hoekstra MJ du Pont JS. Virucidal effect of glycerol as used in donor skin preservation. Burns . 1994;20:S77–80. [CrossRef] [PubMed]
Giovani AM Croci AT Oliveira CR Comparative study of cryopreserved bone tissue and tissue preserved in a 98% glycerol solution. Clinics (Sao Paulo) . 2006;61:565–570. [CrossRef] [PubMed]
Ni S Yu J Bao F Li J Elsheikh A Wang Q. Effect of glucose on the stress-strain behavior of ex-vivo rabbit cornea. Exp Eye Res . 2011;92:353–360. [CrossRef] [PubMed]
Sumida S. Transfusion and transplantation of cryopreserved cells and tissues. Cell Tissue Bank . 2006;7:265–305. [CrossRef] [PubMed]
Armitage WJ. Cryopreservation for corneal storage. Dev Ophthalmol . 2009;43:63–69. [PubMed]
Basile AR. A comparative study of glycerinized and lyophilized porcine skin in dressings for third-degree burns. Plast Reconstr Surg . 1982;69:969–974. [CrossRef] [PubMed]
Kreis RW Vloemans AF Hoekstra MJ Mackie DP Hermans RP. The use of non-viable glycerol-preserved cadaver skin combined with widely expanded autografts in the treatment of extensive third-degree burns. J Trauma . 1989;29:51–54. [CrossRef] [PubMed]
Hoekstra MJ Kreis RW du Pont JS. History of the Euro Skin Bank: the innovation of preservation technologies. Burns . 1994;20:S43–47. [CrossRef] [PubMed]
Marshall L Ghosh MM Boyce SG MacNeil S Freedlander E Kudesia G. Effect of glycerol on intracellular virus survival: implications for the clinical use of glycerol-preserved cadaver skin. Burns . 1995;21:356–361. [CrossRef] [PubMed]
Tomford WW. Bone allografts: past, present and future. Cell Tissue Bank . 2000;1:105–109. [CrossRef] [PubMed]
King JH Jr. Keratoplasty: experimental studies with corneas preserved by dehydration. Trans Am Ophthalmol Soc . 1956;54:567–609. [PubMed]
King JH Jr. The use of preserved ocular tissues for transplantation. Trans Am Ophthalmol Soc . 1958;56:203–211 ; discussion 211–206. [PubMed]
Tan DT Anshu A. Anterior lamellar keratoplasty: ‘Back to the Future'— a review. Clin Experiment Ophthalmol . 2010;38:118–127. [PubMed]
Sutphin JE Goins KM Wagoner MD. Deep anterior lamellar keratoplasty: when should it replace penetrating keratoplasty? Am J Ophthalmol . 2009;148:629–631. [CrossRef] [PubMed]
Zhang X Shen L Jin Y Saban DR Chauhan SK Dana R. Depletion of passenger leukocytes from corneal grafts: an effective means of promoting transplant survival? Invest Ophthalmol Vis Sci . 2009;50:3137–3144. [CrossRef] [PubMed]
Farias RJ Sousa LB Lima Filho AA Lourenco AC Tanakai MH Freymuller E. Light and transmission electronic microscopy evaluation of lyophilized corneas. Cornea . 2008;27:791–794. [CrossRef] [PubMed]
Oh JY Kim MK Lee HJ Ko JH Wee WR Lee JH. Processing porcine cornea for biomedical applications. Tissue Eng Part C Methods . 2009;15:635–645. [CrossRef] [PubMed]
Utine CA Tzu JH Akpek EK. Lamellar keratoplasty using gamma-irradiated corneal lenticules. Am J Ophthalmol . 2011;151:170–174. e1. [CrossRef] [PubMed]
Kalevar V. Preservation and suitability of donor human corneas for keratoplasty. J All India Ophthalmol Soc . 1966;14:75–82. [PubMed]
2010 Eye Banking Statistical Report . Washington DC: Eye Banking Association of America; 2011.
Rojanapongpun P Ritch R. Clear corneal graft overlying the seton tube to facilitate laser suture lysis. Am J Ophthalmol . 1996;122:424–425. [CrossRef] [PubMed]
Singh M Chew PT Tan D. Corneal patch graft repair of exposed glaucoma drainage implants. Cornea . 2008;27:1171–1173. [CrossRef] [PubMed]
Footnotes
 Supported in part by a 2010 Richard Lindstrom Grant from the Eye Bank Association of America, and Grant 2009A151 from the Medicine and Health Foundation of Zhejiang Province, China.
Footnotes
 Disclosure: J. Li, None; S. Shi, None; X. Zhang, None; S. Ni, None; Y. Wang; None; C.A. Curcio, None; W. Chen, None
Figure 1. 
 
The experimental flow chart.
Figure 1. 
 
The experimental flow chart.
Figure 2. 
 
The percent transmission curves for glycerol-based corneas in four groups and one fresh cornea. Each point on each curve represents an average of the five corneal tissues in each group. The entire light spectrum is shown on the horizontal axis. Red curve, G78 group; blue curve, G20 group; green curve, GRT group; magenta curve, SRT group; black curve, one fresh cornea for control.
Figure 2. 
 
The percent transmission curves for glycerol-based corneas in four groups and one fresh cornea. Each point on each curve represents an average of the five corneal tissues in each group. The entire light spectrum is shown on the horizontal axis. Red curve, G78 group; blue curve, G20 group; green curve, GRT group; magenta curve, SRT group; black curve, one fresh cornea for control.
Figure 3. 
 
Slit-lamp images of glycerol-based corneas in four preservation groups show the G78 cornea is transparent just as fresh tissue, but the SRT cornea looks cloudy and extremely edematous.
Figure 3. 
 
Slit-lamp images of glycerol-based corneas in four preservation groups show the G78 cornea is transparent just as fresh tissue, but the SRT cornea looks cloudy and extremely edematous.
Figure 4. 
 
The center thicknesses of glycerol-based corneas in four groups after rehydration. Values of corneal thickness is given as the mean ± SD, scale in μm. Black point, SRT group; red point, GRT group; blue point, G20 group; green point, G78 group.
Figure 4. 
 
The center thicknesses of glycerol-based corneas in four groups after rehydration. Values of corneal thickness is given as the mean ± SD, scale in μm. Black point, SRT group; red point, GRT group; blue point, G20 group; green point, G78 group.
Figure 5. 
 
( A) Comparison of average stress-strain behavior of the four specimen groups. The error bars represent the SDs of stress values. (B) The stress-strain behavior of one fresh corneal specimen. The range of strain levels is much larger than glycerol-based preserved corneas.
Figure 5. 
 
( A) Comparison of average stress-strain behavior of the four specimen groups. The error bars represent the SDs of stress values. (B) The stress-strain behavior of one fresh corneal specimen. The range of strain levels is much larger than glycerol-based preserved corneas.
Figure 6. 
 
Transmission electron micrograph showing cytoarchitecture and organelles in a fresh cornea (A); the integrity of keratocytes was destroyed and condensed in glycerol-preserved corneas, and keratocytes exhibit loss of intracellular organelles and dissolution of cytoplasm (B, SRT; C, GRT; D, G78).
Figure 6. 
 
Transmission electron micrograph showing cytoarchitecture and organelles in a fresh cornea (A); the integrity of keratocytes was destroyed and condensed in glycerol-preserved corneas, and keratocytes exhibit loss of intracellular organelles and dissolution of cytoplasm (B, SRT; C, GRT; D, G78).
Figure 7. 
 
Transmission electron micrograph of collagen fibrils in the midcentral stroma illustrating the arrangement of individual collagen fibrils within various lamellae (A, fresh cornea; B, G78; C, G20; D, SRT) (bar, 500 nm).
Figure 7. 
 
Transmission electron micrograph of collagen fibrils in the midcentral stroma illustrating the arrangement of individual collagen fibrils within various lamellae (A, fresh cornea; B, G78; C, G20; D, SRT) (bar, 500 nm).
Figure 8. 
 
Transmission electron micrograph of collagen fibrils in the midcentral stroma illustrating the parallelism of the collagen fibers (A, fresh cornea; B, G78; C, GRT; D, SRT) (bar, 500 nm).
Figure 8. 
 
Transmission electron micrograph of collagen fibrils in the midcentral stroma illustrating the parallelism of the collagen fibers (A, fresh cornea; B, G78; C, GRT; D, SRT) (bar, 500 nm).
Figure 9. 
 
The glycerol-preserved cornea (G78) show positive reaction (brown) for HLA-ABC (A, limbus; B, epithelium and anterior stroma) and HLA-DR (C, limbus; D, epithelium) (light microscopy; bar, 20 μm).
Figure 9. 
 
The glycerol-preserved cornea (G78) show positive reaction (brown) for HLA-ABC (A, limbus; B, epithelium and anterior stroma) and HLA-DR (C, limbus; D, epithelium) (light microscopy; bar, 20 μm).
Figure 10. 
 
The fresh cornea and glycerol-preserved cornea show positive reaction (brown) for CD45 (A, fresh, limbus; B, fresh, epithelium and anterior stroma; C, G78, limbus; D, G78, epithelium) (light microscopy; bar, 20 μm).
Figure 10. 
 
The fresh cornea and glycerol-preserved cornea show positive reaction (brown) for CD45 (A, fresh, limbus; B, fresh, epithelium and anterior stroma; C, G78, limbus; D, G78, epithelium) (light microscopy; bar, 20 μm).
Table. 
 
Average Percent Transmission of Glycerol-Based Corneas in Four Preservation Groups between Visible Light Wavelength
Table. 
 
Average Percent Transmission of Glycerol-Based Corneas in Four Preservation Groups between Visible Light Wavelength
Group (n = 6) Average Transmittance (%)
SRT 27.4 ± 1.80
GRT 31.9 ± 1.63
G20 51.5 ± 1.03
G78 57.0 ± 0.91
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