Medium-term preservation of human corneas in an enriched culture medium at +37 degrees Centigrade

A study of 36 penetrating keratoplasties using donor corneas stored at +37 degrees C in organ culture incubation for an average of 12 days prior to transplantation is reported. The criteria for the survival of corneal grafts were mean central corneal thickness, clarity of the cornea and mean endothelial cell density. A total of 86% of the grafts remained clear after 12 months follow-up time. The mean central corneal thickness was 0.47 mm. The mean endothelial cell density was 1452 cells/mm2. Based on the results of this study, organ culture reported here appears to be a safe and efficient method of medium to long-term corneal storage.     

A morphometric study of endothelial cells of human corneas stored in MK media and warmed at 37 degrees C.

No study has yet been done to investigate the changes in endothelial cell size, perimeter, and density that may result from the warming of corneas in MK (McCarey-Kaufman) medium for specular microscopy. In the present investigation eye bank eyes were stored in MK medium at 4 degrees C and rewarmed daily for six days at 37 degrees C before specular photography of the endothelium was performed. These photographs were compared with wet mount preparations stained with trypan blue and alizarin red made from the same corneas and those stored without rewarming for six days. In addition all corneas were qualitatively analysed with the scanning electron microscope (SEM). The data from serial specular photography were insufficient to allow significant conclusions to be drawn about day to day changes in cell morphology. However, analysis of wet mount preparations revealed that cell density and perimeter varied significantly between those corneas rewarmed daily and those held in cold storage for six days. SEM studies showed an intact cell monolayer with cell loss along the folds of corneal endothelium. We therefore concluded that repeated rewarming at 37 degrees C of corneas stored in MK medium at 4 degrees has a deleterious effect on cell morphology and that folds induced by swelling of corneal tissue result in endothelial cell damage with some loss.     

Cell death during corneal storage at 4 degrees C.

PURPOSE: To evaluate cell death in human donor corneas stored at 4 degrees C, to determine whether terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end labeling (TUNEL) discriminates between apoptosis and necrosis in corneas stored at 4 degrees C. METHODS: Ten human corneas were stored in Optisol (Chiron Ophthalmics, Irvine, CA) at 4 degrees C for periods ranging from 0 to 21 days and then fixed for histologic examination. Central corneal sections from each cornea were examined by transmission electron microscopy (TEM) and by the TUNEL assay. Electron micrographs of at least 15 keratocytes each from the anterior, middle, and posterior stroma were examined by three masked observers who graded each cell as normal, apoptotic, or necrotic. Central sections from the same corneas were processed by the TUNEL assay and evaluated with a laser scanning confocal microscope to determine the percentage of apoptotic cells. RESULTS: By TEM, apoptosis occurred in 23% of the keratocytes and necrosis in 12%. By TUNEL assay, apoptosis occurred in 11% of the keratocytes, with the results in individual corneas being similar to the findings by TEM for apoptosis, rather than for necrosis. By TUNEL assay, apoptosis occurred in 13% of the epithelial cells and in 8% of the endothelial cells. The percentage of apoptotic cells and storage time correlated significantly for the epithelium, but not for the keratocytes or endothelium in this small sample. CONCLUSIONS: Both apoptosis and necrosis occur in cells during corneal storage at 4 degrees C, with apoptosis appearing to predominate. The TUNEL assay identifies cells undergoing apoptosis, but not necrosis, in corneal tissue. Inhibition of apoptosis in corneas stored at 4 degrees C may prolong acceptable storage times.     

Preservation of human corneas in an enriched culture medium at +37 degrees Centigrade: histological and biochemical analyses

Using the methods of Doughman, Sperling and Pels 66 human corneas were preserved up to five weeks in a modified tissue culture medium at +37 degrees C. The organ culture medium consisted of the following ingredients: Dulbecco Iscov's medium, 2% fetal calf serum, penicillin 100 IU/ml, Streptomycin 0.5 mg/ml, and fungizone 5 mg/ml. After culture the corneal endothelium was examined by light microscopy after staining with trypan blue 0.3% and alizarin red 1%. The number of dead cells was counted and the morphological alterations were described at 7, 14, 21 and 35 days. Biochemical analysis of the medium (pH, lactate, glucose) during storage has allowed the comparison of three kinds of storage: 50 ml, 20 ml, and 20 ml with 10 ml substitution weekly. After culture the number of dead cells did not exceed 1% at each period indicating that no dead cell was present at that time. Alteration of the cell shape and formation of rosette and joint meetings of 5 to 8 cells were the dominating morphological changes of the endothelial cells. The endothelial layer was intact and coherent on to 35 days culture. Endothelial cells loses during culture were not determined. The biochemical studies have shown the better quality (pH stability, anaerobic ratio) of storage in 50 ml medium or 20 ml 50% weekly substitution medium. This last kind of storage has given the best metabolic conditions for the preserved corneas. However for long period of storage up to 3-4 weeks, renewing 50% of the medium is found appropriate. At the end of this study a clinical trial is proposed.     

干燥法长期保存角膜板层移植1267例临床研究

目的：评价干燥脱水法长期保存角膜片在板层治疗性角膜移植术中的效果。方法：保存角膜片选自我院眼库干燥保存符合临床标准、复水透明的完整角膜片，临床病例选自我院角膜病组1976年8月～1999年12月期间行板层治疗性角膜移植患者1267例，从手术前后视力比较、原发病灶控制、植片存活情况进行临床效果综合分析。结果：保存角膜片复水透明合格率为94．9％，临床治愈率：感染性角膜溃疡／穿孔88．8％，血管翳性全角膜白斑98．3％。化学及热烧伤(穿孔，睑球角粘连等)92．1％，圆锥角膜及急性圆锥角膜99．5％，角膜变性(边缘性及前基质层)99．0％。其他88．9％，平均总治愈率93．5％。结论：1．对各种严重的角膜感染、药物治疗无法控制的病人，治疗性角膜移植术是唯一有效的方法。2．采用无水氯化钙一硅胶干燥脱水长期保存的供体角膜能为临床随时提供角膜材料，是一项简单易行、便于推广应用的成功率很高的保存方法。     

Comparison of Chen Medium and Optisol-GS for human corneal preservation at 4 degrees C: results of transplantation

PURPOSE: To compare results after transplantation of donor corneas stored in Chen Medium (containing beta-hydroxybutyrate without sodium bicarbonate or chondroitin sulfate) to corneas stored in Optisol-GS medium (containing sodium bicarbonate and 2.5% chondroitin sulfate). METHODS: We performed 32 consecutive penetrating keratoplasties with donor corneas stored at 4 degrees C in either Chen Medium or Optisol-GS by random assignment. Corneal thickness measurements were made at 1 day, 1 week, 3 weeks, 2 months, and 1 year postkeratoplasty. Specular microscopic images of the donor endothelium were obtained at the beginning of storage and 2 months and 1 year postkeratoplasty. The percentage of intact epithelium 1 day after keratoplasty and the graft epithelialization time were estimated by the surgeons. Donor rim cultures were performed. RESULTS: No statistically significant differences in corneal thickness or endothelial cell loss between the corneas stored in the two media were found at any time, although differences of less than 12% cell loss or 0.09-mm thickness at 2 months or less than 25% cell loss or 0.10-mm thickness at 1 year could not be excluded with 90% certainty in this small series. The mean percentages of intact graft epithelium on day 1, 64% for Chen Medium and 65% for Optisol-GS, were not significantly different. Endothelial cell density 2 months postkeratoplasty was significantly decreased for corneas stored in both media. Endothelial cell loss at 2 months was directly correlated with storage time in both media. CONCLUSIONS: After keratoplasty, no statistically significant differences in corneal thickness, epithelial survival, and endothelial cell loss were found between corneas stored in Chen Medium and Optisol-GS. Endothelial cell loss at 2 months was significantly correlated with storage time in both media.     

Tolerance of corneas to multimolar dimethyl sulfoxide at 0 degrees C. Implications for cryopreservation

Attempts to improve current methods of cryopreservation of corneas, whether by conventional freezing and thawing or by vitrification in the absence of ice, will require the use of high concentrations of cryoprotectants. In this study we extend our previous investigation of the tolerance of rabbit corneas to multimolar concentrations of the cryoprotectant dimethyl sulfoxide (Me2SO) added and removed at 0 degrees C in CPTES, a hyperkalaemic preservation solution containing the impermeant anionic buffer N-Tris(hydroxymethyl)methyl-2-aminoethane sulphonate (TES). Isolated corneas were exposed to 1, 2 or 3 mol/l Me2SO at 0 degrees C to minimize any effect due to temperature-dependent chemical toxicity and attention was given to the procedure for diluting Me2SO from the tissue in order to minimize osmotic stress to the endothelium. Endothelial integrity following these procedures was assessed both by the ability to control stromal hydration during perfusion on the specular microscope and by the structural integrity when examined by light and electron microscopy. The presence of an active endothelial pump and good morphology were demonstrated in corneas exposed to 1 and 2 mol/l Me2SO; serial dilution of the cryoprotectant was more beneficial than a single-step direct dilution. Corneas immersed directly into 3 mol/l Me2SO were irreparably damaged irrespective of the method of dilution. Sequential addition of 1 M, then 2 M and finally 3 M cryoprotectant followed by serial dilution was, however, tolerated by the endothelium and minor alterations to the structural integrity of the endothelial layer were rapidly repaired. The osmotic nature of these observations are analyzed and discussed.     

Cryopreservation of rabbit and cat corneas at -18 to -24 degrees C.

A simple method of corneal cryopreservation, in which corneas were frozen at -18 to -24 degrees C, was examined. Rabbit and cat corneas were placed successively in solutions of 50% fetal calf serum in McCarey-Kaufman medium with an increasing glycerol and glucose content. They were then frozen and stored in a -20 degrees C domestic freezer. Rabbit corneas stored in this way were examined in vitro by light and scanning electron microscopy, and both rabbit and cat corneas were also assessed after orthotopic allotransplantation into adult recipient animals. Functional corneal grafts were obtained with rabbit and cat tissue that had been cryopreserved for 3-4 weeks and 1 week, respectively. Endpoint analysis (by light and scanning electron microscopy) of grafts that had survived for 50 days indicated the presence of an intact corneal endothelial monolayer. The corneal endothelium slowly degenerated as the storage time was increased. Importantly, however, the endothelium appeared to withstand the freezing and thawing processes and we conclude that it may be possible to store corneas at temperatures above -196 degrees C, without the need for complex, low-temperature cryopreservation systems.     

Our experience with myopic implants. Initial optical results

28 patients with high myopia (over-10 dioptries) were implanted in anterior chamber with a myopic IOL. 3 months postoperatively the visual acuity without correction was equal to the preoperative acuity. With a light correction, especially of residual astigmatism, we noticed a gain of acuity of 0.2.     

Recovery of endothelial function after vitrification of cornea at -110 degrees C

PURPOSE: To determine whether endothelial function is retained after ice-free cryopreservation of cornea by vitrification at -110 degrees C. METHODS: Rabbit corneas, mounted on support rings, were exposed to a solution containing 6.8 M propane-1,2-diol (PROH) and cooled at approximately 7 degrees C/min to -110 degrees C, which was below the glass transition temperature (T(g)) of the solution. After rewarming at approximately 12 degrees C/min and removal of the PROH, endothelial function was assessed by monitoring corneal thickness during perfusion at 34 degrees C. RESULTS: Addition and removal of 6.8 M PROH without cooling to -110 degrees C did not markedly impair endothelial function, although corneas were thicker than control samples. There was no visible crystallization of ice during cooling to -110 degrees C; but a few small, discrete sites of crystallization remote from the endothelium, were observed during warming. After removal of the PROH, corneas approximately doubled in thickness during the first 3 hours of perfusion, but they then started to thin, which suggested active control of stromal hydration by the endothelium. This was confirmed in a further set of experiments by removal of bicarbonate ions from the perfusate at this point, which resulted in further swelling at +58 +/- 2 microm/hour (SD; n = 4). Restoring bicarbonate to the perfusate halted this swelling, and the corneas then thinned at -13 +/- 2 microm/hour (n = 4). Morphologically, staining with trypan blue and alizarin red S showed an apparently intact endothelial monolayer. CONCLUSIONS: Rabbit corneal endothelium tolerated exposure to 6.8 M PROH, and endothelial function was evident after vitrification at -110 degrees C. Preliminary morphologic results with vitrified human cornea also showed retention of endothelium.     

Gene expression in keratoconus. Initial results using DNA microarrays

INTRODUCTION: Keratoconus is a non-inflammatory ocular disease characterised by conical deformation, progressive thinning and scarring of the central cornea. Despite intensive investigations, the exact cause of the disease still remains unclear. Clinical studies provide strong indications of a major genetic role in the aetiology. We set out to examine the involvement in the manifestation of keratoconus of any of the 5,600 gene specificities available on the Affymetrix GeneChip HuGeneFL. METHODS: After examination of two corneas they were stored in RNAlater, RNA was extracted and hybridised on the chips. Using a combination of dyes it was possible to read the chips with laser detection and to visualise the gene expression pattern. RESULTS: We found an upregulation of collagens, versican, metalloproteinases and cell adhesion proteins. A downregulation was observed for TIGR protein, cytokeratins, eyes absent homologue (Eab1) and the proteins for radical treatment selenoprotein P and monooxygenase. CONCLUSIONS: Our results indicate that keratoconus is a process in which repair and scar-formation mechanisms operate at the same time. As candidate genes for this mechanism, collagen IV and related proteoglycans were favoured.     

Epikeratophakia for keratoconus. Long-term results using fresh, free-hand made lamellar grafts.

Epikeratophakia using fresh, free-hand made corneal grafts was done in 16 patients with keratoconus. The follow-up period averaged 27.8 months (range 13-45 months). A significant improvement of visual acuity was obtained (p = 0.002), and 14 of the 16 eyes (87.5%) achieved a corrected visual acuity greater than or equal to 6/12. The spherical equivalent and the cylindrical refractive error were reduced (p less than 0.05), and a significant flattening of the central corneal curvature was obtained (p less than 0.002). The mean postoperative central corneal astigmatism was 4.25 D. Postoperatively, the mean central corneal thickness was 0.670 mm, and the mean central thickness of the epithelialized graft was 0.336 mm. Six patients reported some postoperative glare or blurring of vision, despite a visual acuity greater than or equal to 6/9 on the Snellen chart. No significant subjective or objective changes were noticed after the 6-month postoperative follow-up visit.     

A 40 degrees gaze down position for pneumatic displacement of submacular hemorrhage: clinical application and results

PURPOSE: To test the validity of the geometric conclusion that 40 degrees gaze down is optimal for pneumatic displacement of a subretinal hemorrhage (SRH) in the macula. METHODS: Nine consecutive patients with SRH in the macula had an intravitreal injection of perfluorocarbon gas sufficient to cover the macula when the patient gazed down 40 degrees below the horizontal. They were asked to maintain the gaze down position for 20 minutes every hour while awake. RESULTS: The SRH in eight of nine patients was displaced rapidly in the first week. Visual acuity improved in seven patients. Visual recovery was limited by the presence of a subpigment epithelial component. CONCLUSIONS: Gaze 40 degrees below the horizontal will rapidly displace a subretinal hemorrhage that is covered by a gas bubble.