Use of autologous serum in corneal epithelial defects post-lamellar surgery

PURPOSE: To evaluate the efficacy of an autologous serum treatment of post-LASIK (laser in situ keratomileusis) corneal epithelial defects in a rabbit model. METHODS: Five milliliters blood samples from 10 New Zealand rabbits were obtained by venepuncture. The serum was aseptically separated and diluted with saline solution to 20%. The final preparation was placed into 3-mL bottles with ultraviolet protection and maintained at 4 degrees C. Corneas were de-epithelialized using a 7-mm optical zone marker. A 160-microm thick flap was created in both eyes of all rabbits using an automatic corneal shaper microkeratome. Right eyes were treated with serum drops 6 times per day. Left eyes were treated with preservative-free artificial tears. Vital staining of the ocular surface and the area of corneal epithelial defect was measured daily for 1 week. Rabbits were humanely euthanized at postoperative day 7, and corneas were fixed and sectioned. Hematoxylin and eosin staining and immunohistochemical analysis were performed. RESULTS: Corneas treated with autologous serum had a statistically significant increase in the epithelial healing rate compared with those treated with artificial tears. Serum-treated corneas showed significantly less terminal transferase-mediated dUTP nick-end labeling (TUNEL) staining in the interface, minimal inflammatory cell infiltration, and less induced synthesis of stromal chondroitin sulfate than did corneas treated with preservative-free artificial tears. CONCLUSIONS: Treatment with autologous serum could be an efficient way to provide essential components to the ocular surface in the treatment of post-LASIK epithelial defects. Autologous serum induces faster epithelial healing than do artificial tears, leading to (1) a decrease in keratocyte apoptosis and migration of fibroblasts and myofibroblasts in the wound site, (2) a decrease in the migration of inflammatory cells, and (3) a consequent inhibition of cytokine release. This treatment could improve long-term refractive results post-LASIK.     

Mitomycin C alters corneal stromal wound healing and corneal haze in rabbits after argon-fluoride excimer laser photorefractive keratectomy.

PURPOSE: To investigate the effects of mitomycin C (MMC) on rabbit cornea wound healing after photorefractive keratectomy (PRK). MATERIALS AND METHODS: Rabbit corneas were stained with dichlorotriazinyl aminofluorescein immediately after PRK. MMC was applied to the right eye and phosphate-buffered salt solution (PBS) to the left. Corneal epithelial wound healing rate and corneal haze were examined. Ultrasound pachymetry was performed. Stromal collagen regeneration was evaluated by fluorescent microscopy. We used terminal deoxyribonucleotidyl transferase-mediated D-uridine 5'-triphosphated-digoxigenin nick-end labeling (TUNEL) assay and transmission electron microscopy (TEM) to evaluate keratocyte apoptosis. RESULTS: In eyes treated with MMC, there was no delay to the healing rate of corneal epithelial wound, and less haze 4 weeks after PRK. Ultrasound pachymetry showed thinner corneal thickness in MMC-treated eyes at week 4. Corneal stromal thickness regression was less in MMC-treated eyes observed by fluorescent microscope at week 4. Keratocyte apoptosis was noted in both MMC- and PBS-treated eyes by TUNEL assay and TEM observation. This study discovered the phenomenon that MMC prolongs keratocyte apoptosis. CONCLUSIONS: Applying MMC after PRK is an effective method to decrease haze formation and corneal stromal thickness regression in rabbit corneas. The effect may be related to MMC prolonging keratocyte apoptosis.     

In vitro comparison of Chen medium and Optisol-GS medium for human corneal storage

PURPOSE: To compare paired human corneas after storage at 4 degrees C in Chen medium (CM) and Optisol-GS medium (OM) for 7, 10, 14, and 21 days. METHODS: One cornea of each pair from nine human donors was randomly stored in either CM or OM, with its mate cornea stored in the other medium. Three pairs of corneas were stored for 7 days and two pairs each were stored for 10, 14, and 21 days at 4 degrees C. Baseline corneal thickness measurements and endothelial photographs were obtained with a specular microscope. Corneal thickness measurements were also taken on days 7, 10, 14, and 21 of storage. At the end of storage, the corneas were warmed 2 hours before endothelial photographs were taken and were then placed in fixative. A corneal endothelial analysis system was used to compare changes in endothelial size and shape after storage. After fixation, the corneal endothelium was examined by scanning electron microscopy (SEM), and TdT-dUTP terminal nick-end labeling (TUNEL) assays with 4'6-diamidino-2-phenylindole (DAPI) counterstaining were performed on tissue sections of each cornea. A laser scanning confocal microscope and an automated digital analysis system were used to detect the presence of TUNEL-positive apoptotic cells in each cell layer and to determine keratocyte densities. RESULTS: Mean corneal thickness at 0, 7, 10, 14, 21 days of storage was 0.69 +/- 0.05 mm, 0.69 +/- 0.06 mm, 0.73 +/- 0.08 mm, 0.87 +/- 0.04 mm, and 0.87 +/- 0.03 mm, respectively, for CM and 0.65 +/- 0.06 mm, 0.59 +/- 0.07 mm, 0.63 +/- 0.03 mm, 0.60 +/- 0.03 mm, and 0.69 +/- 0.02 mm, respectively, for OM (p < 0.0001). The mean decrease in endothelial cell density at the end of the 7-, 10-, and 14-day storage periods was 11 +/- 10% for the CM corneas and 5 +/- 5% for the OM corneas (p = 0.18). SEM showed an intact endothelial monolayer in all corneas. The mean percentages of TUNEL-positive cells in epithelium, stroma, and endothelium of CM-stored corneas were 4 +/- 4%, 2 +/- 3%, and 0.1 +/- 0.3%, respectively, and did not differ from the OM-stored corneal values of 4 +/- 3%, 2 +/- 4%, and 0.9 +/- 1.5%. The percentage of TUNEL-positive cells did not increase with storage time. Keratocyte density was 368 +/- 130 cells/mm2 for CM-stored corneas and 447 +/- 96 cells/mm2 for OM-stored corneas (p = 0.13). CONCLUSIONS: Corneas stored in CM were thicker during storage than those stored in OM. The two storage media did not differ with respect to endothelial cell loss during storage or to the percentage of TUNEL-positive cells or keratocyte density at the end of the storage period.     

Protective role of corneal epithelium against ultraviolet radiation damage

PURPOSE: It is known that the corneal epithelium strongly absorbs ultraviolet radiation (UVR). The aim of the present study was to examine the protective role of corneal epithelium against UVR damage by comparing the biological effect of UVR exposure on whole corneas with that on de-epithelialized corneas. METHODS: Six New Zealand albino rabbit corneas were exposed to UVR centred around 280 nm at a dose that causes biomicroscopically significant keratitis (012 J/cm(2)). Three corneas underwent manual de-epithelialization prior to UVR exposure. A control group of three rabbits underwent only manual de-epithelialization. The animals were killed 76 hours after treatment. The corneas were stained with haematoxylin and evaluated by light microscopy. RESULTS: Corneas that underwent only the exposure to UVR showed a loss of epithelial cells in the treated area. No damage to keratocytes or the stroma was detected. Corneas that underwent manual de-epithelialization showed a loss of epithelial cells, and also keratocytes in the anterior quarter of the corneal stroma. However, corneas that were exposed to UVR after manual de-epithelialization showed very deep stromal damage. The keratocytes disappeared through the entire thickness of the stroma in the UVR-exposed area. CONCLUSION: Exposure to UVR at 280 nm alone does not result in any deep damage to the corneal stroma and keratocytes. Manual de-epithelialization causes the disappearance of anterior keratocytes. However, the stromal damage caused by UVR in the de-epithelialized corneas was very deep. The corneal epithelium serves to protect the deeper corneal structures against UVR damage, probably by absorbing a substantial amount of the UVR energy applied to the eye.     

Viability of keratocytes in epikeratophakia lenticules.

AIM: To study the influence of cryoprotectant, cooling rate, and warming rate on recovery and viability of keratocytes from corneas for cryolathing. METHODS: Corneas were frozen at -50 degrees C for 2 minutes either after exposure to 10% dimethyl sulphoxide in Eagle's MEM for 15 minutes at room temperature (about 22 degrees C), or without earlier exposure to the cryoprotectant. Corneas were cooled either rapidly (20 degrees C/min) or slowly (1 degree C/min), and they were warmed either rapidly (> 50 degrees C/min) by direct transfer into medium at 22 degrees C or slowly (< 20 degrees C/min) in air at 22 degrees C. The cryoprotectant was removed by dilution in medium containing 0.5 mol/l sucrose. Recovery of keratocytes was determined by using collagenase digestion to release the cells from the stroma and trypan blue staining. Viability was assessed by the outgrowth of cells from stromal explants in primary tissue culture. RESULTS: The use of a cryoprotectant before freezing was beneficial, irrespective of the different cooling and warming regimens. Both collagenase digestion and tissue culture revealed that keratocyte survival was improved when corneas were warmed rapidly rather than slowly. The collagenase digestion assay showed an apparently higher recovery of keratocytes after slow cooling (54.3%) than after rapid cooling (34.1%), but no differences in cell viability could be demonstrated by primary tissue culture. CONCLUSION: Although in these experiments slow cooling apparently provided the best recovery of keratocyte numbers (though not viability), previous work had revealed some disruption of the epithelial basement membrane after slow cooling. If viable keratocytes and good preservation of epithelial basement membrane are considered to be prerequisites for epikeratophakia lenticules then it is suggested that corneas should be prepared for cryolathing by freezing rapidly after exposure to 10% dimethyl sulphoxide and, following cryolathing, they should be warmed rapidly.     

Effect of ectopic epithelial tissue within the stroma on keratocyte apoptosis,mitosis, and myofibroblast transformation

The purpose of this study was to examine the effects of the epithelium on processes involved in stromal wound healing. Lamellar epithelial-stromal flaps were produced in rabbit corneas with a microkeratome. Peripheral corneal epithelial tissue, central corneal epithelial tissue, or no epithelial tissue (control) was introduced beneath the flap. Corneas were removed at time points from 4 hr to 1 month after surgery. Tissue sections were analyzed with immunocytochemistry for Keratin 3 (K3) to detect epithelial antigen, terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling (TUNEL) assay to detect apoptosis, immunocytochemistry for Ki67 to detect cell proliferation, and immunocytochemistry for alpha-smooth muscle actin (SMA) to detect myofibroblasts. K3 was detected at the level of the interface from 4 hr to 1 month after surgery in corneas in which epithelial tissue was introduced, but not control corneas, with the exception of one that developed epithelial in growth. Keratocyte apoptosis was significantly higher at 4 hr after flap formation in both groups in which corneal epithelial tissue was introduced beneath the flap compared with controls. Keratocyte proliferation was significantly greater at 72 hr in corneas in which epithelial tissue was introduced beneath the flap compared to the controls. Corneas in which epithelial tissue was introduced into the interface, but not control corneas, had stromal cells expressing alpha-SMA in the stroma anterior and posterior to the interface at 1 week and 1 month after surgery. This was also noted in the control cornea in which there was epithelial ingrowth. Signals derived from the corneal epithelium promote keratocyte apoptosis. Keratocyte proliferation is higher in corneas that have lamellar surgery when epithelial tissue is introduced into the interface. Epithelium-derived signals also participate in the generation and/or maintenance of myofibroblasts in the corneal stroma.     

Healing after photorefractive keratectomy in cat eyes with a scanning mid-infrared Nd:YAG pumped optical parametric oscillator laser

PURPOSE: To evaluate the healing characteristics of cat corneas treated with a new scanning mid-infrared laser system. METHODS: Six adult cats were treated with 6-mm diameter photorefractive keratectomy (PRK) corrections. One eye in each animal was untreated as a control and the other was treated with either a -3.00 or -6.00 diopter ablation. The laser was a new Nd:YAG pumped optical parametric oscillator laser at 2.94 microm with a new scanning delivery system. The pulse width was 7 nanoseconds, the repetition rate was 10 Hz, the size of the laser spot on the eye was 1.0 mm, and the fluence was 150 mJ/cm2. Healing of the cat corneas was followed for 4 months. Slit-lamp and corneal topography evaluations were done at each follow-up examination. Histology was performed at the end of the study. RESULTS: The corneal epithelium healed within 1 week. There was no stromal haze in any eye after the epithelium healed. After the first 2 weeks, slit-lamp examination could not identify which eye was treated. Corneal topography showed corneal flattening. Light microscopy at 4 months revealed normal epithelium and increased keratocyte density in the anterior third of the cornea. Electron microscopy showed discontinuities in the basement membrane and hemidesmosomes. The deep stroma and endothelium were normal. CONCLUSIONS: Cat corneas treated with the new optical parametric oscillator laser healed normally with no adverse effects. Increased keratocyte activity in the anterior stroma was the only noticeable response besides the flattening shown by topography.     

Neutrophil interactions with keratocytes during corneal epithelial wound healing: a role for CD18 integrins

PURPOSE: To determine the role of keratocytes and leukocyte beta(2) (CD18) integrins in neutrophil (PMN) migration through the corneal stroma after epithelial scrape injury. METHODS: Using C57BL/6 wild-type and CD18(-/-) mice, corneas were excised at 6 hours (wild-type) or 24 hours (CD18(-/-)) after central corneal epithelial abrasion, time points determined previously to have similar levels of emigrated PMNs. Corneas were prepared for ultrastructural morphometric analysis of PMNs, keratocyte networks, and collagen. RESULTS: Transmission electron microscopy revealed intact keratocyte networks within the paralimbus that were morphometrically similar, regardless of epithelial injury or mouse genotype. Secondary to epithelial abrasion, extravasated PMNs within the paralimbus developed close contacts with keratocytes and collagen. In wild-type mice, 40% of the PMN surface was in contact with the keratocyte surface, and this value decreased to 10% in CD18(-/-) mice. PMN contact with collagen was similar in wild-type and CD18(-/-) mice, with approximately 50% of the PMN surface contacting the collagen fibrils. Since corneal edema resulting from scrape injury was similar, regardless of genotype and did not involve structural changes in collagen fibrils, these data favor a direct role for CD18 in mediating PMN contact with keratocytes. CONCLUSIONS: The data show that in response to epithelial scrape injury, PMN migration in the corneal stroma involves close contact between keratocytes and collagen. Although PMN-keratocyte contacts require CD18 integrins, contact with collagen is CD18 independent. Fundamentally, PMN migration along keratocyte networks constitutes the beginning of a new experimental concept for understanding leukocyte migration within the wounded cornea.     

Stiffening of Rabbit Corneas by the Bacteriochlorophyll Derivative WST11 Using Near Infrared Light

Purpose. We evaluated the efficacy and safety of photochemical corneal stiffening by palladium bacteriochlorin 13'-(2-sulfoethyl)amide dipotassium salt (WST11) and near infrared (NIR) illumination, using ex vivo and in vivo rabbit eye models. Methods. Corneas of post mortem rabbits and living rabbits were pretreated topically with 2.5 mg/mL WST11 in saline or in 20% dextran T-500 (WST-D), washed and illuminated with an NIR diode laser (755 nm, 10 mW/cm(2). Studies with corneas of untreated fellow eyes served as controls. Tensile strength measurements, histopathology, electron spin resonance, and optical spectroscopy and fluorescence microscopy were used to assess treatment effects. Comparative studies were performed with standard riboflavin/ultraviolet-A light (UVA) treatment. Results. WST11/NIR treatment significantly increased corneal stiffness following ex vivo or in vivo treatment, compared to untreated contralateral eyes. The incremental ultimate stress and Young's modulus of treated corneas increased by 45, 113, 115%, and 10, 79, and 174% following 10, 20, and 30 minutes of incubation with WST11, respectively. WST-D/NIR had a similar stiffening effect, but markedly reduced post-treatment edema and shorter time of epithelial healing. WST11/NIR and WST-D/NIR generate hydroxyl and superoxide radicals, but no singlet oxygen in the cornea. Histology demonstrated a reduction in the keratocyte population in the anterior half of the corneal stroma, without damage to the endothelium. Conclusions. Treatment of rabbit corneas, with either WST11/NIR or WST-D/NIR, increases their biomechanical strength through a mechanism that does not involve singlet oxygen. The WST-D/NIR treatment showed less adverse effects, demonstrating a new potential for clinical use in keratoconus and corneal ectasia after refractive surgery.     

人脐带血清器官培养液保存猪角膜的实验研究

目的 观察胎牛血清器官培养液和人脐带血清器官培养液对猪角膜内皮细胞形态学、组织学、超微结构、酶活性及代谢等的影响。方法 器官培养方法：4周以内31℃密闭培养,之后脱水24h。选择猪眼113只,其中100只角膜分为两组进行配对器官培养保存。第1组(50只角膜)：应用培养液Ⅰ(含10％胎牛血清);第2组(50只角膜,第1组的对侧角膜)：应用培养液Ⅱ(含10％人脐带血清)。13只角膜作为正常对照。器官培养每周每组各取出12只角膜进行内皮细胞形态学分析、HE染色、酶组织化学染色。保存2周、4周时行扫描电镜检查。检测保存前后培养液的pH值和葡萄糖、乳酸浓度。器官培养过程中行微生物学检测。结果 器官培养期间角膜内皮细胞单层保持完整,多形性增加,两组之间角膜内皮细胞密度、细胞面积的变异系数和六边形细胞比例在保存4周内差异无显著意义。保存4周的猪角膜与正常猪角膜相比,平均内皮细胞丢失率第1组为10．98％,第2组为10．85％。两组角膜器官培养后的组织学、超微结构、酶活性无明显区别。扫描电镜显示内皮细胞层完整,细胞形态改变。酶组织化学染色显示上皮、内皮细胞酶活性旺盛,基质细胞的酶活性随保存时间的延长而降低。角膜代谢状态良好。器官培养液污染率为6％。结论 两种器官培养液均可以保持角膜内皮活性达4周,推测人脐带血清能够替代胎牛血清作为角膜器官培养液的成分。(中华眼科杂志,2004,40：533-538)     

Keratocyte apoptosis after corneal collagen cross-linking using riboflavin/UVA treatment

PURPOSE: Combined riboflavin/UVA treatment inducing collagen cross-links in the cornea has been shown to increase the biomechanical rigidity of the cornea and has been used successfully in the treatment of progressive keratoconus. The current study was undertaken to investigate the possible cytotoxic effect of combined riboflavin/UVA treatment on corneal keratocytes in vivo. METHODS: Thirty-four New Zealand white rabbits were treated with 0.1% riboflavin solution and surface UVA irradiances ranging from 0.75 to 4 mW/cm2 (1.35- 7.2 J/cm2) for 30 minutes. The animals were euthanized either 4 (n = 6) or 24 (n = 28) hours postoperatively. Four additional control eyes underwent epithelial debridement alone. The corneas of the enucleated eyes were evaluated in routine histologic sections. In addition, the TUNEL technique and transmission electron microscopy were used for the detection of keratocyte apoptosis. RESULTS: In the control eyes with corneal epithelial debridement only, apoptotic keratocytes were found in the anterior 50 microm of the corneal stroma 4 hours postoperatively. However, riboflavin/UVA-induced apoptosis was only visible in the rabbit eyes enucleated 24 hours postoperatively. In these eyes, we found apoptosis of keratocytes down to a variable stromal depth depending on the applied UVA irradiance. A cytotoxic UVA irradiance for keratocytes in the range of 0.5-0.7 mW/cm2 could be deduced. CONCLUSIONS: Riboflavin/UVA treatment leads to a dose-dependent keratocyte damage that can be expected in human corneas down to a depth of 300 microm using a surface UVA dose of 5.4 J/cm2. Future studies should be done to examine the keratocyte repopulation and exclude possible adverse sequelae of keratocyte loss like stromal scarring or thinning.     

Keratocyte apoptosis associated with keratoconus.

Keratoconus is an ectatic corneal dystrophy associated with stromal thinning and disruption of Bowman's layer. The purpose of this study was to explore a possible association between keratocyte apoptosis and keratoconus. Keratocyte apoptosis was evaluated in corneas of patients with keratoconus, corneas of patients with stromal dystrophies, and normal donor corneas using the transferase-mediated dUTP-digoxigenin nick and labeling (TUNEL) assay. Keratocyte apoptosis was also studied in keratoconus and normal corneas using transmission electron microscopy. TUNEL-stained keratocytes were detected in 60% of corneas with keratoconus, but only 35% of corneas with stromal dystrophies (P =0.03). The number of TUNEL-positive keratocytes detected in the keratoconus, stromal dystrophy, and normal corneas was 7+/-1 (mean+/-standard error, range 0-20), 2+/-0. 8 (range 0-9), and 0+/-0 (range 0-0) TUNEL-positive cells per section, respectively. The differences between the keratoconus and the stromal dystrophy (P =0.0097) or the normal cornea (P =0.01) groups were statistically significant. The difference between the stromal dystrophy and normal cornea groups was not statistically significant (P =0.45). The stromal dystrophy group was included to account for surgery-associated keratocyte apoptosis. No TUNEL-stained keratocytes were detected in normal corneas. Cell morphologic changes consistent with apoptosis were detected by transmission electron microscopy (TEM) in keratocytes of keratoconus corneas, but not in keratocytes in normal corneas. Chronic keratocyte apoptosis associated with ongoing epithelial injury may link risk factors associated with keratoconus such as chronic eye rubbing, contact lens wear, or atopic eye disease. Similarly, increases that have been detected in several different degradative enzymes in keratoconus corneas could be associated with chronic keratocyte apoptosis and less than perfect control of release of intracellular contents.     

Comparison of corneal preservation media for corneal hydration and stromal proteoglycan loss.

Changes in the composition of stromal proteoglycans (PGs) have been previously demonstrated in corneal edema, wound healing, and disease. To examine if PGs are lost during corneal preservation, rabbit corneal PGs were radiolabeled in situ with 35S-sulfate and 3H-glucosamine, excised and stored in either modified McCarey-Kaufman medium (MMK), K-Sol (Coopervision-Cilco, Bellevue, WA, U.S.A.), corneal storage medium (CSM), or Dexsol (Chiron Ophthalmics, Irvine, CA, U.S.A.) for up to 14 days. The percentage of total radio-label lost was significantly greater from de-epithelialized corneas (p less than 0.05) and from corneas stored in CSM (p less than 0.05) or K-Sol (p less than 0.05). Corneas stored in CSM for 4 and 7 days were significantly more hydrated than corneas stored in MMK, K-Sol, or Dexsol. After 14 days of storage, all corneas were hydrated above control values with the Dexsol-stored cornea showing the least hydration. Results suggest that loss of stromal PGs during corneal storage is reduced with epithelial integrity and with preservation media containing dextran.     

Normal human keratocyte density and corneal thickness measurement by using confocal microscopy in vivo

PURPOSE: To quantify keratocyte density according to stromal region and subject age and to measure the thickness of the normal human cornea and its layers in vivo. METHODS: Seventy normal corneas of 70 subjects were examined by confocal microscopy (contact lens wearers were excluded). Ages of subjects ranged from 12 to 80 years, with 10 subjects per decade. Images were recorded by continuously focusing the optical section through the full-thickness central cornea. Two independent human observers manually identified bright objects (keratocyte nuclei) against a dark background to quantify keratocyte density. This method was validated histologically in three human corneas. Thickness measurements were obtained by plotting mean reflected light intensity in images against corneal depth, and calculating distances between intensity peaks that corresponded to corneal layers. RESULTS: Full-thickness central keratocyte density was 20,522 +/- 2,981 cells/mm(3) (mean +/- SD, n = 69). The number of keratocytes in a full-thickness column of central stroma, which had a cross-sectional area of 1 mm(2), was 9624 +/- 1385 cells. Keratocyte density was highest in the anterior 10% of the stroma. Full-thickness keratocyte density was correlated with age (r = -0.62, P < 0.001), decreasing 0.45% per year. Central corneal thickness was 563.0 +/- 31.1 microm (mean +/- SD) and central epithelial thickness was 48.6 +/- 5.1 microm. CONCLUSIONS: This is the first study to quantify regional keratocyte density comprehensively in vivo across a broad age range of normal human subjects. The method was acceptable to both subject and observer, and may prove useful for quantifying keratocyte density in patients with corneal disorders or after corneal surgery.     

The effects of epithelial viability on stromal keratocyte apoptosis in porcine corneas stored in Optisol-GS

PURPOSE: To investigate the effect of corneal epithelium on the viability of corneal stromal keratocytes in Optisol-GS. METHODS: After sterilization, corneoscleral buttons were excised and stored in Optisol-GS for various time periods. Group 1 corneas (n = 40) underwent mechanical corneal epithelial debridement before storage while group 2 corneas (n = 40) were stored with intact epithelium. Changes in corneal thickness, keratocyte density, and keratocyte apoptosis were investigated immediately, at 4 hours, and on days 1, 2, 3, 5, 7, and 14 in the preservation medium. The differences between group 1 and 2 corneas were analyzed. RESULTS: Corneal thickness increased significantly in the second week of preservation in both groups, though more substantially in group 1. Significant corneal epithelial apoptosis was noticed in the first week in group 2 corneas. Corneal stromal keratocyte density decreased with prolonged preservation time. DNA laddering was detected by ligation-mediated polymerase chain reaction throughout the experiment periods in both groups, but the increase of keratocyte apoptosis was more significant after 5 days of preservation, especially in group 1. CONCLUSIONS: Stromal keratocytes underwent apoptosis in Optisol-GS. The absence of corneal epithelium during preservation further increased the stromal keratocyte apoptosis.     

Structural analysis of normal corneas and diseased corneas by applying second harmonic generation

We have established a second harmonic generation (SHG) microscopy system for imaging of the human cornea with a mode-locked femtosecond laser and a laser confocal microscope. This SHG microscopy system has allowed us to scan corneal tissue noninvasively ex vivo and to obtain three-dimensional images of corneal collagen lamellae. Such three-dimensional imaging of the normal anterior cornea revealed that collagen lamellae at the anterior stroma are inter-woven and adhere to Bowman membrane with these adherent lamellae being designated "sutural lamellae." Sutural lamellae adhere to Bowman membrane at an angle of approximately 19 degrees, whereas the angle of lamellae in the mid-stroma relative to Bowman membrane is smaller. We hypothesize that the structural unit consisting of both Bowman membrane and the sutural lamellae contributes to the rigidity and anterior curvature of the cornea. SHG imaging of keratoconic corneas revealed an either abnormal or a total lack of structure of the sutural lamellae, suggesting that this abnormality might be related to that of the corneal anterior curvature in such corneas. Furthermore, SHG imaging of corneas affected by stromal edema showed that the structure of the sutural lamellae was maintained, although abnormal collagen signals both above and below Bowman membrane were detected in corneas affected by clinical stromal edema for more than 12 months. SHG imaging of the structure of collagen lamellae in normal and diseased corneas thus has the potential to provide insight both into the mechanism for maintenance of corneal curvature as well as into the pathophysiology of corneal diseases.     

Eurosol versus fetal bovine serum-containing corneal storage medium

PURPOSE: To evaluate the usability of Eurosol, a new medium-term corneal storage medium without components of bovine origin. METHODS: Ten pairs of human donor corneas were placed in tissue culture at 31 degrees C for 7, 14, 21, 28, or 35 days. One cornea of each pair was cultivated in conventional storage medium on Earls' minimum essential medium base containing 2% fetal bovine serum; the other one was stored in Eurosol. Corneas were examined with inverse light microscopy; corneal thickness was measured; and scanning electron microscopy was performed. RESULTS: No significant difference in corneal thickness and endothelial cell count was found at any time. Scanning electron microscopy showed a complete endothelial cell layer on all corneas. CONCLUSION. The findings indicate a potential clinical applicability of the tested serum-free medium-term storage medium, offering a safer alternative to conventional media containing fetal bovine serum.     

Changes of MK medium during storage of human cornea.

By comparing the composition of McCarey-Kaufman (MK) medium before and after corneal storage we attempted to identify specific physiological changes in the medium as predictors of tissue damage. We also tried to determine if hydrocortisone (a lysosomal membrane stabiliser) added to the medium could reduce tissue damage during storage. Corneas (human and rabbit) were stored in the MK medium with and without hydrocortisone for 4 days at 4 degrees C. The water and nitrogen contents of the stored cornea were compared with those of the fresh cornea. The medium was analysed before and after corneal storage to determine the concentrations of glucose, protein, and amino acids as well as pH and osmolarity. Scanning electron microscopy (SEM) was used to estimate the degree of the corneal endothelial cell damage. The nitrogen contents and dry weights of the steroid treated and untreated stored corneas were similar to those of the fresh unstored cornea. The steroid treated cornea contained a lesser amount of water than the untreated cornea. The cornea stored in medium without steroid took up a greater amount of glucose from the medium than the cornea stored in medium with steroid. As compared with their concentrations in the fresh unused medium the concentrations of leucine, lysine, and glycine were lower and that of glutamic acid was higher in both the media used for corneal storage. However, the steroid treated storage medium as compared with the untreated storage medium had a greater reduction in the lowering of leucine, lysine, and glycine, and a lesser reduction in the increase of glutamic acid. Steroid treated medium also had a lesser amount of protein released from the stored cornea. Changes in the pH and osmolarity of the media before and after corneal storage were not remarkable. SEM showed that the endothelial cells of the cornea stored in the medium containing steroid were less damaged than those of the cornea stored in the medium without steroid.     

Non-invasive assessment of the donor corneal endothelium using ocular redox fluorometry.

AIMS: To investigate the usefulness of ocular redox fluorometry for evaluating donor corneal endothelial viability. METHODS: Corneas from 42 recipients of penetrating keratoplasty and four donor corneas were examined by ocular redox fluorometry. Autofluorescence from reduced pyridine nucleotides (PN) and oxidised flavoproteins (Fp) of the human corneal endothelium were measured non-invasively, and the PN/Fp ratio was used as a tissue metabolic indicator. Specular microscopy and electron microscopy were also performed. RESULTS: Both the quality of specular microscopic image and the PN/Fp ratio were significantly correlated with the degree of corneal endothelial damage determined by histological examination. Corneas with poor specular microscopic image showed significantly decreased PN/Fp ratio compared with corneas with good or fair specular images (p = 0.041 and 0.027, respectively). The PN/Fp ratio increased in corneas with mildly damaged endothelium but decreased in corneas with severely damaged endothelium determined by histological examination. Evaluation of corneal endothelium by combination of specular microscopy and ocular redox fluorometry showed excellent association with that of histopathological examination (p < 0.0001). CONCLUSION: Ocular redox fluorometry is useful for assessing donor corneal endothelial viability. Combination of ocular redox fluorometry and specular microscopy may increase the ability of donor cornea selection.     

Lysosomal enzyme levels in corneal storage media. K-Sol versus McCarey-Kaufman

Evidence indicates that lysosomal enzymes can carry out corneal autolysis during corneal storage and that they are damaging to the corneal endothelium. The authors investigated the release of lysosomal enzymes into two corneal storage media (K-Sol and McCarey-Kaufman [M-K]) by paired human donor corneas during 4 degrees C storage. The authors also studied the interaction of these media with lysosomal enzymes from human cornea. K-Sol and M-K stimulated (P less than 0.01) both beta-glucuronidase and alpha-galactosidase about equally. beta-N-Acetyl-glucosaminidase, a major catabolic enzyme of the cornea, was inhibited by the chondroitin sulfate in K-Sol by over 90% (P less than 0.01). Corneas stored in M-K released more lysosomal enzymes than corneas stored in K-Sol. At 4 days, the values approached significance (P less than 0.06) and by day 10 significantly higher values were found in the M-K media (P less than 0.01). Both storage methods showed a linear release. Individual corneas were found to vary in their release rates. Whether corneas that release more enzyme will show higher endothelial cell loss or produce less successful penetrating keratoplasty grafts deserves further study.