Corneal Storage Medium Research Articles

Evaluation of the HB&L System for the Microbiological Screening of Storage Medium for Organ-Cultured Corneas

Aims. To compare HB&L and BACTEC systems for detecting the microorganisms contaminating the corneal storage liquid preserved at 31°C. Methods. Human donor corneas were stored at 4°C followed by preservation at 31°C. Samples of the storage medium were inoculated in BACTEC Peds Plus/F (aerobic microorganisms), BACTEC Plus Anaerobic/F (anaerobic microorganisms), and HB&L bottles. The tests were performed (a) after six days of storage, (b) end of storage, and (c) after 24 hours of preservation in deturgescent liquid sequentially. 10,655 storage and deturgescent media samples were subjected to microbiological control using BACTEC (6-day incubation) and HB&L (24-hour incubation) systems simultaneously. BACTEC positive/negative refers to both/either aerobic and anaerobic positives/negatives, whereas HB&L can only detect the aerobic microbes, and therefore the positives/negatives depend on the presence/absence of aerobic microorganisms. Results. 147 (1.38%) samples were identified positive with at least one of the two methods. 127 samples (134 identified microorganisms) were positive with both HB&L and BACTEC. 14 HB&L+/BACTEC− and 6 BACTEC+/HB&L− were identified. Sensitivity (95.5%), specificity (99.8%), and positive (90.1%) and negative predictive values (99.9%) were high with HB&L considering a 3.5% annual contamination rate. Conclusion. HB&L is a rapid system for detecting microorganisms in corneal storage medium in addition to the existing methods.

Identification and evaluation of a thinning agent compatible with MegaCell DCS, an animal product-free corneal storage medium

PurposeMegaCell DCS, an animal product-free culture medium formulated for storing corneas, is superior to the traditionally used MEM (Eagle’s) with Earles salts, Hepes, and supplemented with foetal calf serum (2 %), glutamine and an antibiotic cocktail (EB MEM). Because this medium does not prevent corneal swelling, and Dextran T500, which is traditionally used for reversing this process before transplant may have adverse effects on corneas, the purpose of the current investigation was to identify an alternative polymer that is compatible with MegaCell DCS.MethodsCorneas maintained in MegaCell DCS or EB MEM were transferred to either EB MEM 5 % Dextran T500 or MegaCell DCS containing 5 % Dextran T500, 4 % polyethylene glycol (PEG) 10,000, PEG 35,000 (2 %, 3 %, 4 %) or Poloxamer 188 (4 %). Endothelial cell losses were determined and corneal hydration levels measured. Stromal cell cultures were generated and immunostained with anti α-SMA antibody. Janus Green was used to compare the viability of endothelial cells of corneas maintained in MegaCell DCS and EB MEM and respectively thinned with PEG 35,000 and Dextran T500.ResultsThe rates of endothelial cell loss from corneas held in MegaCell DCS and thinned in MegaCell DCS containing 5 % Dextran T500, 4 % PEG 10,000 and 4 % Poloxamer 188 for 6 days were similar. When explants of these corneas were cultured myofibroblasts were generated. Although at concentrations of 4 % (w/v) both PEG 10,000 and Poloxamer 188 caused excessive dehydration, the hydration levels of corneas held in MegaCell DCS containing 3 % PEG 35,000 were similar to those of corneas held in EB MEM 5 % Dextran T500. Endothelial cell losses after 6 days were negligible, explants of the corneas generated uniform fibroblastic stromal cell cultures and the extents of Janus Green staining were similar. Over 20 days the inclusion of 5 % Dextran T500 in EB MEM but not 3 % PEG 35,000 in MegaCell DCS, increased the rate of endothelial cell loss.ConclusionPEG 35,000 at a concentration of 3 % w/v does not induce endothelial cell loss and is compatible with MegaCell DCS for thinning corneas prior to transplantation.

Corneal Endothelial Autocrine VIP Enhances Its Integrity in Stored Human Donor Corneoscleral Explant

To demonstrate corneal endothelial (CE) integrity enhanced during eye banking by a brief treatment of human donor corneoscleral explant (explant) with CE autocrine trophic factor vasoactive intestinal peptide (VIP). Paired explants were used as control versus VIP (10 nM)-treated before storage in corneal storage medium (4°C). CE ciliary neurotrophic factor receptor (CNTFRα) and CNTF (0.83 nM) responsiveness in connexin 43 upregulation were monitored (Western blot analysis). CE damage in CNTF-modulated explants and corneal buttons from explants was quantified by analysis of panoramic and microscopic images of the alizarin red-stained corneal endothelium. CE cells scraped from the Descemet's membrane were counted. CE VIP receptor was demonstrated (Western blot analysis). CE cells in every VIP-treated, freshly dissected explant demonstrated higher CNTFRα levels than controls (100% vs. 142% ± 15%; P = 0.014; 7 pairs stored for 4 to 25 days). Nine days after VIP treatment of previously preserved explants, CNTF responsiveness was 174% ± 23% (P = 0.023; 4 pairs) of controls. Panoramic images of explants and corneal buttons revealed that VIP treatment reduced CE damage to 75% ± 6% (P = 0.023; 4 pairs) and 71% ± 11% (P = 0.016; 9 pairs) of controls, respectively, whereas CE damage to 39% (2 pairs) and 23% ± 4% (P < 0.001; 7 pairs), respectively, was revealed in microscopic images. Twenty-one days after VIP treatment of previously preserved explants, CE cell retention was 206% ± 38% (P = 0.008; 14 pairs) of the control. CE cells from human donor corneas expressed VIP receptor VPAC1 (not VPAC2). CE integrity during eye banking was enhanced by a brief treatment of the explant with the CE autocrine VIP.

High-Resolution, Noninvasive, Two-Photon Fluorescence Measurement of Molecular Concentrations in Corneal Tissue

To perform high-resolution, noninvasive, calibrated measurements of the concentrations and diffusion profiles of fluorescent molecules in the live cornea after topical application to the ocular surface. An 800-nm femtosecond laser was used to perform two-photon fluorescence (TPF) axial scanning measurements. Calibration solutions consisting of sodium fluorescein (Na-Fl; concentration range, 0.01%-2.5%) and riboflavin (concentration range, 0.0125%-0.1%) were tested in well slides, and TPF signals were assessed. Excised feline eyeballs preserved in corneal storage medium and with either intact or removed corneal epithelia were then treated with Na-Fl, riboflavin, or fluorescein dextran (Fl-d) of different molecular weight (MW) for 30 minutes. Calibrated TPF was then used immediately to measure the concentration of these molecules across the central corneal depth. The axial resolution of our TPF system was 6 μm, and a linear relationship was observed between TPF signal and low concentrations of most fluorophores. Intact corneas treated with Na-Fl or riboflavin exhibited a detectable penetration depth of only approximately 20 μm, compared with approximately 400 to 600 μm when the epithelium was removed before fluorophore application. Peak concentrations for intact corneas were half those attained with epithelial removal. Debrided corneas treated with 2,000,000 MW Fl-d showed a half-maximum penetration depth of 156.7 μm compared with 384 μm for the 3,000 MW dextran. The peak concentration of the high MW dextran was one quarter that of the lower MW dextran. TPF is an effective, high-resolution, noninvasive method of quantifying the diffusion and concentration of fluorescent molecules across the cornea.

Evaluation of Megacell™ MEM as a Storage Medium for Corneas Destined for Transplantation

Purpose: Gibco’s Minimum Essential Medium with Earle’s salts and HEPES supplemented with glutamine, antibiotics (EB MEM) and 2% foetal calf serum (FCS) is used in European eye banks to store corneas. Although FCS is important to endothelial cell survival in this medium, it is a potential biohazard. Megacell MEM, formulated to reduce the FCS requirement of cells by a factor of 5, has therefore been evaluated as a corneal storage medium. Methods: Corneal stromal and epithelial cells were incubated in Megacell MEM (serum-free or 2% FCS) to assess their viability in these media. Endothelial cell densities of paired corneas held in either EB MEM 2% FCS or Megacell MEM (serum-free or 2% FCS) were measured over 5 weeks. Discs subsequently punched from the centre of these corneas were weighed, dried and reweighed to determine hydration levels. Results: Both corneal stromal and epithelial cells proliferated in Megacell MEM 2% FCS. Relative to EB MEM, 2% FCS Megacell MEM prolonged the viability of corneal endothelial cells and improved their morphological appearance, irrespective of whether it contained FCS or not. This was independent of corneal swelling. Conclusion: Serum-free Megacell MEM is a better storage medium than EB MEM 2% FCS for corneas destined for transplantation

Antimicrobial In Vitro Evaluation of Corneal Storage Media using a Closed Chamber Study Model

Purpose: To evaluate in vitro the antimicrobial influence of the corneal storage media containing antibiotics using a closed chamber study model under the closest simulated environment of corneal preservation process. Materials and Methods: Samples of cornea storage media containing streptomycin and gentamicin (Optisol-GS®) were analyzed at different moments after its contamination with Staphylococcus aureus (ATCC25923). Samples were analyzed at 1, 2, 3, 4, 5, 6, 24, 48, 72 hours; 7 days; and 14 days after contamination. The samples were analyzed using a new study model system that consists of two closed coupled chambers. The upper chamber contained two culture media (chocolate agar and Sabouraud agar) and CO2 indicator (indication of bacterial aerobic activity). The inferior chamber contained supplemented solution with an antimicrobial inhibitor. The bacterial growth parameters were analyzed by the presence or absence of bacteria in chocolate agar and by color change of CO2 indicator when positive. First reading was performed after 24 hours, and, in the absence of bacterial growth, a second reading was carried out after 48 hours. Results: Color change in CO2 indicator was found in samples contaminated after 1, 2, and 3 hours on the first reading. On the second reading, we observed color change in all remaining samples, except for samples contaminated after 14 days. Conclusion: Samples of cornea storage media containing gentamicin sulphate and streptomycin sulphate in vitro showed viable Staphylococcus aureus for up to 7 days of contamination.

Upregulation of chemokine expression in murine cornea due to mechanical trauma or endotoxin

Background and aims:Allograft rejection is the commonest cause of corneal transplant failure and is significantly higher in high-risk patients. Corneal tissue is reported to produce chemokines in response to stress/inflammation....

Preserving the cornea: corneal storage media

The aim of this article is to review the recent literature regarding corneal storage media since the last review that discussed this topic in substantial detail in this journal. During the last few years, despite the development of new corneal storage media and the addition of new additives to established corneal storage media, Optisol GS (Bausch & Lomb, Irvine, California, USA) has continued to remain the popular choice among storage media used in the United States, and traditional organ culture methods are still used in Europe. Recognizing that persistent epithelial defects after corneal transplantation can be a serious complication, however, attention has started to focus on not only preserving the endothelium, but also the epithelium. In addition, there has been more research towards antimicrobial prophylaxis. With recent evidence suggesting that longer storage times may allow better outcomes in high-risk grafts because of the depletion of donor T cells from the donor cornea into the storage media, storage media will need to be optimized for preserving the endothelium for longer periods of time. In addition, because the epithelium is typically unable to be sustained for longer than 1 week in storage media, research toward preserving the epithelium will also be essential.

The effect of basic fibroblast growth factor and epidermal growth factor on corneal endothelial cells preserved in mid-term preserving medium

To investigate the effect of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on corneal endothelial cell preserved in mid-term preserving medium. Corneal rings were preserved in mid-term preserving medium in control group. bFGF (5 ng/ml), bFGF (20 ng/ml), EGF and bFGF + EGF were added into the mid-term preserving medium in experimental groups A, B, C, D respectively. At the 3rd, 7th and 14th day after preservation, the viability of corneal endothelial cells (CECs) was assessed by trypan blue and alizarin red stain. The microstructure of corneal endothelial cells was observed by electron microscope (EM). At the 14th days of preservation, viability of CECs was (10.35 +/- 1.32)% (control group), (62.18 +/- 1.56)% (group A), (92.57 +/- 0.90)% (group B), (71.01 +/- 2.67)% (group C) and (82.59 +/- 1.45)% in group D respectively. The viability of CECs in experimental groups was much higher than in control group (P < 0.01). At the 14th days, corneal rings in control group showed edematous, cloud, defect of integrity of endothelial cells and Y type conjunction broken. But in experimental groups, the corneal edema and folds were slightly, furthermore the cornea ring was transparency and cell shape was intact, especially in group B and group D. Electronic microscope (EM) showed that corneal endothelial cells Y type conjunction in experimental groups was completely integrity, endothelial cells had tight conjunction and rich micro-villa on surface, large cells body and obvious nucleus. Addition of bFGF and EGF in corneal storage medium plays a significant role in maintaining CECs structure and viability.

Assessment of the Number of Lamellae in the Central Region of the Normal Human Corneal Stroma at the Resolution of the Transmission Electron Microscope

To determine the total number of lamellae within the central region of the human corneal stroma by using a continuous section through the corneal stroma and at the ultrastructural resolution of the electron microscope. Six human eye bank corneas, from individuals aged between 42 and 82 years, were received in corneal storage medium (Optisol GS) and then processed for transmission electron microscopy with buffered 2% glutaraldehyde fixation. Thin sections were mounted on parallel bar copper grids for an uninterrupted full corneal thickness observation. A sequence of overlapping micrographs were taken at 2,600x magnification, printed at a final magnification of 3,640x, and assembled as a montage. The number of lamellae were counted across the corneal stromal strip by three observers, independently, by using a preagreed set of criteria for identifying individual lamella. The average number of lamellae per stroma was determined to be 242+/-4 (range, 234-247). In the more anterior region of the stroma, the density of the lamellae was 50% greater than in the posterior stroma (mean, 57+/-12 per 100 microm anteriorly vs. 38+/-5 per 100 microm posteriorly). Interobserver differences were modest and generally less than the intersample variance. When assessed at the resolution of the transmission electron microscope, the number of stromal lamellae in the central region of the human cornea are higher (at approximately 240 per cornea) than most previous estimates reported from light microscopy studies.

CTLA4-Ig prevents corneal allograft rejection in mice

Selective inhibition of T cell activation using the B7 specific fusion protein CTLA4-Ig has been shown to block CD28/B7 signal pathway. In this study, the capacity of soluble CTLA4-Ig alone in the suppressing corneal allograft rejection is further tested in mice, and its mechanism of preventing the cornea from immune rejection in local eye is analyzed. The mouse models of corneal allografts were established. In one group (25 mice), the corneal donors from C57BL/6 mice were incubated in corneal storage medium containing 10 microg/ml of CTLA4-Ig for 24 hours, and then transplanted orthotopically into the recipients cornea of BALB/c mice. In another group (25 mice), as a control, no treatment was conducted on the donor corneas before surgery. The condition of the allografts was monitored using slit lamp microscopy every 3 days and the cellar architecture of selected graft was examined by histological and immunohistochemical techniques weekly. Local corneal cytokines expressing in the rejected grafts were examined. Some mice, which were treated with CTLA4-Ig and their grafts survived beyond 6 weeks, were selected as recipients of skin grafts from C57BL/6 donors. Delayed type hypersensitivity (DTH) was measured after the skin grafts rejection. All mice in the control group were rejected within 14 days after transplantation, while grafts incubated in CTLA4-Ig for 24 hours survived well beyond 100 days. The histopathology of surviving allografts had a normal cellular architecture, whereas the rejected allografts were heavily infiltrated with inflammatory cells and T lymphocytes (including CD4+, CD8+, and CD11+ cells). The cytokines including IL-10, TNF-alpha, IFN-gamma, B7-1 and CD40 were detected in the rejected corneas. Skin grafts from the C57BL/6 donor into the BALB/c mice after corneal transplantation were rejected with the expected median survival time of 11 days. DTH ear swelling challenge was induced after skin rejection, and a significant difference was found comparing with negative control mice. The results indicate that CTLA4-Ig could competitively combine with B7, block CD28/B7 T-cell signals, and inhibit immune responses.

Viability of limbal epithelium after anterior lamellar harvesting using a microkeratome

Objective To determine the viability in cold eye bank storage of different layers of central and limbal corneal epithelium, including the limbal basal stem cell population, on days 0, 3, 6, and 9 after harvest using a large diameter microkeratome system. Methods Twenty-two human whole globes not suitable for transplantation were obtained from an eye bank (San Diego Eye Bank, San Diego, California) and used for study. Large-diameter anterior corneal discs were prepared using a large diameter microkeratome and stained with calcein AM and an ethidium homodimer to differentiate live from dead cells, respectively. A laser confocal microscope and digital imaging were used to distinguish live (green) from dead (red) cells. Central and limbal epithelial regions were isolated and the middle and basal epithelial sections were cell counted by 3 independent observers. These sections were stored up to 9 days at 4° C in an eye bank corneal storage medium. Differences were tested using nonparametric methods. Main outcome measures The percentage of live cells in each of these epithelial layers was determined for up to 9 days in cold eye bank corneal storage medium. Results At all time points studied, the better protected basal epithelial layers displayed greater mean viability than the overlying middle epithelial layers. However, the difference was not statistically significant on all days. When comparing the basal epithelial viability of the limbal and central regions, after day 0 in 4° C cold organ culture, the observed viability of the limbal basal epithelium, the purported location of the limbal epithelial stem cell region, was significantly greater than that of the central epithelium. On day 0, median limbal basal versus central basal epithelial viability were 100% (range, 71.7–100%) versus 98.4% (range, 88.9–100%) ( P>0.05); on day 3, 100% (range, 64.3–100%) versus 63.4% (range, 13.6–95.5%) ( P<0.0005); on day 6, 95.0% (range, 35.0–100%) versus 28.0% (range, 0–92.0%) ( P<0.0005); and on day 9, 95.0% (range, 3.7–100%) versus 68.6% (range, 0–100%) ( P<0.0005). Conclusions After microkeratome harvesting, the limbal basal epithelium is significantly longer lived in cold eye bank storage than central basal epithelium and the middle layers of limbal and central epithelium. This longevity not only bodes well for organ storage of limbal grafts, but also confirms the hardiness of the stem cell region.

Development of Novel Corneal Storage Medium: First Report. Examinations of Rabbit Cornea

Purpose: To develop a new corneal storage medium with a simple formula and evaluate it by histological methods. Methods: Two corneal storage media containing minimum essential medium and 2.5% chondroitin sulfate (pH 7.33), with osmolarity of 320 mOsm/kg, were compared to Optisol-GS. The differences in the two media were the molecular weight (MW) and the source of chondroitin sulfate. The MW of Medium I was 27,500 and the MW of Medium II was 33,700. Japanese albino rabbits were used in this study. A cornea with scleral rim obtained from a rabbit was stored in either Medium I or Medium II and the fellow cornea was stored in Optisol-GS for 7 or 14 days at 4°C. Histological examination of corneal endothelial cells was performed both by scanning electron microscopy and by transmission electron microscopy. Results: At day 7, there was no significant difference in histological findings among the rabbit corneas stored in Optisol-GS, Medium I, or Medium II. At day 14, corneas stored in Optisol-GS or Medium I showed similar histological findings. In Medium II, endothelial cells showed marked degeneration. Conclusions: The results of experiments with rabbit cornea indicated that Optisol-GS and Medium I could preserve endothelial cellular structure better than Medium II. The difference between Medium I and Medium II was only the MW of the chondroitin sulfate used. The MW may be an important factor for determining suitable chondroitin sulfate for use in a corneal storage medium.

Nitric oxide generated by corneas in corneal storage media.

The purpose of the study was to quantify nitric oxide release by human corneal buttons in storage media over time. Group 1 consisted of six chambers of Optisol GS corneal storage media, each containing a viable human corneal button with an attached scleral rim (unsuitable for transplantation), sampled at 1-day intervals for at least 17 days (range, 17-28 days). Group 2 consisted of 34 chambers of Optisol GS media, each used to store a corneal button for penetrating keratoplasty, sampled immediately after each surgery. An unused vial of Optisol GS storage medium was sampled daily for 17 days to serve as a background medium control. The total amount of nitrite and nitrate in each sample was determined by a spectrophotometric method based on the Griess reaction. Data from the daily sampling in group 1 showed that nitrite and nitrate concentrations in storage media containing human corneas increase from a baseline level (beginning at the time the corneas are placed in the media) to an equilibrium concentration of 2.77 microM in a mean time of 6.15 days. Seventy-six percent of the data points from group 2 fell within the 80% predictive interval derived from group 1. No nitrite or nitrate was detected in background medium control samples. The progressive increase in nitrite and nitrate in corneal storage media over time suggests that nitric oxide is continuously released by corneas during storage before transplantation. Given the toxic free radical properties of nitric oxide, corneas in storage media may be subjected to the cumulative toxic effects of nitric oxide.

Development of Corneal Storage Medium: Second Report. Examination of Human Cornea

To develop and evaluate by histological methods a new corneal storage medium with a simple formula. We compared a corneal storage medium which contained minimum essential medium and 2.5% chondroitin sulfate (molecular weight 27,500), pH 7.33, osmolality 320 mOsm/kg with OPTISOL-GS. Paired human donor eyes provided by the Lions Eye Bank of Oregon were stored in a moist chamber until the experiment. A cornea with scleral rim was excised and stored in OPTISOL-GS, and its fellow cornea was stored in the test medium for 5, 10, or 14 days at 4 degrees C. Histological examination of corneal endothelial cells was done by both scanning electron microscopy and transmission electron microscopy. On days 5 and 10, there was no significant difference in histological findings between corneas stored in OPTISOL-GS and those in the test medium. Both corneal groups developed degenerative changes with the increase of storage time, but their histological findings were similar for both storage media. On day 14, corneal endothelial cells showed marked degeneration of intracellular organelles such as a swelling of mitochondria in both media. Human corneas stored in the test medium for 14 days maintained their structure as well as those in OPTISOL-GS. This shows that the newly developed corneal storage medium composed can be used for medium-term corneal storage.

Development of Corneal Storage Medium: First Report. Examination of Rabbit Cornea

Development of Corneal Storage Medium: First Report. Examination of Rabbit Cornea

Protection of corneal allografts by CTLA4-Ig.

CTLA4, a high-affinity ligand of B7, can, in soluble form, prevent antigen-driven T-cell activation by blocking CD28-B7 interaction and can thereby prevent immune graft rejection. In this study, we tested the capacity of soluble CTLA4-Ig alone or in combination with UV-B irradiation to suppress corneal allograft rejection in rabbits. Corneas from Dutch belted rabbits were incubated in corneal storage medium containing 0, 1, 10, 25, or 250 microg/ml of CTLA4-Ig for 18 h and were then transplanted into the vascularized or nonvascularized corneas of New Zealand White rabbit recipients. A series of donor corneas were exposed to UV-B irradiation alone or a combination of irradiation and CTLA4-Ig to determine if these two treatments would have an additive effect in prolonging graft survival. The fate and clinical condition of the allografts were evaluated by slit-lamp photomicroscopic observation and corneal-thickness measurements. Grafts that were rejected were processed for histopathologic and immunohistochemical analysis to determine the characteristics of cells infiltrating the grafts. Grafts placed in nonvascularized corneas showed no differences in survival times, regardless of treatment. Among the grafts placed in vascularized corneas, those incubated with CTLA4-Ig at a concentration of 250 microg/ml failed within 7-14 days. Histopathologic and immunocytochemical examination revealed a dense accumulation of immune inflammatory cells, especially class II major histocompatibility complex (MHC)-expressing, antigen-presenting cells, in the failed grafts. Grafts incubated with CTLA4-Ig at concentrations of 1 and 10 microg/ml had mean survival times greater than the control, untreated corneal allografts. Some of the grafts in these two treatment groups survived for the 100-day observation period, whereas none of the grafts in the other treatment groups survived to this end point. UV-B irradiated grafts incubated with CTLA4-Ig at a concentration of 1 microg/ml appeared to have longer survival times and fewer rejections compared with control, untreated grafts and grafts treated with UV-B or CTLA4-Ig alone. The results show that the CTLA4-Ig coreceptor blocking agent can prolong corneal allograft survival in vascularized graft sites and that UV-B irradiation followed by incubation in CTLA4-Ig may prolong graft survival better than either treatment alone. These results suggest that agents that prevent second-signal interaction between antigen-presenting cells and T lymphocytes may be useful for inhibiting corneal allograft rejection.

Free-Electron Laser (FEL) Ablation of Ocular Tissues

The purpose of the present study was to investigate the ablation characteristics of free-electron laser (FEL) pulses at 5.3 μm and 6.0 μm on ocular tissues. The advanced FEL was developed by the Los Alamos National Laboratory. It operates in the 4–7 μm range, with a macropulse duration of 10 μs and repetition rate of 10 Hz. Each macropulse consists of a train of micropulses that are 15 ps in pulse duration separated by 10 ns. The output energy of macropulses can be adjusted from 5 to 120 mJ. Five transplant grade corneal-scleral buttons preserved in corneal storage medium were used. Wavelengths 5.3 and 6.0 μm were selected based on Fourier-transform infrared absorption spectra of a type I collagen film. Ocular tissue cuts made at 6.0 μm revealed a well-defined ablation boundary with a lateral tissue damage at 10±2 μm. The ablation boundary of the corneal cuts made at 5.3 μm revealed tissue disruption with thermally denatured tissue constituents with loss of organised structure. The lateral dimension of this effect extended up to 220 μm. It was concluded first that Fourier-transform infrared absorption spectra may be useful in selecting laser ablation wavelengths, and second that lasers emitting near 6 μm achieve excellent spatial confinement of laser energy secondary to both protein and water energy partitioning. This wavelength may have potential for cutting ocular tissue such as cornea, sclera, vitreous or lens, because of energy partitioning.

Storage of human corneas in dextran and chondroitin sulfate-based corneal storage medium: changes in stromal free sodium.

To evaluate the hydration and the levels of free and total sodium in human corneal stromata preserved for up to 21 days in a dextran and chondroitin sulfate-based corneal storage medium (Optisol-GS, Chiron IntraOptics, Irvine, Calif) and to evaluate the effect of epithelial removal on stromal sodium and hydration parameters. Stromal hydration was evaluated thermogravimetrically. A sodium-specific electrode and an atomic absorption spectrophotometer were used to determine the amounts of free and of total stromal sodium, respectively, of preserved human corneas. In 50% of the corneas, the epithelium was removed prior to placement in the storage medium. After 3, 7, 14, or 21 days at 4 degrees C, corneas were removed from the storage medium and sodium measurements were taken. In corneas with an intact epithelium, the stromal hydration as well as the stromal free sodium and total sodium levels were relatively constant up to 21 days of storage in the preservation medium. In the absence of the epithelium, the water and sodium contents of the stroma increased significantly during storage. The presence of an intact epithelium is required for maintaining the hydration and sodium levels within the corneal stroma during storage. Removal of the epithelium prior to storage results in increased sodium values and hydration, which may affect postkeratoplasty deturgescence.

Distribution of integrins alpha v beta 5, alpha v beta 3 and alpha v in normal human cornea: possible implications in clinical and therapeutic adenoviral infection.

Integrins are heterodimeric cell surface molecules involved in cell-cell and cell-matrix interactions. Adenoviral entry into human cells has been shown to be dependent on integrins alpha v beta 5 and alpha v beta 3 that promote viral internalisation. We studied the distribution of integrins alpha v beta 5, alpha v beta 3 and the alpha v chain in normal human cornea to investigate possible mechanisms of adenoviral entry to specific corneal cell types. We used immunohistochemistry with monoclonal antibodies to study the distribution of alpha v beta 5, alpha v beta 3 and alpha v in normal human corneas maintained for up to 4 days in corneal storage medium (Optisol) at 4 degrees C (n = 9). Both alpha v beta 5 and alpha v were present to a variable extent on the corneal epithelium and corneal endothelium of most specimens. In some specimens staining of both alpha v beta 5 and alpha v in the epithelium was graded, with more basal than superficial staining, alpha v beta 3 was not detectable in either the corneal epithelium or the corneal endothelium in those specimens tested. The integrin alpha v beta 5 is present on both epithelium and endothelium in the normal human cornea. The role of alpha v integrins in clinical infection and in adenoviral entry for gene transfer is discussed.