RESUMEN
Different methods of corneal cryopreservation have been introduced, those employing intracellular cryoprotectants such as Me2SO or glycerol being the most widely favored. We investigated the influence of several freeze-thaw trauma variables on the survival of porcine endothelial monolayers when employing the extracellular cryoprotective agent dextran. We first examined the effects of various dextran concentrations and then, having ascertained the optimal concentration, further investigated the influence of fetal calf serum (FCS) concentration in the cryopreservation medium, the cooling rate, the thawing temperature, and the length of the preincubation in the freezing medium prior to cryopreservation. The numerical densities of endothelial cells were determined at dissection in hypoosmotic balanced salt solution and after organ culture by staining with alizarin red S and trypan blue. Morphological evaluation was not performed directly after thawing but after a subsequent organ culture at 37 degrees C to detect latent cell damage after freeze-thaw trauma. Our data revealed that corneas cryopreserved in minimal essential medium containing 10% dextran but lacking FCS, preincubated for 3 h, frozen at a cooling rate of 1 degrees C/min, and thawed at 37 degrees C incurred the lowest cell losses (22.4%, SD +/- 3.8). We conclude that dextran is an effective cryoprotectant for freezing of porcine corneas. However, variations between species in the results of cryopreservation require further investigation of an in vivo animal model and studies with human corneas before its clinical use can be recommended.
Asunto(s)
Córnea , Criopreservación/métodos , Crioprotectores , Dextranos , Conservación de Tejido/métodos , Animales , Bovinos , Supervivencia Celular , Medios de Cultivo , Endotelio Corneal/citología , Técnicas In Vitro , PorcinosRESUMEN
PURPOSE: To investigate the impact of transportation, simulated under laboratory conditions, on the corneal endothelium preserved by Optisol-GS, Likorol-DX, Likorol, and MK-Medium. METHODS: Three hundred twenty corneas from freshly slaughtered pigs were stored in Optisol-GS. Likorol-DX, Likorol. and MK-Medium for 1, 3, 6, or 10 days. After short-term preservation, the transportation was simulated on a laboratory shaker at 4 degrees C with an acceleration of 0-100 km/h in 16 seconds for 5 hours. Mate corneas served as the control. Corneal endothelial cell density was determined at the time of dissection, directly before the experiment, and after subsequent organ culture. RESULTS: A significant cell loss induced by transportation simulation was not observed in any experimental group. Preservation in MK-Medium starting at 3 days of short-term preservation resulted in a significant cell loss. Storage for up to 6 days in Optisol-GS, Likorol-DX, and Likorol did not result in a significant decrease in cell density. CONCLUSION: Short-term preserved corneas can be routinely distributed without a reevaluation, if the tissue is preserved for a shorter time as recommended by the manufacturers.
Asunto(s)
Córnea/efectos de los fármacos , Criopreservación/métodos , Medio de Cultivo Libre de Suero/farmacología , Medios de Cultivo/farmacología , Soluciones Preservantes de Órganos/farmacología , Preservación de Órganos/métodos , Transportes , Animales , Recuento de Células , Sulfatos de Condroitina , Mezclas Complejas , Córnea/citología , Trasplante de Córnea , Dextranos , Endotelio Corneal/citología , Endotelio Corneal/efectos de los fármacos , Gentamicinas , Técnicas In Vitro , Compuestos Orgánicos , Porcinos , Factores de TiempoRESUMEN
BACKGROUND: Donors who have suffered a traumatic death are, on average, younger than those who have died from other causes, and the time from death to enucleation (DET) also tends to be shorter. Corneae obtained from such donors are therefore considered particularly suitable for grafting. One of the reasons for excluding a donor cornea from transplantation is the occurrence of endothelial cell necrosis during organ culture, and we investigated whether the incidence of this phenomenon bears a relationship to death by traumatic or nontraumatic means. METHODS: Data from 2125 donor corneae were collected using standardized evaluation protocols between January 1991 and December 1995 and included information on cause of death, age and DET, as well as endothelial cell loss and necrosis. Traumatic deaths were recorded in 346 cases, nontraumatic deaths in the other 1779 cases. Since differences in age (P = 0.006) but not in DET occurred within each of these groups, a more refined comparison, with matched data (< 35 years), was also undertaken. RESULTS: Forty (11.6%) of the 346 corneae derived from traumatic death donors manifested total or partial endothelial cell death in organ culture. The corresponding figure in the nontraumatic death group was only 105/1779 (5.8%; P = 0.0002). After matching for age, endothelial cell death during culturing was revealed in 18 (13.5%) of the 133 of the traumatic death corneae and in 3 (2.6%) of the 115 nontraumatic death ones (P = 0.004); the overall incidence of endothelial cell death (total or partial) during organ culture was 6.8% (145/2125). Endothelial cell loss during culturing of the 227 age-matched donor corneae which still had an intact endothelial monolayer at the end of the incubation period was 340 +/- 388 cells/mm2 in traumatic death corneae (n = 115) and 255 +/- 318 cells/mm2 in nontraumatic death ones (n = 112; P = 0.051). CONCLUSION: Corneae obtained from traumatic death donors were more liable to undergo total or partial endothelial cell death during organ culture than were those procured from nontraumatic death ones. However, in corneae which survived the period of culture, there was no significant difference in endothelial cell loss between the two groups. Whilst the mechanism underlying this increased susceptibility of traumatic death corneae to cell death remains elusive, the data gleaned from this investigation nonetheless emphasize the potential importance of being able to perform meaningful in vitro viability tests on donor corneae; this is possible only under organ culture conditions.
Asunto(s)
Causas de Muerte , Muerte Celular , Endotelio Corneal/patología , Técnicas de Cultivo de Órganos , Heridas y Lesiones/etiología , Heridas y Lesiones/patología , Adulto , Recuento de Células , Enucleación del Ojo , Humanos , Persona de Mediana Edad , Estudios Retrospectivos , Donantes de TejidosRESUMEN
Many clinical studies have been performed investigating corneal endothelial cell loss after penetrating keratoplasty using different preservation methods. Most studies, however, included variable follow-up intervals, neglecting the dynamic cell loss over the course of time. The aim of this study was to perform endothelial cell evaluation 5 years after penetrating keratoplasty using two different storage methods. Fifty-four patients were examined 5.2 (+/-0.5) or 4.9 (+/-0.6) years after surgery. Twenty-four patients had received a cornea stored in a moist chamber, 30 patients a cornea preserved by organ culture. All corneas had remained clear in the follow-up period. The follow-up periods did not differ significantly (P = 0.26). The post-mortem time of moist-chamber-stored tissue was significantly lower (P < 0.001), endothelial cell density significantly higher (P < 0.001) in organ-culture-preserved corneas. Donor age (P = 0.64) and patient age (P = 0.046) did not differ significantly. Average corneal endothelial cell density was 1070 (+/-499) cells/mm2 using moist chamber stored and 1095 (+/-497) cells/mm2 using organ-culture-preserved tissue (P = 0.82). Five years after penetrating keratoplasty, we were not able to find significant differences in corneal endothelial cell density using either moist chamber or organ-culture-preserved corneas. We prefer to use organ culture since there is a lower incidence of primary graft failure, tissue with longer postmortem times can be used, and surgery can be scheduled.
Asunto(s)
Endotelio Corneal/patología , Queratoplastia Penetrante/patología , Preservación de Órganos/métodos , Adulto , Anciano , Recuento de Células , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana EdadRESUMEN
PURPOSE: To assess whether the biological quality of corneoscleral tissue dissected in situ is, after organ culture, comparable to that harvested after enucleation. METHODS: Corneoscleral discs were prepared from 23 donor eyes, either after enucleation, under laminar flow conditions (right eyes; group 1) or by direct excision in situ (left eyes; group 2). Endothelial cell counts were made and the degree of tissue contamination assessed both prior to and upon termination of organ culture. RESULTS: Microbial growth was found in 12/22 conjunctival swabs collected from group 1 eyes and in 14/22 of those obtained from group 2 globes (p = 0.76). Bacterial growth was detected in four primary culture media, two from each group, at low colony densities. No significant difference in endothelial cell counts were encountered between the two groups, either immediately after dissection [group 1: 2940 +/- 308 (2100-3500) c/mm2; group 2: 2947 +/- 345 (2200-3700) c/mm2; p = 0.945] or upon termination of organ culture [group 1: 2646 +/- 321 (1895-3200); group 2: 2723 +/- 312 (2100-3650); p = 0.413]. CONCLUSION: Dissection of corneoscleral discs in situ may serve as an alternative to the conventional technique if consent is obtained to remove only the cornea. The risk of contamination is no higher and endothelial cell viability no lower than in tissue derived from enucleated globes, provided that the excision is performed by a skilled surgeon and a rigorous disinfection protocol is instigated.
Asunto(s)
Córnea/cirugía , Trasplante de Córnea , Enucleación del Ojo , Esclerótica/cirugía , Adolescente , Adulto , Anciano , Bacterias/aislamiento & purificación , Recuento de Células , Supervivencia Celular , Recuento de Colonia Microbiana , Conjuntiva/microbiología , Córnea/microbiología , Córnea/fisiología , Endotelio Corneal/citología , Humanos , Persona de Mediana Edad , Técnicas de Cultivo de Órganos , Esclerótica/microbiología , Esclerótica/fisiología , Donantes de TejidosRESUMEN
Endophthalmitis after penetrating keratoplasty is rare but more frequently observed than after other intraocular procedures. The main source may be a contaminated donor cornea. This survey reviews current concepts of donor globe decontamination and avoidance of infection during preservation. Basically, we suggest the use of a 3% PVP-Iodine solution for decontamination of donor globes. Using short-term preservation techniques, the tissue should be stored at ambient temperature after preparation of corneoscleral disc or before transplantation for a while to allow antibiotics to act against decontaminants. Tissue stored by organ culture should be kept in a sealed vial. Alternatively, the medium may be changed every 10 to 14 days under sterile conditions.
Asunto(s)
Desinfección/métodos , Endoftalmitis/prevención & control , Queratoplastia Penetrante , Preservación de Órganos/métodos , Esterilización/métodos , Infección de la Herida Quirúrgica/prevención & control , Humanos , Factores de RiesgoRESUMEN
PURPOSE: To investigate the feasibility to use hydroxyethylstarch as an alternative deswelling additive in short-term preservation media. MATERIALS AND METHODS: Corneoscleral discs were prepared from pairs of eye balls of freshly slaughtered pigs. Corneas were stored in MEM-medium containing either 10% or 20% hydroxyethylstarch 450 000 at 4 degrees C in a refrigerator. Subsequently, the tissue was stored for 24 hours in organ culture at 37 degrees C in MEM-medium containing 10% fetal calf serum to detect latent endothelial cell damage. Mate corneas were treated the same except for being stored in Optisol GS during 4 degrees C storage. We determined corneal endothelial cell density, stromal thickness, and glucose concentration in the medium directly after preparation, after short-term storage at 4 degrees C, and after subsequent organ culture at 37 degrees C. Scanning electron microscopy of corneal endothelium was performed at each step during the experimental course. RESULTS: We did not observe any significant differences in endothelial-cell density between experimental groups and control groups. No decrease in endothelial-cell density was observed during the course of experiments. No increase in stromal thickness was determined in any group after short-term storage at 4 degrees C. Corneas stored in medium containing 20% hydroxyethylstarch showed a decrease in stromal thickness after short-term storage. After subsequent organ culture all corneas displayed a uniform stromal swelling. Glucose concentrations in the media decreased in all groups during the experiment. In scanning-electron microscopy we observed a reversible degeneration of cell borders after storage at 4 degrees C. Additionally, corneas stored in Optisol GS showed a reversible cobblestone appearance at this stage of the experiments. CONCLUSION: Hydroxyethylstarch appears to be an alternative to the use of dextran and chondroitin sulfate as a deswelling additive in corneal preservation media.
Asunto(s)
Trasplante de Córnea/patología , Endotelio Corneal/efectos de los fármacos , Derivados de Hidroxietil Almidón/farmacología , Técnicas de Cultivo de Órganos , Animales , Recuento de Células , Sustancia Propia/efectos de los fármacos , Sustancia Propia/patología , Criopreservación , Medios de Cultivo , Endotelio Corneal/patología , Microscopía Electrónica de Rastreo , PorcinosRESUMEN
We reviewed the results of sterility testing from culture media of 1,134 donor corneas preserved by organ culture at 37 degrees C in our eye bank. All corneas were stored in minimal essential medium containing 2% fetal calf serum, 0.1 mg/ml penicillin G, 0.1 mg/ml streptomycin, and 2.5 micrograms/ml amphotericin B. After removal of ocular adnexal tissue, donor globes were rinsed with sterile saline solution, incubated in 3% polyvinylpyrrolidone-iodine solution for 3-5 min, and subsequently rinsed again with sterile saline solution. Samples for microbiological evaluation were obtained from the initial evaluation medium, at every medium change (every 10 days), and from the medium used for deswelling of the individual cornea 1 day before transplantation. Incidence of contamination was 0.53% (6 of 1,134 corneas). Three corneas were contaminated by Micrococcus species, three by fungi. We conclude from our study that a combination of rinsing donor globes with sterile saline solution, the initial use of a disinfectant, and the employment of penicillin/streptomycin and amphotericin B in the organ culture medium, which have been commonly used in cell culture for decades, result in a low incidence of bacterial and fungal contamination of corneas preserved by organ culture.
Asunto(s)
Bacterias/aislamiento & purificación , Córnea/microbiología , Trasplante de Córnea , Hongos/aislamiento & purificación , Bancos de Tejidos , Trasplante de Córnea/normas , Medios de Cultivo , Desinfectantes/farmacología , Alemania , Humanos , Incidencia , Persona de Mediana Edad , Técnicas de Cultivo de Órganos , Bancos de Tejidos/normas , Donantes de Tejidos , Conservación de Tejido/métodosRESUMEN
Previous experimental studies concerning morphological changes of corneal endothelium after cryopreservation have been performed directly or within hours after thawing. The purpose of this study was to investigate morphological alterations of corneal endothelium up to 1 week after thawing in organ culture. We evaluated (1) pig corneas that were cryopreserved within 4 h of death, (2) pig corneas that had been stored in cryopreservation medium at 31 degrees C for 4 h before cryopreservation, and (3) as a control, corneas that were preserved in organ culture until the specific time of evaluation. Corneas were frozen to -196 degrees C in an automated freezing chamber in a cryopreservation medium containing chondroitin sulfate. Morphologic evaluation after trypan blue and alizarin red staining was performed after 1 day and after 1 week of organ culture preservation after thawing. One day after thawing, computer-assisted morphometric analysis of endothelial cell density and necrotic areas revealed endothelial cell loss of 25.7% in group 1 (8% necrosis), 48.7% in group 2 (17.4% necrosis) and 0% in group 3 (0% necrosis) compared to freshly dissected corneas. After 1 week, endothelial cell density had decreased by another 29% in groups 1 and 2 and by 11% in group 3 compared to results 1 day after thawing. However, necrotic areas could no longer be detected in any of the groups. We conclude that the highest decrease in endothelial cell density can be expected within 1 day after thawing. Further cell loss seems to be due to non-mitotic wound healing resulting in a confluent endothelial monolayer.
Asunto(s)
Supervivencia Celular/fisiología , Criopreservación , Endotelio Corneal/patología , Animales , Recuento de Células , Sulfatos de Condroitina/metabolismo , Necrosis , Técnicas de Cultivo de Órganos , Porcinos , Cicatrización de Heridas/fisiologíaRESUMEN
Ninety porcine corneas were evaluated by vital staining with alizarin red S and trypan blue in a three-step experiment. Central cell densities were counted (a) on freshly dissected corneas (n = 30), (b) on cryopreserved corneas directly after thawing (n = 30), and (c) after a postthawing organ culture interval of 24 h (n = 30). Two freezing methods were used: (a) minimum essential medium--containing 20% fetal calf serum and (b) the same but containing additionally 2% chondroitin sulfate. Directly after thawing neither method showed significant cell loss (3.9% and 3%) compared to fresh tissue. After postthawing organ culture, however, tissue that had been frozen without chondroitin sulfate displayed a cell loss of 73.5% compared to corneas of the same freezing protocol directly after thawing. Corneas in chondroitin sulfate containing medium showed a cell loss of only 33.2%. We conclude that reliable morphologic evaluation should not be obtained from cryopreserved corneas examined directly after thawing.
Asunto(s)
Córnea/patología , Criopreservación/métodos , Endotelio Corneal/patología , Animales , Antraquinonas , Recuento de Células , Supervivencia Celular , Sulfatos de Condroitina , Colorantes , Medios de Cultivo , Técnicas de Cultivo de Órganos , Porcinos , Azul de TripanoRESUMEN
In an experimental study using porcine corneas we investigated the influence of several variables of freeze-thaw trauma on survival of corneal endothelial cells after corneal cryopreservation employing chondroitin sulfate as a cryoprotectant. We examined the influence of: (1) the concentration of chondroitin sulfate in the cryopreservation medium, (2) the concentration of fetal calf serum in the cryopreservation medium, (3) the cooling rate, and (4) the preincubation period in the cryopreservation medium before freezing. Controls consisted of corneas cryopreserved in culture medium without cryoprotectants and corneas frozen in dimethyl sulfoxide (Me2SO) by the method of Capella et al. (Preservation of viable corneal tissue. Cryobiology, 2, 116-121, 1965). Morphological evaluation was performed by determining endothelial cell density after staining with alizarin red S and trypan blue. Morphological evaluation was not performed directly after thawing but after a subsequent storage period in organ culture at 31 degrees C in order to detect latent cell damage after freeze-thaw trauma. The group that yielded the best endothelial cell density was evaluated in perfusion chamber experiments in order to measure the functional integrity of the tissue. Controls included corneoscleral rims from freshly slaughtered pigs, corneas stored in organ culture for 1 day, and fresh corneas mechanically denuded of endothelium. It was demonstrated that corneas that had been cryopreserved in MEM medium containing 2% chondroitin sulfate and 20% fetal calf serum with a cooling rate of 1 degree C/min displayed the highest endothelial cell density (2430 cells/mm2, SO = 383, n = 15) compared with freshly dissected corneas (3395 cells/mm2, SD = 200, n = 48). Control corneas frozen by the method of Capella et al. demonstrated only a poor outcome. We conclude that in our experimental environment, corneal cryopreservation with chondroitin sulfate provides higher corneal endothelial cell densities than corneas preserved by conventional methods. Moreover, even slight changes in variables that affect the tissue in the freeze-thaw cycle have a major impact on corneal endothelial cell survival after cryopreservation.
Asunto(s)
Sulfatos de Condroitina , Córnea , Criopreservación/métodos , Animales , Recuento de Células , Córnea/citología , Crioprotectores , Endotelio Corneal/citología , Estudios de Evaluación como Asunto , Técnicas In Vitro , Perfusión , PorcinosRESUMEN
Clinical and experimental studies with rabbit and human corneas have shown the correlation between short postmortem times and successful corneal cryopreservation. In this experimental study we investigated this phenomenon considering the latent freeze-thaw-induced cell damage. Enucleated eye-balls of freshly slaughtered pigs were stored in moist chambers at 4 degrees C for 2, 4, 8, 16, 32, and 72 h before cryopreservation. After thawing, the corneas were organ-cultured for 1 day. After staining with trypan blue and alizarin red S the tissue was evaluated morphometrically, calculating the amount of necrotic areas on the central corneal surface and the endothelial cell density. Corneas stored up to 32 h before cryopreservation showed no difference regarding the amount of necrosis and endothelial cell density compared to freshly cryopreserved tissue. Corneas stored 72 h before cryopreservation revealed no endothelial cell survival. We conclude that a post-mortem time of up to 32 h before corneal cryopreservation has no influence on endothelial cell survival.
Asunto(s)
Córnea , Criopreservación/métodos , Endotelio Corneal/patología , Preservación de Órganos , Animales , Autólisis/patología , Recuento de Células , Supervivencia Celular , Microscopía de Contraste de Fase , Necrosis , Técnicas de Cultivo de Órganos , Porcinos , Factores de TiempoRESUMEN
It is recommended by the Eye Bank Association of American that the post-mortem time of donor tissue stored in intermediate storage should not exceed 6 h. In an experimental study we investigated the impact of post-mortem time on the storage of corneas kept in McCarey-Kaufman medium. Eyeballs of freshly slaughtered pigs were kept in moist chamber storage at 4 degrees C for 0, 6, 12, 24, and 48 h. Subsequently, corneal-scleral rims were dissected and stored in MK-Medium for 2 days at 4 degrees C. The corneas were stored in organ culture for another 2 days at 31 degrees C in MEM medium containing 10% fetal calf serum. A control group consisted of corneas that had been stored in organ culture for 2 days. Morphometric analysis revealed no significant changes in endothelial cell density compared to control corneas after up to 24 h of moist chamber storage prior to storage in MK medium. A significant decrease in endothelial cell density could only be detected in corneas that had been kept in moist chamber storage for 48 h before MK medium storage. We conclude that the recommendation that only donor corneas with short post-mortem times be used may exclude large amounts of potentially feasible tissue for penetrating keratoplasty.
Asunto(s)
Trasplante de Córnea/patología , Endotelio Corneal/citología , Cambios Post Mortem , Animales , Recuento de Células , Medios de Cultivo , Compuestos Orgánicos , Porcinos , Conservación de TejidoRESUMEN
Clinically employed methods of corneal cryopreservation usually use the intracellular cryoprotectant dimethyl sulfoxide (DMSO). However, it has been demonstrated that extracellular cryoprotectants such as chondroitin sulfate (ChS) also display effective cryoprotection. The purpose of our study was to investigate the effect of combinations of intra- and extracellular cryoprotectants in corneal cryopreservation. Porcine corneas were cryopreserved in a cryopreservation medium consisting of MEM-medium containing 20% fetal calf serum and 2% chondroitin sulfate. The medium was varied by the addition of 2%, 4% and 8% DMSO. Sixty corneas were cryopreserved at -196 degrees C and thawed at 37 degrees C in a water bath. Morphometric evaluation was not performed directly after thawing but after a 24-h storage period in organ culture. Cryopreservation in medium without DMSO revealed the best results concerning endothelial cell density (2581 cells/mm2). Addition of 2% or 4% DMSO revealed no significant changes in endothelial cell density. Addition of 8% DMSO, however, resulted in a significant decrease (1312 +/- 319 cells/mm2) combined with a significantly higher amount of necrotic areas in the central corneal surface. We conclude that combining intra- and extracellular cryoprotectants does not enhance endothelial cell density after corneal cryopreservation. Higher concentrations of DMSO added to the cryopreservation medium appear to have a negative impact on endothelial cell viability.
Asunto(s)
Trasplante de Córnea/patología , Criopreservación/métodos , Crioprotectores/farmacología , Endotelio Corneal/patología , Animales , Sulfatos de Condroitina/farmacología , Dimetilsulfóxido/farmacología , PorcinosRESUMEN
Rabbit corneas were injected intrastromally with Pseudomonas aeruginosa. Sixteen hours after injection, the rabbits were divided randomly into four treatment groups (3 rabbits/6 eyes per group: 1, ciprofloxacin and prednisolone; 2, ciprofloxacin only; 3, prednisolone only; 4, untreated. Ocular signs of inflammation were graded in a masked fashion by slit lamp examination before injection and 16 and 27 hours after injection. Slit lamp examination scores were significantly lower in eyes receiving ciprofloxacin and prednisolone or prednisolone alone, compared with scores in untreated eyes. Slit lamp examination scores were not significantly lower in eyes receiving ciprofloxacin alone, compared with untreated controls. The numbers of viable bacteria in the corneas treated with ciprofloxacin and in the corneas treated with ciprofloxacin and prednisolone were similar and were significantly less (P less than 0.0001) than those in untreated corneas, indicating that the presence of the steroid did not interfere with the bactericidal action of ciprofloxacin.
Asunto(s)
Ciprofloxacina/uso terapéutico , Infecciones Bacterianas del Ojo/tratamiento farmacológico , Queratitis/tratamiento farmacológico , Prednisolona/uso terapéutico , Infecciones por Pseudomonas/tratamiento farmacológico , Administración Tópica , Animales , Recuento de Colonia Microbiana , Sustancia Propia/microbiología , Modelos Animales de Enfermedad , Quimioterapia Combinada , Infecciones Bacterianas del Ojo/patología , Queratitis/microbiología , Queratitis/patología , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/crecimiento & desarrollo , Conejos , Distribución AleatoriaRESUMEN
In corneal cryopreservation and other preservation techniques, the donor tissue post-mortem time is believed to affect endothelial cell survival. In this study, porcine eyes were stored in a moist chamber at 4 degrees C for 2, 4, 8, 32, and 72 h. Then the corneas were subjected to cryopreservation. After thawing, a 24-h interval of organ culture was used as a viability test. The cell density of the central cornea and the percentage of Descemet's membrane denuded of endothelium were determined with vital staining and morphometric methods. Corneas stored 2-32 h before cryopreservation showed no difference in necrotic areas or cell density of surviving endothelium. Corneas stored 72 h before cryopreservation revealed no endothelial cell survival. We conclude that a post-mortem time of up to 32 h before corneal cryopreservation has no influence on endothelial cell survival.
Asunto(s)
Supervivencia Celular/fisiología , Criopreservación , Endotelio Corneal , Cambios Post Mortem , Animales , Endotelio Corneal/patología , PorcinosRESUMEN
We investigated the effect of hyaluronic acid, molecular weight 250,000 and 500,000 as an osmotic additive to corneal preservation media. Porcine corneal specimens were incubated in organ culture for 8 days, followed by endothelial cell count and ultrasonic pachymetry. The preservation medium MEM-Earle with 2% FCS contained either no additives, 6% dextrane, MW 500,000 2% chondroitin sulfate, 1% hyaluronic acid MW 250,000, or 1% hyaluronic acid, MW 500,000. Whereas corneal thickness was estimated to be about 4 mm in the group without osmotic additives, all other media kept the corneas thin between 700 and 760 microns. The endothelial cell counts after preservation were similar in all groups. As hyaluronic acid is a physiological substance of the eye, it may offer some advantages for corneal perservation.
Asunto(s)
Trasplante de Córnea/métodos , Endotelio Corneal/efectos de los fármacos , Ácido Hialurónico/farmacología , Preservación de Órganos/métodos , Equilibrio Hidroelectrolítico/efectos de los fármacos , Animales , Medios de Cultivo , Supervivencia de Injerto/efectos de los fármacos , Peso Molecular , PorcinosRESUMEN
Pneumolysin, a cytolytic protein produced by Streptococcus pneumoniae, has many properties which suggest it may be an important virulence factor in pneumococcal ocular infections. To directly test this possibility, we have constructed pneumolysin-negative strains of S. pneumoniae and compared their virulence with that of the wild type in a rabbit model of intracorneal infection. A pneumolysin-negative strain produced by chemical mutagenesis (probably a point mutant) was found to be no less virulent than the parent strain. However, a strain bearing a deletion in the pneumolysin gene showed greatly reduced virulence. This strain produced less pathology while showing significantly higher bacterial counts. These results suggest that a property of the pneumolysin molecule other than its cytolytic (hemolytic) activity may be involved in its pathogenic mechanism of action. This property may be the ability to activate complement, known to be a function of pneumolysin, which results in influx of PMNs, reducing the bacterial counts but also producing tissue damage.
Asunto(s)
Enfermedades de la Córnea/microbiología , Infecciones Bacterianas del Ojo/microbiología , Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae/patogenicidad , Estreptolisinas/fisiología , Animales , Proteínas Bacterianas , Southern Blotting , Deleción Cromosómica , Enfermedades de la Córnea/patología , Mutagénesis , Plásmidos , Conejos , Especificidad de la Especie , Streptococcus pneumoniae/genética , Estreptolisinas/genética , Transfección/genética , Virulencia/genéticaRESUMEN
Five patients with Down's syndrome underwent penetrating keratoplasty for keratoconus. In three patients, the indication for surgery was acute corneal hydrops, which had not resolved in the three months before surgery. The other two patients had corneal scars. Two patients had combined penetrating keratoplasty, cataract extraction, and intraocular lens insertion. Four of the five patients maintained clear grafts at their most recent follow-up examination. Two of the five patients had one or more graft reaction episodes; one graft was lost. Good results can be obtained in penetrating keratoplasty for keratoconus in patients with Down's syndrome who do not demonstrate a tendency toward excessive eye rubbing and for whom a single observant caretaker can be relied on to provide consistent postoperative care.
Asunto(s)
Síndrome de Down/complicaciones , Queratocono/cirugía , Queratoplastia Penetrante , Adulto , Supervivencia de Injerto , Humanos , Queratocono/complicaciones , Lentes Intraoculares , Masculino , Persona de Mediana Edad , Pronóstico , Agudeza VisualRESUMEN
In order to compare the results after experimental corneal cryopreservation of porcine corneas to human tissue we cryopreserved several human corneas that were not suitable for transplantation. The corneas were frozen in a medium containing 2% chondroitin sulfate, 15% DMSO, and 20% fetal calf serum in MEM-medium to -196 degrees C and thawed at 37 degrees C in a waterbath. Subsequently they were kept in organ culture for 24 h. After vital staining with trypan blue and alizarin red S they were evaluated morphometrically. Endothelial cell densities after the experiment ranged between 47% and 64% of the endothelial cell counts revealed before cryopreservation. Human corneal endothelium showed enlargement of single nucleated cells, and large numbers of giant cells with one or two nuclei but only few multinucleated giant cells and only few necrotic spots compared to porcine tissue in which multinucleated giant cells and large necrotic areas were a frequent finding. We conclude that human corneal endothelium is less sensitive to the freeze-thaw trauma than porcine tissue. Porcine tissue can be used as a susceptible model for the development of new methods of corneal cryopreservation.