Characterization of a stabilized form of microplasmin for the induction of posterior vitreous detachment.

PURPOSE: To investigate the stability and safety of a diluted acidified form of microplasmin and its ability to induce a posterior vitreous detachment (PVD) following intravitreal injection in post-mortem porcine eyes. METHODS: Microplasmin diluted in normal saline (NS) and balanced salt solution (BSS+) was assayed for residual activity by hydrolysis of the chromogenic substrate Glu-Phe-Lys-pNA. Residual activity on vitreous was determined by injecting aliquots of microplasmin reconstituted in balanced salt solution (BSS+) or normal saline (NS) kept at room temperature (RT) for up to 1 hr, then injected in aliquots of porcine vitreous and incubated for 2 hr at 37°C. The breakdown products were submitted to SDS Page electrophoresis and compared to determine the level of enzymatic activity. Pig eyes were incubated with graded concentrations of microplasmin 0.625, 1.25, or 2.50 mg/mL reconstituted in BBS+ or NS. Morphologic alterations and the ability to induce a PVD was assessed by light and electron microscopy. RESULTS: Microplasmin's enzymatic activity in an in vitro assay in BSS+ was 70% of its baseline value after 30 min, and about 50% after 60 min at RT. The corresponding effect on degradation of vitreous gel was 60 and 40% baseline at 30 and 60 min. There was no loss of activity in the microplasmin diluted in normal saline over this time period. Dilution of acidified microplasmin in normal saline did not lead to structural changes within the retina. A dose dependent PVD was observed in eyes treated with microplasmin diluted in NS. CONCLUSIONS: Acidified (stabilized) microplasmin has the same intraocular activity profile as microplasmin at a neutral pH. Better retention of activity at room temperature makes it a better candidate for use in clinical practice.

Microplasmin: ex vivo characterization of its activity in porcine vitreous.

PURPOSE: Microplasmin is a recombinant protein limited to the enzymatic moiety of plasmin without any of its cringle domains. Its enzymatic activity is similar to that of plasmin enzyme. The present study characterizes in a porcine eye model the vitreolytic ability of microplasmin. METHOD: Freshly harvested porcine eyes were used in these trials. Eyes were injected with escalating doses of microplasmin (62.5, 125, 250, 400 microg) for 1 hour or with 125 microg microplasmin with increasing time exposures (15, 30, 60, 120 minutes). Eyes were fixed by a very slow dehydration process to preserve the integrity of the vitreous retinal interface. They were examined by light microscopy to determine the degree of posterior vitreous detachment and by scanning electron microscopy (SEM) to study structural changes. RESULTS: Effective separation of the posterior hyaloid appeared to be dose dependent. After 1 hour, the posterior pole was detached in 100% of porcine eyes exposed to 125 microg microplasmin and in the midperiphery to 250 microg microplasmin. Vitreous at the ora did not detach. At 120 minutes of exposure, midperipheral detachment was observed with 125 microg microplasmin. A smooth retinal surface was seen where the enzyme caused posterior vitreous detachment. There was also significant change to the integrity of the vitreous without any obvious structural alterations to the retina by histology or scanning electron microscopy. CONCLUSIONS: Microplasmin caused vitreolysis and posterior vitreous separation in an ex vivo porcine eye model in an apparent dose- and time-dependent fashion. In this model system, the minimal effective dose appeared to be 125 microg.

The effect of paraformaldehyde fixation and PBS storage on the water content of the human lens.

PURPOSE: Fixation and phosphate buffered saline (PBS) storage are frequently used before studies of the morphological, biochemical, and optical properties of the human lens begin. It is assumed that this does not alter the properties being examined. The present study was undertaken to determine the effects of fixation and PBS storage on the human lens wet weight. METHODS: Human donor lenses were incubated in a buffered paraformaldehyde (PF) solution or in PBS and their wet weights were monitored for up to 44 and 13 days, respectively. RESULTS: PF fixation resulted in a large decrease in wet weight, averaging 25%+/-2.3% at 30 days for 14 human donor lenses, aged 49-80 years. The loss was essentially complete by 21 days. Out of the 10 lenses, aged 52-71 years, which were incubated in PBS alone, six of them increased in weight by an average of 38% over 13 days and four ruptured within four days. Comparison of literature data for a fixed eight-year-old lens with those for an unfixed seven-year-old lens indicated that the decrease in wet weight was due mainly to a loss of water from the cortex, which resulted in virtual disappearance of the water/protein gradient and the formation of a plateau containing 58% water in over 90% of the lens. CONCLUSIONS: Fixation substantially alters the amount and distribution of water in the human lens. Caution should be exercised when interpreting data on water and protein distributions as well as cell dimensions obtained with lenses which have been fixed. In addition, prolonged storage of a lens in PBS will result in substantial water uptake, which may affect measurements of their dimensions and optical properties.

Morphology of age-related cuneiform cortical cataracts: the case for mechanical stress.

We evaluated the gross morphology, location, and fiber cell architecture of equatorial cortical opacities in the aging human lens. Using dark-field stereomicroscopy, we photographed donor lenses in toto and as thick slices. In addition, we investigated the details of the fiber cell architecture using fluorescent staining for membranes and by scanning electron microscopy. We then combined our data with data from recent studies on lens viscoelasticity. We found that small cortical and cuneiform opacities are accompanied by changes in fiber structure and architecture mainly in the equatorial border zone between the lens nucleus and cortex. Because the lens cortex and nucleus have different viscoelastic properties in young and old lenses, we hypothesize that external forces during accommodation cause shear stress predominantly in this border zone. The location of the described changes suggests that these mechanical forces may cause fiber disorganization, small cortical opacities, and ultimately, cuneiform cataracts.

In vitro study on the closure of posterior capsulorrhexis in the human eye.

PURPOSE: An unexplained clinical observation is the development of posterior capsular opacification (PCO), even when the central part of the posterior capsule has been removed. The purpose of this study was to investigate in vitro the mechanisms involved in the closure of the posterior capsulorrhexis in a capsular bag model. METHODS: A sham extracapsular cataract extraction was performed in 71 human donor eyes, followed by a central posterior capsulorrhexis 3 to 4 mm in diameter. Each capsular bag was pinned to a PMMA ring with a central hole of 5 mm and placed in a Petri dish. The capsular bags were cultured and monitored for 3 to 7 weeks by phase-contrast microscopy, after which they were prepared for light, transmission, and scanning electron microscopy. RESULTS: Proliferation of lens epithelial cells (LECs) within the posterior rhexis area was found in 22 cases (31%) of which 3 had a complete closure. In the absence of the posterior capsule, a monolayer of LECs was observed growing on a basal lamina, consisting of loosely arranged fibers. Further observations on noncultured capsular bags revealed that this basal lamina corresponds to the anterior hyaloid membrane. CONCLUSIONS: This study corroborates the clinical observation that LECs that remain after cataract extraction have the potential to proliferate, in the absence of their natural substrate, on a basal lamina of vitreous origin and are able to close the posterior capsulorrhexis partially or totally in approximately one third of cases.

A preliminary study on the prevention of posterior capsule opacification by photodynamic therapy with bacteriochlorin A in rabbits.

PURPOSE: Photodynamic therapy (PDT) with bacteriochlorin a (BCA) has proven to be successful in the treatment of cancers and to be cytocidal for lens epithelial cells (LECs) in culture. The present study aimed to determine whether PDT with BCA is also effective in destroying LECs in the capsular bag in vivo and could therefore be a strategy for prevention of posterior capsule opacification (PCO). MATERIALS AND METHODS: BCA was obtained by saponification and acid hydrolysis of bacteriochlorophyll a and was formulated in 30% polyethylene glycol, 20% ethanol and 50% water. Nine albino rabbits were anesthesized and both pupils dilated. Extracapsular lens extraction by phacoemulsification was performed on both eyes. One eye of each animal served as control. In the other eye, 1.5 ml BCA (10 or 50 microg/ml) was injected in the capsular bag and after 10 min, the eye was illuminated with a diode laser (wavelength 760 nm) for 10 or 15 min. Six weeks after surgery, the rabbits were sacrificed and the globes were enucleated, the capsular bags and the corneas removed, fixed and examined using stereomicroscopy and light microscopy. RESULTS: In the control capsular bags, extensive proliferation of LECs and formation of a complete ring of Soemmering was found, while in the PDT-treated capsular bags, LEC proliferation was markedly diminished and an incomplete irregular and much thinner ring of Soemmering was formed. Using the presently described application, the corneas of the PDT-treated animals were opaque and swollen and had lost their endothelial lining. CONCLUSION: PDT with BCA induces cell death in LECs and greatly reduces the formation of a ring of Soemmering. Therefore, it could be a promising novel means of prevention of PCO, provided the total length of the treatment can be substantially reduced and the negative effects on corneal transparency avoided.

Morphological differences between lens fibers in albino and pigmented rats.

The purpose of this study was to investigate the morphological characteristics of lens fibers in albino and pigmented rats by scanning electron microscopy. In addition to the ubiquitous interdigitating edge protrusions many ball-and-socket junctions were found on the lateral surfaces of lens fibers in pigmented rats. Notable differences in density, shape and size between superficial and deep cortical layers were observed. Especially, in the intermediate equatorial cortex large ball-and-socket junctions were found. In contrast, only few and small ball-and-socket junctions were observed in albino rats and many ruptures of lens fiber membranes were present in the anterior, superficial and intermediate equatorial cortex. The present observations show that different strains of rats have a different morphology of lens fibers. In view of a postulated role of ball-and-socket junctions in calcium homeostasis in the lens this may account for differences in cataractogenesis between albino and pigmented rats.