Ben Willekens

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.

Otoconial alterations after embryonic development in hypergravity

The relation between prolonged hypergravity and structural adaptation of otoconia was studied in hamsters (n = 56). Three groups of hamsters (n = 27), were conceived and born in a centrifuge: group 1 (n = 10) 1 month under 2.5 G, group 2 (n = 9) 5 months under 2.5 G and 4 months under 1 G, group 3 (n = 8) 1 month under 2.5 G and 8 months under 1 G. Control hamsters (n = 29) were conceived and born under 1 G (1 month old, n = 7; 9 months old, n = 22). Histological study of the otoconial layers (energy dispersive x-ray element analysis and scanning electron microscopy) showed similar calcium content, size, and shape in utricular and saccular otoconia in all groups. Different were the utricular otoconial size classes, large, medium-sized, and small. The area with small otoconia increased in group 1 (p = 0.002). In group 2, the large otoconial area decreased (p = 0.001) and the medium-sized one increased (p < 0.001). In group 3, the large otoconial area decreased (p = 0.003) and the medium-sized one increased (p = 0.007). For age-related effects we found group 1 with an increased area of large otoconia (p = 0.001) and a decreased medium-sized one compared to groups 2 (p < 0.001) and 3 (p = 0.02). Hypergravity during formation of otoconia does not affect calcium content, size, or shape, but changes relative size of the areas with large, medium-sized, or small otoconia and the development of these areas. This resulted in a structural adaptation to hypergravity.

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.

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 µg/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.

Corneal decompensation and graft failure secondary to a broken posterior chamber poly(methyl methacrylate) intraocular lens haptic

A 66-year-old man developed an unexplained corneal decompensation 7 years after extracapsular cataract extraction and implantation of a single-piece poly(methyl methacrylate) (PMMA) posterior chamber intraocular lens (IOL). He had penetrating keratoplasty (PKP). Two years later, he developed corneal graft failure secondary to an IOL haptic fragment in the anterior chamber angle. The patient had a repeat corneal graft and IOL exchange. The broken haptic was examined with scanning electron microscopy. The findings were consistent with late fracture of the haptic within the capsular bag, which was presumably weakened by an improper implantation technique. Fracture of a PMMA haptic should be suspected as a cause of corneal decompensation and corneal graft failure after cataract surgery. This case emphasizes the importance of safe implantation techniques.