To determine the sex of individual cells in paraffin sections of the human eye by fluorescence in situ hybridisation (FISH) of the X and Y chromosomes.
METHODS
The authors developed a protocol for FISH of the X and Y chromosomes in paraffin sections of human eyes.
RESULTS
In all the specimens that had been fixed in 10% formalin and with a fixation time of up to 3 days sex determination of individual cells was achieved. The percentage of cells with clearly identifiable signals was up to 98% for corneal epithelium, keratocytes, corneal endothelium, trabecular meshwork, lens epithelium, retina, and optic nerve.
CONCLUSIONS
FISH allows the determination of the sex of single cells in paraffin sections of human eyes without destruction of the tissue structure. Its main application is the histological analysis of sex mismatched corneal, RPE, or neuroretinal transplants to distinguish host and donor cells.
The aim of this study was to assess the biomechanical efficacy of transepithelial collagen crosslinking using the femtosecond laser pocket technique compared with that using the standard crosslinking (CXL) technique.Forty ex vivo porcine eyes were divided into 4 groups with 10 samples each. Group 1 comprised the untreated controls. Group 2 was the standard CXL group with debridement, instillation of 0.1% riboflavin-dextran solution for 15 minutes before and every 5 minutes during the 30 minutes of irradiation with ultraviolet A (UVA) light of 370 nm and an irradiance of 3 mW/cm². Group 3 pertained to the femtolaser pocket control with an intrastromal pocket but without riboflavin/UVA. Group 4 was the femtolaser pocket CXL group with an intrastromal pocket of an 8-mm diameter at a 180-μm depth, riboflavin/dextran application for 15 minutes and subsequent exposure to UVA light for 30 minutes. Postoperatively, biomechanical stress-strain measurements were performed.In the standard CXL group, the stress at 10% strain was 207.8 ± 64.1 × 10 Pa (+79.45% vs. controls; P = 0.021) compared with 115.8 ± 20.8 × 10 Pa in the untreated control group; in the crosslinked femtolaser pocket group, it was 159.5 ± 30.4 × 10 Pa (+37.74%; P = 0.049), in the non-cross-linked femtolaser pocket group, it was 103.5 ± 17.3 × 10 Pa (-10.62%; P = 0.103). The Young modulus was 5.4 MPa (+100% vs. controls) in the standard CXL group, 3.7 MPa (+37.04%) in the crosslinked femtolaser pocket group, and 2.4 MPa (-11.12%) in the non-cross-linked femtolaser pocket group compared with 2.7 MPa in the untreated control group.The biomechanical effect of CXL using the femtolaser pocket technique is about 50% less pronounced than that after standard CXL. Future studies will show whether the efficacy of the technique can still be improved and whether the clinical effect is sufficient for stabilizing ectatic corneas.
To evaluate the biomechanical effect of combined riboflavin-ultraviolet A (UVA) treatment on porcine and human corneas.Department of Ophthalmology, Technical University of Dresden, Dresden, Germany.Corneal strips from 5 human enucleated eyes and 20 porcine cadaver corneas were treated with the photosensitizer riboflavin and irradiated with 2 double UVA diodes (370 nm, irradiance = 3 mW/cm2) for 30 minutes. After cross-linking, static stress-strain measurements of the treated and untreated corneas were performed using a microcomputer-controlled biomaterial tester with a prestress of 5 x 10(3) Pa.There was a significant increase in corneal rigidity after cross-linking, indicated by a rise in stress in treated porcine corneas (by 71.9%) and human corneas (by 328.9%) and in Young's modulus by the factor 1.8 in porcine corneas and 4.5 in human corneas. The mean central corneal thickness was 850 microm +/- 70 (SD) in porcine corneas and 550 +/- 40 microm in human corneas.Riboflavin-UVA-induced collagen cross-linking led to an increase in mechanical rigidity in porcine corneas and an even greater increase in human corneas. As collagen cross-linking is maximal in the anterior 300 microm of the cornea, the greater stiffening effect in human corneas can be explained by the relatively larger portion of the cornea being cross-linked in the overall thinner human cornea.
Editor, Cross-linking treatment of progressive keratoconus (CXL) using the photosensitizer riboflavin and ultraviolet (UV) A was introduced by Wollensak in 2003 and is becoming increasingly popular (Wollensak et al. 2003a). A long-term effect could be demonstrated recently (Wollensak & Iomdina 2008). Extensive studies have been performed in vitro and in animals to evaluate the potential cytotoxicity increasing the safety of the new treatment modality (Wollensak et al. 2003b, 2004; Wollensak 2006). However, no human microscopic specimens have been studied so far. Recently, I had the opportunity to examine histologically the eye of a patient whose cornea had been cross-linked 24 hr before enucleation. The 23-year-old man with a blind painful left eye (caused by status post-severe perforating sclera and lens injury in 1992 and subsequent unsuccessful operations for visual restoration including vitrectomy, encircling band and silicone oil) had a visual acuity of 20/20 in his healthy right eye. With the patient's consent, a standard cross-linking treatment was performed in the left eye one day before the scheduled enucleation. As described previously, the central 8 mm of the epithelium was removed and the cornea soaked with the 0.1% riboflavin solution for 20 min. Irradiation was performed for 30 min using a UVA double diode (3 mW/cm2) at a distance of 1 cm. Antibiotic ointment was administered after the cross-linking procedure and the eye was enucleated 24 hr later. The retrieved eye was fixed in neutral buffered 4% formalin. Paraffin sections (4 μm thin) were stained with haematoxylin and eosin. On light microscopy, absence of the epithelium in the debrided central cornea was noted. Bowman's layer was unremarkable. There was complete loss of keratocytes down to a depth of 250–280 μm with normal keratocyte density beneath. Descemet's membrane and the endothelial cells were complete and intact (Fig. 1). There were no inflammatory cells and only minimal oedema. Apart from the corneal findings, the lens was partially calcified and the retina atrophic with silicone oil inclusions and a funnel-shaped retinal detachment. Photomicrograph of cross-linked human cornea 24 hr after cross-linking. Note the absence of epithelium, loss of keratocytes down to 250 μm (zone A), intact keratocytes in posterior stroma (zone B) forming a 'safety zone', intact endothelium and the absence of inflammatory cells (haematoxylin and eosin stain, × 200). Concurrent findings have been reported after standard CXL applying 3 mW/cm2 of UVA for 30 min: keratocyte damage has been observed down to 300 μm using in vivo confocal microscopy (Mazzotta et al. 2007) like in the animal experiments. Accordingly, a treatment effect as evaluated by hydration studies (Wollensak et al. 2007) and slit-lamp biomicroscopy (Seiler & Hafezi 2006) is also noted only in the anterior stroma down to 250–300 μm. Interestingly, the cytotoxic threshold for the endothelium is only 0.36 mW/cm2 (Wollensak et al. 2003b) compared to 0.5 mW/cm2 (Wollensak et al. 2004) for keratocytes. There is a relatively large safety gap towards the endothelium with still-intact keratocytes: otherwise, the endothelium with its lower cytotoxic threshold would be destroyed despite keratocyte survival directly anterior to the endothelium. In conclusion, the present article confirms earlier findings in animals and demonstrates a high degree of safety for the endothelium in standard CXL as long as a stromal thickness of at least 400 μm is present in the irradiated target area of the cornea.
Photodynamic collagen cross-linking by using ultraviolet A (UVA) irradiation and the photosensitizer riboflavin has been recently introduced as a new possible treatment of progressive keratoconus. This is the first study, to our knowledge, investigating biochemical aspects of the new procedure. Its aim was to analyze the possible changes in the electrophoretic pattern of corneal collagen type I after collagen cross-linking treatment.Twenty fresh postmortem porcine corneas were cross-linked; another 20 porcine corneas treated with physiologic saline were used as controls. After removal of the central 10 mm of the epithelium, the corneas were treated with the photosensitizer riboflavin and UVA irradiation for 30 minutes by using a double UVA diode (370 nm, 3 mW/cm). For biochemical analysis, the central 10-mm corneal buttons were trephined, tissue was homogenized, and collagen type I was extracted. Subsequently, the collagen extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.In the controls, the typical collagen pattern of normal cornea was found with 1 gamma trimer band, 2 beta dimer bands, and 2 alpha monomer bands. In the cross-linked samples, there was an additional intense polymer band in the stacking gel that was resistant to mercaptoethanol, heat, and pepsin treatment. Its molecular size was estimated to be at least 1000 kDa.In the cross-linked corneas, a strong band of high-molecular-weight collagen polymers was shown as the biochemical correlate of the cross-linking effect, showing the efficiency of the new cross-linking procedure. This polymer band complies well with the morphologic correlate of an increased fiber diameter after cross-linking treatment. Its chemical stability supports hopes of a long-term effect of the new treatment.
'/%3e%3cuse xlink:href='%23a' transform='rotate(144 450 300)'/%3e%3cuse xlink:href='%23a' transform='rotate(216 450 300)'/%3e%3cuse xlink:href='%23a' transform='rotate(288 450 300)'/%3e%3c/svg%3e)