Tear film proteins deposited on high water content contact lenses identified with two-dimensional gel electrophoresis and mass spectrometry.

PURPOSE: Tear film proteins adhere to the surface of contact lenses (CLs). While the proteins in the tears have been extensively studied with various proteomic techniques, adhered proteins to CLs are less studied. In this pilot study, we have separated proteins with 2D gel electrophoresis prior to the conventional mass spectrometry (MS) in order to analyse the deposited proteins on hydrogel CLs from myopic patients. METHODS: pHEMA and PVA hydrogel CLs worn by 3 patients for different time lengths were analysed. After wear, the CLs were frozen at -20°C. Proteins were extracted in lysis buffer, separated on 12% polyacrylamide gels and silver-stained. Protein spots were excised and identified with liquid chromatography - tandem MS. RESULTS: Deposited proteins were extracted with a yield of 26-66 µg and separated by 2D gel electrophoresis. The silver-stained gels showed similar protein patterns independent of the patient, hydrogel type and wear time. Seventy-two spots were analysed with MS, representing at least 12 different tear film proteins or protein fragments. CONCLUSIONS: Deposited tear film proteins from a single set of CLs worn for 1 day can successfully be analysed first with 2D gel electrophoresis and subsequently with MS, thus making examination of individual patients possible. The protein composition appeared homogeneous between the test persons which is a necessity for additional comparison analysis. The molecular masses of the identified proteins indicate that protein degradation occurs only as a minor event. Myopic patients were investigated in this pilot study, but the combined techniques can easily be applied to other eye diseases.

Clinical results of 40 years of paediatric keratoplasty in a single university eye clinic.

PURPOSE: To evaluate indications for paediatric keratoplasty in recipients aged ≤16 years and assess long-term clinical outcome. METHOD: Recipients were identified from records of the Danish Cornea Bank. Data were collected from patient journals, clinical follow-up examinations and questionnaires and stratified into pre-, peri- and postoperative variables. Diagnoses were classified into acquired traumatic, acquired nontraumatic and congenital groups. Recipients were divided into groups of <8 and ≥8 years. Data were analyzed using relative percentages and Kaplan-Meier survival plots. RESULTS: Thirty-three out of sixty identified recipients (73 keratoplasties in 63 eyes) were invited. Twenty-four accepted, seven still attended follow-up in our clinic. Follow-up data reached 95% of the eligible recipients (median follow-up 11 years). Twenty-three per cent were <8 years and 77%≤8 years. Diagnoses were mainly acquired nontraumatic (69%), acquired traumatic (12%) and congenital (7%). Indications were primarily optical (52%) or tectonic (41%). Graft survival was best in the acquired nontraumatic group (except regrafts) (median survival 15-20 years) and poorest in the regraft subgroup as well as the acquired traumatic and congenital groups (median survival 1-2 years). Graft failure was higher in the youngest with predisposing risk factors and in combined procedures. In terms of indications, visual improvement and eye preservation was achieved in 70%. CONCLUSION: Paediatric keratoplasty was successful regarding indication. Graft survival was best in the acquired nontraumatic group and poorest in the congenital group. Vascularization and/or combined risk factors, additional surgeries and young recipient age influenced negatively on graft survival.

Metabolic profile of keratoconic cornea.

PURPOSE: To investigate the difference in metabolic profile of keratoconic and normal corneas using two different analysis methods. METHODS: Keratoconic corneas were obtained from patients (aged 19-27) during transplantation surgery. Control samples were obtained from human donors (aged 61-75) 1-8 h post-mortem. The metabolic profile of tissues was investigated with high-resolution magic angle spinning (1)H nuclear magnetic resonance (NMR) spectroscopy and high performance liquid chromatography (HPLC). RESULTS: Nine amino acids and 20 metabolites were assigned with HPLC and NMR spectroscopy, respectively. No significant biochemical difference was revealed between keratoconic and control samples, which represent distant age groups. CONCLUSIONS: It suggests that development of keratoconus might be related to the accelerated ageing of the cornea. This issue warrants further studies.

Nordisk ophthalmologisk tidsskrift 1889-1892: the Nordic Journal of Ophthalmology - a forerunner of Acta Ophthalmologica.

In the second half of the nineteenth century several ophthalmological journals appeared (Germany, England, France, United States). In the northern countries (Denmark, Finland, Norway, Sweden) an initiative lead to 'Nordisk ophthalmologisk Tidsskrift' published in the scandinavian languages in the years 1889-92. The 'driving force' behind the journal was the first professor in Ophthalmology in Copenhagen, Dr. Edmund Hansen Grut. The purpose with this presentation is to give the background for the rise and fall of the journal which was an attempt to promote Nordic Ophthalmology. The authors turned out, however, to be mainly those involved as editors. The journal never gained broad acceptance and it simply stopped in 1892, without any closing remarks. In spite of its short life the journal should be remembered for the very first publication on the arcuate scotoma, the Bjerrum scotoma from 1889. Although shortlived the initiative was not in vain, as can be read in the preface to the very first volume of Acta ophthalmologica.

Impact of organ culturing on metabolic profile of human corneas: preliminary results.

PURPOSE: It is suggested that the quality of corneal graft may depend on modifications that appear in the tissue during culturing. The aim of this study was to investigate the differences in the metabolic profile between cultured and noncultured human corneas. METHODS: Corneas from 12 donors were obtained post-mortem and cultured for 6-20 days. Control corneas were obtained from four patients with malignant melanoma of the chorioidea and were kept frozen at -80 °C until analysed. The metabolic profiles of the samples were investigated using high-resolution, magic angle spinning (1) H nuclear magnetic resonance spectroscopy and special software for: (i) analysis of complex mixtures, (ii) principal component analysis and (iii) specialized statistical analysis. RESULTS: Twenty metabolites were detected and assigned in the corneas. Significant differences in metabolic profiles between cultured and noncultured corneas were revealed. It was also shown in samples kept in culture for 9-14 days that the levels of (i) alanine, formate, lactate and (ii) acetate, alanine, arginine, lactate were elevated in comparison with the samples kept for <9 and more than 14 days, respectively. CONCLUSIONS: Corneal culturing affects the metabolic profile of the tissue. The increases in the levels of some metabolites within the second week of culturing likely result from variations in tissue metabolic or enzymatic activity caused by changed (organ culture) environment. As the mechanisms responsible for these changes are not clear, further research is indicated.

Precision and accuracy of the ICare tonometer - Peripheral and central IOP measurements by rebound tonometry.

PURPOSE: The purpose of this study was to evaluate the ICare tonometers precision and accuracy and the extent to which intraocular pressure (IOP) measurements are influenced by measuring position. METHODS: This was carried out by comparing the central and peripheral ICare-IOP readings and comparing ICare- with the Goldmann applanation tonometer (GAT)-IOP readings. IOP was measured using the ICare rebound tonometer on the right eye of 40 subjects, straight at the centre of the cornea (CS), straight 2 mm from the nasal and temporal limbus (NS and TS), and in 10 degrees nasally and temporally angled positions measured from the same location as CS (NA and TA). The IOP was also assessed with the GAT. RESULTS: Central IOP (CS) was significantly (p < 0.001) greater than peripheral measurements (NS, TS, NA and TA) by approximately 3-4 mmHg. Centre IOP (CS) significantly overestimated by mean 2 mmHg and the peripheral measurements significantly underestimates approximately 1.4-2 mmHg compared with GAT readings. CONCLUSION: The ICare tonometer may be useful in a routine clinical setting for IOP screening, but the ICare measurement is not a substitute for the GAT measurement, when a precise and accurate IOP is desired.

History of Acta Ophthalmologica - the first 50 years.

Acta Ophthalmologica appeared as the scientific journal of the Nordic ophthalmological Societies in 1923. The intention was to expose the clinical and experimental developments among the ophthalmological communities of the four Nordic countries: Denmark, Finland, Norway and Sweden. The collaboration within the field of ophthalmology had been attempted with the publication of 'Nordisk ophthalmologisk Tidsskrift' in the years 1889-1892. Now, once again, the wish for a tighter Nordic cooperation was explored by enthusiastic ophthalmologists in all four countries. One person stands out as the driving force, K.K.K. Lundsgaard, who in spite of his early death in 1931 was remembered by name on the front page of Acta since 1936 by the Latin phrase 'A K.K.K. Lundsgaard edi coepta'. The present review recalls details from the early years when the journal found its place in the ophthalmology and creates the background for some reflections on purpose and goals by publishing a scientific journal.

Attempting to distinguish between endogenous and contaminating cytokeratins in a corneal proteomic study.

BACKGROUND: The observation of cytokeratins (CK's) in mass spectrometry based studies raises the question of whether the identified CK is a true endogenous protein from the sample or simply represents a contaminant. This issue is especially important in proteomic studies of the corneal epithelium where several CK's have previously been reported to mark the stages of differentiation from corneal epithelial stem cell to the differentiated cell. METHODS: Here we describe a method to distinguish very likely endogenous from uncertain endogenous CK's in a mass spectrometry based proteomic study. In this study the CK identifications from 102 human corneal samples were compared with the number of human CK identifications found in 102 murine thymic lymphoma samples. RESULTS: It was anticipated that the CK's that were identified with a frequency of <5%, i.e. in less than one spot for every 20 spots analysed, are very likely to be endogenous and thereby represent a 'biologically significant' identification. CK's observed with a frequency >5% are uncertain endogenous since they may represent true endogenous CK's but the probability of contamination is high and therefore needs careful consideration. This was confirmed by comparison with a study of mouse samples where all identified human CK's are contaminants. CONCLUSIONS: CK's 3, 4, 7, 8, 11, 12, 13, 15, 17, 18, 19, 20 and 23 are very likely to be endogenous proteins if identified in a corneal study, whilst CK's 1, 2e, 5, 6A, 9, 10, 14 and 16 may be endogenous although some are likely to be contaminants in a proteomic study. Further immunohistochemical analysis and a search of the current literature largely supported the distinction.

Influence of suture regularity on corneal astigmatism after penetrating keratoplasty.

PURPOSE: To investigate whether suture regularity affects corneal astigmatism after keratoplasty. METHODS: Twenty-one patients undergoing penetrating keratoplasty for various corneal diseases were included in the study. The grafts were sutured in place using a single-running Nylon 10-0 suture, taking 24 bites. Immediately after surgery, standard calibrated images of the grafted eye were captured and stored. Using a dedicated image analysis programme, stitches and needle points were identified, and a number of suture regularity variables were calculated. Corneal topographic images were obtained before suture removal (12 months after surgery) and 3 months after suture removal (18 months after surgery). Topographic measures of astigmatism [surface regularity (SRI), surface asymmetry index (SAI) and simulated keratometric astigmatism] were calculated and correlated with the computed suture regularity variables. RESULTS: The average stitch length was 3.04 ± 0.28 mm and the distance between the outer needle points was 2.53 ± 0.09 mm. The SRI was 1.26 ± 0.36 and the SAI was 1.59 ± 0.67 after 12 months; these decreased to 1.03 ± 0.48 and 0.92 ± 0.46 after 18 months, respectively. Corneal astigmatism was 6.38 ± 2.99 and 5.87 ± 3.13 dioptres after 12 and 18 months, respectively. Suture regularity did not affect SAI, SRI or corneal astigmatism significantly 12 months after surgery. Eighteen months after surgery (3 months after suture removal), the standard deviation on the original stitch length was found to significantly increase corneal astigmatism. In addition, the size of the counter-clockwise angle between stitch and graft radian was correlated significantly with a lower SRI. CONCLUSION: The origin of corneal astigmatism after penetrating keratoplasty is multifaceted. Regular stitch length and stitch advancement on the surface appears to improve the optical quality of the graft after suture removal. Factors such as stitch depth, suture tension and variations in wound construction might also be important predictors of corneal astigmatism after penetrating keratoplasty.

Morphological evaluation of normal human corneal epithelium.

PURPOSE: The human corneal epithelium is usually described as a 50-µm-thick layer of regular stratified squamous non-keratinized cells with a thickness of 5-7 cells. The purpose of this study is systemically to revisit the histopathological appearance of 100 corneas. METHODS: 5-µm-thick sections of corneas from 100 consecutively selected paraffin-embedded eyes were stained with hematoxylin-eosin and Periodic Acid-Schiff (PAS). All specimens were evaluated by light microscopy. The eyes were enucleated from patients with choroidal melanoma. Corneas were considered to be normal. RESULTS: Ninety of 100 eyes were evaluated. The central epithelial, stromal and total corneal thickness was measured as 36.0 µm, 618 µm and 651µm, respectively, with a variation coefficient from 0.21 to 0.22. Pathological appearances were found in 27% of corneas, including thickened basement membrane and alterations in Bowman's membrane. No intraepithelial microcysts, as found in Meesmann corneal dystrophy, were observed. CONCLUSION: The total corneal thickness was higher than reported in in vivo studies and with a wider variation. This may be an effect of uncontrolled swelling and dehydration during preparation. The high number of pathological observations suggests that 'normal' eyes harbour and potentially accumulate considerable pathology.

Longterm follow-up of penetrating keratoplasty for keratoconus.

PURPOSE: This study aims to describe the current visual and refractive status of patients who underwent penetrating keratoplasty (PK) for keratoconus > 20 years ago and to report on the current status of their grafts. METHODS: A total of 138 eyes in 103 patients were grafted for keratoconus between August 1968 and December 1985. Patients who had not undergone retransplantation were invited to attend a clinical examination. Forty-eight patients (with 61 grafts) accepted the invitation and were examined. RESULTS: The average length of time since PK was 26.9 years (standard deviation [SD] 4.2 years, range 20.8-38.0 years, n = 61). The average graft age at examination was 82.1 years (SD 19.9 years, range 41-115 years). A total of 80% (49 of 61 grafts) of the examined eyes had a clear graft and 46% (28 of 61 eyes) had best spectacle-corrected visual acuity (BSCVA) > or = 0.5. The mean endothelial cell density per mm(2) was 894 (SD 4.6, range 470-1775). The mean central corneal thickness of the clear grafts was 0.565 mm (SD 0.048 mm). CONCLUSIONS: Penetrating keratoplasty for keratoconus has a good longterm prognosis; half of the eyes examined in this study had BSCVA > or = 0.5 at > 20 years after surgery.

Riboflavin-UVA treatment in the management of edema and nonhealing ulcers of the cornea.

PURPOSE: Riboflavin-ultraviolet A (UVA) treatment induces cross-linking and stiffens the corneal stroma. A parallel reduction in stromal swelling and increased resistance to microbial and enzymatic degradation has been suggested. The purpose of this study was to evaluate the potential of riboflavin-UVA treatment in the management of corneal disorders, in particular edema due to endothelial decompensation and non-healing ulcers. METHODS: Two clinical series are reported, 11 eyes with endothelial decompensation and 14 eyes with non-healing ulcers. Treatment comprised a) abrasion of epithelium (if present), b) instillation of 0.1% riboflavin in saline, and c) irradiation at 365 nm UVA light over approximately 30 minutes (3 mW/cm(2)). Four eyes in the first group were treated twice. Postoperatively, all eyes were followed for at least 3 months. RESULTS: Reduction in corneal thickness was observed in 10 of 11 eyes with stromal edema and the majority also experienced improvement in vision. The effect occurred over weeks and lasted for months. Fourteen patients with non-healing ulceration were similarly treated; 6 healed, 8 showed no clear effect. CONCLUSIONS: In addition to the stiffening of keratoconic and ectatic cornea, riboflavin-UVA treatment is effective in reducing corneal edema and has the potential to heal corneal ulcers.

Corneal grafting and banking.

Corneal transplantation was conceptualized at the end of the 18th century, but it took more than 100 years before human corneal grafting was introduced. The greatest step forward was the demonstration by Filatov that corneal tissue can be collected and used post mortem. The history of eye banking includes the development of preservation techniques. Storage in cold to minimize microbial growth and tissue disintegration was first choice but during the last 30 years this has been taken over by warm storage (organ culture) where the donor cornea proves its sterility and vitality before being transferred to the recipient. The long-term organ culture storage makes exchange between centres possible and allows for histocompatibility matching. The internationalization led to the establishing of the European Eye Bank Association but also to an increasing number of governmental regulations. Developments in years to come may lead to control of graft biomechanics and optics. This technical development tends to favour a centralization.

Descemet's stripping automated endothelial keratoplasty and penetrating keratoplasty for Fuchs' endothelial dystrophy.

PURPOSE: To compare the outcome of Descemet's stripping endothelial keratoplasty (DSAEK) to that of penetrating keratoplasty (PK) in patients with Fuchs' endothelial dystrophy. METHODS: The first 20 patients who underwent DSAEK at the Department of Ophthalmology, Aarhus University Hospital were compared to 20 patients treated with classic PK. Best-corrected visual acuity, subjective spectacle refraction and corneal thickness were registered before surgery and 1, 3, 6 and 12 months after DSAEK surgery; they were also measured before surgery and 12 months and 2-3 years after PK. Endothelial cell density was measured 12 months after surgery in both groups. RESULTS: Two primary graft failures were observed in the DSAEK group; no failures were seen in the PK group. Best spectacle-corrected visual acuity (BSCVA) at 12 months after surgery was significantly better in the DSAEK group (0.56 +/- 0.04) than in the PK group (0.33 +/- 0.06). At this time, 70% of the DSAEK-treated eyes but only 25% of PK-treated eyes had obtained a BSCVA of 0.5 or better. Two to three years after surgery, BSCVA was 0.5 or better in 55% of PK-treated eyes. Refractive ametropia and astigmatism were significantly smaller in DSAEK-treated eyes than in PK-treated eyes, even after suture removal and arcuate keratotomy. Endothelial cell density (cells/mm2) after 1 year was lower in DSAEK-treated (1.338 +/- 113) than in PK-treated eyes (1.610 +/- 124), but the difference was not statistically significant. CONCLUSION: DSAEK seems to be superior to PK in treating Fuchs' endothelial keratoplasty, although primary graft failure may be more common. Visual recovery is faster, and major ametropia and astigmatism is not induced. Long-term follow-up studies are essential to assess whether this conclusion also holds true more than 1 year after surgery.

Re-examination of organ-cultured, cryopreserved human corneal grafts after 27 years.

PURPOSE: To determine the long-term fate of cryopreserved corneas. Review of 17 organ-cultered cryopreserved corneas grafted in 1978-1979. METHODS: We measured visual acuity and refraction and performed biomicroscopy, applanation tonometry and optical pachometri (CCT). Endothelial photos were taken, cells were counted and morphology was studied. RESULTS: Four of 16 grafted corneas were still clear after 27 years. Mean CCT was 0.52 mm, endothelial cell density was 882 cells/mm(2) and visual acuity was 0.25 or better with an average of 0.6 in the four patients. Cell morphology showed irregularity in shape and size. CONCLUSION: This study shows that cryopreserved endothelium can function as well as non-frozen corneas and that a regular hexagonal pattern is not essential for corneal clarity. The four grafts showed long-term durability despite the irregularity in shape and size.

Comparison of two partial coherence interferometers for corneal pachymetry in high myopia and after LASIK.

PURPOSE: We aimed to compare the Haag-Streit optical low-coherence reflectometry (OLCR) pachymeter and the Zeiss Anterior Chamber Master (ACMaster) for measuring central corneal thickness (CCT) in high myopes and after laser in situ keratomileusis (LASIK) for myopia. METHODS: Central corneal thickness was measured in 55 eyes of 30 myopic subjects (spherical equivalent refraction of - 5.25 D to - 10.75 D, maximal astigmatism of - 2 D), and in 37 eyes of 21 patients 3 months after LASIK for myopia (preoperative spherical equivalent refraction of - 6.0 D to - 10.75 D, maximal astigmatism of - 2 D). All measurements were performed with the Haag-Streit OLCR pachymeter and the Zeiss ACMaster, using group refractive indices of 1.376 and 1.3851, respectively. Thickness measurements were compared using paired t-tests, Pearson's correlation, linear regression and Bland-Altman plots. RESULTS: In myopic subjects, CCT measured 531 +/- 28 microm and 533 +/- 27 microm with the OLCR pachymeter and the ACMaster, respectively (p < 0.01); all measurements correlated closely (r = 0.99, p < 0.01). In LASIK-treated eyes, CCT measured 472 +/- 24 microm using the OLCR pachymeter and 475 +/- 23 microm using the ACMaster (p < 0.01), again with close correlation between the two instruments (r = 0.99, p < 0.01). CONCLUSIONS: Measurements of CCT in high myopes and after myopic LASIK were very similar with the Haag-Streit OLCR pachymeter and the Zeiss ACMaster. Using the current group refractive indices, the observed difference between the two instruments of < 3 microm is of little clinical importance. Thus, it would seem safe to use the OLCR pachymeter and the ACMaster interchangeably for CCT measurements in myopia as well as after myopic LASIK.

Riboflavin-ultraviolet light induced cross-linking in endothelial decompensation.

PURPOSE: To evaluate the potential of collagen cross-linking in the treatment of corneal oedema caused by endothelial decompensation. METHODS: Riboflavin-ultraviolet (UV) treatment induces cross-linking and reduces stromal swelling. Eleven patients with corneal oedema were treated. The technique comprised: epithelial abrasion; instillation of 0.1% riboflavin in saline, and 5.4 J/cm(2) illumination with 365 nm UV-A light over approximately 30 mins (3 mW/cm(2)). RESULTS: A reduction in corneal thickness was observed in 10 patients. The majority also experienced improvement in vision. The effect occurred over weeks and lasted for months. CONCLUSIONS: The study shows a potential application of collagen cross-linking in the management of patients with corneal oedema. Experimental and additional clinical studies are necessary in order to define the precise indications for this type of treatment.

Differences in the protein expression in limbal versus central human corneal epithelium--a search for stem cell markers.

In the search for potential limbal stem cell protein markers, the purpose of this study was to characterize differences in protein expression between human central and limbal corneal epithelium by a proteomic approach using two-dimensional polyacrylamide gel electrophoresis (2D PAGE) combined with mass spectrometry (LC-MS/MS). The results were subsequently confirmed by Western blotting and immunohistochemistry. We detected more than 1000 protein spots in each gel. Thirty-two spots were significantly over-expressed in the central part and 70 spots were significantly over-expressed in the limbal part. We identified 25 different proteins. Among these 11 proteins representing different cellular locations and functions were selected for further investigations. Most interestingly, superoxide dismutase 2 (SOD2), was expressed in clusters of cells in the basal limbal epithelium. Heat shock protein 70 protein 1 (HSP70.1) and annexin I were highly abundant in limbal epithelium, although they were also present in the central epithelium to a minor extent. Among the proteins primarily expressed in the limbal fraction we further identified cytokeratin (CK) 15, CK19 and alpha enolase, which have been reported previously to be related to the limbal basal epithelium. The basal limbal epithelium consists of clusters of slow cycling limbal stem cells and rapid cycling transient amplifying cells. Ideally, proteins exclusively expressed in the limbal part of the epithelium may serve as markers for the basal limbal cells. SOD2 and CK15 identify clusters of limbal basal cells and therefore they may serve as markers for limbal stem cells in conjunction with the earliest transient amplifying cells.