Healthy Human Corneas Research Articles

Assessing microstructures of the cornea with Gabor-domain optical coherence microscopy: pathway for corneal physiology and diseases.

Gabor-domain optical coherence microscopy (GD-OCM) was applied ex vivo in the investigation of corneal cells and their surrounding microstructures with particular attention to the corneal endothelium. Experiments using fresh pig eyeballs, excised human corneal buttons from patients with Fuchs' endothelial dystrophy (FED), and healthy donor corneas were conducted. Results show in a large field of view (1 mm×1 mm) high definition images of the different cell types and their surrounding microstructures through the full corneal thickness at both the central and peripheral locations of porcine corneas. Particularly, an image of the endothelial cells lining the bottom of the cornea is highlighted. As compared to healthy human corneas, the corneas of individuals with FED show characteristic microstructural alterations of the Descemet's membrane and increased size and number of keratocytes. The GD-OCM-based imaging system developed may constitute a novel tool for corneal imaging and disease diagnosis. Also, importantly, it may provide insights into the mechanism of corneal physiology and pathology, particularly in diseases of the corneal endothelium.

Imaging shear stress distribution and evaluating the stress concentration factor of the human eye.

Healthy eyes are vital for a better quality of human life. Historically, for man-made materials, scientists and engineers use stress concentration factors to characterise the effects of structural non-homogeneities on their mechanical strength. However, such information is scarce for the human eye. Here we present the shear stress distribution profiles of a healthy human cornea surface in vivo using photo-stress analysis tomography, which is a non-intrusive and non-X-ray based method. The corneal birefringent retardation measured here is comparable to that of previous studies. Using this, we derive eye stress concentration factors and the directional alignment of major principal stress on the surface of the cornea. Similar to thermometers being used for monitoring the general health in humans, this report provides a foundation to characterise the shear stress carrying capacity of the cornea, and a potential bench mark for validating theoretical modelling of stresses in the human eye in future.

Sağlıklı İnsan Korneasında Hücre ve Sinir Liflerinin İn Vivo Konfokal Mikroskopi ile Değerlendirilmesi

Amaç: Sağlıklı insan korneasında hücre dansiteleri ve sinir lifi parametrelerinin in vivo konfokal mikroskopi ile değerlendirilmesi, ve bu bulguların yaş ve cinsiyet ile ilişkisinin incelenmesi. Gereç ve Yöntemler: Herhangi bir sistemik hastalığı bulunmayan 77 olgu (46 erkek, 31 kadın) çalışmaya dâhil edildi. Olguların santral korneaları lazer tarayıcı in vivo konfokal mikroskop ile değerlendirildi. Analizler için gözlerden biri rastgele seçildi. Bazal epitel hücreleri, ön ve arka stromal keratosit ve endotel hücre dansiteleri ile sub-epitelyal sinir lifi yapısı incelendi. Bulgular: Olguların ortalama yaşı 44,5±18,1 yıl (15-80 yıl) olup, kadın ve erkek cinsiyet arasında yaş ortalaması yönünden anlamlı fark izlenmedi (p=0,715). Ortalama bazal epitel hücre dansitesi 5846±393 hücre/mm2, ön stromal keratosit dansitesi 795±151 hücre/mm2, arka stromal keratosit dansitesi 318±56 hücre/mm2, endotel hücre dansitesi 2743±329 hücre/mm2, sub-epitelyal sinir lifi dansitesi 32,9±8,9 major sinir lifi/mm2, sinir dalı dansitesi 67,5±34,3 sinir dalı/mm2 ve sinir lifi uzunluğu 23,5±3,7 mm/mm2 olarak tespit edildi. Kadın ve erkek cinsiyet arasında ölçülen parametrelerin hiçbirinde anlamlı farklılık izlenmedi (hepsi için p>0,05). Yaş ile ön stromal keratosit dansitesi (r=-0,248; p=0,03), arka stromal keratosit dansitesi (r=-0,557; p<0,001) ve endotel hücre dansitesi (r=-0,590; p<0,001) arasında negatif korelasyon izlenirken, bazal epitel hücre dansitesi ve sinir lifi parametrelerinde ise anlamlı bir korelasyon saptanmadı (hepsi için p>0,05). Sonuç: Lazer tarayıcı in vivo konfokal mikroskopi, korneanın yapısal olarak incelenmesine imkan sağlayan, invaziv olmayan, güvenilir bir yöntemdir. Bu çalışmada sağlıklı kornealardaki hücre dansitesi ve sinir lifi parametrelerine ait normal veriler sunulmuştur.

Ex vivo test bench for preclinical assessment of intra corneal new medical devices is needed more than ever

AbstractPurpose Intra corneal medical devices (ICMD) currently invade the refractive surgery market, particularly for presbyopia. Proposed to correct a simple loss of accommodation and not a disease, they should have very long‐term efficacy and safety objectives. It becomes evident that pre‐clinical tests must be highly efficient in determining the balance of benefits and risks. Despite the European directive 93/42/EEC that defines this balance, several CE marked ICMD, with a quasi‐cosmetic action, are implanted in healthy human corneas after, at best, non‐representative animal experiments. Aim: to demonstrate the urgent need to develop efficient suitable preclinical assessment methods of these ICMDMethods Exhaustive review of the literature about animal models and human experimentations used until now for ICMD assessmentResults None of the usual animal models of ICMD assessment seemed acceptable for various reasons: primate for ethical reasons (presbyopia is not a disease), rat, rabbits and cats for biological reasons especially because of their spontaneous endothelial regeneration, or financial reason especially for long‐term monitoring after implantation in primate, pig, calf or ovines. One ICMD (CE marked since 2005) was tested in 24 rabbits, without any endothelial viability testing, before being implanted in human corneas. Several ICMD (one CE marked since 2009) were never submitted to pre‐clinical assessment and were directly implanted in human corneaConclusion New pre‐clinical assessment, for example ex vivo test bench with very long term stored and monitored human corneas need to be rapidly developed and validated in order to improve patient safety

Translationally Controlled Tumour Protein (TCTP) is present in human cornea and increases in herpetic keratitis

BackgroundTranslationally Controlled Tumour protein is a multifunctional calcium binding protein which has an important role in apoptosis, calcium levels balance and immunological response. The aim of this study was to evaluated the presence and distribution of TCTP in healthy human corneas and to identify and characterize the presence and distribution of this protein in human normal cornea. Since recent studies suggest that apoptosis, calcium levels and immunological mechanisms play a role in the pathogenesis of herpetic stromal keratitis, we studied TCTP expression in this disease.MethodsWe evaluated the expression of TCTP at both RNA messanger and protein level by using reverse transcriptase analysis, immunoblotting and immunohistochemistry in 10 healthy samples cornea: four obtained after penetrating keratoplasty and six from eyes enucleated for other pathologies. Finally, we analysed by immunohistochemistry ten paraffin-embedded samples of Herpes simplex virus keratitis collected at Siena Department of Human Pathology and Oncology: 5 had clinically quiescent disease and 5 had active corneal inflammation.ResultsReverse transcriptase and immunoblotting demonstrated TCTP expression in cornea as a 22,000 Da molecular weight band corresponding to the molecular weight of this protein. Immunohistochemically, all the layers of normal corneal epithelium showed TCTP cytoplasmic expression. TCTP was, also, observed in keratocytes and in the endothelium. In Herpes simplex virus keratitis samples, strong expression of TCTP was evident in stromal cells, in the inflammatory infiltrate and in neo-vessels.ConclusionsIn this preliminary study we demonstrated, for the first time, the presence of TCTP in human cornea, suggesting a potential role in the pathogenesis of herpes virus keratitis.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/3306813447428149

Human Cornea Proteome: Identification and Quantitation of the Proteins of the Three Main Layers Including Epithelium, Stroma, and Endothelium

Diseases of the cornea are common and refer to conditions like infections, injuries and genetic defects. Morphologically, many corneal diseases affect only certain layers of the cornea and separate analysis of the individual layers is therefore of interest to explore the basic molecular mechanisms involved in corneal health and disease. In this study, the three main layers including, the epithelium, stroma and endothelium of healthy human corneas were isolated. Prior to analysis by LC–MS/MS the proteins from the different layers were either (i) separated by SDS-PAGE followed by in-gel trypsinization, (ii) in-solution digested without prior protein separation or, (iii) in-solution digested followed by cation exchange chromatography. A total of 3250 unique Swiss-Prot annotated proteins were identified in human corneas, 2737 in the epithelium, 1679 in the stroma, and 880 in the endothelial layer. Of these, 1787 proteins have not previously been identified in the human cornea by mass spectrometry. In total, 771 proteins were quantified, 157 based on in-solution digestion and 770 based on SDS-PAGE separation followed by in-gel digestion of excised gel pieces. Protein analysis showed that many of the identified proteins are plasma proteins involved in defense responses.

Expression of Membrane-Associated Mucins in Limbal Stem Cell Deficiency and after Transplantation of Cultivated Limbal Epithelium

Purpose: Membrane-associated mucins are important components of the ocular tear film. Our objective was to characterize the expression of membrane-associated mucins MUC1, MUC4, and MUC16 in healthy cornea, healthy conjunctiva, fibrovascular tissue (pannus) covering the corneal surface in limbal stem cell deficiency (LSCD), human limbal epithelial cells cultivated on intact amniotic membrane (HLEC-AM), and corneal epithelium after transplantation of cultivated limbal epithelium (TCLE). Methods: Total RNA was isolated from six samples of healthy human cornea, healthy conjunctiva, pannus, limbal epithelial cell cultures, and four corneal samples after TCLE. The expression of MUC1, MUC4, and MUC16 was evaluated by real-time polymerase chain reaction (PCR), Western blotting, and immunofluorescence. Results: Conjunctiva showed a significantly higher expression of MUC1 and MUC4 than cornea, but the expression of MUC16 was similar in both tissues. The expression of MUC1 in HLEC-AM increased, MUC16 decreased, and MUC4 showed no significant difference when compared to cornea. The expression of all studied mucins in conjunctiva was significantly higher than in HLEC-AM. Pannus revealed a similar expression pattern of membrane-associated mucins to conjunctiva. The expression of all studied mucins in pannus was significantly higher than in cornea or HLEC-AM. A corneal expression pattern of membrane-associated mucins was found in all four samples of cornea after TCLE. Conclusion: A tissue-specific expression of MUC1 and MUC4 but not MUC16 was found in human cornea and conjunctiva. LSCD led to a conjunctival expression pattern of mucins on the corneal surface. A corneal phenotype could be restored after TCLE, confirming the potential of this method to reconstruct the corneal surface.

Expression of Toll-Like Receptors in the Fusarium solani Infected Cornea

Purpose: To investigate the expression of toll-like receptors (TLRs) in the human cornea with Fusarium solani (F. solani) keratitis. Methods: Five human corneas with F. solani keratitis and 5 healthy human corneas were evaluated for TLR1–10 mRNA by reverse transcribed polymerase chain reaction (RT-PCR) and relative real-time PCR. The proteins of TLR2, 4, and 9 were also compared by Western blot. The mean times between these corneas were collected, and the onset of treatment to surgery was 34.4 ± 12.4 days, ranging from 15 to 47 days. The data were analyzed with SPSS15.0. Results: TLR1–10 mRNA was expressed in both healthy and F. solani infected human corneas. The expression of TLR2, 4, 6, and 9 mRNA in the F. solani infected human corneas were upregulated. The Western blot showed the protein expression of TLR2, 4, and 9 was also upregulated in the corneas with F. solani keratitis than that of the healthy corneas. Conclusions: TLRs are expressed diversely in the F. solani infected human cornea, and TLR2, 4, and 9 may be implicated in the pathogenesis of F. solani infection in the cornea.

Characterization of Antigen-Presenting Cells in Fresh and Cultured Human Corneas Using Novel Dendritic Cell Markers

Adult healthy human corneas bear a distinctive number of antigen-presenting cells (APCs) important for the fate of a graft. The purpose of this study was to differentiate between Langerhans cells (LCs) and other dendritic cells (DCs) and between mature and immature APCs in fresh and cultured human corneas using specific markers. Immunofluorescence double staining was performed for Langerin/CD207, CD1a, DC-SIGN/CD209, DC-LAMP/CD208, CD45, CD11c, CD11b and HLA-DR. Langerin(+)/CD1a(+)/HLA-DR(+) LCs (approximately 100 cells/mm(2) in fresh corneas) were found in the limbal and peripheral regions of corneal epithelium and the anterior stroma up to 83 days of culture. All these cells coexpressed CD45 and CD11c. DC-SIGN(+)/CD45(+) DCs (approximately 150 cells/mm(2) in fresh corneas) were detected mainly peripherally and in the anterior stroma, even in long-term cultured corneas. Most of these cells were HLA-DR(-). Few mature DCs (DC-LAMP(+)/HLA-DR(+)) were found in fresh and cultured corneas. Macrophages (CD11c(-)/CD11b(+)) were seen in the peripheral, paracentral, and even central regions of the posterior stroma. This is the first demonstration that human corneas harbor populations of Langerin(+)/CD1a(+)/HLA-DR(+) LCs and DC-SIGN(+) DCs in a distribution pattern similar to that in the skin. Few APCs are in a mature state (DC-LAMP(+)). Given the reduced but not complete depletion of APCs during organ culture, these grafts still bear a potential risk for rejection.

Detection and Localization of the Hydrophobic Surfactant Proteins B and C in Human Tear Fluid and the Human Lacrimal System

Purpose: To evaluate the expression and presence of the surfactant proteins (SP) B and C in the lacrimal apparatus at the ocular surface and in tear fluid. Methods: Expression of SP-B and SP-C was analyzed by RT-PCR in healthy lacrimal gland, conjunctiva, meibomian gland, accessory lacrimal glands, cornea, and nasolacrimal ducts. The deposition of the hydrophobic proteins SP-B and SP-C was determined by Western blot and immunohistochemistry in healthy tissues, tear fluid, and aqueous humor. Results: The presence of both SP-B and SP-C on mRNA and protein level was evidenced in healthy human lacrimal gland, conjunctiva, cornea, and nasolacrimal ducts. Moreover, both proteins were present in tear fluid but were absent in aqueous humor. Immunohistochemical investigations revealed production of both peptides by acinar epithelial cells of the lacrimal gland and additionally by accessory lacrimal glands of the eyelid as well as epithelial cells of the conjunctiva and nasolacrimal ducts. Immunohistochemically, healthy cornea and goblet cells revealed no reactivity. Conclusions: Besides the recently detected surfactant-associated proteins SP-A and SP-D, our results show that SP-B and SP-C are also peptides of the tear film, the ocular surface, and the lacrimal apparatus. Based on the current knowledge of lowering surface tension in alveolar lung cells, a similar effect of SP-B and SP-C may be assumed concerning the tear film.

Effect of gatifloxacin 0.3% and moxifloxacin 0.5% ophthalmic solutions on human corneal epithelium following 2 dosing regimens

To determine whether gatifloxacin 0.3% ophthalmic solution or moxifloxacin 0.5% ophthalmic solutions are toxic to the corneal epithelium when used with 1 of 2 dosing regimens in healthy human eyes. Price Vision Group, Indianapolis, Indiana, USA. In this double-masked randomized fellow-eye comparison study, gatifloxacin 0.3% was instilled in 1 eye and moxifloxacin 0.5% in the other eye either 4 times a day for 7 days or hourly for 10 hours. Before and after dosing, all eyes were examined with a slitlamp and the cell layers in the central cornea were evaluated by confocal microscopy. Subject discomfort with study drop instillation was also assessed. There was no statistically significant increase in the incidence or severity of superficial punctuate keratitis following use of gatifloxacin 0.3% or moxifloxacin 0.5% when instilled 4 times a day for 7 days or hourly for 10 hours. Hourly use of gatifloxacin 0.3% for 10 hours resulted in a mild but statistically significant increase in conjunctival hyperemia (P = .029). Use of moxifloxacin 0.5% resulted in a small but statistically significant deterioration of the corneal epithelial surface as assessed by confocal microscopy (P = .045). The incidence of subject discomfort with study drop instillation was comparable for the 2 antibiotics (P = .67). Use of ophthalmic solutions of gatifloxacin 0.3% or moxifloxacin 0.5% did not result in clinically significant epithelial toxicity in healthy human corneas after dosing regimens of 4 times a day for 7 days or hourly for 10 hours dosing regimens.

A Dataset of Human Cornea Proteins Identified by Peptide Mass Fingerprinting and Tandem Mass Spectrometry

Diseases of the cornea are extremely common and cause severe visual impairment worldwide. To explore the basic molecular mechanisms involved in corneal health and disease, the present study characterizes the proteome of the normal human cornea. All proteins were extracted from the central 7-mm region of 12 normal human donor corneas containing all layers: epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. Proteins were fractionated and identified using two different procedures: (i) two-dimensional gel electrophoresis and protein identification by MALDI-MS and (ii) strong cation exchange or one-dimensional SDS gel electrophoresis followed by LC-MS/MS. All together, 141 distinct proteins were identified of which 99 had not previously been identified in any mammalian corneas by direct protein identification methods. The characterized proteins are involved in many processes including antiangiogenesis, antimicrobial defense, protection from and transport of heme and iron, tissue protection against UV radiation and oxidative stress, cell metabolism, and maintenance of intracellular and extracellular structures and stability. This proteome study of the healthy human cornea provides a basis for further analysis of corneal diseases and the design of bioengineered corneas.

Expression of HLA-G in human cornea, an immune-privileged tissue

Human leukocyte antigen (HLA)-G retains the capacity to modulate immune responses, favoring the establishment of tolerance in solid-tissue allotransplants. To better understand the mechanisms that promote corneal allograft survival, we investigated whether HLA-G was an immunoregulatory factor involved in corneal immunology. We therefore sought HLA-G expression in corneal tissues. Corneal transplantation consists in replacing the center of a diseased cornea with normal corneal tissue. Two corneal parts are not used in such surgery: diseased central corneal tissue and peripheral normal cornea. For this study, we used healthy corneas obtained from deceased donors and diseased corneas obtained from patients with pseudophakic bullous keratopathy or keratoconus who had undergone corneal transplantation. Immunohistochemical analysis carried out on the cryopreserved corneas showed a positive immunohistochemical staining with anti–HLA-G, anti–HLA-A, -B, and -C, and anti–HLA class I monoclonal antibodies. Staining was obtained for keratocytes, epithelial cells, and endothelial cells from both healthy and pathologic human corneas, revealing the presence of HLA class I proteins, including HLA-G. HLA-G transcripts were detected in normal cornea by reverse transcriptase–polymerase chain reaction with a classical pattern of alternative splicing. The detection of HLA-G protein in adult corneas leads to the conclusion that this protein may contribute to the maintenance of the privileged immune status of cornea.

Depth and age-dependent distribution of keratocytes in healthy human corneas: a study using scanning-slit confocal microscopy in vivo

Purpose: To document keratocyte distribution and changes with age in the cellular network of the human cornea in vivo. Setting: Department of Ophthalmology, University of Rostock, Rostock, Germany. Methods: Forty-nine eyes of 31 healthy subjects of various ages were examined with a modified Microphthal® scanning-slit confocal microscope (SSCM) (Hund) to document keratocyte distribution in the intact living cornea. Optical sections made by confocal microscopy were recorded on videotape, and the keratocyte density was determined for the total volume of the cornea and for the stromal sublayers. Results: The highest cell density was in the anterior stroma of the cornea immediately posterior to Bowman’s membrane (24 320 cells/mm 3 ± 6740 [SD]), the lowest in the central area (11 610 ± 4290 cells/mm 3), and an intermediate density in the posterior stroma immediately adjacent to Descemet’s membrane (18 850 ± 4610 cells/mm 3). The differences were statistically significant ( P < .005). The keratocyte density was significantly lower in the anterior and posterior regions in the group older than 50 years: Cell density at 4% depth was 20 960 ± 8200 cells/mm 3 and at 96%, 15 520 ± 4290 cells/mm 3 ( P < .05). Conclusions: In healthy living corneas, the keratocyte density was high in the areas adjacent to Bowman’s and Descemet’s membranes and was lower in patients older than 50 years than in those younger than 50 years. Further studies are needed to document the rate of change with age and to better understand the role and capacity of aging keratocytes in regenerative processes following corneal diseases or surgical procedures.

Microarray analysis of gene expression in human donor corneas.

To use microarray analysis to identify genes expressed in human donor corneas and to create a preliminary, comprehensive database of human corneal gene expression. A complementary DNA (cDNA) library was constructed from transplant-quality, human donor corneas. Biotin-labeled RNA was transcribed from the cDNA library and hybridized in duplicate to microarrays containing approximately 5600 human genes. Results were analyzed using a gene database of the National Institutes of Health, Bethesda, Md. Reverse transcriptase polymerase chain reaction analysis was performed to confirm corneal expression of genes identified by microarray analysis. Duplicate microarrays identified the expression of 1200 genes in human donor corneas. Chromosomal loci had been assigned to 1025 (85%) of these genes. A preliminary database of human corneal gene expression was compiled. A Web site containing these genes was created. Six collagen genes were identified that had not previously been localized within the cornea. Five apoptosis-related genes were identified, 4 of which had not previously been localized within the cornea. Three genes previously shown to cause corneal diseases were identified. Reverse transcriptase polymerase chain reaction analysis of genes identified by microarray analysis confirmed the corneal expression of 2 apoptosis-related genes and 1 collagen gene. Microarray analysis of healthy human donor corneas has produced a preliminary, comprehensive database of corneal gene expression. Large-scale analysis of gene expression has the potential to generate large amounts of data, which should be made readily accessible to the scientific community. The Internet offers many potential advantages as a medium for the maintenance of these large data sets. Identification of structural, apoptosis-related, and disease-causing genes within the cornea by microarrays may increase the understanding of normal and abnormal corneal function with likely relevance to corneal diseases and transplants.

Normal human corneal cell populations evaluated by in vivo scanning slit confocal microscopy.

To analyze cellular populations in healthy human corneas. The study group consisted of 58 eyes of 45 patients with normal corneas. The age distribution was 45 +/- 17 years (mean +/- SD; range, 20-84). Scanning slit confocal microscopy of the central corneas was performed. The images were analyzed visually for cell morphology, and the densities and areas of cells were measured. No statistically significant differences were measured in cell densities or cell areas of any corneal layer between female and male patients (p = 0.22-0.50) nor between right and left eyes (p = 0.16-0.45). The area of superficial epithelial cells was 913 +/- 326 microm2 (mean +/- SD; range, 518-2,112), and the superficial epithelial cell density was 1,213 +/- 370 cells/mm2 (mean +/- SD; range, 473-1,929). The area of basal epithelial cells was 177 +/- 19 microm2 (mean +/- SD; range, 138-242), and the basal epithelial cell density was 5,699 +/- 604 cells/mm2 (mean +/- SD; range, 4,135-7,267). The average apparent keratocyte density was 1,058 +/- 217 cells/mm2 (mean +/- SD; range, 604-1,599) in the anterior stroma, and 771 +/- 135 cells/mm2 (mean +/- SD; range, 493-1,145) in the posterior stroma. The difference in apparent keratocyte densities between the anterior and posterior stroma was statistically significant (p < 0.001). The average endothelial cell area was 334 +/- 51 microm2 (range, 273-553), and the cell density was 3,055 +/- 386 cells/mm2 (mean +/- SD; range, 1,809-3,668). The endothelial cell density had a negative, statistically significant correlation with age (r = -0.68, p < 0.001). The densities of the other corneal cell layers did not have a statistically significant correlation with age. In vivo scanning slit confocal microscopy is a useful tool for studying corneal cell populations. Central corneal cell densities were found to decrease significantly with age only in the endothelium. For the first time in human corneas using in vivo confocal microscopy, this study statistically confirms a higher apparent number of keratocytes in the anterior stroma than in the posterior stroma.

Quantitative analysis of immunogold labellings of collagen types I, III, IV and VI in healthy and pathological human corneas.

We studied the distribution of collagen types I, III, IV and VI in one healthy human cornea and in seven pathological human corneas, in which the disorders were three cases of pseudophakic bullous keratopathy (two severe, one moderate) and one case each of stage IV keratoconus, chronic ulcer, vascularized cornea and disciform keratitis. Transmission electron microscopy examinations were performed on post-embedding immunogold-labelled sections. The staining was evaluated by gold particle count in the different tissues. The presence or absence of a given antigen was determined by statistical analysis, using a d-value test. Our results on healthy corneal tissues corroborate the data available from previous studies, except for collagen type VI, which we found to be absent in Bowman's layer. In pathological corneas with a collagenous layer posterior to Descemet's membrane, collagen types I, III and especially IV were detected in this collagenous layer. Collagen types I, III and VI were detected in the anterior healed stroma of other pathological corneas, except for the keratoconus cornea, in which intense collagen III staining was observed. The presence of collagen types I and III in the posterior collagenous layer of our pseudophakic bullous keratopathy corneas suggests that this layer corresponds to scar tissue secreted by stimulated endothelial cells.

Observations of human corneal epithelium by tandem scanning confocal microscope

We applied the tandem scanning confocal microscope (TSCM) to 30 healthy human corneas of 3 normal volunteers and 27 patients with cataract and retinal detachment to observe normal corneal epithelial cells in vivo. All corneas were normal under slit lamp microscopic examination. The superficial and basal epithelial cells close to the basal lamina in the central cornea were recorded on videotape and analyzed by a computer-assisted digitizer. The mean cell areas of superficial cells exposed at the surface and basal cells at the horizontal section were 624 +/- 109 microns2 and 66 +/- 5 microns2, respectively. The ratio of superficial to basal mean cell area was 11.0 +/- 4.5. TSCM was thus useful in evaluating the relationship between superficial and basal cells in human corneal epithelium in vivo.

Modification, Calibration, and Comparative Testing of an Automated Surgical Keratometer

A new digital surgical keratometer (SK-1, Canon Inc) designed to fit existing operating microscopes and provide quantitative measurements at a nominal 2.0- and 3.6-mm diameter optical zone of the central cornea was modified for single foot-switch operation by the surgeon. The instrument was tested against three calibrated steel spheres, an astigmatic test-jig, and compared with a standard clinical keratometer (CLC, A.O. Co) in two studies involving six eye bank eyes and five healthy human corneas. Assessed with the steel spheres, the SK-1 calibration error was -0.042 diopters at the 3.6-mm diameter and +0.207 D at the 2.0-mm chord, while it was -0.120 D for the CLC. With cylindrical power greater than 0.50 D, the astigmatic angle was accurate to +/- 1 degree. After correction for calibration error, the CLC readings on eye bank eyes were 1.00 D higher in average (range +0.45 to +1.90) than those obtained with the SK-1 which gave lower keratometric values at the 2-mm chord diameter (-0.254 D, range: +0.05 to -0.7). The human subject corrected readings at the 3.6-mm chord were within 0.137 D (range -0.09 to +0.41) from the CLC values. A mean corrected difference of +0.068 D (range -0.27 to +0.30) was found between the two chords. The SK-1 instrument was easier to use than the CLC. A more accurate calibration and the addition of automated averaging and video display of readings as well as of a motorized Placido disc would further augment its clinical usefulness.