Cohesive tensile strength of human LASIK wounds with histologic, ultrastructural, and clinical correlations

PURPOSE: To measure the cohesive tensile strength of human LASIK corneal wounds. METHODS: Twenty-five human eye bank corneas from 13 donors that had LASIK were cut into 4-mm corneoscleral strips and dissected to expose the interface wound. Using a motorized pulling device, the force required to separate the wound was recorded. Intact and separated specimens were processed for light and electron microscopy. Five normal human eye bank corneas from 5 donors served as controls. A retrospective clinical study was done on 144 eyes that had LASIK flap-lift retreatments, providing clinical correlation. RESULTS: The mean tensile strength of the central and paracentral LASIK wounds showed minimal change in strength over time after surgery, averaging 2.4% (0.72 +/- 0.33 g/mm) of controls (30.06 +/- 2.93 g/mm). In contrast, the mean peak tensile strength of the flap wound margin gradually increased over time after surgery, reaching maximum values by 3.5 years when the average was 28.1% (8.46 +/- 4.56 g/mm) of controls. Histologic and ultrastructural correlative studies found that the plane of separation always occurred in the lamellar wound, which consisted of a hypocellular primitive stromal scar centrally and paracentrally and a hypercellular fibrotic stromal scar at the flap wound margin. The pathologic correlations demonstrated that the strongest wound margin scars had no epithelial cell ingrowth-the strongest typically being wider or more peripherally located. In contrast, the weakest wound margin scars had epithelial cell ingrowth. The clinical series demonstrated the ability to lift LASIK flaps without complications during retreatments up to 8.4 years after initial surgery, correlating well with the laboratory results. CONCLUSIONS: The human comeal stroma typically heals after LASIK in a limited and incomplete fashion; this results in a weak, central and paracentral hypocellular primitive stromal scar that averages 2.4% as strong as normal comeal stroma. Conversely, the LASIK flap wound margin heals by producing a 10-fold stronger, peripheral hypercellular fibrotic stromal scar that averages 28.1% as strong as normal comeal stromal, but displays marked variability.     

Biomechanical and wound healing characteristics of corneas after excimer laser keratorefractive surgery: is there a difference between advanced surface ablation and sub-Bowman's keratomileusis?

PURPOSE: To describe the biomechanical and wound healing characteristics of corneas after excimer laser keratorefractive surgery. METHODS: Histologic, ultrastructural, and cohesive tensile strength evaluations were performed on 25 normal human corneal specimens, 206 uncomplicated LASIK specimens, 17 uncomplicated sub-Bowman's keratomileusis (SBK) specimens, 4 uncomplicated photorefractive keratectomy (PRK) specimens, 2 uncomplicated advanced surface ablation (ASA) specimens, 5 keratoconus specimens, 12 postoperative LASIK ectasia specimens, and 1 postoperative PRK ectasia specimen and compared to previously published studies. RESULTS: Histologic and ultrastructural studies of normal corneas showed significant differences in the direction of collagen fibrils and/or the degree of lamellar interweaving in Bowman's layer, the anterior third of the corneal stroma, the posterior two-thirds of the corneal stroma, and Descemet's membrane. Cohesive tensile strength testing directly supported these morphologic findings as the stronger, more rigid regions of the cornea were located anteriorly and peripherally. This suggests that PRK and ASA, and secondarily SBK, should be biomechanically safer than conventional LASIK with regard to risk for causing keratectasia after surgery. Because adult human corneal stromal wounds heal slowly and incompletely, all excimer laser keratorefractive surgical techniques still have some distinct disadvantages due to inadequate reparative wound healing. Despite reducing some of the risk for corneal haze compared to conventional PRK, ASA cases still can develop corneal haze or breakthrough haze from the hypercellular fibrotic stromal scarring. In contrast, similar to conventional LASIK, SBK still has the short- and long-term potential for interface wound complications from the hypocellular primitive stromal scar. CONCLUSIONS: Ophthalmic pathology and basic science research show that SBK and ASA are improvements in excimer laser keratorefractive surgery compared to conventional LASIK or PRK, particularly with regard to maintaining corneal biomechanics and perhaps moderately reducing the risk of corneal haze. However, most of the disadvantages caused by wound healing issues remain.     

A new surgical technique for deep stromal, anterior lamellar keratoplasty

AIMS: To describe a new surgical technique for deep stromal anterior lamellar keratoplasty. METHODS: In eye bank eyes and sighted human eyes, aqueous was exchanged by air, to visualise the posterior corneal surface--that is, the "air to endothelium" interface. Through a 5.0 mm scleral incision, a deep stromal pocket was created across the cornea, using the air to endothelium interface as a reference plane for dissection depth. The pocket was filled with viscoelastic, and an anterior corneal lamella was excised. A full thickness donor button was sutured into the recipient bed after stripping its Descemet's membrane. RESULTS: In 25 consecutive human eye bank eyes, a 12% microperforation rate was found. Corneal dissection depth averaged 95.4% (SD 2.7%). Six patient eyes had uneventful surgeries; in a seventh eye, perforation of the lamellar bed occurred. All transplants cleared. Central pachymetry ranged from 0.62 to 0.73 mm. CONCLUSION: With this technique a deep stromal anterior lamellar keratoplasty can be performed with the donor to recipient interface just anterior to the posterior corneal surface. The technique has the advantage that the dissection can be completed in the event of inadvertent microperforation, or that the procedure can be aborted to perform a planned penetrating keratoplasty.     

Corneal opacity in lumican-null mice: defects in collagen fibril structure and packing in the posterior stroma

PURPOSE: Gene targeted lumican-null mutants (lum(tm1sc)/lum(tm1sc)) have cloudy corneas with abnormally thick collagen fibrils. The purpose of the present study was to analyze the loss of transparency quantitatively and to define the associated corneal collagen fibril and stromal defects. METHODS: Backscattering of light, a function of corneal haze and opacification, was determined regionally using in vivo confocal microscopy in lumican-deficient and wild-type control mice. Fibril organization and structure were analyzed using transmission electron microscopy. Biochemical approaches were used to quantify glycosaminoglycan contents. Lumican distribution in the cornea was elucidated immunohistochemically. RESULTS; Compared with control stromas, lumican-deficient stromas displayed a threefold increase in backscattered light with maximal increase confined to the posterior stroma. Confocal microscopy through-focusing (CMTF) measurement profiles also indicated a 40% reduction in stromal thickness in the lumican-null mice. Transmission electron microscopy indicated significant collagen fibril abnormalities in the posterior stroma, with the anterior stroma remaining relatively unremarkable. The lumican-deficient posterior stroma displayed a pronounced increase in fibril diameter, large fibril aggregates, altered fibril packing, and poor lamellar organization. Immunostaining of wild-type corneas demonstrated high concentrations of lumican in the posterior stroma. Biochemical assessment of keratan sulfate (KS) content of whole eyes revealed a 25% reduction in KS content in the lumican-deficient mice. CONCLUSIONS: The structural defects and maximum backscattering of light clearly localized to the posterior stroma of lumican-deficient mice. In normal mice, an enrichment of lumican was observed in the posterior stroma compared with that in the anterior stroma. Taken together, these observations indicate a key role for lumican in the posterior stroma in maintaining normal fibril architecture, most likely by regulating fibril assembly and maintaining optimal KS content required for transparency.     

Comparative study of human keratocyte density after corneal grafting by using confocal microscopy in vivo

PURPOSE: In a case control study we determined keratocyte density and size of nuclear area in the central segment of the corneal stroma. METHODS: We compared 20 corneas after keratoplasty with 24 eyes of normal healthy individuals. Both groups were matched according to age. Keratocyte density and nuclear area were analyzed using Nidek Confoscan 2 separately for each group. In corneal graft recipients we studied how the mentioned variables were influenced by age of corneal graft donor (ranged from 4- to 71-years old) and by the time from surgery (8 to 77 months). RESULTS: The comparison of healthy controls and patients with keratoplasty revealed no changes in keratocyte density and the size of nuclear area in central stromal layers. In patients after keratoplasty donor age influenced an increase in keratocyte nuclei area only in the posterior stroma layer (p = 0.042). No such changes were observed in anterior and midstroma layers. Donor age was not found to be significant for keratocyte density in any of the layers. Time from surgery neither influenced changes in keratocyte density nor in keratocyte nuclei area. CONCLUSIONS: In our study using confocal microscopy in vivo we found that corneal grafting does not influence keratocyte density and nuclear area in individual layers of the central corneal stroma segment (anterior, midstroma and posterior layers). Donor age influenced an increase in keratocyte nuclei area only in the posterior stroma layer.     

Location of the stress-bearing layers of the cornea

In order to determine which layers of the corneal stroma bear the stress of the intraocular pressure, 6.0-mm nonpenetrating trephine incisions were made centrally in one eye of each of 16 adult albino rabbits. After epithelial healing, the central corneal thickness was measured over 3 hr at 50 mmHg intraocular pressure in both eyes of the anesthetized rabbits. The animals were then killed and the uniformity and depth of trephine cut determined histologically. The mean differences in swelling rates between the cut and the opposite uncut eyes for trephine incisions of different depths were as follows: 1 +/- 2 micron/hr for 8-20% depth, 5 +/- 2 micron/hr for 21-40% depth, and 14 +/- 3 micron/hr for 41-60% depth (P less than 0.01 for all groups). These results indicate that the intraocular tension is probably distributed across all the corneal stromal lamellae rather than being borne primarily by the anterior or posterior layers.     

In-vivo slit scanning confocal microscopy of normal corneas in Indian eyes

OBJECTIVE: To study the cellular populations of healthy corneas of Indian eyes using confocal microscopy and to evaluate the correlation with age, gender and laterality. METHODS: The central corneas of 100 eyes of 50 healthy subjects were examined using an in-vivo slit scanning confocal microscope (Confoscan 2). Images were analysed for cell densities of the epithelium, stroma and endothelium. RESULTS: Good quality images enabling analysis of all cell layer populations were obtained in 74 eyes of 43 healthy subjects (22 males and 21 females) with a mean age of 31.89 +/- 13.47 (range 19-71 years). The basal epithelial cell density was 3601.38 +/- 408.19 cells/mm2 (range 3017.3-4231.1 cells/mm2). The mean keratocyte nuclei density in the anterior stroma was 1005.02 +/- 396.86 cells/mm2 (range 571.6-1249.6 cells/mm2) and in the posterior stroma was 654.32 +/- 147.09 cells/mm2 (range 402.6-1049.1 cells/mm2). Posterior keratocyte nuclei density was 30.76% less than the anterior stromal keratocyte nuclei density. The difference in keratocyte nuclei density was statistically significant (P=0.001). The mean endothelial cell density was 2818.1 +/- 361.03 cells/mm2 (range 2118.9-4434 cells/mm2) and the mean endothelial cell area was found to be 385.44 +/- 42.66 mm2 (range 268.9-489.2 mm2). Hexagonal cells formed 22.5-69.4% of the endothelial cell populations (mean 42.04 +/- 11.81%). Mean coefficient of cell size variation was 32.29 +/- 3.06 (range 27.2-39.2). No statistically significant differences were found in cell densities of any corneal layer either between female and male patients or between right and left eyes. Basal epithelial cell density, anterior stromal keratocyte nuclei and posterior stromal keratocyte nuclei density were unaffected by age (r=0.12, 0.07, -0.12 respectively) (P=0.001). There was a statistically significant negative correlation between mean endothelial cell density and increase in age (r=-0.42, P=0.001). Coefficient of cell size variation and age were positively correlated (r=0.73, P=0.001). CONCLUSION: In-vivo slit scanning confocal microscopy is useful for the study of corneal cell populations. Our study provides normative data of these cell populations.     

The keratocyte network of human cornea: a three-dimensional study using confocal laser scanning fluorescence microscopy

PURPOSE: Keratocytes of the living human cornea were examined to compare quantitatively spatial arrangement and cell volume of the stromal layers. This knowledge is required for further studies toward a quantitative understanding of cellular alterations in corneal pathology. METHODS: Three human corneas were stained with calcein AM and ethidium homodimer (Live/Dead Kit) directly after enucleation. The fluorescent cells were examined with confocal laser scanning fluorescence microscopy. High-resolution three-dimensional (3-D) volumes of < or =270 microm in the z-axis were reconstructed. Cell density and volume density were determined by computer-aided morphometry. RESULTS: Three keratocyte subpopulations were distinguished. Their spatial arrangement was visualized by 3-D reconstructions of the scanned volumes. Whereas cell density decreased progressively from the anterior (100%) to posterior (53.7%) stroma, volume density was highest in the posterior stroma (17.03 +/- 5.05%) and lowest in the central stroma (9.31 +/- 1.09%). In the anterior stroma, volume density was found to be 10.19 +/- 4.37%. CONCLUSION: Confocal laser scanning fluorescence microscopy allowed quantitative analysis and the visualization of the spatial arrangement of the keratocyte network in the living human corneal tissue for the first time. The results provide a basis for further studies of alterations of the normal cellular arrangements in corneal disease.     

Collagen fibril characteristics at the corneo‐scleral boundary and rabbit corneal stromal swelling

Background: The aim of this investigation was to reassess the impact of the scleral rim on the swelling of the mammalian corneal stroma and to investigate the ultrastructural features of the scleral rim and corneal stromal tissues. Methods: The epithelium and endothelium were removed from corneas excised from three‐month‐old female rabbits. The resulting preparations consisted of the corneal stroma plus a surrounding scleral rim, excised corneal stroma or a nine‐millimetre button of central corneal stroma. These preparations were immersed in a 35 mM bicarbonatebuffered mixed salt solution (equilibrated with five per cent CO₂‐air, pH 7.54 at 37 degrees Centigrade for nine hours. Some sclero‐corneal preparations were fixed for light or transmission electron microscopy. Results: The initial rate of swelling of corneal stromal buttons was greatest at 127 ± 8 per cent per hour, less for complete stromal preparations (118 ± 9 per cent per hour) and least for sclerostromal preparations (76 ± 12 per cent per hour). The swelling continued over three to nine hours but sclera preparations swelled up to 40 per cent with no further swelling. Light microscopy demonstrated that the sclero‐corneal rim tissue limited the swelling of the posterior corneal stroma. TEM sections of the episclera and sclera indicate that most fibril bundles show a radial orientation to the cornea. There are marked anterio‐posterior differences in the collagen fibrils of the scleral surround that are distinctly different from previous reports. Average fibril diameters were 62.8 ± 7.9 nm in the episclera, 122.4 ± 18.9 nm, 133.5 ± 51.9 nm and 56.5 ± 11.2 nm in the anterior, mid‐ and posterior scleral stroma, compared to an average fibril diameter of 33.5 ± 3.5 nm for the posterior corneal stroma. Conclusions: When there is a scleral rim in place, the swelling of the corneal stroma is substantially less than for isolated corneal stroma. The effect can be attributed to the a b sence of a cut‐edge effect for the sclero‐corneal stromal preparation but the unique and largely radial arrangement of the collagen fibrils in the scleral rim plays a part in limiting the swelling of the adjacent corneal stroma. The heterogeneous nature of this sclero‐corneal interface requires further investigation to define the mechanism of the effect.     

Transparency, swelling and scarring in the corneal stroma

PURPOSE: This paper briefly reviews current explanations for corneal transparency and uses a well-developed model to try to explain the increased light scattering either accompanying corneal swelling or following phototherapeutic keratectomy (PTK). METHODS: The direct summation of fields (DSF) method was used to compute light transmission as a function of wavelength. The method requires input of a number of structural parameters. Some of these were obtained from electron micrographs and others were calculated from X-ray diffraction data. RESULTS: By swelling sections of stroma cut from different depths in the tissue, we have shown that fluid entering the cornea causes more swelling in the posterior lamellae than in the anterior lamellae. Furthermore, posterior lamellae can reach a higher final hydration than anterior lamellae. Collagen-free regions ('lakes') exist in corneas swollen in vitro and in Fuch's dystrophy corneas, many of which may be caused by the death of cells. The DSF method shows that local fibril disordering, increased refractive index mismatch, and increased corneal thickness together can account for a 20% increase in light scattering in a Fuch's dystrophy cornea at H=5.8 compared to the normal cornea. Additional scattering is probably caused by 'lakes'. The DSF method applied to PTK rabbit stroma with high levels of haze suggests that the newly deposited collagen is not the cause of the increased light scattering. CONCLUSIONS: Fluid is not uniformly distributed within the corneal stroma when the cornea swells. Increased hydration of posterior lamellae may be because of known differences in the glycosaminoglycans between the anterior and posterior stroma. Lamellar interweave in the anterior stroma probably limits the extent to which the constituent lamellae can swell. The DSF method can be used to account for increased light scattering in oedematous corneas but cannot account for haze following PTK.     

Corneal cross-linking-induced stromal demarcation line

PURPOSE: Corneal collagen cross-linking by UVA/riboflavin (X-linking) represents a new method for the treatment of progressive keratoconus and currently is under clinical study. To avoid UVA irradiation damage to the corneal endothelium, the parameters for X-linking are set in a way that effective treatment occurs only in the first 300 microm of the corneal stroma. Here, X-linking not only strengthens the biomechanical properties of the cornea but also induces keratocyte apoptosis. To date, the effectiveness of treatment could be monitored only indirectly by postoperative follow-up corneal topographies or using corneal confocal microscopy. Here we describe a corneal stromal demarcation line indicating the transition zone between cross-linked anterior corneal stroma and untreated posterior corneal stroma. The demarcation line is biomicroscopically detectable in slit-lamp examination as early as 2 weeks after treatment. METHODS: X-linking was performed in 16 cases of progressive keratoconus, and corneas were examined biomicroscopically and by means of corneal topography and pachymetry before and after treatment. RESULTS: In 14 of 16 cases, a thin stromal demarcation line was visible at a depth of approximately 300 microm over the whole cornea after X-linking treatment. CONCLUSION: This newly observed demarcation line may result from differences in the refractive index and/or reflection properties of untreated versus X-linked corneal stroma and represents an effective tool to biomicroscopically easily monitor the depth of effective X-linking treatment in keratoconus.     

Lamellar corneal dissection for visualization of the anterior chamber before triple procedure

PURPOSE: Open-sky cataract extraction during triple procedure can be associated with higher risk of complications owing to the missing counterbalance by the cornea. Herein, we present a fast and easy technique for visualization of the anterior chamber and the lens in eyes with opaque corneas planed for triple procedure. MATERIALS AND METHODS: Four patients with corneal oedema due to Fuchs' endothelial dystrophy and cataract underwent triple procedure. As the anterior chamber view was limited, the central 7.0 mm of the cornea was marked. Then, 60-80% of the corneal thickness was removed by lamellar dissection and filled with hydroxypropylmethylcellulose. Continuous curvilinear capsulorhexis (CCC) and phacoemulsification with intraocular lens implantation through a corneoscleral tunnel were then performed and at the end the remaining corneal tissue was excised and the donor tissue fixed with a single running continuous suture. RESULTS: Lamellar corneal dissection enhances the anterior chamber view and CCC can be performed under stable anterior chamber condition. Phacoemulsification via sclerocorneal tunnel could be easily performed under good anterior chamber view in all cases. The operation time was 60-75 min in all cases. CONCLUSION: Lamellar corneal dissection in opaque corneas before cataract extraction is a useful technique for enhancing anterior chamber view in cases of triple procedure.     

Normal human keratocyte density and corneal thickness measurement by using confocal microscopy in vivo

PURPOSE: To quantify keratocyte density according to stromal region and subject age and to measure the thickness of the normal human cornea and its layers in vivo. METHODS: Seventy normal corneas of 70 subjects were examined by confocal microscopy (contact lens wearers were excluded). Ages of subjects ranged from 12 to 80 years, with 10 subjects per decade. Images were recorded by continuously focusing the optical section through the full-thickness central cornea. Two independent human observers manually identified bright objects (keratocyte nuclei) against a dark background to quantify keratocyte density. This method was validated histologically in three human corneas. Thickness measurements were obtained by plotting mean reflected light intensity in images against corneal depth, and calculating distances between intensity peaks that corresponded to corneal layers. RESULTS: Full-thickness central keratocyte density was 20,522 +/- 2,981 cells/mm(3) (mean +/- SD, n = 69). The number of keratocytes in a full-thickness column of central stroma, which had a cross-sectional area of 1 mm(2), was 9624 +/- 1385 cells. Keratocyte density was highest in the anterior 10% of the stroma. Full-thickness keratocyte density was correlated with age (r = -0.62, P < 0.001), decreasing 0.45% per year. Central corneal thickness was 563.0 +/- 31.1 microm (mean +/- SD) and central epithelial thickness was 48.6 +/- 5.1 microm. CONCLUSIONS: This is the first study to quantify regional keratocyte density comprehensively in vivo across a broad age range of normal human subjects. The method was acceptable to both subject and observer, and may prove useful for quantifying keratocyte density in patients with corneal disorders or after corneal surgery.     

Posterior amorphous corneal dystrophy. An ultrastructural study of a variant with histopathological features of an endothelial dystrophy.

Posterior amorphous corneal dystrophy (PACD) is a rare autosomal-dominant disease, generally classified with the pre-Descemet's dystrophies. It is characterized by deep stromal corneal opacification, flat corneas with low keratometry values, and central thinning. To our knowledge, only one previous ultrastructural study has been published on this disease. This 5-year-old white boy presented with best corrected vision (20/50 right and 20/60 -2 left). The corneas had dense opacities, bilaterally, deep in the corneal stroma. Keratometry was 39.50/40.50, bilaterally. The patient's father had 20/20 vision, bilaterally, with minimal opacifications in the deep corneal stroma. A penetrating keratoplasty was performed. In contrast to the previously reported case of PACD, in which the abnormalities were largely limited to the stroma, our patient had subepithelial deposits, only mild stromal abnormalities, and a thick collagenous layer posterior to Descemet's membrane, thus suggesting that this variant of PACD is a generalized corneal disease including endothelial and epithelial abnormalities, rather than a pure stromal dystrophy.     

Thermomechanical behavior of collagen-cross-linked porcine cornea

PURPOSE: Collagen cross-linking using combined riboflavin/UVA treatment has been shown to increase the biomechanical rigidity of the cornea and has been used successfully for the treatment of progressive keratoconus. From morphological and biochemical investigations, a different degree of cross-linking for the anterior and posterior stroma by the treatment is suggested. The present study was undertaken to better evaluate this effect by testing the thermomechanical behavior. METHODS: Ten 10 x 5 mm corneal strips from porcine cadaver eyes enucleated within 5 h post mortem were cross-linked using the photosensitizer riboflavin and UVA irradiation (370 nm, irradiance = 3 mW/cm(2)) for 30 min and compared to ten untreated corneal strips and ten corneal strips cross-linked with 0.1% glutaraldehyde. The temperature in a water bath was raised from 60 to 95 degrees C with temperature increments of 1 degrees C per minute. The hydrothermal shrinkage of the corneal strips was measured in 2.5 degrees C steps using a micrometer. In addition, six 10-mm whole corneal buttons were cross-linked with riboflavin/UVA and immersed into water at 70 or 75 degrees C. RESULTS: The maximal hydrothermal shrinkage for the untreated control specimens and the posterior portion of the riboflavin/UVA-treated corneas was at 70 degrees C, for the anterior portion of the cornea cross-linked by riboflavin/UVA at 75 degrees C and for glutaraldehyde-cross-linked cornea at 90 degrees C. In the cross-linked corneal buttons, a typical mushroom-like shape was observed at 70 degrees C and a cylinder shape at 75 degrees C. CONCLUSIONS: The different degree of collagen cross-linking in the corneal stroma after riboflavin/UVA treatment is reflected by the differences in the maximal shrinkage temperature of the anterior and posterior portion. Therefore, in the corneas cross-linked with riboflavin/UVA a higher shrinkage temperature was observed for the anterior portion of the cornea (75 degrees C) compared to the posterior stroma (70 degrees C) due to the higher degree of cross-linking of the anterior stroma. The anterior localization of the cross-linking effect is advantageous for the endothelium and for the preservation of the anterior corneal curvature.     

Rabbit cornea microstructure response to changes in intraocular pressure visualized by using nonlinear optical microscopy

PURPOSE: To characterize the microstructural response of the rabbit cornea to changes in intraocular pressure (IOP) by using nonlinear optical microscopy (NLOM). METHODS: Isolated rabbit corneas were mounted on an artificial anterior chamber in series with a manometer and were hydrostatically pressurized by a reservoir. The chamber was mounted on an upright microscope stage of a custom-built NLOM system for corneal imaging without using exogenous stains or dyes. Second harmonic generation in collagen was used to image through the full thickness of the central corneal stroma at IOPs between 5 and 20 mm Hg. Microstructural morphology changes as a function of IOP were used to characterize the depth-dependent response of the central cornea. RESULTS: Regional collagen lamellae architecture through the full thickness of the stroma was specifically imaged as a function of IOP. Hypotensive corneas showed gaps between lamellar structures that decreased in size with increasing IOP. These morphologic features appear to result from interwoven lamellae oriented along the anterior-posterior axis and parallel to the cornea surface. They appear throughout the full thickness and disappear with tension in the anterior but persist in the posterior central cornea, even at hypertensive IOP. CONCLUSIONS: NLOM reveals interwoven collagen lamellae sheets through the full thickness of the rabbit central cornea oriented along the anterior-posterior axis and parallel to the surface. The nondestructive nature of NLOM allows 3-dimensional imaging of stromal architecture as a function of IOP in situ. Collagen morphologic features were used as an indirect measure of depth-dependent mechanical response to changes in IOP.     

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.     

Effects of contact lens wearing on keratoconus: a confocal microscopy observation

AIM: To evaluate the corneal cell morphology of new keratoconus patients wearing two different types of rigid gas-permeable (RGP) contact lenses for 1y. METHODS: Thirty nine eyes of 39 new keratoconus patients were selected and randomly fitted with two types of RGP contact lenses. Group 1 had 21 eyes with regular rigid gas-permeable (RRGP) contact lens and rest 18 eyes were in group 2 with specially designed rigid gas-permeable (SRGP) contact lens. Corneal cell morphology was evaluated using a slit scanning confocal microscope at no-lens wear and after 1y of contact lens wearing. RESULTS: After 1y of contact lens wearing in group 1, the mean anterior and posterior stromal keratocyte density were significantly less (P=0.006 and P=0.001, respectively) compared to no-lens wear. The mean cell area of anterior and posterior stromal keratocyte were also significantly different (P=0.005 and P=0.001) from no-lens wear. The anterior and posterior stromal haze increased by 18.74% and 23.81%, respectively after 1y of contact lens wearing. Whereas in group 2, statistically significant changes were observed only in cell density & area of anterior stroma (P=0.001 and P=0.001, respectively) after 1y. While, level of anterior and posterior stromal haze increased by 16.67% and 11.11% after 1y of contact lens wearing. Polymegathism and pleomorphism also increased after 1y of contact lens wearing in both the contact lens groups. CONCLUSION: Confocal microscopy observation shows the significant alterations in corneal cell morphology of keratoconic corneas wearing contact lenses especially in group 1. The type of contact lens must be carefully selected to minimize changes in corneal cell morphology.     

20%乙醇处理兔角膜后上皮增生和细胞凋亡的研究

目的探讨准分子激光角膜上皮瓣下磨镶术(LASEK)中采用20%乙醇浸润兔角膜40s后角膜上皮增生和角膜细胞凋亡情况与机械刮除角膜上皮后的异同。方法实验组42只新西兰大白兔,用直径为8mm的LASEK专用角膜上皮刀切割角膜上皮,20%的乙醇浸润单眼40s,机械刮除对侧眼中央8mm直径的角膜上皮,随机分7组,于术后0、4h,1、3、5、8、30d取材;6只兔眼为空白对照。角膜冰冻切片,行Ki67免疫组化检查和TUNEL检测,计数角膜中央前基质细胞。结果乙醇浸润后5d中央角膜上皮增生达峰值,术后1d周边角膜上皮增生达峰值;术后4h上皮刀口下方局限的角膜基质细胞TUNEL染色阳性,数量最多;各组角膜中央前基质细胞计数和空白对照比差异无统计学意义(P=0.68)。机械刮除角膜上皮后3d周边角膜上皮增生达峰值,其高于乙醇浸润后角膜上皮的增生峰值;术后4h可见大量中央前基质细胞TUNEL阳性;术后1d中央前基质细胞数量最少(P<0.05)。结论与机械刮除角膜上皮相比,20%乙醇浸润40s对角膜损伤轻,恢复快,乙醇浸润后的角膜上皮对基质细胞有保护作用。     

Interlamellar adhesive strength in human eyebank corneas

The interlamellar biomechanical properties of stromal collagen are relatively unknown, yet may be highly significant with respect to wound healing and the efficacy of certain keratorefractive surgical procedures. Interlamellar adhesive strength was measured as the tearing force required to separate corneal lamellae at a 50% stromal depth in 16 human eyebank corneas. The mean value for the central cornea was found to be 14.2 (+/- 0.5 SEM) g-wt/mm of tissue width. Histology showed a smooth separation between the lamellae along the tearing plane in the central cornea. We believe that the adhesive strength measured in the central cornea may be primarily the force needed to break interlamellar proteoglycan bonds between collagen lamellae, because no torn lamellae were found in this region. The mean adhesive strength and the SEM increased toward the periphery in a symmetrical fashion. The mean adhesive strength in the far periphery was 31.6 (+/- 3.7 SEM) g-wt/mm at 5 mm nasally, and 28.4 (+/- 3.2 SEM) g-wt/mm at 5 mm temporally, and was approximately twice the mean central value. The rising value of the mean adhesive strength with increasing distance from the central cornea was believed to be due to a more highly disorganized collagen network in which greater numbers of lamellae passed obliquely in depth through the tearing plane. These lamellae would contribute their tensile strength to the adhesive strength measurement along the tearing plane. Histology from the peripheral cornea confirmed the existence of depth-varying collagen lamellae and the torn ends of lamellae that passed across the tearing plane.