Keratocyte Density Research Articles

Mitomycin C reduces scar formation after excimer laser (193 nm) photorefractive keratectomy in rabbits.

Sixteen eyes of eight rabbits were randomised to either mitomycin C or Balanced Salt Solution (BSS) application after photorefractive keratectomy (PRK). Regular examinations of wound healing and haze were performed with the slit lamp. The animals were killed between 1 and 26 weeks after treatment, and the corneas examined by light and electron microscopy. While the grade of haze showed no relevant differences between the two groups, scar tissue was found histologically in the mitomycin group in only 1 of 8 corneas compared with 5 of 8 in the BSS group. A marked reduction in keratocytes in all mitomycin-treated corneas and a normal density of keratocytes in the BSS group was observed. Mitomycin reduced the number of keratocytes in the treated corneas, leading to less scar formation but not to a reduction in haze. Since no morphological correlate has been found, haze remains unexplained in the mitomycin-treated corneas.

The in vitro effect of platelet-derived growth factor isoforms on the proliferation of bovine corneal stromal fibroblasts depends on cell density.

Recently, it has been shown that corneal stromal fibroblasts express the mRNA for PDGF-beta-type receptors, while corneal epithelial cells express the mRNA for the PDGF B-chain, suggesting a role of PDGF isoforms in the regulation of corneal homeostasis and wound healing via an unidirectional epithelial to stromal paracrine interaction. The purpose of this study was to characterize the proliferative response of cultured bovine corneal stromal fibroblasts to PDGF isoforms. Bovine corneal stromal fibroblasts were seeded at a cell density of 60 cells/mm2 (low density) and 120 cells/mm2 (high density) and were cultured under serum-free conditions. Except for corresponding controls, PDGF AA, BB and AB (obtained by separate expression of cloned genes in E. coli) were added in concentrations ranging from 3.125 to 100 ng/ml. Cell numbers were determined after an incubation period of 6 days using a cell counter. Stromal fibroblasts, when cultured at a high density, revealed constant cell numbers during the whole incubation period. Under these culture conditions, stimulation with PDGF AA, BB and AB led to a significant dose-dependent increase in cell proliferation. When cultured at a low cell density, stromal fibroblasts revealed a significant reduction of cell numbers after 6 days of incubation. This reduction was prevented by PDGF AA and AB isoforms in a dose-dependent manner. In contrast, PDGF BB was not effective. The results of the "high-density" assays suggest that PDGF isoforms act as mitogens for stromal fibroblasts during wound healing, when density of fibroblasts is high. The results of the "low-density" assays support the idea that PDGF AA and AB can prevent cell loss during corneal homeostasis when density of keratocytes is low.

The corneal stroma: an inhomogeneous structure.

This study was conducted to determine the elemental composition of the human cornea. Special attention was paid to corneal stroma inhomogeneity. Seventy human corneas were examined by means of energy-dispersive X-ray analysis. Epithelium, subepithelium, middle stroma, sub-Descemet layer, Descemet's membrane and endothelium were subjected to repeated measurements. In the cellular layers the phosphorus concentrations were high [0.35 mol/kg dry weight (dw) in the epithelium and 0.403 mol/kg dw in the endothelium]. Similar concentrations were found for sulphur (0.38 mol/kg dw in the epithelium). Stromal layers showed high contents of sulphur: 0.26 mol/kg dw. The phosphorus concentration was found to be higher in the subepithelium than in the middle stroma. Sulphur concentrations were highest in Descemet's membrane, followed by the subepithelium and the middle stroma. Nucleic acids and energy-containing phosphates explain the high levels of phosphorus in the cellular layers. The high sulphur concentrations may be related to the phosphoadenosinphosphosulfate and protein turnover in the epithelium. We interpret the inhomogeneous distribution of phosphorus in the stroma as a function of the density of keratocytes. An evaluation of all known sulphur-containing biochemical components of the stroma (0.217 mol sulphur/kg dw) corresponds to our measurements. In contrast to former results we find the corneal stroma to be an inhomogeneous structure.

A Comparative Study of Human Corneal Keratocyte and Endothelial Cell Density During Aging

To investigate whether the human corneal keratocyte density changes during aging. Comparative data on keratocyte and endothelial cell density (ECD) were obtained from 178 normal corneas (89 persons ranging in age from 30 weeks of gestation to 90 years). Keratocyte density was quantified by using biochemical measurements of the stromal DNA/ mass content within the central 7-mm diameter zone (sDNA) (1 U equals 1 microgram DNA per milligram of dry tissue weight), whereas central ECD was assessed after alizarin red staining. In the first decade of life, there was a mean sDNA of 1.64 +/- 0.29 U, corresponding to 6.22 +/- 1.1 x 10(4) keratocytes per mm3. A direct correlation between keratocyte density and donor age was found (r = -0.49; p < 0.0001) with a physiologic decline of 0.3% per year throughout life (density = 6.30 x 10(4) keratocytes per mm3-190 x age). A similar decrease of 0.3% per year was observed in the ECD during adulthood, whereas the annual decline was 2.9% during infancy and childhood. The interindividual variation in keratocyte density was of the same magnitude as that seen in ECD. Moreover, keratocyte density was positively correlated with ECD (r = 0.23; p < 0.001); however, after correcting for age by multiple regression analysis, this correlation disappeared, indicating that keratocytes and endothelial cells form distinct cell populations. The study demonstrates a linear loss of human corneal keratocytes as a function of age, a loss that parallels the well established decline in ECD.

Ultraviolet-B Enhances Corneal Stromal Response to 193-nm Excimer Laser Treatment

The purpose of the study was to evaluate the biomicroscopic, light microscopic, and electron microscopic effects of ultraviolet-B (UV-B) exposure on the outcome of photorefractive keratectomy (PRK). A total of 24 pigmented rabbits were used in the study. One eye of 16 rabbits received a 193-nm, 45-micron deep (-5.0 diopter) excimer laser PRK. Twenty-one days after PRK, eight of the laser-treated eyes were exposed to 100 mJ/cm2 UV-B (280-315 nm) UV radiation by placing the rabbits in a standard clinically used dermatologic chamber for 7 minutes. Eight PRK-treated rabbits received no further treatment. The remaining eight non-PRK-treated rabbits received 100 mJ/cm2 UV-B only to one eye. Subepithelial haze was assessed before and after UV irradiation. Corneal morphology was assessed 4, 8, 12, and 16 weeks after UV-B exposure, using light microscopic and transmission electron microscopic (TEM) techniques. Untreated eyes exposed to 100 mJ/cm2 UV-B only exhibited photokeratitis for 2 days, but showed no haze and were normal histologically at all intervals. The PRK-treated UV-B irradiated eyes exhibited a significant increase of stromal haze compared to eyes receiving PRK alone. Histologically, the main difference between the UV-B irradiated and nonirradiated post-PRK eyes was the presence of anterior stromal extracellular vacuolization in the UV-B-exposed eyes. The vacuolated foci were confined to the PRK treatment area and showed increased keratocyte density and disorganization of normal collagen lamellae. TEM showed activated keratocytes containing abundant rough endoplasmic reticulum, prominent Golgi zones, and extracellular vacuoles filled with amorphous material. The haze and morphologic changes showed a tendency to incomplete resolution over the period of 16 weeks. The UV-B exposure during post-PRK stromal healing exacerbates and prolongs the stromal healing response and is manifest biomicroscopically by augmentation of subepithelial haze. The findings suggest that excessive ocular UV-B exposure should be avoided during the period of post-PRK stromal repair and that UV-B may modulate the response of tissues to 193-nm excimer laser and perhaps other laser energy in general.

Changes in the human corneal keratocyte and endothelial cell density during aging and during corneal dystrophies

Changes in the human corneal keratocyte and endothelial cell density during aging and during corneal dystrophies

2316 Changes in the human corneal keratocyte and endothelial cell density during aging and during corneal dystrophies

2316 Changes in the human corneal keratocyte and endothelial cell density during aging and during corneal dystrophies

P 176 Changes in keratocyte density of the human cornea during organ culture

P 176 Changes in keratocyte density of the human cornea during organ culture

Changes in keratocyte density of the human cornea during organ culture

Changes in keratocyte density of the human cornea during organ culture

Confocal Microscopy of Corneal Graft Rejection

Corneal allografts were transplanted into inflamed and vascularized graft beds in rabbit eyes. The grafts were examined every 4 days by slit-lamp biomicroscopy and scanning confocal microscopy. Confocal images were recorded with a video camera and computer enhanced in real-time. Layers of the cornea were visualized in serial optical sections parallel to the epithelium. In the third postoperative week, signs of graft rejection were observed; slit-lamp examination revealed a circumferential line of epithelial rejection, along with cloudiness and edema. Vessels were observed growing into the graft. By confocal microscopy, infiltrating cells were seen in the graft stroma. Foci of cells were especially pronounced around the sutures. Scattered leukocyte infiltrates were prominent at capillary terminals. There was an accompanying reduction in the stromal keratocyte density in the region of the infiltrate. Additionally, various degrees of fibrosis were noted around each suture and at the host-graft interface. Confocal microscopy may provide a valuable clinical tool for determining the earliest indicators of an antigraft immune response, and as an aid in the differential diagnosis of other inflammatory conditions of the cornea.

Basic science and applications of in vivo microscopy.

Confocal microscopy creates a scanned image from a point light source and point detection or a scanning slit to remove scattered light and improve optical resolution. This also results in optical sectioning of tissues. These capabilities can be employed to image structures in the human cornea, in vivo, both for research and for the diagnosis and treatment of human disease. Optical sections, when recombined, can lead to three-dimensional reconstructions from which very useful information is obtained. Investigators have found keratocyte density decreases from anterior to posterior in the stroma of the rabbit cornea. Surface epithelial desquamation can also be studied and the effects of contact lens use can be demonstrated. The instrument is also useful for diagnosing and guiding therapy for some human diseases such as Acanthamoeba keratitis. Colonies of bacteria may also be observed and treatment evaluated in patients with infectious crystalline keratopathy. Confocal microscopy can also image the retina.

Quantitative Assessment of Anteroposterior Keratocyte Density in the Normal Rabbit Cornea

The anteroposterior keratocyte density distribution in the rabbit cornea was measured. Unsectioned tissue blocks from the central cornea of five rabbits were stained with propidium iodide and imaged using a Leica laser scanning confocal microscope. A z-series of images was acquired confocal microscope. A z-series of images was acquired in each sample, from anterior to posterior stroma in either 3- or 8-microns steps. Software was developed to allow interactive marking of the keratocyte nuclei within each section of the z-series and for calculating cell density. For convenience, cell density was expressed as the number of cells per corneal volume element (CVE), where CVE is a newly defined volume unit with x, y, and z dimensions of 250, 250, and 10 microns, respectively. The calculated keratocyte density was 20.2 +/- 1.0 cells/CVE (n = 5), which is equivalent to 32,360 +/- 1,660 cells/mm3. The greatest density was underneath the epithelium (26.3 +/- 2.5 cells/CVE), the density then decreased linearly with depth to 15.2 +/- 1.4 cells/CVE; there was a slight increase in density pre-Descemets membrane to 18.5 +/- 3.5 cells/CVE. A 30% decrease in cell density over the entire anteroposterior stromal thickness was observed. To facilitate statistical analysis, the cell density was averaged over 5% thickness intervals from anterior to posterior cornea. A significant difference in mean cell density of these intervals was found (ANOVA, n = 20, p < 0.01). To further assess the density distribution, linear regression analysis was performed. A significant correlation was found between keratocyte density and stromal depth (R = -0.94, n = 20, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

A three-dimensional study of the human corneal keratocyte density.

The population of stromal keratocytes represents a rarely examined topic. In the present paper we investigated the three-dimensional distribution of keratocytes in human donor corneas. The keratocyte density was calculated from biochemical measurements of the DNA content in samples of the corneal (and scleral) stroma obtained from well-defined regions. The DNA content of the central stroma (0-2 mm from apex) had a mean (+/- SD) of 1.25 +/- 0.30 micrograms DNA/mg dry tissue weight, corresponding to a cellularity of 4.6 +/- 1.1 x 10(4) cells/mm3 (n = 13). Towards the corneal periphery, the cellularity gradually increased to a 60-70% higher cell density at limbus. In the central stroma, an anterior-posterior cell gradient was found with a 30% lower cellularity in the subendothelial region compared to the subepithelial stroma (n = 8). In the four main quadrants of the central stroma, a uniform cell density was found while the peripheral areas showed a 10% higher cellularity in the superior quadrant. A close intraindividual correlation was observed between data obtained from 40 paired corneas ('cornea 1' versus 'cornea 2'); the stromal cellularity within 0-3.75 mm from apex (r = 0.81), the stromal cellularity within 3.75-5.5 mm (r = 0.92), the stromal cellularity within 5.5-8.0 mm (r = 0.85), and the endothelial cell density (r = 0.84). However, within a given cornea no correlation was found between the density of endothelial cells and keratocytes. These data define normal values for the regional density of keratocytes in the human cornea.

The keratocyte density of human donor corneas.

The cellularity of the human corneal stroma has not been described in the literature. In the present study we calculated the density of keratocytes in human donor corneas using a new method for biochemical measurement of the stromal DNA content (sDNA). The DNA measurements were compared to morphological counts of the number of keratocyte nuclei per area (KNPA) obtained from histological sections. A significant correlation was found between the data achieved by the two methods (r = +0.52, p < 0.001, n = 46). No significant change in either sDNA or KNPA was found during 28 days of organ culture, and no influence of donor age, sex, or post mortem time was found on either sDNA or KNPA. Both sDNA and KNPA approximated a normal distribution with a mean sDNA of 1.10 +/- 0.25 micrograms DNA/mg dry tissue weight and an average KNPA of 200 +/- 53 nuclei/mm2 (n = 35). Between paired corneas the sDNA were closely correlated (r = +0.83, p < 0.005, n = 11 pairs) with an intra-individual variation of only 0.5%. Using the sDNA data, the keratocyte density in the central region of human donor corneas was calculated to be 129,000 +/- 29.000 per mg dry tissue weight (n = 35). Thus, when corneal grafting is performed (using a 7 mm trephine) an average of 818,000 +/- 186,000 donor keratocytes are transplanted. Assuming a uniform cellularity throughout the stroma, the average number of keratocytes was calculated to be 2,430.000 +/- 551,000 per human donor cornea.(ABSTRACT TRUNCATED AT 250 WORDS)

Wound Healing After Excimer Laser Keratomileusis (Photorefractive Keratectomy) in Monkeys

Laser myopic keratomileusis (photorefractive keratectomy) was performed on 29 rhesus monkey corneas with an argon fluoride (193-nm) excimer laser and a computer-controlled, moving slit delivery system. The 4-mm-diameter central ablation zone ranged in depth from 11 microns (-2 diopters effect) to 46 microns (-8 diopters effect). Corneas were studied for the 9 months postoperatively by clinical slit-lamp microscopy, and periodically with light and transmission electron microscopy. By 6 weeks, mild to moderate subepithelial haze was apparent in 93% of the corneas, with considerable variability in density. Progressive clearing occurred so that by 6 to 9 months 12 of 13 surviving corneas (92%) were either completely clear (4 corneas) or trace hazy (8 corneas). The epithelium was thickened at 21 days after ablation and returned to normal thickness by 3 months. At 3 weeks, subepithelial fibroblasts were three times the density of normal keratocytes and returned to nearly normal numbers by 9 months. We concluded that the anterior monkey cornea demonstrated a mild, typical wound healing response after excimer laser keratomileusis.

Corneal Ablation in Rabbits Using an Infrared (2.9-μm) Erbium:YAG Laser

The authors used a 2.9-μm infrared erbium:YAG (ER:YAG) laser to ablate the central corneas (a 3.5-mm wide, 180-μm deep area) of ten albino rabbits. In group 1(10 eyes), the central epithelium was removed by mechanical scrubbing to ablate the anterior stroma. In group 2 (10 eyes), a central anterior corneal cap was removed to ablate the midstroma. In group 1, epithelial wound healing occurred in 2 to 3 days. A mild anterior haze was observed with the slit lamp, but it gradually cleared in most eyes of both groups. Histologic specimens were obtained immediately and at 2 and 6 months after ablation for light and transmission and scanning electron microscopy. This acute histopathologic study did not show any endothelial damage even after ablations at 300-μm depth, although there was thermal damage more prominent at the borders of the ablated area for an extension of 40 μm. After 2 months, in the group 1 eyes, the regenerated epithelium appeared normal except for a thickening at the margins of the ablated area. Six months postoperatively there was still epithelial thickening at the margins of the remodeled zone. The basement membrane was present with anchoring filaments and hemidesmosomes. The healing of the anterior (group 1) and midstroma (group 2) occurred with some degree of disorganization of the lamellae and an increase in the population of keratocytes. The density of keratocytes at the ablated areas at 6 months was reduced in comparison with the 2-month observation. By photokeratoscopy, the corneas were smooth and slightly flattened. The authors conclude that corneal ablation using an ER:YAG laser at 2.9 μm may meet the criteria for photorefractive keratectomy (PRK).