Keratocyte Density Research Articles

Prevention of stromal cell loss after superfitial corneal injury in rabbits

For the study of the effects of trauma on the cornea, usually a rabbit eye is used as a model. The aim of this experimental study of corneal surface injury was to evaluate stromal cell loss after epithelial scraping and to indicate the possibilities of preventing it by applying contact lenses or topical heparin. The study was conducted on 21 Chinchilla gray rabbits, which were divided into five groups. The central portion measuring 6mm of corneal epithelium was removed from one eye of 18 experimental animals. Three rabbits in the fifth group, with intact corneas, served as controls. Postoperatively, the first experimental group (n=3) was treated for 24 hours with topically applied heparin. The second group (n=3) was treated for seven days with subconjunctivally applied heparin. In the third group, (n=6) soft contact lenses were placed for five days. The fourth group of (n=6), has not been treated after injury. During the first postoperative day, anterior stromal keratocytes were lost after epithelial removal, but the cell repopulation was completed within two weeks. Twenty four hours after deepithelization the corneas showed decreased density of keratocytes within the anterior stroma compared with the posterior storma. Topically applied heparin for 24 hours after deepithelization prevented keratocyte loss (p 0.05). The eyes treated with contact lenses had a significantly higher number of keratocytes than the control corneas and corneas treated with heparin. We were able to conclude that topical application of heparin during the first day and contact lens application during the first week, can minimize keratocyte loss and may thus be beneficial in the healing process.

In vitro human corneal model to investigate stromal epithelial interactions following refractive surgery

To develop an in vitro human corneal model to evaluate stromal epithelial interactions following corneal refractive surgical procedures. Department of Academic Ophthalmology, Rayne Institute, St. Thomas' Hospital, London, United Kingdom. Fifty-six human donor corneas procured from the eye bank were placed in a specially designed acrylic corneal holder and were cultured using the air-interface organ culture technique for up to 4 weeks. Corneal refractive surgical procedures such as a simple epithelial defect, 4 diopter (D) and 9 D photorefractive keratectomy (PRK), 4 D and 9 D laser-assisted subepithelial keratectomy (LASEK), and 9 D laser in situ keratomileusis (LASIK) were performed on the model. Temporal events in epithelial and keratocyte cell kinetics were evaluated using digital imaging, confocal microscopy, and light microscopy. Two-way analysis of variance and Student t tests were used to assess statistical significance. Epithelial healing following PRK was completed by 92 hours +/- 10 (SD) at a rate of 0.58 +/- 0.45 mm2/hour. In LASEK, the epithelial flap was replaced by regenerating peripheral epithelium that showed significant delay in epithelial closure (120 +/- 5 hours) with prolonged latency (24 +/- 4 hours, P<.0001) in comparison with PRK. The magnitude of keratocyte loss corresponded to ablation depth, and keratocyte regeneration was dependent on epithelial closure. In comparison, LASIK corneas showed a lesser percentage of keratocyte loss with poor recovery of keratocyte density in the stromal flap. Epithelial viability and keratocyte density were well preserved in the in vitro human model as observed in control corneas for up to 4 weeks. The temporal events in stromal epithelial interactions in the in vitro human model closely mimicked in vivo observations. The human model further avoided species-specific variations and provided a suitable test bed for evaluating newer algorithms and therapeutic regimens following refractive surgery.

Spatial Distribution of Keratan Sulfate in the Rabbit Cornea Following Photorefractive Keratectomy

To examine the keratan sulfate content of the stroma and to assess its correlation with the healing process (epithelialization and keratocyte density) after photorefractive keratectomy (PRK). Using an Aesculap Meditec MEL 70(G-scan) excimer laser, -6.0 diopters (6.0-mm diameter, 82 microm photoablation depth), PRK was carried out on the right eye of 32 New Zealand pigmented rabbits. After enucleation (at days 1, 4, 7, 14, and 28 and months 2, 3, and 7; sub-groups of 4 animals), fluorescent immunohistochemistry was performed on sections from the central comea using monoclonal mouse anti-keratan-sulfate antibody, immunohistochemistry with proliferative cell nuclear antigen antibody, and hematoxylin-eosin histology. The left, untreated eyes served as controls. Cellular morphology and spatial distribution of keratan sulfate were recorded, stromal thickness measured, and keratocyte density calculated. Keratan sulfate was found on the surface of migrating epithelial cells in the early stage (from days 1 to 7). In the stroma, three phases were noted. (Phase 1) Day 1 to 14, intense granular fluorescence appeared in the anterior stroma with hypocellularity. (Phase 2) Month 1 to 2, newly synthesized lamellar keratan sulfate restored the repopulating anterior stroma. Endothelial cells became keratan sulfate positive, while in the posterior stroma, lamellar-form keratan sulfate increased from week 1 and peaked at month 1 (100% increase). (Phase 3) Month 2 to 7, remodeling and deposition of keratan sulfate was noted, which was produced in phase 2. Keratan sulfate was found in the epithelium, stroma, and endothelium. By controlling the interlamellar spacing, keratan sulfate plays a role in postoperative edema, remodeling of the corneal stroma, and simultaneous regulation of inflammation after PRK.

Amiodarone Keratopathy

To evaluate the characteristics and evolution of corneal morphologic changes induced by systemic amiodarone treatment by using in vivo slit scanning confocal microscopy in an attempt to understand the pathogenesis of corneal and other systemic side effects of this drug. Forty-nine eyes of 25 consecutive subjects receiving amiodarone therapy (group A) and 26 eyes of 13 age- and sex-matched healthy control subjects (group B) were enrolled in the study. In group A, the mean dosage of amiodarone was 224.0 +/- 71.3 mg, and the mean duration of treatment was 21.3 +/- 20.9 months. In eight (8 of 49,16.3) eyes of four patients, no deposition was observed, neither by slitlamp nor by confocal microscopy. The deposition could be detected as early as 2 months after the onset of treatment in 41 (41 of 49, 83.7%) eyes, by slitlamp and confocal microscopy. Deposition was significantly correlated with the duration of treatment and, therefore, the cumulative dose of the drug ingested. Deposits were observed as bright, hyper-reflective spots that were localized intracellularly and were present at the level of basal lamina of all 41 (100%) corneas in which deposition could be observed at confocal microscopy. Deposits were also observed in the superficial epithelium, anterior stroma, mid stroma, and subepithelial nerves in eyes with grade 2 to 4 keratopathy. Additionally, morphologic abnormalities were observed in anterior stromal keratocytes, subepithelial and stromal nerves, and endothelium. The mean anterior and posterior keratocyte densities were statistically significantly higher than those in group B. In addition to showing drug deposition does not seem to occur before 2 months of treatment and does not seem to be correlated with tear function. Although the clinical significance of morphologic changes induced by the drug is known and although deposition could not be detected before it was evident with slitlamp biomicroscopy, with some improvement in instrumentation, the authors believe that confocal microscopy will also prove to be useful in early diagnosis and in understanding the pathophysiology of amiodarone keratopathy. This may help grow insight to the mechanism of other, sometimes lethal, systemic side effects of this drug.

In vivo corneal confocal microscopy in keratoconus

To evaluate the corneas of keratoconic subjects using in vivo confocal microscopy. Slit scanning confocal microscopy was used to evaluate the central cornea of one eye of each of 29 keratoconic subjects (mean age 31 +/- 10 years; range 16-49 years). Quantitative aspects of corneal morphology were compared against data from control subjects. Compared with normal control corneas, epithelial wing cell nuclei were larger (p < 0.0001) and epithelial basal cell diameter was larger (p < 0.05) in the keratoconic cornea. Many of the keratoconic corneas investigated showed increased levels of stromal haze and reflectivity, which appeared to be related to the presence of apical scarring on slit lamp examination. A grading scale was devised to quantify the levels of haze. This scale was shown to provide a measure of the level of scarring present. The anterior keratocyte density (AKD) and posterior keratocyte density were 19% lower (p < 0.0001) and 10% lower (p = 0.004) than in controls, respectively. The reduction in AKD was significantly associated with three factors: a history of atopy, eye rubbing and the presence of corneal staining. The mean endothelial cell density in keratoconus was 6% greater than that of normal controls (p = 0.05). The level of endothelial polymegethism was shown not to be different between keratoconic subjects and matched controls (paired t-test: t = 1.82, p = 0.08). Confocal microscopy demonstrates significant quantitative alterations of corneal morphology in keratoconus.

Observations of Banding Patterns (Vogt Striae) in Keratoconus

To investigate Vogt striae in keratoconus using confocal microscopy. The central cornea of 51 eyes of 29 subjects with keratoconus was observed using a slit-lamp biomicroscope, slit-scanning confocal microscope (TOMEY Confoscan 1), and corneal topographer (EyeSys 2000). Alternating dark and light bands were seen in the stromal images of 23 eyes examined. The bands corresponded with the appearance of Vogt striae on slit-lamp biomicroscopy examination. Bands were found most commonly in the posterior stroma. Posterior bands varied in width, ran mainly in a nearly vertical direction, and appeared to run a straight course through individual image frames. Keratocyte nuclei were located in between the bands. Posterior keratocyte density was unaffected by the presence of bands. Nerve fibers appeared to run a straight course through the bands. When present, bands in the anterior stroma showed greater variability in width and direction within a single frame. Bands were only present in the anterior stroma in more severe levels of keratoconus. The difference in banding pattern noted between the anterior and posterior stroma parallels the known collagen fiber arrangement in the anterior and posterior stroma. The bands apparent on confocal microscopy of the stroma of the keratoconic cornea correspond with Vogt striae on slit-lamp microscopy. It appears that these bands (and hence Vogt striae) represent collagen lamellae under stress. The stress pattern appears to radiate from the center of the cone and is consistent with the direction of striae when viewed with the confocal microscope.

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

目的 观察Confoscan 2.0共焦显微镜下正常活体角膜影像表现,测量基质细胞密度与各层厚度.方法检查34例(48眼)正常人.记录图像,并计算基质细胞密度和各层厚度.结果基质细胞密度从前到后逐渐降低,前基质比后基质细胞密度明显增高(t=-9.016,P=0.000),Bowman膜下密度最高,为(1113.2±227)个/mm2.全基质细胞密度为(806.5±57)个/mm2.角膜中央厚度为(568.3±53.8)μm,基质层为(465.5±60.2)μm,上皮层为(58.5±20.4)μm.各层厚度均与全基质细胞密度无显著相关性(P＞0.05).结论Confoscan 2.0共焦显微镜能检测角膜基质细胞密度和各层厚度。

Effect of exogenous keratinocyte growth factor on corneal epithelial migration after photorefractive keratectomy

Purpose: To investigate the effect of topical keratinocyte growth factor (KGF) on wound healing after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK). Setting: Department of Ophthalmology, Rayne Institute, St. Thomas' Hospital, London, United Kingdom, St. Erick's Eye Hospital, Stockholm, Sweden, and the University of Regensberg, Regensberg, Germany. Methods: In a placebo-controlled trial, 24 New Zealand white female rabbits were divided into 3 equal groups. Group 1 (n = 8) had myopic PRK (6.0 diopters [D]) using the Technolas® 217z laser (Bausch & Lomb). Group 2 and Group 3 had myopic LASIK (6.0 D) with a flap depth of 140 μm and 180 μm, respectively. Topical KGF (20 μg/mL) was administered to half the treated eyes in each group intraoperatively and postoperatively; the other half received placebo eyedrops. Epithelial closure, corneal haze, and keratocyte activation in the rabbit eyes were analyzed and compared with those in placebo-controlled eyes for 5 weeks postoperatively. Results: In Group 1, the mean reepithelialization after PRK was 0.10 mm 2/h ± 0.02 (SD) in the KGF group and 0.33 ± 0.05 mm 2/h in the control group ( P = .001). There was no significant difference in the mean backscatter between the KGF eyes (154 ± 45.95) and the control eyes (141 ± 38.45) after PRK ( P = .42). Histology revealed reduced epithelial cell layers in the KGF group and comparable keratocyte density as in the control group. In Groups 2 and 3, there was no significant difference in backscatter, epithelial layers, and keratocyte density between KGF and control eyes after LASIK. Conclusions: Topical KGF (20 μg/mL) delayed reepithelialization after PRK. It had no effect on stromal wound healing in LASIK eyes with an intact epithelial barrier.

Long-term keratocyte deficits in the corneal stroma after LASIK

Purpose To determine changes in keratocyte density up to 3 years after LASIK. Design Prospective, nonrandomized, comparative trial. Participants Seventeen eyes of 11 patients received LASIK with a planned 180-μm flap to correct refractive errors between −2.0 diopters (D) and –11.0 D (mean, −6.56±2.44). Methods Corneas were examined by using confocal microscopy before LASIK and 1, 3, 6, 12, 24, and 36 months after LASIK. Bright objects that resembled keratocyte nuclei were manually counted by a masked observer. Cell densities were determined in anterior and posterior halves of the stromal flap, anterior and posterior halves of the 100-μm-thick layer immediately behind the ablation (retroablation layer), and the posterior third of the stroma. The region of stroma that was ablated (as measured 1 month after LASIK) was omitted from preoperative analysis. Cell densities after LASIK were compared (using1-factor repeated-measures analysis of variance) with densities in the corresponding layer of the normal preoperative stroma (which served as its own control). Main outcome measure Corneal keratocyte density. Results Before LASIK, keratocyte densities in the anterior and posterior stromal flap and the anterior retroablation layer were 34 818±5108 cells/mm 3 (mean ± SD), 25 390±4045 cells/mm 3, and 21 328±2980 cells/mm 3, respectively, and densities in these layers decreased 14% to 20% at 1 month after LASIK ( P<0.001). Keratocyte densities in these layers remained stable at 3 and 6 months, and then gradually decreased further ( P<0.001) to 26% to 36% below pre-LASIK densities by 3 years. Keratocyte densities in the remaining stromal layers did not change after LASIK. Conclusion Keratocyte densities in the stromal flap and in the anterior retroablation layer decrease during the first 6 months after LASIK and then decrease further during the next 2.5 years. Further studies on these patients and others are warranted to confirm these findings and learn their significance.

Morphologic characteristics of cornea in Fuchs endothelial dystrophy observed by confocal microscopy

To analyze the image of corneas with Fuchs endothelial dystrophy using confocal microscopy in vivo. Central corneas of 38 eyes (19 patients aged 33 approximately 76 years) were examined using scanning slit confocal microscopy in vivo after being diagnosed with Fuchs endothelial dystrophy. They were divided into two groups namely symptomatic (19 eyes) and asymptomatic (19 eyes). The control group comprised of 30 normal eyes. The images were analyzed visually for morphology of corneas and the densities of cells, as well as the diameter of the guttae and the corneal nerves were counted by NAVIS software. (1) In the symptomatic groups, confocal microscopy revealed guttae in 19 eyes. The diameter of the guttae ranged from 20 to 60 micro m. The density of endothelial cells was statistically significant compared with the control group (t = 18.74, P < 0.01). Descemet's membrane was thickened in 9 eyes. Dark bands 6.0 approximately 10.8 micro m in diameter were detected in 14 eyes. All eyes showed a diffuse increased light reflection in the stroma.17 eyes exhibited an abnormal Bowman's layer: multiple focal bright reflection. 10 eyes revealed normal corneal nerves. The differences between posterior and anterior keratocyte densities in the control and study groups were not statistically significantly different (t = 0.854, 1.173; P = 0.38, 0.24 respectively). (2) In the asymptomatic groups, less number of guttae could be seen in the endothelial layer. The diameter of the guttae was 15 approximately 40 micro m. The densities of endothelial cells were not significantly different compared with control group (t = 1.998, P = 0.053). Other layers of the cornea exhibited no other abnormal phenomenon. The densities of endothelial cells between symptomatic and asymptomatic groups were statistically significantly different (t = 8.352, P < 0.01). Morphologic characteristics of cornea are observed by confocal microscopy in Chinese patients with Fuchs endothelial dystrophy. Preliminary results demonstrate that there is no significant difference between Chinese and western patients. Confocal microscopy allows to diagnose Fuchs dystrophy and visualize endothelial cells within the swollen cornea. And it is especially useful for patients whose endothelial cells can not be seen with non-contact specular microscopy.

Confocal microscopy of the corneas of long-term rigid contact lens wearers

<h2>Abstract</h2> Slit scanning confocal microscopy (Tomey Confoscan P4) was used to evaluate the central cornea of 22 subjects who had been wearing rigid lenses on a long-term, daily wear basis. Anterior and posterior keratocyte densities appeared unaffected by rigid lens wear (<i>P</i>=0.10 and 0.34 for anterior and posterior keratocyte densities, respectively). Subjects with a previous history of polymethyl methacrylate (PMMA) lens wear showed a reduction in anterior keratocyte density (AKD) (<i>P</i><0.0001) and an increased level of haze in the anterior stroma. This may represent previous hypoxic damage. Endothelial cell density (ECD) was unaffected by rigid lens wear (<i>P</i>=0.36) although an increase in endothelial polymegethism was evident (<i>P</i><0.0001). Subjects who had only worn rigid lenses with no history of PMMA lens wear did not show an increase in endothelial polymegethism (<i>P</i>=0.10). An increased number of microdot opacities compared to the non-lens wearing eye was apparent (<i>P</i>=0.05). The number of microdot opacities induced by rigid lenses, as reported in this study, appears to be less than that reported by others in respect of soft lenses.

Sutureless lamellar keratoplasty by microkeratome combined with fibrin tissue adhesive in rabbits

To evaluate the feasibility and safety of sutureless lamellar keratoplasty by microkeratome combined with fibrin tissue adhesive. Twenty-four New Zealand white rabbits were divided into two groups, the donor grafts and recipient beds were made by the microkeratome, the grafts were glued over the stoma bed using the commercial product Tisseel in one group; and grafts without tissue adhesive were used as the control group. Corneal refractive power was measured by automated keratometer preoperatively and in 3 days, 2 weeks, 1 and 3 months postoperatively. Rejection and cornea transparency were observed. Confocal microscopy was used to observe corneal wound healing response and to measure the keratocyte and endothelium densities in vivo. Corneal wound healing was also evaluated using light and fluorescence microscopy. Ninety-two percent (11/12 eyes) of the glued grafts were retained in the Tisseel group, whereas all grafts were lost in the control group. All survived grafts were clear 1 month after surgery. However, in the control group, severe haze in the grafts occurred 2 weeks postoperatively. Confocal microscopy showed that there was a significant decrease of the keratocyte density surrounding the lenticule-host interface, and no changes occurred in the posterior keratocyte and endothelium. Histopathologic observations demonstrated the presence of a line of amorphous eosinophilic substance in the lenticule-host interface at 3 days after surgery, but the line disappeared after 1 month. Fluorescence microscopy showed no detectable regenerated stromal tissue. This initial study demonstrates sutureless optical lamellar keratoplasty performed by microkeratome combined with fibrin tissue adhesive is a simple and safe technique. Stromal wound healing response to this surgery is minimal. Fibrin tissue adhesive has no influence on the cornea optical property.

Confocal microscopy of the corneas of long-term rigid contact lens wearers

Slit scanning confocal microscopy (Tomey Confoscan P4) was used to evaluate the central cornea of 22 subjects who had been wearing rigid lenses on a long-term, daily wear basis. Anterior and posterior keratocyte densities appeared unaffected by rigid lens wear ( P=0.10 and 0.34 for anterior and posterior keratocyte densities, respectively). Subjects with a previous history of polymethyl methacrylate (PMMA) lens wear showed a reduction in anterior keratocyte density (AKD) ( P<0.0001) and an increased level of haze in the anterior stroma. This may represent previous hypoxic damage. Endothelial cell density (ECD) was unaffected by rigid lens wear ( P=0.36) although an increase in endothelial polymegethism was evident ( P<0.0001). Subjects who had only worn rigid lenses with no history of PMMA lens wear did not show an increase in endothelial polymegethism ( P=0.10). An increased number of microdot opacities compared to the non-lens wearing eye was apparent ( P=0.05). The number of microdot opacities induced by rigid lenses, as reported in this study, appears to be less than that reported by others in respect of soft lenses.

Effects of benoxinate hydrochloride 0.4% on the morphological appearance of the cornea using confocal microscopy

Purpose: To investigate whether benoxinate hydrochloride 0.4% used to make confocal microscopy more comfortable alters the morphology of the cornea as viewed with the confocal microscope. Methods: Confocal microscopy was performed on both eyes of 10 subjects prior to instillation of either topical anaesthetic or non-preserved sterile saline, on two randomly ordered occasions. Images of all corneal layers were analysed quantitatively and qualitatively in a masked fashion. Results: The images were similar in appearance in 5/10 subjects, there was greater clarity when anaesthetic was instilled in 4/10 subjects, and in the remaining subject there was greater clarity when saline was used. Anaesthetic had no influence on anterior keratocyte density (AKD), posterior keratocyte density (PKD) or endothelial cell density (ECD). Conclusions: Local anaesthetic does not affect corneal morphology as imaged using the confocal microscope. However, failure to use anaesthetic may lead to a degradation of image quality due to patient discomfort and excessive eye movements.

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

In a case control study we determined keratocyte density and size of nuclear area in the central segment of the corneal stroma. 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). 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. 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.

Change to corneal morphology after refractive surgery (myopic laser in situ keratomileusis) as viewed with a confocal microscope.

This study aimed to look at morphological changes induced by myopic laser in situ keratomileusis (LASIK) in the human cornea using the confocal microscope and to investigate the link between these changes and alterations to corneal sensitivity. An in vivo slit-scanning real-time confocal microscope (Tomey ConfoScan P4, Erlangen, Germany) fitted with an Achroplan 40x/0.75 NA immersion objective and a Cochet-Bonnet esthesiometer were used to examine the morphology and sensitivity of the central corneas of six subjects (12 eyes) at an initial visit (before surgery), and at 1 week, 1 month, 3 months, and 6 months after LASIK for myopia. Keratocyte density anterior to the flap interface showed differences between visits (p < 0.0001) and was found to be lower than at the initial visit at 1 week, 1 month, 3 months, and 6 months. Microfolds were noted at the level of the anterior limiting membrane in 11 of 12 eyes after surgery at all visits. Highly reflective flap interface particles were seen in all eyes at all visits after surgery. The subepithelial nerve fiber layer was clearly visible before surgery but could not be imaged in any of the eyes after surgery. Short, unconnected nerve fibers were observed 3 months after surgery; these appeared to form anastomosing interconnections after 6 months. Postsurgical corneal sensitivity was reduced during the first 3 months and recovered to presurgical levels after 6 months. LASIK showed a decrease in anterior keratocyte density and microfolds in the anterior limiting membrane, and reflective particles were observed at the flap interface. Corneal sensitivity was depressed during the first 6 months after LASIK surgery; this time course paralleled the appearance of nerve regeneration during this period. Confocal microscopy is capable of providing interesting new insights into the effects of refractive surgery on corneal morphology.

Histologic evaluation of corneal stroma in rabbits after intrastromal corneal ring implantation.

Previous refractive corneal implants have produced histologic changes characteristic of nonspecific lipid keratopathy. Intacs intrastromal corneal ring segments are polymethylmethacrylate (PMMA) corneal inserts approved for the correction of low myopia by the U.S. Food and Drug Administration. The purpose of this study was to describe histologic changes associated with these corneal implants in rabbit eyes. Two 150-degree PMMA arc segments, 0.30 mm thick, were surgically implanted through a single radial incision at two-thirds stromal depth in the mid-peripheral cornea of five New Zealand white rabbits. We collected corneas 6 months after implantation that were prepared for light and electron microscopy. We analyzed tissue with oil red O, filipin, and periodic acid-Schiff (PAS) staining. Additional studies with energy-dispersive x-ray analysis and autofluorescent microscopy were conducted. In all samples, we observed new collagen formation with lamellar organization adjacent to the implant and increased keratocyte density. Intracellular accumulations of osmophilic and saturated lipid material corresponded to stromal opacification visible by slit-lamp microscopy. These same regions were autofluorescent and stained positively with oil red O, and filipin but negatively with PAS. Granular lipid inclusions characteristic of lipofuscinosis were absent on transmission electron microscopy. Energy-dispersive x-ray analysis demonstrated elevated levels of calcium compared with adjacent tissue. Tissue response to these corneal inserts includes keratocyte activation, intracellular lipid accumulation, and new collagen formation. Our histologic findings suggest that these deposit formations are not lipofuscin accumulations.

Neonatal corneal stromal development in the normal and lumican-deficient mouse.

The purpose of this study was to characterize temporally stromal growth and transparency in lumican-deficient and normal neonatal mice. Lumican-deficient mice and CD1 wild-type mice were evaluated by in vivo confocal microscopy through-focusing (CMTF) to quantify stromal and epithelial thickness and corneal light-scattering and by laser scanning CM to determine density of keratocytes from 1 day to 12 weeks after birth. CD1 corneas showed a rapid loss of light-scattering, decreasing by 50% from day 1 to day 12, that paralleled a 60% decrease in density of keratocytes. By contrast, the stroma demonstrated a marked swelling from day 8 to day 12, followed by thinning at day 14. Compared to corneas from CD1 mice, lumican-deficient corneas showed significantly increased (P < 0.05) light-scattering beginning at week 3 that remained elevated above wild-type levels for the duration of the study. Stromal development was also markedly altered, with thinning detected at week 3, followed by no detectable stromal growth for the duration of the study. Density of keratocytes was significantly increased, but the total cell number was similar compared with that in the wild-type cornea, suggesting no effect on keratocyte differentiation. Development of normal neonatal corneal transparency appears related to changes in density of keratocytes. The stroma, however, undergoes a marked swelling and thinning at the time of eyelid opening (days 8-14). In the lumican-deficient mouse, stromal swelling is abolished, indicating that this critical phase in stromal development is lumican dependent and essential for normal stromal growth and maintenance of stromal transparency.

Confocal Microscopic Features of Amiodarone Keratopathy

In order to evaluate the clinical and confocal microscopic features of amiodarone keratopathy, we examined 12 eyes of six patients receiving amiodarone and 12 eyes of six healthy control subjects. The duration of amiodarone therapy ranged from 6 to 24 months (16 ± 5.9 months). According to slit‐lamp findings, seven eyes were graded as grade 1 and five as having grade 2 amiodarone keratopathy. In the basal cell layer there were highly reflective, bright, intracellular inclusions in the epithelial layer of all patients on amiodarone. In advanced cases, there were bright intracellular inclusions even in the endothelial cell layer, as well as the anterior and posterior stroma. The keratocyte density of the anterior stroma was reduced in cases with amiodarone keratopathy compared to the control group, and irregularity in stromal nerve fibers was significant in advanced cases (p < 0.001). Ultrasonic pachimetry showed a corneal thickness of 538 ± 17.9 µm in the amiodarone group and 519 ± 16.7 µm in the controls (ns). We suggest that in amiodarone‐induced keratopathy, confocal microscopy is a useful, noninvasive technique for detecting deposits in the epithelial basal membrane early on, and which later appear in deeper layers.

Association of Type XII Collagen with Regions of Increased Stability and Keratocyte Density in the Cornea

The anterior avian cornea possesses several distinct cellular and extracellular regions including the epithelial basal lamina, Bowman's layer and the interfacial matrix that separates Bowman's layer from the stroma. These unique regions differ biochemically, physically and morphologically but all contain type XII collagen. Previously, the collagen fibrils of several of these interfacial regions were shown to be stable to thermal and enzymatic denaturation. We reasoned that type XII collagen, a fibril-associated collagen, would be a good candidate to confer such stabilizing properties. The studies described herein were performed to localize type XII collagen and to assess its role in the interfacial matrices (IM). Using antibodies that react with both the short and long type XII collagen isoforms and that react specifically with the long isoform, we demonstrate that it is the short isoform that is present in Bowman's layer and the associated interfacial matrix lying between Bowman's and the stroma proper. In situ hybridization analyses demonstrate that both the epithelial and endothelial cells synthesize type XII collagen. In vitro cell culture analyses, however, demonstrate that in addition to epithelial cell synthesis, the stromal fibroblasts are capable of synthesizing type XII collagen as well. Immunofluorescence analyses performed at elevated temperature demonstrate that type XII collagen is thermally stable in Bowman's layer, but not in the anterior interfacial matrix or Descemet's layer. In addition, we observed that the distribution of type XII collagen during the development of the anterior extracellular matrices correlates precisely with an elevated density of keratocytes populating the interfacial matrix just deep to Bowman's layer. We show that this cellular density is developmentally regulated and does not arise from a localized increase in cell proliferation. These data demonstrate that Bowman's layer and the anterior interfacial matrix have unique biochemical and morphologic properties. Type XII collagen is thermally stable in Bowman's layer and, as a surface component of type I collagen fibrils, may contribute to the stability of the fibrils in this region. Neither type XII nor type I collagen is stable in the adjacent interfacial matrix, suggesting that differences in the type I–XII collagen fibril organization may exist between Bowman's layer and IM.