Keratocyte Density Research Articles

Chloroquine Keratopathy of Rheumatoid Arthritis Patients Detected by In Vivo Confocal Microscopy

Aim: To investigate microscopic changes in corneal morphology in chloroquine (CQ)-treated patients without corneal abnormalities being diagnosable by slit lamp microscopy.Methods: Thirty-six eyes of 36 patients with rheumatoid arthritis (RA) were divided into two groups: group 1 included 26 patients receiving chloroquine therapy; group 2 included the other 10 patients, not receiving chloroquine therapy. The control group included 15 healthy subjects. Best spectacle-corrected visual acuity (BSCVA), intraocular pressure (IOP) and slit lamp microscopy and in vivo confocal microscopy examinations were performed on all subjects.Results: In group 1, hyperreflective abnormal particles were found in different layers of the cornea in 19 (75%) of 26 eyes by confocal microscopy. The deposits were present within the superficial epithelium (14/19), basal epithelium (8/19) and anterior stroma (5/19). In both RA groups, more beaded and tortuous fibers were seen in the sub-basal corneal nerve bundles, and the sub-epithelial and stromal nerves had much more branches, than did normal controls. The anterior keratocyte density in group 1 was lower than in group 2 (P < 0.05) and the number of sub-basal nerves was larger than in group 2 (P < 0.05). Cumulative dosage had a statistically significant correlation with anterior keratocyte density, the number of sub-basal nerves and the density of abnormal particles.Conclusion: Corneal microdeposits are an important manifestation of chloroquine-induced keratopathy. Corneal stroma cell density and corneal nerve number and morphology changes are also useful for detecting early chloroquine-induced keratopathy. This would be proved in our future longitudinal studies.

Mitomycin C: Biological Effects and Use in Refractive Surgery

To provide an overview of the safety and efficacy of mitomycin C (MMC) as adjuvant therapy after refractive surgery procedures. Literature review. Over the past 10 years, MMC has been used by refractive surgeons to prophylactically decrease haze after surface ablation procedures and therapeutically in the treatment of preexisting haze. Development of MMC treatments has had a significant role in the revival of surface ablation techniques. We reviewed the literature regarding mechanism of action of MMC, its role in modulating wound healing after refractive surgery, and its safety and efficacy as adjuvant therapy applied after primary photorefractive keratectomy surgery or after photorefractive keratectomy re-treatment after laser in situ keratomileusis and other corneal surgeries and disorders. The drug is a potent mitotic inhibitor that effectively blocks keratocyte activation, proliferation, and myofibroblast differentiation. Many studies have suggested that MMC is safe and effective in doses used by anterior surface surgeons, although there continue to be concerns regarding long-term safety. After initial depletion of anterior keratocytes, keratocyte density seems to return to normal 6 to 12 months after the use of MMC when corneas are examined with the confocal microscope. Most clinical studies found no difference between preoperative and postoperative corneal endothelial cell densities when MMC 0.02% was applied during refractive surgery, with exposure time of 2 minutes or less. After more than 10 years of use, MMC has been found to be effective when used for prevention and treatment of corneal haze. Questions remain regarding optimal treatment parameters and long-term safety.

Confocal Microscopic Observations of Stromal Keratocytes in Soft and Rigid Contact Lens Wearers

To determine the density of corneal stromal cells in wearers of soft contact lenses (SCLs) and rigid gas-permeable (RGP) contact lenses (CLs). The keratocyte density (KD) was measured at different depths of the stroma by confocal microscopy. In study 1, 32 wearers of rigid gas-permeable (RGP) lenses and 30 wearers of SCLs were studied. Forty volunteers with no history of CL wear were studied as controls. In study 2, 16 volunteers with no history of CL wear were divided into 2 groups; 7 subjects wore RGP lenses (oxygen transmissibility, Dk/L, 35) and 9 subjects wore SCLs (Dk/L, 34). All subjects were asked to wear the CLs daily for 6 months. In study 1, the KDs in the anterior stroma (AST) and the posterior stroma (PST) of the cornea were significantly lower in the RGP lens group than in the control group. The KD in the SCL group was significantly lower at all depths of the cornea than that of the control group. In study 2, the KD in the AST of the RGP lens group was significantly lower after 1 month of CL wear. The KD in the AST and PST of the SCL group was decreased significantly at 1 month, and all layers were decreased by 10% to 20% 6 months after wearing CLs. At 5 weeks after discontinuation of SCL wear, the KD in all layers was not significantly different from that at the baseline. The change in the KD was greater in CL wearers than in volunteers with no history of CL wear and also greater in SCL wearers than in RGP lens wearers. Analysis of the KD by confocal microscopy may be a useful method for evaluating the effect of CL wear.

Effects of Contact Lenses on the Ocular Surface in Patients With Keratoconus: Piggyback Versus ClearKone Hybrid Lenses

Because patients with keratoconus tend to wear contact lens for a long period of time, they are more prone to ocular surface changes induced by the lenses. This study aimed to compare immunohistochemical changes induced by two different types of contact lenses in patients with keratoconus. Twenty-four contact lens-naive keratoconus patients (30 eyes) were included in this prospective study. Group 1 comprised 14 eyes (12 patients) wearing piggyback lenses, and group 2 comprised 16 eyes (12 patients) wearing ClearKone hybrid lenses. The patients were analyzed for bulbar conjunctival impression cytology, tear interleukin-6 (IL-6) and IL-8 levels, and confocal microscopic changes of the cornea before and 6 months after wearing contact lenses. Six months after wearing contact lenses, the groups demonstrated similar epithelial metaplasia rates, tear IL-6 and IL-8 levels, and similar confocal microscopy findings (P>0.05 for all intergroup comparisons). Among the parameters tested in this study, only IL-6 and IL-8 levels and posterior keratocyte density on confocal microscopy showed an increase after 6 months when compared with baseline values but at a similar degree in the two groups. This small sample was not able to demonstrate a difference between the two types of lenses with regard to the variables examined, and further larger trials would be required to determine if differences truly exist or not. However, clinicians may still consider patient comfort and vision in selecting the lens type in patients with keratoconus.

Corneal Confocal Microscopy in Patients with Vernal Keratoconjunctivitis

To compare corneal morphologic features using in vivo confocal microscopy in vernal keratoconjunctivitis (VKC) patients compared with normal subjects. Prospective, comparative study. Thirty-two VKC patients (26 males, 6 females; mean age, 17.1 years) and 40 normal subjects (20 males, 20 females; mean age, 19.3 years) were included. All subjects underwent a full ophthalmologic examination. Confoscan CS4 (Nidek, Gamagori, Japan) images of the central cornea were obtained with a ×40 noncontact lens and Z-ring device. The superficial and basal epithelium, subbasal nerve plexus, anterior stroma, stromal nerves, and endothelium of the central cornea were studied. The VKC patients had increased diameter, reflectivity, and presence of nuclear activation of superficial epithelial cells; reduced density of the basal membrane; lower density of keratocytes, increased presence of activated keratocytes, and inflammatory cells in the anterior stroma; and lower density and number of fibers, lower number of beadings, and higher grade of tortuosity of fibers in the subbasal nerve plexus. Increased alterations in thickness, deflections, and tortuosity were observed in stromal corneal nerves. An increased number of inflammatory cells in close proximity to the subbasal and stromal nerve fibers also was observed in VKC subjects. Corneal involvement in VKC is associated with alterations of the epithelium and subbasal and stromal corneal nerves. These changes may relate to the tear dysfunction and nonspecific hyperreactivity typical of these patients. Corneal confocal microscopy is a useful tool for studying in vivo pathologic corneal changes in VKC.

In vivo confocal microscopy: corneal changes of hydrogel contact lens wearers

To evaluate the corneal findings in hydrogel contact lens wearers by in vivo confocal scanning microscopy. One hundred and forty-two eyes of 71 myopic contact lens wearers (group 1) and 142 eyes of 71 non-contact lens wearers (group 2), whose age, gender and refractive error matched, were enrolled in order to detect the corneal changes by in vivo confocal microscopy through the central cornea. The average age was 25.5±5.7 (16-52) and 25.6±5.6 (17-49) in groups 1 and 2, respectively. The mean duration of contact lens wear was 43.9±15.3 (6-240) months. Anterior keratocyte density was 667.5±128.3 cells/mm(2) in group 1 and 821.4±136.7 cells/mm(2) in group 2 (P=0.001). Posterior keratocyte densities of groups 1 and 2 were 540.2±87.6 cells/mm(2) and 628.2±72.4 cells/mm(2), respectively (P<0.001). Endothelial cellular density was 2611.2±298.4 cells/mm(2) in group 1 and 2643±218.2 cells/mm(2) in group 2 (P=0.52). Ratio of polymegethism was 44.6±8.8% in group 1 and 31.3±4.7% in group 2 (P<0.001). Epithelial cellular enlargement was observed in eyes wearing contact lenses with a mean Dk/t ratio of 26.5×10(-9)±5.9 (8.9-32×10(-9)). Stromal microdots occurred with contact lenses with a mean Dk/t ratio of 13.2×10(-9)±17.5×10(-9) (8.9-20×10(-9)). In vivo examination of the cornea with confocal microscopy revealed a number of changes. These changes can be attributed both to the mechanical and the hypoxic effects of soft contact lenses. In soft contact lenses with a high Dk/t ratio, these changes would be less frequent.

Mechanical Versus Alcohol-assisted Epithelial Debridement During Photorefractive Keratectomy: A Confocal Microscopic Clinical Trial

To compare the effects of mechanical versus alcohol-assisted epithelial debridement on corneal cellular elements after photorefractive keratectomy (PRK) using confocal microscopy. This randomized, clinical trial included 66 eyes from 33 patients with spherical equivalent refraction <-4.00 diopters (D). Mechanical versus alcohol-assisted epithelial debridement was performed during PRK. The right eye of each patient was randomly assigned to one group (mechanical group or alcohol-assisted group) and the fellow eye to the alternate group. Confocal examination was performed preoperatively and at 3 and 6 months postoperatively. Main outcome measures were keratocyte density and maximum anterior stromal light reflectivity. Mean epithelial healing time was 3.2 ± 0.4 and 3.0 ± 0.3 days in the mechanical and alcohol-assisted groups, respectively (P=.001). Anterior retroablation stromal keratocyte density was 704.3 ± 119.9 cells/mm² and 734.3 ± 103.7 cells/mm² at 3 months (P=.05) and 643.8 ± 134.4 cells/mm² and 696.7 ± 129.6 cells/mm² at 6 months (P=.02) in the mechanical and alcohol-assisted groups, respectively. No significant difference was noted in midstromal and posterior keratocyte density between the two groups. Maximum reflectivity was 61.56 ± 12.64 international units (IU) and 56.93 ± 7.86 IU in the mechanical and alcohol-assisted groups, respectively, 3 months after surgery (P=.018). Corresponding values were 49.46 ± 4.97 IU and 48.98 ± 4.60 IU, respectively, 6 months after surgery (P=.628). Due to more adverse effects of mechanical epithelial debridement on anterior keratocyte density and anterior stromal reflectivity, alcohol-assisted epithelial debridement is recommended as the procedure of choice for epithelial removal during PRK in patients with mild myopia.

Assessment of Corneal Thickness and Keratocyte Density in a Rabbit Model of Laser In Situ Keratomileusis Using Scanning Laser Confocal Microscopy

To determine the repeatability of corneal thickness and keratocyte density using in vivo confocal scanning laser microscopy in a rabbit model of laser in situ keratomileusis. Prospective, experimental animal study. En face tomographic images of corneal tissue were captured from 5 New Zealand white rabbits. Central corneal thickness was compared with conventional ultrasonic pachymetry. Keratocyte density was measured as a function of stromal depth at baseline and 6 weeks after a 130-μm lamellar incision in the following regions: first countable stromal image (30 to 39 μm), anterior stroma (40 to 75 μm), incision zone (76 to 150 μm), mid stroma (151 to 250 μm), and deep stroma (251 to 400 μm). The mean residual difference between ultrasonic and confocal corneal thickness measurements was 2.1 μm (95% confidence interval [CI], -7.0 to 11.2 μm; P = .61). Before the lamellar incision, keratocyte density was highest in the first countable frame of the anterior stroma, 53 800 cells/mm(3) (95% CI, 35 000 to 72 000 cells/mm(3)) and was least in deep stroma, 27 100 cells/mm(3) (95% CI, 22 400 to 32 000 cells/mm(3)). Six weeks after stromal lamellar incision, keratocyte density was unchanged in the first countable frame of the anterior stroma, 43 700 cells/mm(3) (95% CI, 31 800 to 55 500 cells/mm(3); P = .29). There were no changes in cell density in deeper stromal regions. There was excellent agreement between ultrasonic and confocal microscopy measurements of corneal thickness. In vivo repeatability of keratocyte density estimation using scanning laser confocal microscopy is comparable with the results of previous reports using tandem-scanning confocal microscopy. Keratocyte density was more varied, but not significantly different, in the anterior-most corneal stroma 6 weeks after a lamellar incision.

Keratocyte Density 3 Months, 15 Months, and 3 Years After Corneal Surface Ablation With Mitomycin C

To study the effects of surface ablation with mitomycin C (MMC) on keratocyte population. Prospective, nonrandomized, interventional, comparative case series. Thirty two eyes treated with surface ablation with 0.02% MMC were compared with nontreated eyes at Vissum Santa Hortensia, Madrid, Spain. Keratocyte density was measured with the Heidelberg Retina Tomograph II (Rostock Cornea Module) 3, 15, and 36 to 42 months after the surgery in the anterior, mid, and posterior stroma, and compared with control eyes. Three months postoperatively, we found a lower stromal bed density compared to controls (16 993 ± 8001 vs 29 660 ± 5904 cells/mm(3), P = .0001), while there was a significantly higher cell density in the mid (30 783 ± 9300 vs 18 505 ± 1996 cells/mm(3), P = .0001) and deep stroma (30 268 ± 8321 vs 18 438 ± 2139 cells/mm(3), P = .0001). Three years after the surgery, the cellularity in the stromal bed had not significantly changed from the 3-month follow-up, but the density in the mid (18 889 ± 3474 cells/mm(3)) and posterior stroma (18 992 ± 3402 cells/mm(3)) had decreased to show no difference from controls. The mean cell density between the anterior, mid, and posterior stroma was not significantly different from controls 15 months and 3 years after the surgery. Our study suggests that there is a reorganization of the stromal cell population soon after surface ablation with MMC, with a decrease in the stromal bed compensated initially with an increase in the mid and posterior stroma. Corneal cellularity tends to normalize over time, and 3 years postoperatively the mean cell density throughout the cornea seems to maintain normal values.

Evaluation of bFGF effect on corneal collagen and nerve recovery following LASIK with confocal microscopy

Background Basic fibroblast growth factor(bFGF)secreted by cornea after injury is important to cytothesis,collagen fibers reconstruction and axons recovery.However,the local bFGF is not enough for the reparation process.Objective This study aimed to observe the findings of corneal collagen and nerve recovery under the confocal microscopy through focusing(CMTF) in the eyes with intervene of exogenous recombinant bFGF(rbFGF) after excimer laser in situ keratomileusis(LASIK).Methods LASIK rabbit models were binocularly created in 34 clean New Zealand white rabbits.The tobramycin combined with dexamethasone were dropped after operation for 10 days in bilateral eyes.rbFGF was topically administered in the right eyes of rabbits from 1 day through 3 months after LASIK,and lubricant was used in the left eyes at the same way.The corneal collagen and nerve recovery,keratocyte and endothelial cell counting were observed with CMTF at the 1st week,2nd week,2nd month,3rd month and 5th month after LASIK.Results Total 19 rabbits were meted the request of LASIK models.The keratocyte densities in anterior stroma of both groups reached the lowest level at the 2nd week and the highest level at the 3rd month.Otherwise,haze changed on the contrary.No statistically significant differences were found in anterior stroma keratocyte densities,haze grade,grey value between rbFGF group and lubricant group at various time points after operation(P＞0.05).The nerve cord densities of both groups were increased gradually,and those under the epithelial basement membrane were more dominant.The nerve density of the anterior stroma of rbFGF group was significant higher than the lubricant one in the 2nd group(P=0.038).The considerably elevated the subepithelial nerve density value was also seen in rbFGF group compared with lubricant at 5 months after operation(Z=-2.060,P=0.039).No any corneal neovascularization occurred in both groups through experiment duration.The positive correlation was found between grey value with haze grade in rbFGF group(b=22.687,F=37.975,P=0.000) and lubricant group(b=20.410,F=18.516,P=0.000).However,haze grade was not significant correlated with stromal keratocyte density(rbFGF group:b=0.001,F=0.164,P=0.668;lubricant group:b=-0.002,F=1.896,P=0.178).Conclusion Exogenous bFGF can improve the recovery of corneal nerve and regeneration of keratocyte after LASIK.No evidence of bFGF promoting corneal neovascularization is found in this experiment. Key words: Basic fibroblast growth factor; Laser assisted in situ keratomileusis; Confocal microscopy; Reparation

Assessment of Keratocyte Density in Patients With Keratoconus Not Using Contact Lenses

To assess the keratocyte density (KD) in the cornea of patients with keratoconus not using contact lenses. In this pilot study, the eyes (n = 68) of 35 patients with the diagnosis of keratoconus, who did not use contact lenses and the eyes(n = 70) of 35 healthy controls were prospectively examined using the Heidelberg Retinal Tomography Rostock cornea module (HRT3/RCM). The cases with keratoconus and controls were compared with regard to KD in the anteroposterior stromal layers. Of the cases with keratoconus, 15 (42.9%) were men and 20 (57.1%) were women. The mean age was 21.1 ± 11.1 (range =14-41) years. Of the controls, 26 (73.9%) were men and 9 (26.1%)were women. The mean age was 23.9 ± 12.9 (range = 14-35) years.Of the 68 eyes with keratoconus, 22 (28.2%) had mild keratoconus, 23 (35.9%) had moderate keratoconus, and 23 (35.9%) had severe keratoconus. The mean anterior stromal KD was 651.4 ± 89 cells per square millimeter in cases with keratoconus and 879.4 ± 75 cells per square millimeter in controls (P < 0.05). The mean anterior stromal KD decreased by 25.9% in corneas with keratoconus. The mean posterior stromal KD was found to be 363.6 ± 74 cells per square millimeter in cases with keratoconus and 469.7 ± 56 cells per square millimeter in controls (P< 0.01). The mean posterior stromal KD decreased by 22.6% in corneas with keratoconus. The anterior stromal KD was higher than the posterior stromal KD (P < 0.001). No significant relationships were found between the stromal KD data and central corneal thickness or steepest keratometric. The KD in the anterior and posterior corneal stroma was decreased in patients with keratoconus, but without the use of contact lens, compared with healthy controls. This density difference was independent from the central corneal thickness and the stage of keratoconus.

Short-Term Corneal Response to Cross-Linking in Rabbit Eyes Assessed by In Vivo Confocal Laser Scanning Microscopy and Histology

Corneal cross-linking for the treatment of keratoconus has been tested in animal trials and proven clinically. A combination of in vivo confocal laser scanning microscopy (CLSM) and histology was used in rabbit corneas to assess early modifications at the cellular level after corneal cross-linking. Twelve New Zealand male rabbits were tested; in each case, the right eye was the study eye and left eye was the control eye. In vivo CLSM was performed on both eyes before and at 3 days and 1 week after cross-linking. Keratocyte and endothelial cell densities were determined by CLSM before and after cross-linking. After CLSM, the corneas were excised and processed for histology and immunohistochemistry. Massive edema was observed 3 days after cross-linking. The corneal epithelium had already closed again by day 3. No cellular structures were detected in the stroma and endothelium. One week after cross-linking, normal corneal transparency and thickness were restored. The anterior stroma still lacked nuclei. The number of nuclei in the posterior stroma was significantly lower than that in the intact corneas. Highly reflective spindle-shaped structures were detected in the posterior stroma. The endothelial monolayer had closed again but still showed significantly decreased cell density. At 1 week after cross-linking, immunohistochemical staining revealed the presence of proliferating cells in the corneal epithelium, posterior stroma, and endothelium. The early response of the rabbit cornea to cross-linking was successfully characterized at the cellular level by in vivo CLSM and histology, and the results obtained with both techniques correlated positively.

Keratocyte Density after Laser-Assisted Subepithelial Keratectomy with Mitomycin C

Keratocyte Density after Laser-Assisted Subepithelial Keratectomy with Mitomycin C

Evaluation of Prostaglandin Analogue Effects on Corneal Keratocyte Density Using Scanning Laser Confocal Microscopy

To determine if treatment with prostaglandin (PG) analogues affects keratocyte density as an indirect measure of extracellular matrix in the corneal stroma. In this case control study, 129 eyes from 68 patients were examined: 52 eyes had only PG analogue therapy for at least 3 years, 37 eyes had only β-adrenergic blocker therapy for at least 3 years, and 40 control eyes were without therapy. Scanning laser confocal microscopy was carried out, and each corneal stroma was subdivided into the anterior, midstroma, and posterior stroma. Keratocyte density was determined from the images by manual counting. Keratocyte densities in the entire stroma and in 2 stromal layers were significantly higher in patients with PG analogue therapy compared with control patients (entire stroma, P<0.001; anterior stroma, P=0.001; posterior stroma, P=0.016). Keratocyte density in the PG analogue therapy patients was also greater compared with the β-adrenergic blocker patients (entire stroma, P<0.001; anterior stroma, P<0.001; mid-stroma P=0.023, posterior stroma P=0.007). There were no significant differences between the control and β-adrenergic blocker groups. Overall, the density increase for the PG analogue group was greatest in the anterior stroma. PG analogue therapy increased the keratocyte density in each layer of corneal stroma, but more significantly in the anterior stroma than in the midstroma or the posterior stroma. The increased keratocyte density might be the result of diminished extracellular matrix, possibly owing to the known activation of matrix metalloproteinases and inhibition of tissue inhibitors of the metalloproteinases.

Comparative evaluation in the use of topical corticosteroid in the management of corneal alkali burn ulcers in rabbits

This study was performed to evaluate the addition of steroidal or non-steroidal, anti-inflammatory agents or topical medroxyprogesterone acetate to tetracycline and vitamin C in the management of ocular alkali burns. Alkali wounds were created on the central corneas of rabbits by applying a round filter paper, 12 mm in diameter, soaked in 1 M NaOH for 30 s. Only one eye in each rabbit was treated. A total of 25 rabbits were divided into five groups with five animals each. Five rabbits without treatment after the alkali burn were designated as the control group. In the remaining four groups, topical tetracycline and systemic vitamin C treatment were included. In group A, 1% topical medroxyprogesterone acetate, in group B, 1% topical prednisolone acetate and in group C, topical diclofenac were used. In group D, corneal ulcers were treated with only tetracycline and vitamin C. Clinical outcome was monitored daily by corneal opacity, duration of blepharospasm, corneal vascularization, and duration of ocular discharge. After 3 weeks, corneas were excised for histopathological analysis. Samples were monitored by evaluating corneal thickness (μm), numbers of epithelial rows, keratocyte density, stromal vascularization, stromal inflammation, and stromal collagen arrangement. Comparison between groups showed that groups A, B, and C had significantly lower discharge days, and groups B and C had significantly shorter duration of blepharospasm than the control group. In the microscopic evaluation of the corneas, groups B and C had a significantly lower degree of corneal vascularization, and group B had significantly lower degree of corneal inflammation than the control group. In conclusion, the topical application of 1% prednisolone combined with vitamin C and tetracycline may be therapeutically valuable in the early treatment (first 3 weeks) of corneal alkali burns.

Comparative in Vivo Confocal Microscopical Study of the Cornea Anatomy of Different Laboratory Animals

Purpose: The aim of the present study was to analyze and compare in vivo morphology of healthy cornea of six different laboratory animals.Matherials and Methods: One Pomeranian Coarsewool sheep, 5 Beagle dogs, 1 Norwegian and 2 Domestic Short-haired cats, 20 New Zealand White rabbits, 6 Wistar rats, and 10 Balb/c mice were included. The examination was performed bilaterally, using Heidelberg Retina Tomograph equipped with Rostock Cornea Module. The morphology of living corneal layers was visualized and compared between species. The central corneal thickness, density of keratocytes, and endothelial cells were quantified.Results: The epithelial multilayer showed a similarity in morphology between animal types, displaying three clearly distinguishable layers: superficial, intermediate, and basal. Subbasal nerve fibers were displayed as hyperreflective structures underneath basal cells. The subbasal fibers were confirmed in all species, however, the density varied between species. A pronounced Bowman’s membrane was visualized in sheep. In all other species, however, a thin acellular layer with overlying nerve fibers could be seen between basal epithelial cells and anterior stroma. The keratocytes nuclei could be demonstrated in all species except for mice, where no nuclei but only reflective structures resembling keratocytes cell bodies were detected. Overall, the density of keratocytes nuclei was significantly higher in the anterior than in the posterior stroma. Besides endothelial cells density, the endothelial cells morphology was very similar among all species, except for sheep. The endothelial cells were displayed as polygonal structures with bright cytoplasm and dark borders. In sheep, the appearance of the endothelium was very poor because of a thick hyperreflective Descemet’s membrane.Conclusions: The present study will help researchers consider appropriate models for animal experiments, depending on focus of investigation. In vivo CLSM can be used for the characterization of the living cornea over time, thus, reducing the number of animal experiments.

A comparison of the effect on the keratocyte population of mechanical vs femtosecond‐assisted LASIK

Abstract Purpose To analyze the changes in the corneal keratocyte population three months after mechanical (M‐Lasik) versus femtosecond‐assisted (FS‐Lasik) laser in situ keratomileusis Methods This study included 22 eyes treated with M‐Lasik to correct a mean refractive error of ‐2,3+1,1 D sphere and ‐0,9+0,4 D cylinder, and 23 eyes treated with FS‐Lasik to correct a mean refractive error of ‐3,2+2,2 D sphere and ‐0,8+‐0,7 D cylinder. Both groups were compared with a control group comprised of healthy eyes. Corneas were examined using confocal microscopy three months after the procedures. Keratocyte densities were determined in the anterior stroma, stromal bed, central and posterior stroma Results Keratocyte density in the anterior stroma was 23263, 8 + 9030,2 cells/mm3 in the M‐Lasik group, and 25959,4 + 8007 cells/mm3 (p= 0,2) in the FS‐Lasik group. In the stromal bed it was 25269,4+ 8438,6 cells/mm3 and 20856,9 + 5012,2 cells/mm3 (p= 0,03) in M‐Lasik and FS‐Lasik respectively. In the mid stroma it was 29311,1+ 10606 cells/mm3 in M‐Lasik and 27173,9 + 8709,7 cells/mm3 in FS‐Lasik (p= 0,4), and in the posterior stroma it was 29350 + 9395,8 cells/mm3 and 27515,9 + 98284 cells/mm3 (p= 0,5) in M‐Lasik and in FS‐Lasik respectively. The comparison between the average keratocyte density throughout the whole cornea showed no statistically significant difference between M‐Lasik and FS‐Lasik 3 months after surgery, but it was significantly increased in both groups when compared with the control group (p=0.001) Conclusion Both M‐Lasik and FS‐Lasik seem to cause a similar change in the corneal keratocyte population 3 months after surgery

Confocal microscopy after implantation of intrastromal corneal ring segments

Abstract Purpose To evaluate with in vivo confocal microscopy (IVCM) the corneal microstructure changes due to Intrastromal Corneal Ring Segments (ICRS; KERARING Mediphacos Inc., Belo Horizonte, Brazil) implantation. Methods Nine eyes of keratoconic patients were prospectively examined with the HRT 3 confocal microscope with the Rostock cornea module before and 1 month after ICRS implantation. Epithelial basal cell density, endothelial cell density and keratocyte density in the anterior, mid and posterior stroma were measured in the center of the cornea and around ICRS (approximately 3.5 mm off the center of the cornea). Results In the central cornea, we found normal morphologic features at all layers. In peripheral section (near the ICRS) epithelial basal cell density and endothelial cell density were unchanged. However, around the ICRS, keratocyte density increased in the mid stroma (6% increase) and in the posterior stroma (9% increase)(P&lt;.05) but no increase was noticed in the anterior stroma (P=.13). This increase may be due to the best visualisation of activated keratocytes. Hyper‐reflective keratocyte nuclei are thought to indicate the presence of fibroblastic cells. Increased haze adjacent to the ICRS is found to be due to fibroblastic accumulation and irregular collagen fibres. Conclusion Whereas the central corneal zone appears unchanged, the corneal stroma adjacent to the ICRS displays a slight, but distinct, activation of wound healing. IVCM offers a potential insight into the pathophysiology of the microstructural changes in keratoconus after ICRS implantation.

Corneal keratocyte population after laser subepithelial keratectomy (LASEK) with mitomycin C (MMC): 3 months vs 15 months comparison

Abstract Purpose To study corneal keratocyte population after LASEK with mitomycin C (MMC) 15 months vs 3 months after surgery Methods Sixty eyes were included in this study, all of them treated with LASEK with intraoperative 0.02% MMC, divided into 2 groups. 32 eyes had LASEK performed 3 months before, versus 28 in the 15 months post‐op group. Keratocyte density was examined by using confocal microscopy in the anterior, mid and posterior stroma in both groups, and the mean density was compared with a control group of healthy eyes Results Keratocyte density in the anterior stroma was 16993,75+ 8001,7 cells/mm3 and 18529,1 + 2917,7 cells/mm3(p=0,3) in the 3 months and in the 15 months groups respectively, in the mid stroma it was 30783,3+ 9300 cells/mm3 in the 3 months and 20754,7 + 3615,3 cells/mm3 in the 15 months groups (p= 0,0001) and 30286,75+ 8321 cells/mm3and 19994+ 3085,9 cells/mm3 (p= 0,0001) in the posterior stroma in the 3 months and in the 15 months groups respectively. The comparison between the average densities through the whole cornea showed statistically significant difference between the 3 months vs 15 months group and 3 months vs normals, but it was not statistically significant different between 15 months vs normals Conclusion The changes in the corneal keratocyte population after LASEK + MMC seen in the early post‐op, seem to change towards the normal values 15 months after the procedure.

Experimental study of cornea reparation after epipolis laser in situ keratomileusis

目的 探讨微型角膜刀法准分子激光上皮下角膜磨镶术(Epi-LASIK)术后角膜的愈合规律,并与准分子激光屈光性角膜切削术(PRK)比较.方法 建立兔近视性Epi-LASIK及近视性改良PRK动物模型,用锥虫蓝-茜素红活性染色法观察Epi-LASIK术后角膜上皮细胞活性,裂隙灯显微镜下观察角膜瓣变化、上皮修复过程和上皮下雾状混浊(haze)形成情况,通过角膜组织病理学观察角膜上皮及基质的愈合反应.结果 Epi-LASIK术后角膜上皮愈合时间为(4.67±0.41)d,而PRK术后为(2.75±0.27)d,差异有统计学意义(t=9.550,P=0.000).PRK术后即刻和3d、5d角膜上皮瓣细胞活性率分别为(85.83±2.07)%、(48.67±3.41)%、(91.33±3.10)%,差异有统计学意义 (F=215.060,P=0.000).Epi-LASIK术后角膜瓣逐渐融解并被新生上皮取代,上皮愈合过程较改良PRK缓慢.PRK术后haze分级高于Epi-LASIK,差异有统计学意义(Z=2.27,P=0.02).角膜组织病理学检查显示,术后1个月时2组上皮均明显增厚,3个月时上皮细胞排列仍不规则,6个月时上皮细胞形态基本恢复正常,术后3～6个月时Epi-LASIK组角膜上皮厚度已基本接近正常,与PRK组比较差异有统计学意义(P＜0.05).术后3～6个月时Epi-LASIK组角膜前基质细胞数明显低于PRK组(P＜0.05).结论 Epi-LASIK术后上皮瓣活性随时间的推移而降低,上皮修复较慢.与改良PRK比较,Epi-LASIK术后上皮细胞形态变化和上皮增厚程度较轻,角膜基质细胞恢复快,haze程度明显减轻。