Altered Corneal Nerves in Chinese Thyroid-Associated Ophthalmopathy Patients Observed by In Vivo Confocal Microscopy.

BACKGROUND Thyroid-associated ophthalmopathy (TAO) is a common endocrine autoimmune disease. The present study explored corneal nerve changes in TAO patients. MATERIAL AND METHODS Thirty-eight Chinese TAO patients and 20 healthy individuals were included in the study. Central corneal subbasal nerve density and morphology were evaluated with in vivo laser scanning confocal microscopy and quantified using automated CCmetrics software. RESULTS The values of the central corneal subbasal nerve plexus parameters of both active and inactive TAO patients were significantly decreased compared with those of controls, including corneal nerve fiber density (CNFD) (P<0.001 for both), corneal nerve branch density (CNBD) (P<0.001 for both), corneal nerve fiber length (CNFL) (P<0.001 for both), corneal nerve fiber total branch density (CTBD) (P<0.001 for both), corneal nerve fiber area (CNFA) (P<0.001 for both), corneal nerve fiber width (CNFW) (P=0.046, P=0.027, respectively), and corneal nerve fiber fractal dimension (ACNFrD) (P<0.001 for both). In addition, CNFD and ACNFrD values were significantly lower in the active TAO patients compared with those in the inactive TAO patients (P=0.020, P=0.002, respectively). There were significant correlations between CNFD, CNBD, CNFL, CTBD, CNFA, and ACNFrD and the ocular surface parameters and activity assessment items. CONCLUSIONS Abnormal corneal subbasal nerves were observed in both active and inactive Chinese TAO patients, suggesting that nerve degeneration is associated with the disease. However, the exact underlying mechanisms remain to be elucidated.

Observation of Corneal Langerhans Cells by In Vivo Confocal Microscopy in Thyroid-Associated Ophthalmopathy.

PURPOSE: To examine the density and morphology of Langerhans cells (LCs) in the cornea of patients with thyroid-associated ophthalmopathy (TAO). METHODS: Forty patients with TAO and 20 healthy volunteers were studied. All subjects underwent a thorough ophthalmic examination of both eyes. The ocular surface status was assessed by Ocular Surface Disease Index (OSDI) symptom questionnaires, tear break-up time (BUT), fluorescein staining and the Schirmer test. Laser scanning in vivo confocal microscopy was applied to evaluate the LC density and morphology in both central and peripheral cornea. The correlations between confocal microscopy data and clinical data were also analyzed. RESULTS: The OSDI and fluorescein staining values were significantly higher, while BUT and Schirmer test scores were lower in both active and inactive TAO patients compared to the controls. Central LC densities of patients with active TAO (76.38 ± 67.77 cell/mm(2)) and inactive TAO (47.49 ± 38.58 cell/mm(2)) were both significantly higher than those of the controls (21.46 ± 21.74 cell/mm(2)). The number of LCs in the peripheral cornea was also significantly increased in the active TAO group (131.53 ± 74.18 cell/mm(2)) compared to the control group (70.21 ± 37.76 cell/mm(2)). Central LC morphology (LCM) values were significantly higher in both active (1.77 ± 0.63) and inactive (1.51 ± 0.63) TAO groups compared to the control group (1.01 ± 0.80), whereas peripheral LCM scores of the two TAO groups were increased without statistical significance. There were significant correlations between both central LC density and central LCM scores and clinical data, including clinical activity score, OSDI and Schirmer test scores, and between peripheral LC density and OSDI and Schirmer test scores. No significant correlations were found between peripheral LCM scores and clinical data. CONCLUSIONS: The increase of corneal LCs in density and maturation in patients with TAO reflects an activated state of the local immune system, which indicates an inflammatory process in the cornea of TAO.

Inhibition of corneal fibrosis by Smad7 in rats after photorefractive keratectomy.

BACKGROUND: Haze or corneal subepithelial fibrosis is one of the common complications after refractive surgery procedures, such as photorefractive keratectomy (PRK), laser epithelial keratomileusis, and epipolis laser in situ keratomileusis, which would result in refractive regression, decreased visual quality, and corneal opacification. Haze directly resulted from corneal fibrosis mediated by transforming growth factor ß (TGFß). Smad7, an inhibitory Smad, can inhibit TGFß signal transduction. Recently, the effects of Smad7 on the inhibition of fibrosis in several organs have been studied, while little is known about the effects on cornea after PRK. This study was aimed to determine the effects of lentiviral-mediated Smad7 gene expression on corneal fibrosis in rats after PRK. METHODS: Four different experimental groups were established using right eyes of Sprague-Dawley rats. Thirty-two eyes underwent de-epithelialization only and served as a sham operation group (group 1). Ninety-six eyes underwent PRK operation and were further divided into group 2 (the PRK group) without lentivector administration, group 3 (the Lv-blank group) with control lentiviral vector without Smad7 administration, and group 4 (the Lv-Smad7 group) with Smad7 expressing lentiviral vector Smad7 administration. At 1 day, 1 week, 1 month, and 3 months after PRK, the transfection efficiency was determined by measuring the fluorescence signal as well as Smad7 protein and mRNA levels. Corneas were further processed for immunoblotting to assess the phosphorylation of Smad2 as a downstream event of TGFß/Smad signaling. The expression of fibrotic markers, such as α-smooth muscle actin (α-SMA), Type III collagen (collagen III), and cell cycle-related marker Ki67, was measured by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Lentivirus-mediated exogenous Smad7 gene expression in rat corneal tissue resulted in reduced activation of TGFß/Smad signaling caused by downregulation of phosphorylation of Smad2. Smad7 also downregulated the expression of TGFß2. Markers of cell proliferation and fibrosis, including Ki67, α-SMA, and collagen III, were inhibited by Smad7 up to 3 months after PRK operation. CONCLUSION: Smad7 gene transfer inhibits fibrogenic responses of cornea in rats after PRK.

[Purification and characterization of adult optic nerve head astrocytes].

OBJECTIVE: To establish a method of purifying and characterizing adult astrocytes from optic nerve head (ONH). METHODS: Experimental study. The lamina cribrosa tissue from ONH of human eye was isolated under anatomic microscopy, and then 4 to 6 little explants were incubated in each culture plate containing culture medium DMEM/F12. After 8 to 10 weeks, the cells were removed by digesting cells with 0.25% trypsogen. Selective astrocyte culture medium is subsequently used. After two passages, astrocytes were identified by the observation of cell morphology and immunofluorescent staining of GFAP and NCAM. RESULTS: After 2 to 3 weeks of explants planting, cells showed an obvious migration procession by crawling in succession from the verge of the explants and rapidly splitting. Most cells displayed a flat star shape or polygon after digested with trypsogen. Several cells are long fusiformis. Almost all cells presented a flat star shape and simultaneously expressed GFAP and NCAM when the cells cultured with selective astrocyte culture medium. CONCLUSIONS: Cultured human ONH astrocytes can be obtained by precisely separating lamina cribrosa and placing the explants on the margin of culture medium, a method that promotes cell adherence. Using selective astrocyte culture medium is very effective and convenient in purifying primary astrocytes.

Expression of Smad7 inhibits fibrogenic responses of keratocytes to transforming growth factor ß2.

BACKGROUND: Transforming growth factor ß (TGFß) is one of the most important growth factors in the development of fibrosis and scarring on cornea. Smad7, an inhibitory Smad, can inhibit TGFß signal transduction. In recent years, effects of lentiviral-mediated Smad7 on inhibition of fibrosis on some organs have been studied, while little is known about the effects on cornea. This study aimed to determine the effects of lentiviral-mediated Smad7 gene expression on keratocyte proliferation and fibrosis induced by TGF ß2 in vitro. METHODS: Keratocytes were cultured from corneal tissue isolated from Sprague-Dawley (SD) rats and transfected with Smad7 expressing lentiviral vector (Lv-Smad7) or non-functioning control vector (Lv-blank). Following the exposure to TGFß2, keratocytes were processed for immunoblotting to assess the phosphorylation of Smad2 as down-stream event of TGFß/Smad signaling. Expression of fibrotic markers α-smooth muscle actin (α-SMA), type III collagen (collagen III) were measured by Western blotting and quantitative real time polymerase chain reaction (PCR). Overall cell proliferation was determined by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the expression of cell cycle-related marker Ki67 at both mRNA and protein levels. RESULTS: The Smad7 gene transfer suppressed TGFß/Smad signaling in keratocytes by down-regulating phosphorylation of Smad2. Markers of cell proliferation and fibrosis including Ki67, α-SMA, collagen III were inhibited by introduction of Smad 7 into TGFß exposed keratocytes. Consequently, the rate of cell proliferation was attenuated. CONCLUSION: Smad7 gene transfer inhibited fibrogenic responses of keratocytes to TGFß2.

[The effects of protein-free calf blood extract for recovery of corneal nerve after LASEK and LASIK].

OBJECTIVE: To evaluate the effects of protein-free calf blood extract for recovery of corneal nerve after LASEK and LSEIK. METHODS: A prospective, randomized, control and double-blind study was carried out from January through February 2009 at Department of Ophthalmology, Eye Ear Nose and Throat Hospital of Fudan University. Forty-nine eyes of 25 patients who underwent LASEK were randomly divided into two groups. One group with 16 patients (32 eyes) was treated by protein-free calf blood extract eye gel which was defined as drug treated group and the other group with 9 patients (17 eyes) was not treated by protein-free calf blood extract eye gel which was defined as no drug treated group. Forty-four eyes of 23 patients who underwent LASIK were also randomly divided into two groups. One group with 13 patients (24 eyes) was treated by protein-free calf blood extract eye gel which was defined as drug treated group and the other group with 10 patients (20 eyes) was not treated by protein-free calf blood extract eye gel which was defined as no drug treated group. Protein-free calf blood extract eye gel was delivered in both drug treated groups 3 times per day for three months after surgery. Laser scanning confocal microscopic examinations were performed on 48 eyes in vivo. Central corneal sensitivity and tear break-up time (BUT) were tested on 93 eyes preoperatively and 1, 3, 6 months, and 1 year after surgery. The obtained dates in the study were analyzed using independent samples t-test, paired t-test and Mann-Whitney Test. RESULTS: The morphology observed by confocal microscope of sub-basal nerve fibers was not different between drug treated group and no drug treated group until the last follow up after LASIK or LASEK (Z = 0.0000, P = 1.00) and (Z = 0.0000, P = 1.00). Nerve fibers with interconnections were observed in drug treated group at 3 months after LASEK. The morphology of sub-basal nerve fibers had not recovered completely until 1 year after surgery. The central corneal sensitivity was better in drug treated group than in no drug treated group at 1 month after LASEK [(4.95 ± 0.84) µm, (3.62 ± 1.38) µm; t = 4.23, P < 0.01] and at 1 and 3 months after LASIK [(3.29 ± 1.40)µm, (2.35 ± 1.51) µm; t = 2.10, P < 0.05], [(4.31 ± 1.61) µm, (3.18 ± 1.62) µm; t = 2.31, P < 0.05]. At 6 months postoperatively, the central corneal sensitivity of both drug treated group and no drug treated group which underwent LASEK was not significantly different from pre-operation [(5.81 ± 0.35) µm; t = -1.26, P > 0.05], [(5.79 ± 0.36) µm; t = -0.70, P > 0.05]. At 6 months postoperatively, the central corneal sensitivity of drug treated group which underwent LASIK was not significantly different from pre-operation [(5.25 ± 0.91) µm; t = -1.87, P > 0.05]. No significant difference was seen in BUT between drug treated group and no drug treated group after LASEK or LASIK until 1 year after surgery [(8.13 ± 2.18) µm, (8.76 ± 1.64) µm; t = -0.90, P > 0.05], [(7.71 ± 2.14) µm, (7.45 ± 2.37) µm; t = 0.30, P > 0.05]. CONCLUSION: Protein-free calf blood extract could significantly promote the recovery of corneal nerve in the early period after LASEK and LASIK.

[Morphological characteristics of cornea in patients with vernal keratoconjunctivitis by in vivo laser scanning confocal microscopy].

OBJECTIVE: To explore the morphological characteristics on cornea in patients with vernal keratoconjunctivitis (VKC) by the application of in vivo laser scanning confocal microscopy (LSCM). METHODS: The experimental design was retrospective observation case series (case control study). Twenty-six patients, each diagnosed as bilateral VKC, were enrolled in the study, among which 13 were tarsal form, 5 were bulbar form and the rest were mixed form. Nine patients had the clinical course less than one year, eight subjects longer than three years, and the rest between them. Another twenty-six healthy volunteers with matching age and gender were selected as normal control. All participants had their right eyes examined with the in vivo confocal microscopy (HRT II/RCM). Central cornea and superior peripheral cornea were chosen as the examination points. The images were recorded automatically and cellular density of each layer was analyzed by installed software. Software ImageJ was utilized to analyze the density, diameter, branch number and tortuosity of subbasal nerve fiber in VKC patients. Independent t test was performed to assess the differences on cellular density between VKC patients and normal control, as well as those between central and peripheral cornea in VKC patients. Fisher chi-square test was used to compare the infiltration rate of Langerhans cells in corneal epithelium between VKC patients and controls. ANOVA was applied to assess the differences in cellular density among three subtypes, as well as among different duration of VKC. Independent t-test and chi-square test were applied to analyze the parameters of subbasal nerve fiber. RESULTS: The morphological changes in cornea included the absence of superficial hyperreflective polygonal epithelial cells, infiltration of Langerhans cells in and(or) underneath corneal epithelium and activation of keratocytes in anterior stroma. Corneal epithelium conjunctivalization and stromal neovascularization could be identified in patients with corneal neovascular epithelium. Longitudinal or oblique dark striae could be found in the posterior stroma in patients with complicated keratoconus. The density of epithelial cells at central and peripheral cornea in healthy controls were (6033.1 ± 998.7) cells/mm(2) and (6098.4 ± 298.3) cells/mm(2), while that in VKC patients were (5972.2 ± 1148.2) cells/mm(2) and (6178.5 ± 318.9) cells/mm(2) respectively, the differences being no statistical significant between them (t = 1.191, 1.011; P = 0.238, 0.318). However, it's found in VKC patients that cellular density at peripheral cornea was significantly higher than that at central area (t = 2.249, P = 0.03). The density of anterior stromal cells at central and peripheral cornea in healthy controls was (1001.4 ± 125.3) cells/mm(2) and (924.6 ± 201.4) cells/mm(2), while that in VKC patients was (1184.5 ± 115.3) cells/mm(2) and (1101.4 ± 151.1) cells/mm(2), the difference bearing no statistical significance (t = 6.617, 3.439; P = 0.001). The density of posterior stromal cells in normal subjects and VKC patients was (537.7 ± 42.6) cells/mm(2) and (548.7 ± 79.8) cells/mm(2), that of endothelial cells was (2985.7 ± 401.2) cells/mm(2) and (3021.5 ± 383.3) cells/mm(2), respectively, neither difference had statistical significance (t = 0.174, 1.112; P = 0.864, 0.282). Langerhans cell infiltration could be identified in 61.5% (16 cases) VKC patients, which was significantly higher than normal control (2 cases, 7.7%) (χ(2) = 12.49, P = 0.001). Furthermore, much intense Langerhans cells infiltration was found in bulbar form and mix form than tarsal form. (t = 6.617, P = 0.001). The density and diameter of subbasal nerve fiber in VKC patients decreased significantly than those in normal subjects, whereas the tortuosity increased significantly. CONCLUSIONS: The morphological changes of cornea in VKC patients mainly involve corneal epithelium, subbasal nerve fiber and anterior stroma. In vivo LSCM is helpful in discriminating the subtypes of VKC.

An in vivo confocal microscopy and impression cytology analysis of goblet cells in patients with chemical burns.

PURPOSE: To evaluate goblet cell density (GCD) on conjunctiva and cornea in patients with ocular chemical burns by in vivo laser scanning confocal microscopy (LSCM) and impression cytology (IC) and to explore the correlation between two methods. METHODS: Fifty-four patients (58 eyes) with chemical burn were enrolled in the study. LSCM was applied to identify the goblet cells on conjunctiva and cornea under in vivo conditions, and GCD was analyzed with the customized software. Impression cytology was then performed, and the biopsy specimens were stained to visualize goblet cells in vitro and to measure the density. Statistical software was used to analyze the correlation between GCD taken by two methods. RESULTS: Conjunctival goblet cells could be discriminated in 55 eyes and 57 eyes by in vivo LSCM and IC. They could be identified on the cornea in nine eyes and eight eyes by two methods. The positive rate of two methods had no significant difference. GCDs on conjunctiva measured by in vivo LSCM and IC were 136 +/- 79 cells/mm(2) and 121 +/- 66 cells/mm(2). Median GCDs on cornea detected by two methods were 30 cells/mm(2) and 23 cells/mm(2), respectively. A significant positive correlation was found between the GCDs on conjunctiva measured by these two methods as well as the GCDs on cornea. CONCLUSIONS: GCD decreased in patients with chemical burns. A positive correlation was found between GCD measured by in vivo LSCM and IC after chemical burns. In vivo LSCM was a promising device to study goblet cells in vivo under pathologic conditions.

[Clinical assessment of oral ganciclovir capsules on the treatment of herpes simplex keratitis].

OBJECTIVE: To evaluate the clinical values of oral ganciclovir on the treatment of herpes simplex keratitis (HSK). METHODS: A randomized, controlled, single-blind and prospective study was carried out from May in 2008 to June in 2009 at Department of Ophthalmology, Eye Ear Nose and Throat Hospital of Fudan University. 60 patients (60 eyes) with HSK, including stromal keratitis and corneal endotheliitis, were enrolled in the study and were randomly arranged into two groups in average. Oral ganciclovir was orally administered 1000 mg 3 times per day for 8 weeks, 0.15% ganciclovir ophthalmic gel, 4 times per day, and 0.1% fluorometholone eye drops, 3 times per day, in the test group, meanwhile, the control group was adopted the same ophthalmic gel and eye drops without the oral capsules. The symptoms and signs were evaluated before and after the therapy 1st week, 2nd week, 4th week, 6th week and 8th day respectively with the side effects observed. RESULTS: There was no significant difference between the control and test group in the mean scores of symptoms (control 10.70 ± 3.61, test 11.87 ± 3.47) and signs (control 13.83 ± 3.74, test 15.27 ± 3.83) respectively before the treatment (Z = -1.269 and -1.419; P > 0.05). After the administration, the total scores of symptoms and signs in the test group were 8.37 ± 4.31, 2.70 ± 2.65, 0.70 ± 1.44, 0.33 ± 0.92 and 0.17 ± 0.65 respectively at each follow-up time point, which were obviously lower than those in the control group, 13.63 ± 7.64, 10.53 ± 7.18, 7.83 ± 6.49, 5.37 ± 5.33 and 4.37 ± 5.11 respectively (Z = -2.801, -4.895, -5.260, -4.758, and -4.292; P < 0. 05). The efficacy rates in the test group were all 100.0% after the administration, but those in the control group were 50.0%, 73.3%, 86.7%, 93.3% and 96.6%. Furthermore, the cure rates in the test group were 0.0%, 36.7%, 76.7%, 90.0% and 93.3% respectively at each follow-up time point, which were significantly higher than those in the control group with 0.0%, 3.3%, 16.7%, 30.0% and 43.3% respectively (χ(2) = 20.00, 16.433, 22.571, 22.636 and 17.330; P < 0. 001). There was no obvious discomfortableness and adverse reaction observed in the test group. Unfortunately, 5 patients in the control group and 3 patients in the test group underwent the recurrence of HSK after the course of treatment, but there was no significant difference between the groups in the recurrence rate. CONCLUSIONS: Oral ganciclovir can effectively assist to relieve the symptoms and signs and shorten the pathogenesis of herpes simplex stromal keratitis and corneal endotheliitis. And short-term oral ganciclovir has confirmed good safety.

[Normal human bulbar conjunctiva on confocal microscopy in vivo].

OBJECTIVE: To analyze the morphology of human bulbar conjunctiva by in vivo laser scanning confocal microscopy. METHODS: This research was a cross-sectional study. From February to July 2008, 50 eyes of 50 healthy subjects were enrolled in this study. They had no history of ocular trauma, infection or contact lens wear and had no found after routine slit-lamp examinations. In vivo laser scanning confocal microscopic examinations were performed on the superior, inferior, nasal and temporal bulbar conjunctiva and the images were recorded. The morphology of bulbar conjunctiva was analyzed and the density of epithelial cells, dendritic cells and goblet cells were calculated. One-way analysis of variance (ANOVA) was used to compare the means of epithelial cell densities in different layers and goblet cell densities in different positions. Subsequently the datum between two groups were analyzed by least significant difference (LSD). RESULTS: Superficial epithelial cells of bulbar conjunctiva were characterized as large loose-arranged cells with a hyporeflective nucleus. The mean density is (1643 +/- 206) cells/mm(2). Intermediate epithelial cells were captured with features of oval small tight-arranged cells with a punctiform hyperreflective nucleus. The mean density is (4693 +/- 228) cells/mm(2). Basal epithelial cells appeared to be polygonal and regular-arranged within hyperreflective cell borders. The mean density is (4420 +/- 230) cells/mm(2). There was a significant difference among three kinds of conjunctival epithelium (F = 1160.312, P = 0.000). The presumed goblet cell was defined as a large hyperreflective oval-shaped cell with relatively homogeneous brightness, crowded in groups or mainly dispersed. The mean density is (432 +/- 72) cells/mm(2). The dendritic cell appeared to be hyperreflective corpuscular particles with dendritic processes scattered among conjunctival epithelial cells. The mean density is (22 +/- 25) cells/mm(2). The basement membrane, a prominent hyperreflective band, separated epithelial cells from subepithelial structure. Bulbar conjunctival substantia propria, beneath the basement membrane, was mainly composed of highly vascularized, loose connective tissues which were irregularly arranged fibers or a network of fibers, punctiform hyperreflective immune cells and sharp flows of blood vessels. CONCLUSION: In vivo laser scanning confocal microscopy is a useful tool in the analysis of the bulbar conjunctival morphology, which provided a fast and noninvasive method for the diagnosis of ocular surface diseases.

[Proliferative ability and ultramicrostructure of basal limbal epithelial cells in different age group of normal subjects].

OBJECTIVE: To demonstrate the difference in proliferative ability and ultramicrostructure of basal limbal epithelial cells in different age group of normal subjects. METHODS: It is a case-control study.40 specimens of limbus from 40 eyes provided by the eye bank of Eye Ear Nose and Throat Hospital of Fudan University in 2007 - 2008 were enrolled in this study. Only one eye was enrolled from the same donner. Specimens were divided into 4 groups according to the donners' age: A (0 - 19 years), B (20 - 39 years), C (40 - 59 years) and D (60 - 79 years). Immunohistochemistry of proliferating cell nuclear antigen (PCNA) was performed on 10 specimens of each group. The staining status of limbal epithelium was observed and the staining positive rate of the limbal basal epithelial cells was graded. And transmission electric microscopy was performed on specimens of each group. The morphologic feature of the ultramicrostructure of limbal basal epithelial cells were observed. RESULTS: 50% of specimens in group A showed a strong PCNA staining of (++++), while a weak staining of (+) took a main part in all the other 3 groups (B 40%, C 60% and D 50%, respectively). Furthermore, positive nuclear staining of limbal superficial cells was observed in 3 specimens and positive cytoplasm staining was observed in 1 specimen, all in group A. Transmission electric microscopy showed that the stem cell-like cells in group A possessed 3 morphologic features: extremely small, stretching along the basal membrane, with densely packed heterochromatin. In contrast, the stem cell-like cells in the other 3 groups did not show great disparity in cell size with their neighbor epithelial cells, and they stretched vertically to the basal membrane, with dispersed euchromatin. CONCLUSION: Difference in proliferative ability and ultramicrostructure of basal limbal epithelial cells was found in different age group of normal subjects.

In-vivo confocal microscopy of iridocorneal endothelial syndrome.

BACKGROUND: We carried out a study by in-vivo confocal microscopy to investigate the appearance of iridocorneal endothelial (ICE) syndrome, and discuss its diagnostic potential. METHODS: Twelve patients, each with unilateral ICE syndrome, had both their eyes examined by in-vivo confocal microscopy. The images were recorded and analyzed by the use of proprietary software. Endothelium density, average endothelial area, coefficient of variation of cell size, percentage of hexagonal cells, and nerve fiber diameter were measured in both the anterior and posterior stroma. Corneal thickness was also measured for both eyes. A non-parametric test was used to compare differences between the affected eye and the contralateral healthy one. RESULTS: In-vivo confocal microscopy highlighted two main patterns of abnormal "epithelioid-like" endothelium, both characterized by marked hyperreflective nuclei and loss of regularity in cellular size and shape. The first pattern was relatively regular cell size and shape, conserving a pattern similar to that of normal endothelial cells. However, the cells lost normal hexagonality and presented prominent uniform "cobblestone-like" nuclei occupying the central area of the cells. The second type was more irregular in cellular size and shape, with hyperreflective diversely shaped nuclei adjacent to the boundaries of the cells. Cells with two nuclei could be found in both types. Compared with the contralateral eye, the stromal nerve fibers in affected eyes were unusually thicker and distorted. Nerve diameters in the anterior stroma of affected eyes and contralateral eyes were 5.7 +/- 0.5 microm and 3.2 +/- 0.2 microm, respectively; those in the posterior stroma were 10.8 +/- 0.3 microm and 6.6 +/- 0.4 microm, respectively (both P < 0.001). CONCLUSIONS: Application of confocal microscopy indicates that ICE syndrome is characterized by pleomorphic epithelioid-like endothelial cells with hyperreflective nuclei. The technique has great potential in diagnosing ICE syndrome, especially in cases with corneal edema.

[Expression of regulatory T cells and related cytokines in mice allogenic corneal transplantation rejection].

OBJECTIVE: To study the changes of CD4(+)CD25(+) and CD8(+)CD103(+) regulatory T cells and their relevant cytokines in corneal allograft transplantation in mice. METHODS: BABL/c (H-2(d)) mice received corneal allografts from C3H/He (H-2(k)) mice were served as the experimental group. BABL/c (H-2(d)) mice received corneal from BABL/c (H-2(d)) mice were used as the control group. Corneal graft survival time was recorded pre-, 3rd day, 7th day, 4th week, 8th week post-operatively. Infiltration of inflammatory cells and corneal neovascularization were evaluated by histopathologic examination. The percentage of CD4(+)CD25(+) and CD8(+)CD103(+) T cell in peripheral blood and spleen was determined by flow cytometry. The expression of interleukin-10 (IL-10), IL-4, gamma-interferon (IFN-gamma) and IL-1beta in serum and aqueous humor was measured by enzyme-linked immunospecific assay (ELISA). RESULTS: The graft rejection in experimental group occurred at 7 days to 4 weeks after the operation, averaged (14.79 +/- 1.02) days. The grafts in the control group remained clear within 8 weeks observation and the survival time is much longer than that of the allografts (chi(2) = 46.934, P = 0.000). Flow cytometry showed that the percentage of CD4(+)CD25(+) T cell in peripheral blood in the control group after surgery was (3.36 +/- 0.29)%, (4.09 +/- 0.44)%, (5.44 +/- 0.35)%, (5.73 +/- 0.53)% at day 3, day 7, 4th week, and 8th week, respectively, which was significantly higher than that in the experimental group [(2.50 +/- 0.39)%, (3.24 +/- 0.25)%, (4.20 +/- 0.45)%, (4.18 +/- 0.14)%; t = 3.828, 2.898, 3.780, 4.892; P < 0.05]. On the other hand, CD8(+)CD103(+) T cell in peripheral blood in the experimental group after surgery was (2.20 +/- 0.33)%, (2.79 +/- 0.57)%, (4.55 +/- 1.03)%, (4.31 +/- 0.07)% at different periods, which was much higher than that in the control group (t = 7.133, 4.876, 5.196, 19.960; P < 0.05). The changes of CD4(+)CD25(+) and CD8(+)CD103(+) T cells in the spleen was earlier than those in peripheral blood. ELISA showed the expression of IL-10 and IL-4 in the serum in experimental group after surgery was much lower than that in the control group (t = 3.203, 3.141, 3.012, 2.869 and 2.340, 6.681, 8.839, 8.574; P = 0.011, 0.012, 0.013, 0.019 and 0.053, 0.000, 0.000, 0.000). The serum levels of IFN-gamma and IL-1beta in experimental group were higher than that in the control group (t = 3.508, 3.265, 4.402, 5.539 and 3.630, 5.796, 1.728, 0.660; P = 0.006, 0.011, 0.002, 0.000 and 0.005, 0.000, 0.115, 0.524). Furthermore, the cytokine levels in the aqueous humor behaved similarly with those in the serum. CONCLUSION: The down-regulation of CD4(+)CD25(+) Treg, IL-10 and IL-4 and the up-regulation of CD8(+)CD103(+) Treg, IFN-gamma and IL-1beta in mice allograft corneal rejection may play an important role in allograft rejection.

[The influence of lens and vitreous resection on anterior chamber associated immune deviation in mice model].

OBJECTIVE: To explore the impact of lens and vitreous resection on the induction of the anterior chamber associated immune deviation (ACAID) in the mice, and its relationship with the time duration postoperatively. METHODS: The current study was an experimental research. Fifty-one Wistar mice were chosen to involve this study. After the surgeries, 50 Wistar mice were randomly divided into 5 groups. Other 5 SD mice and 1 Wistar mouse were used as the providers of epithelium removed corneal grafts to induce ACAID. Group A: corneal grafts from wistar mouse were implanted into the anterior chamber 1 week after the lens and vitreous resection. Group B: the spleen cells were injected into the nape of neck 1 week after the lens and vitreous resection. The corneal grafts from SD mice were implanted into the anterior chamber 1 week, 4 week and 8 week after the lens and vitreous resection in group C, D and E respectively. To induce the delayed type hypersensitivity (DTH), spleen cells were injected into the right ear pinna 1 week after the neck injection in group B, 2 weeks after the corneal grafts anterior chamber implantation in other groups. At the same time point as the DTH inducement, aqueous humor was collected to measure the concentration of transforming growth factor (TGF) -beta2 and interleukin (IL)-10; heart blood was collected to detect the concentration of IL-4 and IL-10. And the spleens were removed to evaluate the expression of the GATA-3 mRNA by RT-PCR. The eyeballs were enucleated to evaluate the histopathologic changes. RESULTS: (1) The evaluation of DTH. The DTH were found in group B, C, D and E. (2) The analysis of the TGF-beta2 and IL-10 concentrations in aqueous humor and the IL-4, IL-10 concentrations in serum. The concentrations of IL-4 in serum were (4.073 +/- 0.198) ng/L in group A, (5.806 +/- 0.635) ng/L in group B, (5.535 +/- 0.278) ng/L in group C, (4.102 +/- 0.344) ng/L in group D and (5.313 +/- 0.317) ng/L in group E. The concentrations of IL-10 in serum in group A to group E were (7.854 +/- 2.349) ng/L, (25.633 +/- 6.307) ng/L, (40.103 +/- 16.010) ng/L, (14.321 +/- 2.983) ng/L and (28.620 +/- 5.251) ng/L. The IL-10 concentrations in the aqueous humor were (8.857 +/- 0.401) ng/L, (22.882 +/- 3.315) ng/L, (21.548 +/- 0.477) ng/L, (7.742 +/- 0.952) ng/L and (12.119 +/- 0.477) ng/L, respectively. The concentrations of TGF-beta in the aqueous humor were (5.800 +/- 2.899) ng/L in group A, (60.010 +/- 0.000) ng/L in group B, (57.055 +/- 4.179) ng/L in group C, (28.490 +/- 4.144) ng/L in group D and (36.370 +/- 3.169) ng/L in group E. Significant differences were found in concentrations of TGF-beta2 and IL-10 in aqueous humor and the concentrations of IL-4 and IL-10 in serum in group C, D and E. The concentrations of TGF-beta2, IL-4 and IL-10 were significantly lower in group D than those in group C and E. The trend that the concentrations of TGF-beta2, IL-4 and IL-10 increased after one week postoperatively, then decreased, and finally increased again was observed. (3) The expression of GATA-3 mRNA in spleen. The expression values of GATA-3 mRNA were 662.5 +/- 114.4 in group A, 730.7 +/- 53.8 in group B, 881.9 +/- 10.7 in group C, 1288.3 +/- 258.0 in group D and 1129.7 +/- 95.7 in group E. An obvious up-regulation of the GATA-3 mRNA was noted in group D and E. CONCLUSIONS: The eye after the lens and vitreous resection lost the ability to induce ACAID in the research duration of this study. But with the gradually decrease of the intraocular inflammation, the ability to induce ACAID might recover with the time.

[Variations of the microstructure in human limbus by confocal microscopy in vivo].

OBJECTIVE: To demonstrate the variations in the niche of limbal stem cells and the limbal basal epithelial cells in vivo by confocal microscopy. METHODS: One hundred and twenty eyes of 120 healthy subjects were enrolled in this study after a routine slit-lamp examination. They were divided into four groups according to the age: group A (0-), group B (20-), group C (40-) and group D (60 - 79). Confocal microscopic examination in vivo was performed and the images of the inferior limbus were recorded. The morphologic features of each group were analyzed and the average size of limbal basal epithelial cells of each group were measured. RESULTS: The morphologic features of limbus varied among groups. 96.7% (29/30) of group A showed clear Vogt Palisades, while 3.3% (1/30) showed no Vogt Palisades. 96.7% (29/30) of group B also showed clear Vogt Palisades, while 3.3% (1/30) showed no Vogt Palisades.70.0% (21/30) of group C showed clear Vogt Palisades, while 13.3% (4/30) showed atrophic Vogt Palisades and 16.7% (5/30) showed no Vogt Palisades at all. 33.3% (10/30) of group C showed clear Vogt Palisades, while 10.0% (3/30) showed atrophic Vogt Palisades and 56.7% (17/30) showed no Vogt Palisades at all. The average size of limbal basal epithelial cells in each group was (9.89 +/- 1.12), (10.65 +/- 1.45), (10.70 +/- 1.39) and (12.22 +/- 1.42) microm in sequence. CONCLUSIONS: There is variations in the microenvironment of niche in human limbus. Meanwhile, limbal basal epithelial cells showed different quantity and cell size, demonstrating varied proliferative potential.

[Morphological changes of cornea in iridocorneal endothelial syndrome under the confocal microscopy].

OBJECTIVE: To evaluate the morphological changes of cornea in iridocorneal endothelial syndrome (ICE) under the examination of in vivo confocal microscopy. METHODS: The experimental design was retrospective observation case series. Twenty-three eyes of 23 consecutive patients, each diagnosed as ICE, had their both eyes examined with the in vivo confocal microscopy (NIDEK, confoscan 3.0). The images were recorded and analyzed by software NAVIS. Measurements were performed on endothelium density, average endothelial area, the percentage of hexagonal cells and the percentage of endothelium with nuclei, and the ANOVA was done to assess the differences. RESULTS: In vivo confocal microscopy highlighted two main patterns of endothelial changes: "kite-like" and "epithelial-like" abnormal endothelium, characterised by marked hyperreflective nuclei and loss of regularity in cellular size and shape. With the progression of disease, the endothelium density and the percentage of hexagonal endothelial cells decreased, which were (1687.1 +/- 122.6), (1210.6 +/- 168.7), (947.3 +/- 145.2), (856.8 +/- 73.4) cells/mm2 and (51.5 +/- 6.3)%, (39.8 +/- 9.2)%, (32.7 +/- 8.1)%, (24.1 +/- 5.6)% respectively in detail. In contrast, the average endothelial area the percentage of endothelium with nuclei increased, which were (678.3 +/- 56.3), (928.7 +/- 96.2), (1188.5 +/- 72.6), (1337.5 +/- 60.8) microm2 and (12.6 +/- 1.4)%, (56.8 +/- 3.7)%, (78.7 +/- 5.6)%, (84.3 +/- 2.8)%. The differences all had statistical significance (F = 7.158, 7.736, 6.876, 14.452 respectively, P = 0.000). The morphology of keratocyte and endothelium were normal, compared with the contralateral healthy eyes. However, the stromal nerves became thicker and more tortuous with the disease advancement. CONCLUSIONS: The application of confocal microscopy indicates that the ICE is characterised by epithelial-like endothelial cells with hyperreflective nuclei. The technique has great potential in diagnosing ICE, especially in evaluating the disease progression.

[Comparative study on deep lamellar keratoplasty and penetrating keratoplasty for keratoconus].

OBJECTIVE: To investigate the therapeutic outcomes of deep lamellar keratoplasty (DLKP) and penetrating keratoplasty (PKP) for keratoconus. METHODS: Retrospective review of 29 eyes of 29 keratoconus patients, who underwent surgery in Eye & ENT Hospital of Fudan University from April in 2003 to April in 2006. Eleven eyes underwent DLKP and the rest had PKP. Pre- and post-operative visual acuity, astigmatism, complications and post-operative graft status were assessed. RESULTS: 82% of patients (9 eyes) in DLKP group gained BCVA better than 0.5, while in PKP group the proportion was just 78% (14 eyes). Postoperative astigmatism of two groups had no statistical significant difference with the value (-4.03 +/- 1.87) D and (-3.43 +/- 2.31) D respectively (DS: t = 2.135, P = 0.460, DC: t = -0.643, P = 0.528). The confocal image of epithelial cell, basal epithelium and Bowman's membrane was similar to that of normal cornea. The stromal cell was a little bit smaller and disordered. The endothelium in DLKP-treated eyes had normal cellular size and shape. There was no statistical significance between the density of operated eyes and contralateral unoperated eyes (2311.72 +/- 439.73) cells/mm2 and (2477.81 +/- 535.92) cells/mm2 respectively (t = 1.060, P = 0.780). However, the endothelial cells in PKP-treated eyes were highly pleomorphic with a decreased cellular density of (1642.17 +/- 583.41) cells/mm2, whereas the contralateral unoperated eyes had endothelium density of (2739.05 +/- 401.77) cells/mm2. The difference was statistical significance (Z = 7.32, P = 0.006). Complication rates were similar for DLKP and PKP, although the classification of the complications varied, being less severe in the DLKP group. CONCLUSIONS: DLKP seems to be a safe alternative for patients with keratoconus because of its equivalent effect to PKP. DLKP is more technically challenging but allows the risk of endothelial rejection to be avoided and may reduce the risk of late endothelial failure.

Effect of pirenzepine ophthalmic solution on form-deprivation myopia in the guinea pigs.

BACKGROUND: Nonselective muscarinic receptor antagonist, atropine, was believed to inhibit myopic progression. The purpose of this study was to determine the efficacy, through topical administration, of the M1-selective muscarinic antagonist pirenzepine in preventing experimentally induced form-deprivation myopia in guinea pigs. METHODS: Fifty-three guinea pigs, which underwent monocular deprivation with their eyelids sutured, were divided into 6 groups. Three groups were treated with 1%, 2% or 4% pirenzepine ophthalmic solutions; the fourth group with atropine; the fifth with saline and the last group left untreated. Ocular refraction, in vivo biometric measurements and wet eye weight were collected before and after the experiment. All the eyes were finally enucleated for histopathological examination to evaluate the possible toxic effects on ocular structures. RESULTS: Animals untreated or treated with saline produced (-2.31+/-1.47) D and (-2.25+/-0.88) D of axial myopia respectively. Those treated with 1% pirenzepine ophthalmic solution produced relative myopia of (-1.63+/-0.48) D, and those under the treatment of 2% and 4% pirenzepine ophthalmic solution only developed a relative myopia of (-0.89+/-0.42) D and (-0.70+/-0.41) D (F=9.56, P<0.05). The significant reduction in myopia in 2% and 4% pirenzepine treated animals was caused by significantly less vitreous chamber elongation and axial elongation of the deprived eyes [2% group: (0.009+/-0.052) mm, 4% group: (0.006+/-0.078) mm] when compared with untreated, saline treated or 1% pirenzepine treated guinea pigs (0.057+/-0.056) mm, (0.064+/-0.053) mm and (0.033+/-0.035) mm, respectively]. Histological examinations revealed no obviously toxic effects on the eyes treated with pirenzepine. CONCLUSION: Topical administration of the M1-selective muscarinic antagonist, pirenzepine, can prevent induced form-deprivation myopia in guinea pigs by inhibiting axial elongation without obvious damage to ocular tissues.