Classifications of anterior segment structure of congenital corneal opacity in infants and toddlers by ultrasound biomicroscopy and slit-lamp microscopic photographs: an observational study.

BACKGROUND: The structural features have an impact on the surgical prognosis for congenital corneal opacity (CCO). The structural classification system of CCO, however, is lacking. Based on data from ultrasound biomicroscopy (UBM) findings in infants and toddlers with CCO, this research proposed a classification system for the anterior segment structure severity. METHODS: Medical records, preoperative UBM images and slit-lamp photographs of infants and toddlers diagnosed with CCO at University Third Hospital between December 2018 and June 2022 were reviewed. According to the anterior segment structural features observed in UBM images, eyes were classified as follows: U1, opaque cornea only; U2, central anterior synechia; U3, peripheral anterior synechia combined with angle closure; and U4, aniridia or lens anomaly. The opacity appearance and corneal vascularization density observed in slit-lamp photographs were assigned grades according to previous studies. The extent of vascularization was also recorded. The corresponding intraocular anomaly classifications and ocular surface lesion severity were analysed. RESULTS: Among 81 eyes (65 patients), 41 (50.6%) were right eyes, and 40 (49.4%) were left eyes. The median age at examination was 6.91 months (n = 81, 1.00, 34.00). Two (2.5%) of the 81 eyes were classified as U1, 20 (24.7%) as U2, 22 (27.2%) as U3a, 11 (13.6%) as U3b and 26 (32.1%) as U4. Bilateral CCO eyes had more severe UBM classifications (P = 0.019), more severe dysgenesis (P = 0.012) and a larger angle closure (P = 0.009). Eyes with more severe UBM classifications had higher opacity grades (P = 0.003) and vascularization grades (P = 0.014) and a larger vascularization extent (P = 0.001). Eyes with dysgenesis had higher haze grades (P = 0.012) and more severe vascularization (P = 0.003 for density; P = 0.008 for extent), while the angle closure range was related to haze grade (P = 0.013) and vascularization extent (P = 0.003). CONCLUSIONS: This classification method based on UBM and slit-lamp photography findings in the eyes of CCO infants and toddlers can truly reflect the degree of abnormality of the ocular surface and anterior segment and is correlated with the severity of ocular surface anomalies. This method might provide meaningful guidance for surgical procedure design and prognostic determinations for keratoplasty in CCO eyes.

A New Treatment for Recalcitrant Neurotrophic Keratopathy of Ocular Graft-Versus-Host Disease with Virus Infection.

INTRODUCTION: Neurotrophic keratopathy (NK) is a rare degenerative ocular disease that can be difficult to treat. There were no effective resolutive treatments for severe NK caused by ocular graft-versus-host disease (oGVHD) along with virus infection. To address this question, we designed a prospective cohort study to evaluate the efficacy and safety of topical recombinant human nerve growth factor (rhNGF) in patients with recalcitrant NK of oGVHD and viral infection. METHODS: This prospective cohort study enrolled patients with recalcitrant NK diagnosed with oGVHD and treated with rhNGF. Clinical evaluations included the range of epithelial defects, best corrected visual acuity, intraocular pressure, slit-lamp examination, and corneal fluorescein staining. Examinations of the central corneal thickness, corneal sensitivity, and nerve fiber regeneration were performed at each visit at 4, 8, 12, 20 weeks and 6 months, respectively, after initiating rhNGF treatment. RESULTS: All enrolled patients were diagnosed with NK at stage 2 (7 eyes, 63.6%) or stage 3 (4 eyes, 36.4%) and responded to rhNGF treatment. Five of 11 (45.5%) and 9 of 11 eyes (81.8%) achieved complete corneal epithelial healing after 4 and 8 weeks, respectively. All 11 eyes (100%) achieved complete corneal healing after 12 weeks. There was also a significant reduction in the corneal ulcer area during each visit (P < 0.001), as well as in the corneal fluorescein staining score (P < 0.010). There was a significant improvement in corneal sensation when compared to the baseline (P < 0.050). CONCLUSION: Topical treatment with rhNGF effectively promoted the complete corneal healing of persistent epithelial defects and corneal ulcers in patients with recalcitrant NK in oGVHD and viral infection.

DALK combined intralamellar tectonic patch graft: an alternative approach to treat frank corneal perforation.

BACKGROUND: Deep anterior lamellar keratoplasty (DALK) has gained popularity in cases of corneal thinning and leaking descemetocele. In this study, we introduced an intralamellar tectonic patch graft in addition to conventional DALK procedures to treat frank cornea perforation. METHODS: This retrospective case series included 13 patients (13 eyes) with frank corneal perforations who underwent DALK combined with intralamellar tectonic patch graft between December 2015 and December 2021. In addition to the standard DALK procedure, the perforation site was repaired with an extra intralamellar tectonic patch graft. The collected data included patient demographics, aetiology, size and location of the corneal perforation, visual acuity, surgical details, and postoperative complications. RESULTS: Seven patients underwent autologous intralamellar patch grafts, whereas six received allogeneic ones. Anatomical success was achieved in all patients. The mean postoperative follow-up was 33.31 ± 25.96 months (6-73 months). The postoperative visual acuity (0.90 ± 0.65 logMAR) was significantly improved (P = 0.003) compared to the preoperative score (1.74 ± 0.83 logMAR). Best corrected visual acuity (BCVA) improved in 12 eyes (92.3%). The mean endothelial cell density was 2028 ± 463 cells/mm2, 6-12 months postoperatively. There was no recurrence of perforation, and the anterior lamellar graft remained transparent in 12 patients (92.3%). Postoperative complications included epithelial defects (23.1%), ocular hypertension (15.4%), and cataract (7.7%). CONCLUSIONS: DALK combined with intralamellar tectonic patch graft may serve as a secure and effective alternative in treating frank corneal perforation, with reduced complications compared to conventional penetrating keratoplasty.

Three-dimensional in vivo evaluation of the cornea in patients with unilateral posterior interstitial keratitis.

Purpose: The purpose of this study was to investigate the in vivo morphologic features of the cornea in patients with unilateral posterior interstitial keratitis. Methods: Seven eyes of 7 patients with unilateral posterior interstitial keratitis were examined by slit-lamp biomicroscopy, anterior segment optical coherence tomography (AS-OCT), and in vivo confocal microscopy (IVCM). The imaging features of the cornea were evaluated and analyzed. Results: By slit-lamp examination, the posterior corneal stromal opacities were observed in all 7 eyes, and deep neovascularization in 4 eyes. The posterior stromal opacities showed higher reflectivity with an intact overlying epithelium by AS-OCT and did not invade the Bowman's layer in all cases. IVCM revealed highly reflective dispersed microdots, needle-shaped bodies, and increased reflectivity of keratocytes in the lesion site in all patients. Active Langerhans cells and an attenuated subbasal nerve plexus were observed in 5 eyes. After treatment, the active Langerhans cells disappeared; however, highly reflective microdots and needle-shaped bodies remained. Conclusion: The three-dimensional evaluation of slit-lamp biomicroscopy, AS-OCT, and IVCM may help in the early diagnosis of patients with posterior interstitial keratitis.

Assessing abnormal corneal endothelial cells from in vivo confocal microscopy images using a fully automated deep learning system.

BACKGROUND: The goal of this study is to develop a fully automated segmentation and morphometric parameter estimation system for assessing abnormal corneal endothelial cells (CECs) from LASER in vivo confocal microscopy (IVCM) images. METHODS: First, we developed a fully automated deep learning system for assessing abnormal CECs using a previous development set composed of normal images and a newly constructed development set composed of abnormal images. Second, two testing sets, one with 169 normal images and the other with 211 abnormal images, were used to evaluate the clinical validity and effectiveness of the proposed system on LASER IVCM images with different corneal endothelial conditions, particularly on abnormal images. Third, the automatically calculated endothelial cell density (ECD) and the manually calculated ECD were compared using both the previous and proposed systems. RESULTS: The automated morphometric parameter estimations of the average number of cells, ECD, coefficient of variation in cell area and percentage of hexagonal cells were 257 cells, 2648 ± 511 cells/mm2, 32.18 ± 6.70% and 56.23 ± 8.69% for the normal CEC testing set and 83 cells, 1450 ± 656 cells/mm2, 34.87 ± 10.53% and 42.55 ± 20.64% for the abnormal CEC testing set. Furthermore, for the abnormal CEC testing set, Pearson's correlation coefficient between the automatically and manually calculated ECDs was 0.9447; the 95% limits of agreement between the manually and automatically calculated ECDs were between 329.0 and - 579.5 (concordance correlation coefficient = 0.93). CONCLUSIONS: This is the first report to count and analyze the morphology of abnormal CECs in LASER IVCM images using deep learning. Deep learning produces highly objective evaluation indicators for LASER IVCM corneal endothelium images and greatly expands the range of applications for LASER IVCM.

Fully automated grading system for the evaluation of punctate epithelial erosions using deep neural networks.

PURPOSE: The goal was to develop a fully automated grading system for the evaluation of punctate epithelial erosions (PEEs) using deep neural networks. METHODS: A fully automated system was developed to detect corneal position and grade staining severity given a corneal fluorescein staining image. The fully automated pipeline consists of the following three steps: a corneal segmentation model extracts corneal area; five image patches are cropped from the staining image based on the five subregions of extracted cornea; a staining grading model predicts a score for each image patch from 0 to 3, and automated grading score for the whole cornea is obtained from 0 to 15. Finally, the clinical grading scores annotated by three ophthalmologists were compared with automated grading scores. RESULTS: For corneal segmentation, the segmentation model achieved an intersection over union of 0.937. For punctate staining grading, the grading model achieved a classification accuracy of 76.5% and an area under the receiver operating characteristic curve of 0.940 (95% CI 0.932 to 0.949). For the fully automated pipeline, Pearson's correlation coefficient between the clinical and automated grading scores was 0.908 (p<0.01). Bland-Altman analysis revealed 95% limits of agreement between the clinical and automated grading scores of between -4.125 and 3.720 (concordance correlation coefficient=0.904). The average time required for processing a single stained image during pipeline was 0.58 s. CONCLUSION: A fully automated grading system was developed to evaluate PEEs. The grading results may serve as a reference for ophthalmologists in clinical trials and residency training procedures.

A Fully Automated Segmentation and Morphometric Parameter Estimation System for Assessing Corneal Endothelial Cell Images.

PURPOSE: To develop a fully automated segmentation and morphometric parameter estimation system for assessing corneal endothelial cells from in vivo confocal microscopy images. DESIGN: Artificial intelligence (neural network) study. METHODS: First, a fully automated deep learning system for assessing corneal endothelial cells was developed using the development set (from 99 subjects). Second, 184 images (from 97 subjects) were used to construct the testing set to evaluate the clinical validity and usefulness of the automated segmentation and morphometric system. Third, the automatically calculated endothelial cell density (ECD) values, Topcon's cell density, and manually calculated ECD were compared. RESULTS: After slit lamp examination, 88 healthy subjects, 2 Fuchs endothelial dystrophy patients, and 7 corneal endotheliitis patients were identified among the 97 subjects in the testing set. The automatedly estimated morphometric parameters for the testing set were an average number of 234 cells, an ECD of 2592 cells/mm2, a coefficient of variation in the cell area of 32.14%, and a percentage of hexagonal cells of 54.16%. Pearson's correlation coefficient between the automated ECD and Topcon's cell density and between the manually calculated ECD and Topcon's cell density was 0.932 (P < .01) and 0.818 (P < .01), respectively. The Bland-Altman plot of Topcon's cell density and the automated ECD yielded 95% limits of agreement between 271.94 and -572.46 (concordance correlation coefficient = 0.9). CONCLUSIONS: A fully automated method for segmenting corneal endothelial cells and estimating morphometric parameters using in vivo confocal microscopy images is more efficient and accurate for assessing the normal corneal endothelium.

Severe Corneal Edema Increases ECL From Grafts After DSAEK-Corneal Edema and ECL After DSAEK.

OBJECTIVES: To determine the relationship between the preoperative degree of corneal edema in the recipient and the endothelial cell density in grafts after Descemet stripping automated endothelial keratoplasty (DSAEK). METHODS: This retrospective case series enrolled 111 eyes of 107 patients who underwent DSAEK. The preoperative and postoperative central corneal thickness (CCT) was measured by anterior-segment optical coherence tomography. Eyes were divided into three groups according to the preoperative recipient CCT: group A (mild edema): 550 µm

A short-term study of corneal collagen cross-linking with hypo-osmolar riboflavin solution in keratoconic corneas.

AIM: To report the 3mo outcomes of collagen cross-linking (CXL) with a hypo-osmolar riboflavin in thin corneas with the thinnest thickness less than 400 µm without epithelium. METHODS: Eight eyes in 6 patients with age 26.2±4.8y were included in the study. All patients underwent CXL using a hypo-osmolar riboflavin solution after its de-epithelization. Best corrected visual acuity, manifest refraction, the thinnest corneal thickness, and endothelial cell density were evaluated before and 3mo after the procedure. RESULTS: The mean thinnest thickness of the cornea was 408.5±29.0 µm before treatment and reduced to 369.8±24.8 µm after the removal of epithelium. With the application of the hypo-osmolar riboflavin solution, the thickness increased to 445.0±26.5 µm before CXL and recover to 412.5±22.7 µm at 3mo after treatment, P=0.659). Before surgery, the mean K-value of the apex of the keratoconus corneas was 57.6±4.0 diopters, and slightly decreased (54.7±4.9 diopters) after surgery (P=0.085). Mean best-corrected visual acuity was 0.55±0.23 logarithm of the minimal angle of resolution, and increased to 0.53±0.26 logarithm after surgery (P=0.879). The endothelial cell density was 2706.4±201.6 cells/mm(2) before treatment, and slightly decreased (2641.2±218.2 cells/mm(2)) at last fellow up (P=0.002). CONCLUSION: Corneal collagen cross-linking with a hypo-osmolar riboflavin in thin corneas seems to be a promising treatment. Further study should be done to evaluate the safety and efficiency of CXL in thin corneas for the long-term.

Corneal collagen cross-linking with hypoosmolar riboflavin solution in keratoconic corneas.

PURPOSE: To report the 12-month outcomes of corneal collagen cross-linking (CXL) with a hypoosmolar riboflavin and ultraviolet-A (UVA) irradiation in thin corneas. METHODS: Eight eyes underwent CXL using a hypoosmolar riboflavin solution after epithelial removal. The corrected distance visual acuity (CDVA), manifest refraction, the mean thinnest corneal thickness (MTCT), and the endothelial cell density (ECD) were evaluated before and 6 and 12 months after CXL. RESULTS: The MTCT was 413.9 ± 12.4 µm before treatment and reduced to 381.1 ± 7.3 µm after the removal of the epithelium. After CXL, the thickness decreased to 410.3 ± 14.5 µm at the last follow-up. Before treatment, the mean K-value of the apex of the keratoconus corneas was 58.7 ± 3.5 diopters and slightly decreased (57.7 ± 4.9 diopters) at 12 months. The mean CDVA was 0.54 ± 0.23 logarithm of the minimal angle of resolution before treatment and increased to 0.51 ± 0.21 logarithm at the last follow-up. The ECD was 2731.4 ± 191.8 cells/mm(2) before treatment and was 2733.4 ± 222.6 cells/mm(2) at 12 months after treatment. CONCLUSIONS: CXL with a hypoosmolar riboflavin in thin corneas seems to be a promising method for keratoconic eyes with the mean thinnest corneal thickness less than 400 µm without epithelium.

[Immunocompetence effects of polysaccharide of snakegourd root on human peripheral blood mononuclear cells in vitro].

OBJECTIVE: To establish the method of promoting human peripheral blood mononuclear cell proliferation by polysaccharide of snakegourd root and identify the effects of polysaccharide of snakegourd root on lymphocyte proliferation, T lymphocyte subsets and the different levels of TNF-alpha and IL-6. METHOD: The polysaccharide of snakegourd root preparations were purified with dialysis and ethanol precipitation. The healthy human PBMC were used as the target cells for screening potency of the drugs. MTT colorimetry was established to examine the levels of lymphocyte proliferation on human PBMC by polysaccharide of snakegourd root in vitro. The percents of lymphocyte subsets (CD3+, CD4+, CD8+ T lymphocyte) and the different levels of TNF-a and IL-6 in PBMC were analysed by FCM and ELISA, respectively. RESULT: 1.0-50.0 mmol x L(-1) of polysaccharides of snakegourd root showed the significant effects of promoting proliferation of human PBMC (P < 0.05). The percents of CD3+, CD4+, CD8+ T lymphocytes in PBMC treated with 5.0 and 10.0 mmol x L(-1) of polysaccharides of snakegourd root were significantly higher than those of the control group (P < 0.05). The levels of TNF-alpha and IL-6 were significantly higher than those of the control group after 1.0, 5.0, 10.0 mmol x L(-1) of polysaccharides of snakegourd root stimulation on the human PBMC at 8 hours (P < 0.05). CONCLUSION: The significant effects on promoting lymphocyte proliferation and activation of the polysaccharide of snakegourd root are confirmed in this study. The percents of lymphocyte subsets are increased in different degrees by the polysaccharide of snakegourd root. The high levels of TNF-alpha and IL-6 are secreted after the polysaccharides of snakegourd root stimulation on the human PBMC, which lays a foundation for further elucidating the immunocompetence effects and mechanism of the polysaccharide of snakegourd root.

Differentiation of rabbit bone marrow mesenchymal stem cells into corneal epithelial cells in vivo and ex vivo.

PURPOSE: To examine whether bone marrow mesenchymal stem cells (MSCs) could be differentiated into corneal epithelial cells in vivo and ex vivo. METHODS: In vivo, BrdU labeled rabbit MSCs (Rb-MSCs) were suspended in the fibrin gels and transplanted onto the surface of the damaged rabbit corneas. Histology and molecular phenotype were studied on postoperative day 28. In vitro, labeled Rb-MSCs were cultured for three days in two different systems: (1) Group A: Rb-MSCs were co-cultured with rabbit limbal stem cells (Rb-LSCs) by the Transwell culture system. A suspension of Rb-LSCs was added to the upper membrane surface, and the inserts were positioned in the culture wells, which were incubated with Rb-MSCs; (2) Group B: Supernatant medium that had first been used to culture Rb-LSCs and then filtered with a 0.45 mum filter was used to culture Rb-MSCs. For both groups, immunofluorescence and flow cytometric analysis were used to examine the expression of cytokeratin 3 (CK3) in differentiated Rb-MSCs. RESULTS: In vivo, the data showed that following transplantation of Rb-MSCs, the rabbit's damaged corneal surface was successfully reconstructed and that some Rb-MSCs participated in the healing of the injured corneal epithelium and expressed CK3. In vitro, the data showed that Rb-MSCs rapidly differentiated into cells with a morphological and molecular phenotype of corneal epithelial-like cells. For both groups, the differentiated Rb-MSCs were positive for corneal epithelial-specific marker CK3. In Group A, flow cytometry analysis showed that at day one, only 3.46+/-1.9% of cells expressed CK3. This increased to 7.24+/-3.80% at day two and decreased slightly (5.50+/-3.33%) at day three. The proportion of CK3 in Group B was 4.09+/-1.84% at day one, rising to 9.31+/-5.92% after 24 h, but falling (4.37+/-2.61%) at day three. The mean differences are significant between each group and the negative control, but was not significant between Group A and Group B. CONCLUSIONS: MSCs could differentiate into corneal epithelial-like cells in vivo and ex vivo.

[Experimental research of small-incision Descemet's stripping endothelial keratoplasty].

OBJECTIVE: To investigate the surgical procedure, clinical efficacy, complications, density of endothelial cells and histological changes after Descemet's stripping endothelial keratoplasty (DSEK) surgery. METHODS: It was a experimental study. Twenty four New Zealand rabbits were divided into 3 groups, 8 rabbits per group. Donor grafts were dissected from 16 New Zealand rabbit eyes. Group A was experimental group, a 5 mm limbal tunnel incision was made. Descemet's membrane was striped off at 10 mm diameter, then the same diameter donor cornea (including Descemet's membrane and endothelium with a little of posterior stroma) was inserted into the recipient's anterior chamber. Air was injected into the anterior chamber to press the graft up against the recipient cornea. Group B was the control group, only striped the Descemet's membrane at the recipient cornea. Group C was the experiment control group, the procedure was similar to the group A, but the donor graft was without endothelial cells. RESULTS: All corneas of group A were transparent, and the mean density of the endothelial cells was (2195 +/- 77)/mm2 (t = 12.455, P < 0.001). Endothelial grafts attached to the recipients well and no scar formation between them under histological observation. The corneas were severe edema in groups B and C one month after surgery. CONCLUSIONS: DSEK is a safe surgery, can be recovered rapidly with little damages, and without interface scar formation after surgery. DSEK may be the first choice for the treatment of bullous keratopathy.

[The proapoptotic effect of the homogenate of the tissue of different parts of pig's full thickness dermal wounds on cultured fibroblasts].

OBJECTIVE: To observe the proapoptotic effect of the homogenate of different parts of pig's full thickness dermal wounds on cultured fibroblasts. METHODS: The tissues were dissected from the wound center and sub-neoepithelium separately 15 days after homogenization and sterilization, the specimens stored at -70 degrees C. The forth passage of the fibroblasts were cultured for 16 hours in different culture solutions and were grouped into 7 groups: DMEM containing 5% fetal bovine serum as Group I, DMEM containing 5% homogenate of tissue from wound center as Group II, DMEM containing 5% homogenate of tissue from sub-neoepithelium as Group III, the culture solution of Group I mixed with 10 microg/ml GM6001 in Group IV, with the culturing medium of Group III plus 10 microg/ml GM6001 as Group V, the culture solution of Group II mixed with 10 ng/ml aFGF as Group VII, and the culture solution of Group III mixed with 10 ng/ml aFGF as Group VII. In all groups except Group I, the fibroblasts of the 6 pigs were treated with the homogenate derived from the same animal respectively. After being incubated in Annexin V-FITC and PI, cells were analyzed by Flow Cytometry and the rate of apoptotic cells was acquired. The data were analyzed by SPSS 11.0 using Least-significant Difference test (LSD). RESULTS: The apoptotic rate of the 7 groups were as follows: 4.39% +/- 0.41% in Group I, 10.98% +/- 1.42% in Group II, 13.47% +/- 1.44% in Group III, 7.2% +/- 0.46% in Group IV, 12.1% +/- 0.85% in Group V, 3.9% +/- 0.63% in Group VI, 9.8% +/- 0.50% in Group VII; there were significant differences between every two groups except Group I and Group VI. CONCLUSION: Homogenate of the tissue derived from the sub-neoepithelium has greater proapoptotic effect than that from the wound center; the proapoptotic effect of homogenate of the tissue both under neoepithelium and in wound center can be significantly alleviated by acid fibroblast growth factor, partly because of MMPs.

[Isolation and purification of eccrine sweat glands in human skin].

OBJECTIVE: To investigate the method of isolation and purification of epithelial cells of human eccrine sweat gland in vitro. METHODS: Through digesting human skin with collagenase type II, cells of human eccrine sweat gland were isolated. Highly purified gland cells were obtained through transferring into the conditioned medium with a micropipette for at least three times under an inverted microscope. Primary culture was started immediately after purification. Cells could be further purified by enzyme-digestion to eradicate the fibroblasts. RESULTS: Collagenase type II could digest dermal collagen with little damage to gland cells. Isolated cells from human sweat glands were adherent to the wall of culture flask, and they grew well in cultures. The problem of contamination by tissue debris and other cells such as fibroblast could be overcome. CONCLUSION: Isolation and purification of human sweat gland cells in vitro are still facing tough problems. Gland cells are successful to isolate and cultivate without contamination.

[Culture and identification of human skin fibroblasts].

OBJECTIVE: To explore the method of isolation, cultivation, and identification of human skin fibroblasts in vitro. METHODS: By digesting human skin with collagenase type II to isolate human eccrine sweat glands. The fibroblasts grew along with the growth of eccrine sweat gland cells,and they were separated by digesting with 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA). Dulbecco's modified Eagle's medium (DMEM) was the basic culture medium, being supplemented with fetal bovine serum (10%), penicillin (100 kU/L), and streptomycin(100 mg/L) Fibroblasts were incubated at 37 centigrade in a humidified atmosphere of 5% CO2 and 95% air in the incubator. Cellular morphologies were observed by inverted phase contrast microscopy and hematoxylin-eosin staining, and the cultured cells were identified by vimentin immunostaining and chromosome analysis. RESULTS: The isolated fibroblasts could grow and proliferate in vitro, and immunostaining of vimentin was positive in cultured fibroblasts and the number of chromosome was 46. CONCLUSION: Acquired human skin fibroblasts can be cultured in stable condition in vitro, and sufficient and reliable target cells can be obtained for the study of the mechanisms of wound healing at molecular level.