Mini review: human clinical studies of stem cell therapy in keratoconus.

Treatment of keratoconus is one of the most interesting research fields for researchers in the world. Regenerative medicine based on human stem cells in the treatment of keratoconus has recently received attention. Despite extensive laboratory and animal studies in regenerative medicine of cornea, there are limited clinical studies in keratoconus. These studies showed promising results of stem cell therapy. In initial studies, the transplantation of these cells into stroma was associated with increased vision and improved corneal parameters without side effects. In this article, we tried to review different aspects of keratoconus stem cell therapy, including cell extraction and culture, surgical procedure, effectiveness and safety of this method in human clinical studies.

Diagnostic accuracy of confocal scan in detecting acanthamoeba keratitis and fungal keratitis: a systematic review and meta-analysis.

PURPOSE: Acanthamoeba keratitis (AK) and fungal keratitis (FK) are two microbial keratitis that cause serious damage and, without early accurate diagnosis and treatment, may lead to blindness. In vivo corneal confocal scan, as an emerging ocular diagnostic method in comparison with microbiological smears and cultures as the gold standard, may assist in accelerating appropriate diagnosis. OBJECTIVE: To determine the diagnostic accuracy of confocal scan for the diagnosis of AK and FK. METHODS: Data were collected via a comprehensive literature search of PubMed, Web of Science, Cochrane Library, Embase and Scopus using keywords related to diagnostic accuracy of confocal scan in AK and FK up to October 2022. Pooled data underwent meta-analysis in terms of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall diagnostic odds ratio (DOR) of confocal scan for the diagnosis of AK and FK. RESULTS: The final 14 relevant studies were identified, including 1950 eyes. Meta-analysis in AK group revealed 94% sensitivity, 87% specificity, 89% PPV, 92% NPV, and DOR of 143.32, and in FK group disclosed 88% sensitivity, 85% specificity, 85% PPV, 88% NPV, and DOR of 75.98. CONCLUSION: The accuracy of confocal scan for the diagnosis of AK was significantly more than that for detecting FK; despite the limitations such as limited numbers of available retrospective studies for the detection of FK, confocal scan had an acceptable performance in detecting FK eyes. The overall performance of NCS was similar with that of HRT-RCM for the detection of both types of keratitis.

Impression cytology for detection of clinically suspected ocular surface disorders: A cross-sectional study.

PURPOSE: Investigating impression cytology (IC) results of various types of clinically suspected ocular surface lesions over a 14-year period in a referral center in Iran. METHODS: IC findings obtained from patients with different types of ocular surface disorders between 2005 and 2018 were reviewed. Agreement between clinical suspicions and IC results was evaluated by calculating Cohen's Kappa coefficient (CKC). RESULTS: Clinical suspicions in 688 surveyed eyes were ocular surface squamous neoplasia (OSSN, 42.0%), limbal stem cell deficiency (LSCD, 36.3%), dry eye-related disorders (DERD, 11.5%), Acanthamoeba keratitis (AK, 7.2%), benign pigmented lesions (BPL, 1.9%), immune-related conjunctivitis (IRC, 0.7%), and malignant pigmented lesions (MPL, 0.4%). General agreement between clinical suspicions and IC results was 0.68 for all groups. This agreement was almost perfect in AK (CKC = 0.966) and BPLs (CKC = 0.843), and was substantial in MPLs (CKC = 0.749), OSSNs (CKC = 0.684), and LSCD (CKC = 0.612). CKC in IRC (0.567) and DERDs (0.443) was moderate. Histopathologic results were available in 22 eyes and were well-correlated with corresponding IC results (CKC = 0.86). Multiple post-treatment follow-up sessions of IC were performed in 51 eyes (11.4%) that had diagnosis of LSCD (31), OSSN (17), and MPL (3) at the first IC session. CONCLUSION: Our survey not only demonstrated an overall substantial agreement between IC results and primary clinical suspicions, but also showed an almost perfect correlation between IC results and existent histopathologic data. Therefore, IC as a non-invasive diagnostic modality can be of great importance in proper diagnosis of various ocular surface diseases especially when distinguishing malignant from benign lesions is required.

A mutation in DOP1B identified as a probable cause for autosomal recessive Peters anomaly in a consanguineous family.

Purpose: Peters anomaly (PA) is a heterogeneous developmental disorder characterized by central corneal opacity and iridocorneal or corneolenticular adhesions. Although many causative genes have been identified, most screened patients do not have mutations in the known genes. We aimed to identify the genetic cause of Peters anomaly in a pedigree with three affected individuals. Methods: Slit-lamp biomicroscopy and ultrasound biomicroscopy were performed for definitive diagnosis. Exome sequencing was conducted on the DNA of all three patients. After identification of a candidate causative gene, expression of the gene was assessed with real-time PCR in various ocular tissues of three human embryos and three adults. Results: The patients were affected with isolated PA. The parents of the patients were related to one another. Inheritance of PA was autosomal recessive. After appropriate filtering of the exome data, a homozygous variation in DOP1B remained as the only candidate genetic cause of PA in the pedigree. The variant segregated with disease status in the pedigree and was absent among 800 control Iranians. The variant has been reported in various databases at frequencies of 0.006 or less only in the heterozygous state in some cohorts of African origin. The p.Val1660 amino acid affected by the mutation is completely conserved in mammals and birds during evolution. Expression of DOP1B was shown in all adult and embryonic lens, iris, cornea, sclera, and retina tissues that were tested. Conclusions: DOP1B that encodes DOP1 leucine zipper like protein B was identified as the putative PA-causing gene in pedigree PA-101. As DOP1B is positioned within the Down syndrome chromosomal region on chromosome 21, until now this gene has mostly been studied with respect to brain functions. However, members of the Dopey gene family have been shown to have roles in development in other organisms. Evidence of the expression of DOP1B in various PA-relevant eye tissues, which, to the best of our knowledge, is shown here for the first time, is to be noted. However, this finding does not necessarily implicate a specific role for DOP1B in eye development as the gene is expressed in many tissues. Ultimately, definitive assessment of the contribution of DOP1B to PA pathology awaits identification of mutations in the gene in unrelated patients with PA and functional studies.

Comparison of toluidine blue 1% staining patterns in cytopathologically confirmed ocular surface squamous neoplasias and in non-neoplastic lesions.

PURPOSE: To evaluate the role of toluidine blue (TB) staining patterns in diagnosis of ocular surface squamous neoplasia (OSSN) in comparison to that of impression cytology. METHODS: TB 1% dye was applied to different ocular surface lesions, followed by impression cytology (IC). Dye distribution, intensity, and pattern of uptake by the lesion were scored and total score ≥5 was considered "positive TB staining". The TB results were then compared with those using IC to determine the presence of cellular atypia. RESULTS: The study enrolled 88 eyes of 82 patients. IC demonstrated cellular atypia in 50 (56.8%) cases. Forty-three of 45 "TB-positive" eyes (95.51%) had cellular atypia on IC (p < 0.001). Sensitivity and specificity of TB in identifying OSSN were 86% and 94.74%, respectively, with positive and negative predictive values of 95.56% and 83.72%. TB staining intensity of dark blue and/or mixed types and stippled pattern of TB staining were strongly correlated with dysplastic changes in IC (P ˂ 0.001). TB staining distribution whether in form of diffuse, patchy or scattered eyes with atypia did not significantly differ from those without atypia in IC (P = 0.172). CONCLUSION: The sensitivity and specificity of TB vital dye in diagnosing OSSN can be increased by focusing on color intensity and a stippled pattern.

Microbiological Evaluation of Bandage Soft Contact Lenses Used in Management of Persistent Corneal Epithelial Defects.

PURPOSE: To investigate the rate and agents of contamination in bandage soft contact lenses fitted for management of persistent corneal epithelial defects. METHODS: This prospective comparative case series enrolled 57 consecutive eyes fitted with bandage contact lenses for treatment of persistent corneal epithelial defects. The lenses were collected at the time of epithelial closure or when it was necessary to exchange contact lenses and were immediately placed in sterile tubes containing an enriched thioglycolate liquid medium. When contamination of the contact lens was detected, the microorganism was cultured in different media and identified based on various tests. All isolates were tested for susceptibility to various antibiotics. Univariate analyses were used to evaluate the influence of different variables (duration of contact lens use, use of topical corticosteroids, presence of blepharitis, contact lens deposits, and presence of sutures) on bandage contact lens contamination. RESULTS: Seventeen of the contact lenses (29.8%) were contaminated. The most commonly isolated pathogen was Staphylococcus epidermidis (n = 10), followed by Enterobacter cloacae (n = 3), Staphylococcus aureus (n = 1), Streptococcus viridans (n = 1), and Alcaligenes spp. (n = 1). One contact lens yielded a mixed infection with E. cloacae and Candida spp. Infectious keratitis was not observed in any eyes. Correlations between contact lens contamination and patient- and lens-related variables were not statistically significant. CONCLUSIONS: Most bandage contact lenses (70.2%) used for treatment of persistent corneal epithelial defects did not show bacterial growth. Staphylococcus epidermidis was the most common microorganism isolated from the contaminated contact lenses.

Factors Influencing Graft Endothelial Cell Density after Descemet Stripping Automated Endothelial Keratoplasty.

PURPOSE: To determine the factors that influence the endothelial cell density (ECD) of donor grafts after Descemet stripping automated endothelial keratoplasty (DSAEK). METHODS: This retrospective, interventional case series comprised 77 eyes of 64 patients who underwent DSAEK. Confocal microscopy was performed at the final follow-up examination to evaluate the endothelial cell count, cell morphology, and graft thickness. Univariate and multiple linear regression analyses were used to investigate recipient-, donor-, surgical-, and postoperative related variables capable of influencing graft endothelial cell counts after DSAEK. RESULTS: The mean patient age was 62.3 ± 15.6 years; patients were followed-up for 26.2 ± 20.9 months postoperatively. Forty-six eyes (59.7%) underwent stand-alone DSAEK; 31 eyes (40.3%) underwent DSAEK combined with cataract surgery. The donor trephination size was 8.0 ± 0.21 mm. The mean donor age was 30.4 ± 11.2 years, and the mean preoperative endothelial cell density was 3127.4 ± 315.1 cells/mm2, which decreased to 1788.6 ± 716.5 cells/mm2 postoperatively (P < 0.001). The mean postoperative central graft thickness was 102.4 ± 31.6 µm. Univariate analysis revealed that postoperative ECD was significantly associated with death to preservation time (P = 0.046), graft thickness (P = 0.016), follow-up duration (P = 0.005), and graft non-attachment (P = 0.049). Multiple regression analyses identified graft thickness (ß = 10.62, P = 0.003) and follow-up duration (ß = -22.09, P = 0.001) as the significant characteristics influencing postoperative ECD. CONCLUSION: The primary predictors of ECD after DSAEK were graft thickness and duration of follow-up. Surgeons' requests for ultrathin DSAEK donor grafts to improve visual outcomes might not have the desired postoperative outcome with respect to ECD.

Ocular Safety of Intravitreal Connective Tissue Growth Factor Neutralizing Antibody.

PURPOSE: To detect the safety of intravitreal injection of anti-connective tissue growth factor (CTGF) (IVAC) in rat eyes in order to apply this neutralizing antibody for experimental animal studies. METHODS: Forty-five Lister Hooded male pigmented rats were divided into five groups that received IVAC (2 µl) corresponding to the doses of 10 (B), 20 (C), 50 (D), and 100 µg/ml (E), equal to 1.25, 2.5, 6.25, and 12.5 µg/ml of antibody concentration in rat vitreous, respectively. The sham group (A) received 2 µl of normal saline. Full field electroretinography (ERG) was performed at baseline and on days 7 and 28 after IVAC. The animals were euthanized and the corresponding eyes were subjected to routine histopathology, immunohistochemistry for glial fibrillary acidic protein (GFAP), and terminal transferase dUTP nick end-labeling (TUNEL) assay. RESULTS: Scotopic rod b-wave amplitude and maximal combined b-wave amplitude were 111.89 ± 71.2 and 178.57 ± 55.58 µV, respectively, at baseline which significantly reduced to 79.31 ± 52.59 and 128.73 ± 41.61 µV, respectively, after 28 days in group E (p < 0.05). There was no significant reduction of amplitudes in other groups with lower doses of anti-CTGF antibody. Retinal ganglion cells were significantly decreased in group E as compared to other groups. GFAP immune reactivity was not significant in any of the groups. TUNEL test showed inner retinal neural cell apoptosis only in group E. CONCLUSIONS: ERG, histopathologic, and apoptotic assays revealed no toxic effects of 10-50 µg/ml of IVAC in rat eyes. Using 100 µg/ml IVAC led to a significant toxic effect in terms of functional, histopathologic, and TUNEL findings.

Impression Cytology in a Series of Clinically Diagnosed Ocular Surface Melanocytic Lesions.

PURPOSE: To report impression cytology (IC) results of clinically diagnosed ocular surface melanocytic lesions. METHODS: Ten patients with a clinical diagnosis of an ocular surface melanocytic lesion underwent IC using cellulose acetate strips and Periodic acid Schiff-Papanicolaou staining. Excisional biopsy of lesions was performed in case of observing atypical cells on IC or at the patient's request, and excised specimens were subjected to histopathological analysis. Agreement between clinical diagnoses and IC results and between IC results and histopathology were evaluated. RESULTS: Clinical diagnoses were nevi in 6, primary acquired melanosis (PAM) with atypia/melanoma in 2, and atypical nevus versus pigmented conjunctival intraepithelial neoplasia (CIN) in 2 cases. IC results were suggestive of a benign nevus in 7, PAM with atypia/melanoma in 2 and CIN versus an atypical epithelioid type melanocytic lesion in 1 case. IC results were consistent with the clinical diagnoses in 9 cases (Cohen's kappa index of 0.83) and excluded CIN in 1. Histopathology in 6 cases disclosed benign melanonevus in 3, malignant melanoma in the context of PAM with atypia in 2, and CIN in 1 case. Histologic results were well correlated with the IC features (Cohen's kappa index of 0.74). CONCLUSION: By demonstrating typical cytomorphological features of ocular superficial layers IC diagnosed the true nature of melanocytic ocular surface lesions in the majority of cases. Although IC does not substitute histopathology, given the high correlation between IC results and histopathology, it can be of great assistance in diagnosis and management of ocular surface melanocytic lesions.

Retinal Toxicity of Intravitreal Granulocyte Colony-Stimulating Factor in Rabbit Eyes.

PURPOSE: Granulocyte colony-stimulating factor (G-CSF) has potential ocular neuroprotective effects. The aim of this study was to evaluate the retinal toxicity of intravitreal G-CSF in rabbit eye. METHODS: Eight New Zealand albino rabbits, weighing between 2 and 3 kg, were selected for this study. The initial concentration of G-CSF (300 µg/0.5 ml) was titrated to obtain different concentrations of 45 µg, 30 µg, 15 µg, and 7.5 µg in 0.1 ml. Each concentration was injected into two rabbit eyes. For each dose, dextrose was injected in one contralateral eye and the other fellow eye remained non-injected. Electroretinographic (ERG) testing was performed before and 4 weeks after injections. The rabbits were euthanized and the eyes were enucleated 4 weeks after injections and examined using light microscopy and immunohistochemistry. RESULTS: One rabbit with the injected dosage of 7.5 µg died at the first post-injection day. No sign of intraocular toxicity was found in clinical examination in other rabbits. A significant decrease in at least one of the a- or b-wave measurements of scotopic or photopic responses was found in 45 µg, 15 µg, and 7.5 µg injected eyes. Eyes with an intravitreal injection dosage of 30 µg G-CSF did not have significant changes compared to the baseline values. Histologic and immunohistochemistric studies were unremarkable for pathologic changes in all injected eyes. CONCLUSION: While histologic and immunohistochemistric examinations revealed no toxicity in all G-CSF-injected eyes, significant ERG changes were observed in all doses except for the dose of 30 µg/0.1 ml.

Cultivation of Retinoblastoma Cells: Correlation Between In Vitro Growth Pattern and Histopathology.

PURPOSE: To investigate the correlation between floating versus adherent growth pattern of cultivated retinoblastoma (RB) cells from three patients with RB and their histopathologic features. METHODS: RB cells from three Iranian patients (MM, NR, and MS) were cultivated in Dulbecco's modified eagle medium (DMEM) supplemented with 15% fetal bovine serum (FBS) for four weeks in each passage. Fresh medium was added on a weekly basis and immunocytochemistry for Synaptophysin was performed. All experiments were performed in duplicate. Growth pattern of the cultivated RB cells was studied during the three consecutive passages and compared among three cases in the light of histopathologic data. RESULTS: Cultivated RB cells from MM and NR demonstrated an adherent growth pattern in the 2nd week and the pattern was enhanced by the 4th week. The RB tumorspheres adhered to the bottom of the flask while surrounded by fibroblasts. Histopathologic diagnosis in MM and NR was a well-differentiated RB without optic nerve involvement. Such an adherent growth was not observed in cultivated RB cells from MS, in which the histopathologic analysis revealed a poorly-differentiated RB with optic nerve intrusion and prominent choroidal invasion. CONCLUSION: The adherent growth pattern of cultivated RB cells might be associated with tumor differentiation and the lack of optic nerve involvement in histopathology.

Determination of the toxicity of intravitreal minocycline in rabbit eyes.

PURPOSE: To evaluate the retinal toxicity of intravitreal minocycline in rabbit eyes. METHODS: Intravitreal injection of minocycline with concentrations of 1000, 500, 250, 125 and 62.5 µg in 0.1 ml was performed in 10 New Zealand albino rabbits. Each concentration was injected into two rabbit eyes. For each dose, normal saline was injected in one contralateral eye and the other fellow eye remained non-injected. Electrophysiologic testing was performed before and 4 weeks after injections. The eyes were enucleated 4 weeks after injections and examined using light microscopy. RESULTS: The clinical examination was unremarkable after injections. Electroretinography recordings were significantly affected at all doses in at least one of the a- or b-waves of photopic or scotopic responses. Histopathologic examination revealed marked atrophy and loss of integrity in all retinal layers in all minocycline injected eyes. Contralateral eyes were normal. CONCLUSION: In our study, intravitreal minocycline was toxic to the retina in albino rabbits even at a concentration of 62.5 µg/0.1 ml.

Safe dose of intravitreal imatinib and its effect on laser-induced choroidal neovascularization: a rat-model experiment.

BACKGROUND: This two-phase experimental study was conducted to determine the maximum safe dose of intravitreal imatinib (IVI) and its inhibitory effect on a rat model of choroidal neovascularization (CNV). METHODS: In phase I, 60 rats were divided into six groups (A to F); five of which received IVI with concentrations of 330 (A), 250 (B), 165 (C), 80 (D), and 40 (E) µg/5 µl, and the control group (F) received balanced salt solution (BSS). In addition to electroretinography (ERG), routine histopathological analysis and immunohistochemistry for glial fibrillary acidic protein were performed. In phase II, CNV was induced by laser photocoagulation in 25 rats and the animals were divided into two groups. One group received the maximum safe dose of IVI, determined in phase I, and the other received intravitreal BSS. After 4 weeks, the groups were compared in terms of mean scores of fluorescein leakage in fluorescein angiography and the mean CNV areas in histopathological sections. RESULTS: In phase I, ERG and the histopathological findings revealed retinal toxicity in groups A to D and A to C, respectively; therefore, a dose of 40 µg/5 µl imatinib was specified as the maximum safe dose for phase II. In phase II, late phase fluorescein leakage and the CNV areas were not significantly different between the imatinib-treated eyes and the controls (p = 0.62 and p = 0.5, respectively). CONCLUSIONS: Despite the safety of IVI with a dose of 40 µg/5 µl, no inhibitory effect on laser-induced CNV was observed. Further studies are required to investigate the possible synergistic effects of Imatinib with conventional anti-CNV drugs.

Intravitreal Topotecan Inhibits Laser-induced Choroidal Neovascularization in a Rat Model.

PURPOSE: A two-phase preclinical study was designed to determine the safe dose of intravitreal topotecan and its inhibitory effect on experimental choroidal neovascularization (CNV) in a rat model. METHODS: In phase I, 42 rats were categorized into 6 groups, 5 of which received intravitreal topotecan injections of 0.125 µg, 0.25 µg, 0.5 µg, 0.75 µg, and 1.0 µg/5 µl, respectively; the control group received an injection of normal saline. Ophthalmic examination and electroretinography (ERG) were performed on days 7 and 28, and enucleated globes were processed for histopathology and immunostaining for glial fibrillary acidic protein. In phase II, CNV was induced via laser burns in 20 rats and the animals were divided into 2 groups. One group received topotecan and the other received normal saline intravitreally. Four weeks later, mean scores of fluorescein leakage on fluorescein angiography as well as mean CNV areas on histology sections were compared. RESULTS: In phase I, clinical, ERG and histopathologic results were unremarkable in terms of retinal toxicity in all groups. Based on the results of phase I, a dose of 1 µg/5 µl topotecan was chosen for phase II. Leakage scores obtained from late-phase fluorescein angiography were significantly lower in topotecan-treated than control eyes (P < 0.01) four weeks after induction of CNV. Compared to control eyes, topotecan-treated eyes showed a significantly lower incidence of fibrovascular proliferation (8.7% vs. 96.2%) and significantly smaller areas of CNV (P < 0.01). CONCLUSION: Intravitreal injection of topotecan at a dose of 1 µg/5 µl is safe and may be a promising treatment for CNV.

Donor Descemet-off versus Descemet-on deep anterior lamellar keratoplasty: a confocal scan study.

PURPOSE: To compare confocal features of grafts following deep anterior lamellar keratoplasty (DALK) using a donor without Descemet membrane (DM) versus a full-thickness donor with intact DM and endothelium. METHODS: This retrospective comparative study examined 45 eyes from patients with keratoconus who underwent DALK using the big-bubble technique. The big-bubble technique yielded a bared DM in all keratoconic eyes. Twenty-seven eyes received tissue from a donor without DM (group 1), while 18 received tissue from a full-thickness donor with an intact DM and endothelium (group 2). A group of normal eyes (n = 28, group 3) served as controls. Confocal microscopy was used to determine keratocyte density, explore the donor-recipient interface including clarity and reflectivity, evaluate endothelial cell density and morphology, as well as measure interface depth and central corneal thickness. RESULTS: Mean follow-up duration was 20.2 ± 8.6 months and 29.6 ± 17.0 months in groups 1 and 2, respectively (p = 0.13). Confocal scan demonstrated that the keratocyte profiles and distribution were more similar to normal corneas in group 2. Significantly more severe interface haziness was observed when donor DM and endothelium was retained (mean interface reflectivity value of 102.7 ± 22.1 versus 161.7 ± 30.0 light reflectance units in groups 1 and 2, respectively, p<0.001). CONCLUSIONS: Graft cellular profiles and healing response at the donor-recipient interface can be profoundly affected depending on whether donor DM and endothelium is removed or retained.