Exome sequencing and genome-wide association analyses unveils the genetic predisposition in hydroxychloroquine retinopathy.

OBJECTIVES: To unveil the candidate susceptibility genes in chloroquine/hydroxychloroquine (CQ/HCQ) retinopathy using whole exome sequencing (WES) and genome-wide association study (GWAS). METHODS: Patients with a diagnosis of CQ/HCQ retinopathy based on the comprehensive demographic and ocular examination were included. The peripheral blood was extracted for WES and GWAS analyses. The Chinese Han Southern database from 1000 genomes was used as control group to compare the affected percentage. Multivariate logistic regression analysis adjusted for age, HCQ dose, duration and renal disease were used to analyze the correlation between genetic variants and visual outcome. A poor vision outcome was defined as visual acuity <6/12. An abnormal anatomical outcome was defined as disruption of ellipsoid zone in the fovea. RESULTS: Twenty-nine patients with an average age of 60.9 ± 13.4 years, treatment duration of 12.1 ± 6.2 years, daily dose of 8.5 ± 4.1 mg/kg, and the cumulative dose of 1637.5 ± 772.5 g, were genotyped. Several candidate genes associated with CQ/HCQ retinopathy were found, including RP1L1, RPGR and RPE65, with a difference of affected percentage over 50% in mutation between the case and control groups. New foci in CCDC66: rs56616026 (OR = 63.43, p = 1.63 × 10-8) and rs56616023 (OR = 104.7, p = 5.02 × 10-10) were identified significantly associated with HCQ retinopathy. Multivariate analysis revealed increased genetic variants were significantly associated with poor functional (OR = 1.600, p = 0.004) and structural outcome (OR = 1.318, p = 0.043). CONCLUSIONS: Several candidate susceptibility genes including RP1L1, RPGR, RPE65 and CCDC66 were identified to be associated with CQ/HCQ retinopathy. In addition to disease susceptibility, patients with increased genetic variants are more vulnerable to poor visual outcomes.

Dry eye syndrome model established in rabbits via mitomycin C injection in the lacrimal gland.

PURPOSE: To develop a new dry eye syndrome (DES) animal model by injecting mitomycin C (MMC) into the lacrimal glands (LGs) of rabbits evaluated by clinical examinations. MATERIALS AND METHODS: A volume of 0.1 mL of MMC solution was injected in the LG and the infraorbital lobe of the accessory LG of rabbits for DES induction. Twenty male rabbits were separated into three groups, the control group, and different concentration of MMC, (MMC 0.25: 0.25 mg/mL or MMC 0.50: 0.5 mg/mL) were tested. Both MMC-treated groups received MMC twice injection on day 0 and day 7. Assessment of DES included changes in tear production (Schirmer's test), fluorescein staining pattern, conjunctival impression cytology, and corneal histological examination. RESULTS: After MMC injection, no obvious changes in the rabbit's eyes were noted by slit-lamp examination. Both the MMC 0.25 and the MMC 0.5 groups revealed decreased tear secretion after injection, and the MMC 0.25 group showed a continuous decrease in tear secretion up to 14 days. Fluorescent staining showed punctate keratopathy in both MMC-treated groups. In addition, both MMC-treated groups demonstrated decreased numbers of conjunctival goblet cells after injection. CONCLUSION: This model induced decreased tear production, punctate keratopathy, and decreased numbers of goblet cells, which are consistent with the current understanding of DES. Therefore, injecting MMC (0.25 mg/mL) into the LGs is an easy and reliable method to establish a rabbit DES model which can apply in new drug screening.

Overnight orthokeratology-associated Acanthamoeba keratitis at a tertiary referral hospital in Taiwan: A retrospective case-control study.

BACKGROUND: Acanthamoeba keratitis (AK) is a vision-threatening disease, usually associated with contact lens (CL) wear. As overnight orthokeratology (OOK) is increasingly used to control myopia, we have found incidence of OOK-associated AK is increasing. This study aimed to investigate the clinical presentation and visual outcomes of OOK-associated AK. METHODS: Demographic characteristics, clinical features, and treatment outcomes were collected by reviewing the medical charts of CL-associated AK patients (n = 35) diagnosed at Taipei Veterans General Hospital from 2001 to 2016. Cases were OOK-associated AK patients (n = 13), and controls were all other CL-associated AK patients (n = 22). Student t tests and chi-square tests were used to compare cases and controls. Linear regression analyses were used to identify factors associated with the final visual outcome in CL-associated AK. RESULTS: OOK-associated AK accounted for half of all CL-associated AK after 2010. OOK-associated AK patients and other CL-associated patients had similar best-corrected logarithm of the minimum angle of resolution visual acuity (BCLVA) before treatment (1.10 ± 0.75 vs 1.13 ± 0.76, p = 0.893), but OOK-associated AK patients were younger (17.15 ± 3.21 vs 26.36 ± 12.81 years, p = 0.004), had less severe disease (ring infiltration, 0% vs 31.82%, p = 0.023), and had better post-treatment BCLVA (0.06 ± 0.15 vs 0.51 ± 0.95, p = 0.041). Multiple linear regression analysis showed that better BCLVA after treatment in CL-associated AK was associated with initial presentation without ring infiltration (p = 0.002) but not with OOK use itself (p = 0.793). Twenty-six of 35 CL-associated AK patients had final BCLVA equal to or better than 0.10 (Snellen visual acuity of 6/7.5). All 13 OOK-associated AK cases were treated with chlorhexidine 0.02% ± voriconazole 1% ± oral voriconazole, and 12 of these patients had final BCLVA equal to or better than 0.10. CONCLUSION: Most CL-associated AK patients had satisfactory visual outcomes. Half of AK at our hospital is OOK-associated since 2010. Early diagnosis and correct treatment may be the reason why OOK-associated AK patients had better vision prognosis.

Bilateral corneal edema in an alcoholic male.

Pseudophakic bullous keratopathy and Fuchs' endothelial dystrophy are the two most common causes of corneal edema after cataract surgery. We report a 61-year-old alcoholic male with bilateral corneal edema that improved after his alcohol abstinence. He had uneventful bilateral cataract surgery 3 years ago and blurred vision in both eyes developed for weeks. As he had no history of endothelial dystrophy, the treatment for viral endotheliitis was used initially yet in vain. We asked him to stop alcohol and adjusted his psychiatric drugs, but he lied about stopping drinking. The corneal edema progressed, and finally, he underwent penetrating keratoplasty in his left eye 1 year later. During hospitalization for surgery, alcohol withdrawal syndrome was noted because he could not drink alcohol in our hospital. After he quit drinking for months, corneal edema in the right eye disappeared. Our case highlights that alcoholism can result in corneal edema, and stopping drinking is necessary in these patients.

Comparison of the iCare, Tono-Pen, non-contact airpuff, and Goldmann applanation tonometers in eyes with corneal edema after penetrating keratoplasty.

BACKGROUND: To compare the utility of the iCare, Tono-Pen, and non-contact airpuff (NCT) tonometers with the Goldmann applanation tonometer (GAT) for measuring intraocular pressure (IOP) in patients with corneal edema after penetrating keratoplasty (PKP) and to assess the effects of central corneal thickness (CCT) and corneal curvature (CC) on IOP measurements. METHODS: Thirty-two eyes of 27 patients with corneal edema after PKP due to corneal abnormalities and 43 control eyes of 30 patients with normal corneas were recruited. Before IOP measurements, all patients underwent a baseline examination, including auto-refraction, keratometry, slit lamp biomicroscopy, and CCT measurement. IOP was measured using the devices in the same order: first the NCT, followed by the iCare, Tono-Pen, and GAT. The differences between the iCare, Tono-Pen, NCT, and GAT were calculated with repeated-measures analysis of variance. The Bland-Altman method was used to assess the agreement between the iCare, Tono-Pen, and NCT versus the GAT. The influences of CCT and CC on IOP measurement were evaluated by correlation analysis using Pearson's correlation coefficient. RESULTS: Mean IOP measurements were significantly higher with the NCT and Tono-Pen than with the GAT in the PKP and control groups. When compared with GAT, iCare showed significantly higher IOP readings in the control group, but the IOP readings did not differ between the iCare and GAT in the PKP group. Poor agreement was noted between the NCT and GAT in both groups. The Tono-Pen showed clinically acceptable agreement with GAT in control eyes and poor agreement in PKP eyes. The agreement between the iCare and GAT appeared to be clinically acceptable in both groups. Correlation analysis of the results from control eyes showed that the IOP measurements with the GAT and NCT were weakly related to CCT and moderately correlated with CC. The iCare IOP readings were weakly correlated with CCT and CC. CONCLUSION: In the PKP group, the NCT and Tono-Pen significantly overestimated IOP, whereas the iCare IOP readings were similar to those obtained using the GAT. Poor agreement was noted between the NCT and GAT as well as between the Tono-Pen and GAT, but the iCare showed clinically acceptable agreement with GAT. In normal corneas, the GAT, NCT, and iCare were affected by CCT and CC. The iCare tonometer was less affected by corneal edema than were the NCT and the Tono-Pen. The iCare appears to be a useful device for IOP measurement in eyes with corneal edema after PKP.

Topical Application of Hyaluronic Acid-RGD Peptide-Coated Gelatin/Epigallocatechin-3 Gallate (EGCG) Nanoparticles Inhibits Corneal Neovascularization Via Inhibition of VEGF Production.

Neovascularization (NV) of the cornea disrupts vision which leads to blindness. Investigation of antiangiogenic, slow-release and biocompatible approaches for treating corneal NV is of great importance. We designed an eye drop formulation containing gelatin/epigallocatechin-3-gallate (EGCG) nanoparticles (NPs) for targeted therapy in corneal NV. Gelatin-EGCG self-assembled NPs with hyaluronic acid (HA) coating on its surface (named GEH) and hyaluronic acid conjugated with arginine-glycine-aspartic acid (RGD) (GEH-RGD) were synthesized. Human umbilical vein endothelial cells (HUVECs) were used to evaluate the antiangiogenic effect of GEH-RGD NPs in vitro. Moreover, a mouse model of chemical corneal cauterization was employed to evaluate the antiangiogenic effects of GEH-RGD NPs in vivo. GEH-RGD NP treatment significantly reduced endothelial cell tube formation and inhibited metalloproteinase (MMP)-2 and MMP-9 activity in HUVECs in vitro. Topical application of GEH-RGD NPs (once daily for a week) significantly attenuated the formation of pathological vessels in the mouse cornea after chemical cauterization. Reduction in both vascular endothelial growth factor (VEGF) and MMP-9 protein in the GEH-RGD NP-treated cauterized corneas was observed. These results confirm the molecular mechanism of the antiangiogenic effect of GEH-RGD NPs in suppressing pathological corneal NV.

Fish-Scale Collagen Membrane Seeded with Corneal Endothelial Cells as Alternative Graft for Endothelial Keratoplasty Transplantation.

The human corneal endothelium has limited regeneration capacity. Several methods have been developed in an attempt to repair it. Descemet stripping automated endothelial keratoplasty (DSAEK) is commonly performed on patients with endothelial dysfunction. However, donor demand far exceeds donor supply. Here, we prepared fish-scale collagen membrane (FSCM) and seeded it with CECs in preparation for corneal endothelial transplantation. The fish scales were decellularized, decalcified, and curved. The FSCM was inspected by fluorescence microscopy, SEM, and TGA to validate decellularization, microstructure, and decalcification, respectively. The cytotoxicity of FSCM and the viability of the cells in contact with it were evaluated by LDH and WST-1, respectively. CEC tight junctions and ZO-1 structure were observed by SEM and confocal microscopy. FSCM seeded with CECs were implanted to rabbit anterior chambers to evaluate host tissue reactions to it. FSCM biocompatibility and durability were also assessed. The results showed that FSCM has excellent transparency, adequate water content, and good biocompatibility. The cultivated CECs mounted on the FSCM were similar to normal CECs in vivo. The FSCM plus CECs developed here have high potential efficacy for endothelial keratoplasty transplantation.

Development of Kaempferol-Loaded Gelatin Nanoparticles for the Treatment of Corneal Neovascularization in Mice.

Cornea is the transparent layer in front of the eye that does not contain blood vessels. Among eye diseases, corneal neovascularization (NV) is one of the major causes of vision loss, since it can also lead to blindness. An herbal extraction containing flavonoid, kaempferol (KA), with antiangiogenic effect was chosen as a candidate drug for inhibited vessel formation. The use of nanomedicine has led to higher drug bioavailability and slow release of the drug as an effective therapeutic formulation in ocular drug delivery. In this study, we prepared gelatin nanoparticles (GNP) with kaempferol encapsulation (GNP-KA) for corneal NV treatment by topical delivery, i.e., eye drops. We found that GNP with/without KA loading was in the size of 85-150 nm, and its zeta potential was around 22-26 mV. The KA entrapment rate of GNP-KA was around 90-98%, and the loading rate was about 4.6%. The TEM results clearly indicated the GNP-KA NPs to be round spheres. The in vitro test involved the adoption of human umbilical vein endothelial cells (HUVECs) for coculture with these nanoparticles. From WST-8 assay, and cell migration examinations, it was evident that GNP-KA had the capacity to inhibit the cell viability and function of HUVECs. The results from in vivo tests such as ocular vessels observation, hematoxylin & eosin (H&E) stain, and metalloproteinases (MMP)/vascular endothelial growth factor (VEGF) quantification revealed the mice's eyes with corneal NV treated by eye drops containing GNP-KA once daily for 7 days had better therapeutic effects with less vessels in-growths in the cornea, compared to the KA solution group by reducing the production of MMP and VEGF in the cornea. Therefore, we expected to achieve a comfortable treatment with a simple method using nanomedicine (GNP-KA) as ophthalmological agent delivered as eye drops.

Dry Eye Disease: A Review of Epidemiology in Taiwan, and its Clinical Treatment and Merits.

Dry eye disease (DED) has become common on a global scale in recent years. There is a wide prevalence of DED in different countries based on various ethnicities and environment. DED is a multifactorial ocular disorder. In addition to advanced age and gender, such factors as living at high altitude, smoking, pterygium, prolonged use of consumer electronics or overingesting of caffeine or multivitamins are considered to be the major risk factors of DED. We report the DED epidemiology in Taiwan firstly in this article. According to the pathophysiological factors and changes inthe composition of the tear film in DED, it can be categorized into several subtypes, including lipid anomaly dry eye, aqueous tear deficiency, allergic and toxic dry eye among others. Each subtype has its own cause and disease management; therefore, it is important for ophthalmologists to identify the type through literature review and investigation. The management of DED, relies not only on traditional medications such as artificial tears, gels and ointments, but also newer treatment options such as acupuncture, SYL1001, and nanomedicine therapy. We also conducted a comprehensive literature review including common subtypes and treatment of DED. Clearly, more clinical trials are needed to assess the efficacy and safety of the various treatments and common subtypes of DED.

Gelatin-epigallocatechin gallate nanoparticles with hyaluronic acid decoration as eye drops can treat rabbit dry-eye syndrome effectively via inflammatory relief.

INTRODUCTION: Dry-eye syndrome (DES) is a general eye disease. Eye drops are the common ophthalmological medication. However, the ocular barrier makes it difficult to attain high drug bioavailability. Nanomedicine is a promising alternative treatment for ocular diseases and may increase drug content in the affected eye. METHODS: To explore this potential, we constructed nanoparticles (NPs) containing an anti-inflammatory agent for DES treatment. The NPs were made of gelatin-epigallocatechin gallate (EGCG) with surface decoration by hyaluronic acid (HA) and designated "GEH". The particle size, surface charge, and morphology were evaluated. The in vitro biocompatibility and anti-inflammation effect of nanoparticles were assayed via culturing with human corneal epithelium cells (HCECs) and in vivo therapeutic effect was examined in a DES rabbit's model. RESULTS: The synthesized GEH NPs had a diameter of approximately 250 nm and were positively charged. A coculture experiment revealed that 20 µg/mL GEH was not cytotoxic to HCECs and that an EGCG concentration of 0.2 µg/mL downregulated the gene expression of IL1B and IL6 in inflamed HCECs. Large amounts of GEH NPs accumulated in the cytoplasm of HCECs and the ocular surfaces of rats and rabbits, indicating the advantage of GEH NPs for ocular delivery of medication. Twice-daily topical treatment with GEH NPs was performed in a rabbit model of DES. The ocular surface of GEH-treated rabbits displayed normal corneal architecture with no notable changes in inflammatory cytokine levels in the cornea lysate. The treatment improved associated clinical signs, such as tear secretion, and fluorescein staining recovered. CONCLUSION: We successfully produced GEH NPs with high affinity for HCECs and animal eyes. The treatment can be delivered as eye drops, which retain the drug on the ocular surface for a longer time. Ocular inflammation was effectively inhibited in DES rabbits. Therefore, GEH NPs are potentially valuable as a new therapeutic agent delivered in eye drops for treating DES.

Preparation of arginine-glycine-aspartic acid-modified biopolymeric nanoparticles containing epigalloccatechin-3-gallate for targeting vascular endothelial cells to inhibit corneal neovascularization.

Neovascularization (NV) of the cornea can disrupt visual function, causing ocular diseases, including blindness. Therefore, treatment of corneal NV has a high public health impact. Epigalloccatechin-3-gallate (EGCG), presenting antiangiogenesis effects, was chosen as an inhibitor to treat human vascular endothelial cells for corneal NV treatment. An arginine-glycine-aspartic acid (RGD) peptide-hyaluronic acid (HA)-conjugated complex coating on the gelatin/EGCG self-assembly nanoparticles (GEH-RGD NPs) was synthesized for targeting the αvß3 integrin on human umbilical vein endothelial cells (HUVECs) in this study, and a corneal NV mouse model was used to evaluate the therapeutic effect of this nanomedicine used as eyedrops. HA-RGD conjugation via COOH and amine groups was confirmed by 1H-nuclear magnetic resonance and Fourier-transform infrared spectroscopy. The average diameter of GEH-RGD NPs was 168.87±22.5 nm with positive charge (19.7±2 mV), with an EGCG-loading efficiency up to 95%. Images of GEH-RGD NPs acquired from transmission electron microscopy showed a spherical shape and shell structure of about 200 nm. A slow-release pattern was observed in the nanoformulation at about 30% after 30 hours. Surface plasmon resonance confirmed that GEH-RGD NPs specifically bound to the integrin αvß3. In vitro cell-viability assay showed that GEH-RGD efficiently inhibited HUVEC proliferation at low EGCG concentrations (20 µg/mL) when compared with EGCG or non-RGD-modified NPs. Furthermore, GEH-RGD NPs significantly inhibited HUVEC migration down to 58%, lasting for 24 hours. In the corneal NV mouse model, fewer and thinner vessels were observed in the alkali-burned cornea after treatment with GEH-RGD NP eyedrops. Overall, this study indicates that GEH-RGD NPs were successfully developed and synthesized as an inhibitor of vascular endothelial cells with specific targeting capacity. Moreover, they can be used in eyedrops to inhibit angiogenesis in corneal NV mice.

Synergistic Effect of Artificial Tears Containing Epigallocatechin Gallate and Hyaluronic Acid for the Treatment of Rabbits with Dry Eye Syndrome.

Dry eye syndrome (DES) is a common eye disease. Artificial tears (AT) are used to treat DES, but they are not effective. In this study, we assessed the anti-inflammatory effect of AT containing epigallocatechin gallate (EGCG) and hyaluronic acid (HA) on DES. Human corneal epithelial cells (HCECs) were used in the WST-8 assay to determine the safe dose of EGCG. Lipopolysaccharide-stimulated HCECs showing inflammation were treated with EGCG/HA. The expression of IL-1ß, IL-6, IL-8, and TNF-α was assessed by real-time PCR and AT physical properties such as the viscosity, osmolarity, and pH were examined. AT containing EGCG and HA were topically administered in a rabbit DES model established by treatment with 0.1% benzalkonium chloride (BAC). Tear secretion was assessed and fluorescein, H&E, and TUNEL staining were performed. Inflammatory cytokine levels in the corneas were also examined. The non-toxic optimal concentration of EGCG used for the treatment of HCECs in vitro was 10 µg/mL. The expression of several inflammatory genes, including IL-1ß, IL-6, IL-8, and TNF-α, was significantly inhibited in inflamed HCECs treated with 10 µg/mL EGCG and 0.1% (w/v) HA (E10/HA) compared to that in inflamed HCECs treated with either EGCG or HA alone. AT containing E10/HA mimic human tears, with similar osmolarity and viscosity and a neutral pH. Fluorescence examination of the ocular surface of mouse eyes showed that HA increased drug retention on the ocular surface. Topical treatment of DES rabbits with AT plus E10/HA increased tear secretion, reduced corneal epithelial damage, and maintained the epithelial layers and stromal structure. Moreover, the corneas of the E10/HA-treated rabbits showed fewer apoptotic cells, lower inflammation, and decreased IL-6, IL-8, and TNF-α levels. In conclusion, we showed that AT plus E10/HA had anti-inflammatory and mucoadhesive properties when used as topical eye drops and were effective for treating DES in rabbits.

Cytomegalovirus and herpes simplex virus as causes of bilateral anterior uveitis in an immunocompetent patient.

We report a case with two distinct clinical manifestations of bilateral anterior uveitis caused by two different members of the herpes virus group. A 72-year-old immunocompetent man, who had a documented history of two episodes of Posner-Schlossman syndrome in the left eye, presented with multiple mutton-fat keratic precipitates and elevated intraocular pressure (IOP) in his right eye. Herpes simplex virus Type I DNA was detected by polymerase chain reaction in the aqueous humor of the right eye. One year later, the patient appeared with a few round and whitish keratic precipitates and elevated IOP in his left eye. Polymerase chain reaction analysis showed positive for cytomegalovirus in the aqueous humor of the left eye. During both episodes, the anterior uveitis subsided and IOP returned to normal after systemic and topical antiglaucomatous medication as well as topical steroid.

An injectable oxidated hyaluronic acid/adipic acid dihydrazide hydrogel as a vitreous substitute.

Vitrectomy is a common procedure for treating ocular-related diseases. The surgery involves removing the vitreous humor from the center of the eye, and vitreous substitutes are needed to replace the vitreous humor after vitrectomy. In the present study, we developed a colorless, transparent and injectable hydrogel with appropriate refractive index as a vitreous substitute. The hydrogel is formed by oxidated hyaluronic acid (oxi-HA) cross-linked with adipic acid dihydrazide (ADH). Hyaluronic acid (HA) was oxidized by sodium periodate to create aldehyde functional groups, which could be cross-linked by ADH. The refractive index of this hydrogel ranged between 1.3420 and 1.3442, which is quite similar to human vitreous humor (1.3345). The degradation tests demonstrated that the hydrogel could maintain the gel matrix over 35 days, depending on the ADH concentration. In addition, the cytotoxicity was evaluated on retina pigmented epithelium (RPE) cells cultivated following the ISO standard (tests for in vitro cytotoxicity), and the hydrogel was found to be non-toxic. In a preliminary animal study, the oxi-HA/ADH hydrogel was injected into the vitreous cavity of rabbit eyes. The evaluations of slit-lamp observation, intraocular pressure, cornea thickness and histological examination showed no significant abnormal biological reactions for 3 weeks. This study suggests that the injectable oxi-HA/ADH hydrogel should be a potential vitreous substitute.

Protection of thymosin beta-4 on corneal endothelial cells from UVB-induced apoptosis.

Cornea absorbs most of daily ultraviolet (UV) light. An excess of UV damages results in not only keratopathy and cataract but also maculopathy. It has been reported that thymosin beta-4 (Tbeta4) promotes wound healing, decreases inflammatory response and prevents apoptosis of corneal epithelial cells. However, it is not clear whether Tbeta4 protects UVB-induced corneal injury, particularly in corneal endothelial cells because of its non-proliferation in nature. The purpose of this study is to compare the protective effects of Tbeta4 on bovine corneal endothelial (BCE) cells from low- and high-dose UVB damage. In this study, 1 microg/ml of Tbeta4 was added to BCE cells 2 h before low (12.5 mj/cm2) or high dosage (100 mj/cm2) UVB exposure. Using a fluorogenic substrate cleavage assay, we found that Tbeta4 diminished the reactive oxygen species level in BCE cells elicited by UVB. However, the protection of viability by Tbeta4 could only be detected under low-dose UVB exposure. Moreover, both caspase-9 activity and annexin V/propidium iodine staining demonstrated that Tbeta4 only protected BCE cells from low-dose UVB-induced apoptosis but not high-dose UVB-induced necrosis. Together, Tbeta4 protected corneal endothelial cells from UVB-induced oxidative stress and apoptosis after low-dose UVB exposure. The results support further investigation towards topical use or anterior chamber injection of this small hydrophilic peptide in treating and preventing UVB-induced corneal endothelial damage.

Identification of monoclinic calcium pyrophosphate dihydrate and hydroxyapatite in human sclera using Raman microspectroscopy.

Raman microspectroscopy was first used to determine the composition of a calcified plaque located at the pterygium-excision site of a 51-year-old female patient's left nasal sclera after surgery. It was unexpectedly found that the Raman spectrum of the calcified sample at 1149, 1108, 1049, 756, 517, 376 and 352/cm was similar to the Raman spectrum of monoclinic form of calcium pyrophosphate dihydrate (CPPD) crystal, but differed from the Raman spectrum of triclinic form of CPPD. An additional peak at 958/cm was also observed in the Raman spectrum of the calcified plaque, which was identical to the characteristic peak at 958/cm of hydroxyapatite (HA). This is the first study to report the spectral biodiagnosis of both monoclinic CPPD and HA co-deposited in the calcified plaque of a patient with sclera dystrophic calcification using Raman microspectroscopy.

Tissue-engineered human corneal endothelial cell sheet transplantation in a rabbit model using functional biomaterials.

BACKGROUND: This study was performed to investigate whether transplantation of bioengineered human corneal endothelial cell (HCEC) sheet grafts into corneas denuded of endothelium could restore corneal function and clarity in a rabbit model. METHODS: After being labeled with PKH26 fluorescent dye, the adult HCECs derived from eye bank corneas were cultivated on the thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm)-grafted surfaces for 3 weeks at 37 degrees C, and were harvested as transplantable cell sheets after incubation for 45 min at 20 degrees C. Attached by gelatin hydrogel discs, the bioengineered cell monolayers were transplanted to rabbit corneas denuded of endothelium (HCEC sheet group). Traumatized rabbit corneas were served as controls. Postsurgical corneas underwent clinical observations and histological examinations for 6 months. RESULTS: By transmission electron microscopy and Western blot analysis of zonula occludens-1 and Na+,K+ -adenosine triphosphatase proteins, the structure and function of HCEC sheets resembled those of native corneal endothelium. After endothelial cells were removed, corneas of each group turned severe edematous and opaque. In the HCEC sheet groups, corneal clarity was gradually restored and corneal thickness was significantly less than that in the control groups (P<0.05). The attached PKH26-positive HCECs spread on rabbit Descemet's membrane after receiving cell sheet grafts. Intraocular delivery of HCEC sheets by means of a minimally invasive technique (i.e., small-incision surgery using biodegradable hydrogels) demonstrated long-term graft integration with damaged corneas. CONCLUSIONS: These results indicate that using cultured HCECs and functional biomaterials, PNIPAAm and gelatin, an effective cell sheet-based therapy can be developed for the treatment of corneal endothelium deficiency.

Preliminary identification of Beta-carotene in the vitreous asteroid bodies by micro-Raman spectroscopy and HPLC analysis.

beta-carotene was first identified from the vitreous asteroid bodies (ABs) excised from one patient with asteroid hyalosis (AH) by confocal Raman microspectroscopy and was also verified by high performance liquid chromatography (HPLC). Two patients had been diagnosed with AH and intervened by surgical vitrectomy due to blurred vision. The morphology and components of both AB specimens were observed by optical microscopy and determined by using confocal Raman microspectroscopy and HPLC analysis, respectively. Surprisingly, two unique peaks at 1528 and 1157 cm(-1) were found in the Raman spectrum for the AB specimen of patient 1 alone, which were in close agreement with that of the Raman peaks at 1525 and 1158 cm(-1) for beta-carotene and/or lutein. However, HPLC analytical data clearly indicated that the retention time for the extracted sample from the AB specimen of patient 1 was observed at 13.685 min and just identical to that of beta-carotene (13.759 min) rather than lutein (2.978 min). In addition, the lack of any peak in the HPLC profile for the AB specimen of patient 2 also confirmed the absence of Raman peaks at 1525 and 1158 cm(-1). Thus this preliminary study strongly suggests that beta-carotene as a unique component of ABs was specifically detected from the AB specimen of one AH patient by using confocal Raman microspectroscopy and HPLC analysis.

Bioengineered human corneal endothelium for transplantation.

OBJECTIVE: To investigate whether the bioengineered human corneal endothelial cell (HCEC) monolayers harvested from thermoresponsive culture supports could be used as biological tissue equivalents. METHODS: Untransformed adult HCECs derived from eye bank corneas were cultivated on a thermoresponsive poly-N-isopropylacrylamide-grafted surface for 3 weeks at 37 degrees C. Confluent cell cultures with a phenotype and cell density similar to HCECs in vivo were detached as a laminated sheet by lowering the culture temperature to 20 degrees C. In vitro characteristics of the HCEC sheets were determined evaluating their viability and by scanning electron microscopy, immunohistochemistry, and histological studies. RESULTS: After separation from culture surfaces via a thermal stimulus, the HCEC sheets remained viable. Polygonal cell morphology and multiple cellular interconnections were observed throughout the HCEC sheets. Immunolocalization of zonula occludens-1 and Na+,K+-adenosine triphosphatase (ATPase) indicated the formation of tight junctions and the distribution of ionic pumps at the cell boundary. In addition, we ascertained that cultured HCECs have a monolayered architecture that mimics native corneal endothelium. CONCLUSION: These data suggest that a well-organized and functional HCEC monolayer can feasibly be used as tissue equivalents for replacing compromised endothelium.Clinical Relevance Bioengineered human corneal endothelium fabricated from thermoresponsive supports can potentially offer a new therapeutic strategy for corneal endothelial cell loss.

Siderosis oculi: visual dysfunctions even after iron removal: a role of OCT.

Four males with sidersosis oculi were reviewed. Vitreous/anterior chamber angle irons (cases 1 and 3) were misdiagnosed initially and discovered later. In case 2, the retina-incarcerated iron was long ignored. Exceptionally in case 4, the iron was encapsulated by using optical coherence tomography (OCT). Preoperatively, in cases 1 and 4, the injured eye's vision, electro-oculogram, and electroretinogram were reduced compared with the other eye. In three cases, field defects were relevant to their iron locations. Postremoval, iron-impaired retinal functions didn't obviously improve. Early iron removal seems vital. OCT identified iron encapsulation, ameliorating iron toxicity. Consistently, field defect in case 4 was nonprogressive.