Ex Vivo Pig Eye Model for Studying Diffusion of Acylcarnitines with Different Physicochemical Properties.

Purpose: To explore the diffusion capacities between the anterior and vitreous chambers in a novel ex vivo pig eye model using a mix of stable isotope-labeled acylcarnitines representing metabolites with different physical and chemical properties, and analysis using mass spectrometry (MS). Methods: Enucleated pig eyes were injected in the anterior or vitreous chamber of the eye with a stable isotope-labeled acylcarnitine mix (free carnitine, C2, C3, C4, C8, C12, and C16-having an increasing size and hydrophobicity in that order). Samples were collected from each chamber at 3, 6, and 24 h postincubation for analysis using MS. Results: After injection into the anterior chamber, the concentration of all acylcarnitines increased in the vitreous chamber over the observation period. After injection in the vitreous chamber, the acylcarnitines diffused to the anterior chamber with the highest concentration observed at 3 h postinjection, followed by a decrease in concentration possibly due to an elimination from the anterior chamber despite continued diffusion from the vitreous chamber. C16, the most hydrophobic and longest chain molecule, showed slower diffusion in both experimental settings. Conclusion: We hereby show a distinct diffusion pattern of molecules with different molecular size and hydrophobicity within and between the anterior and vitreous chamber. This model can be useful for optimizing choices and design of therapeutic molecules with higher retaining or depot properties into the two chambers of the eye for future intravitreal, intracameral, and topical treatment purposes.

Anterior chamber enhancement predicts optic nerve infiltration in retinoblastoma.

OBJECTIVES: As described recently, intravenously injected gadolinium-based contrast agent (GBCA) penetrates into the anterior eye chamber (AC) and is drained from the retina to the distal optic nerve (ON) along perivascular spaces, which serves retinal homeostasis and was termed the orbital glymphatic system (GS). Independently, AC enhancement predicted ON infiltration, a major risk factor for advanced retinoblastoma (RB), in a small RB patient cohort. We aimed to review the supposed imaging biomarker for ON infiltration in a large RB cohort and with respect to the recently described orbital GS. METHODS: This IRB-approved retrospective single-center study encompassed 539 orbital MRIs performed with an orbital coil and with the children under general anesthesia. Differences of signal intensity ratios (∆SIRs) of the AC to the lens were determined between non-contrast and GBCA-enhanced T1-weighted images and were correlated with histopathologic presence of ON infiltration. RESULTS: ∆SIR of the RB eye was an independent, significant predictor for ON invasion in multivariate analysis with adjustment for tumor size (p < 0.05) and increased with infiltration level. CONCLUSIONS: GBCA enhancement of the AC predicts ON infiltration. This might be caused by impairment of the orbital glymphatic system, which is supposed to clear toxic metabolites from the retina to the postlaminar ON. In RB with ON infiltration, this efflux path is likely to be inhibited, which is supposed to result in disturbed retinal homeostasis, release of vascular endothelial growth factor, and iris neovascularization, which increases penetration of GBCA into the AC. KEY POINTS: • Infiltration of the optic nerve can be predicted by anterior chamber enhancement after intravenous MRI contrast agent administration. • Increased anterior chamber enhancement in retinoblastoma with optic nerve infiltration might result from dysfunction of the orbital glymphatic system with disturbance of retinal homeostasis and consecutive iris neovascularization.

Secondary glaucoma with anterior chamber cholesterolosis: Case series.

RATIONALE: The presence of cholesterol crystals in the anterior chamber is extremely rare, and secondary glaucoma with cholesterol crystals in the anterior chamber, reported in the literature, is even rarer. This paper reports 3 cases of secondary glaucoma with cholesterol crystals in the anterior chamber. PATIENT CONCERNS: Three patients were admitted to the hospital because of ocular distension and blindness. Ocular examination on admission indicated high intraocular pressure, and crystalline gold substances were observed in the anterior chamber. DIAGNOSIS: Based on clinical manifestations and an aqueous fluid smear, absolute glaucoma and anterior chamber cholesterol crystals were diagnosed. INTERVENTIONS: In the first case, transscleral ciliary photocoagulation was performed; in the last 2 cases, trabeculectomy combined with extracapsular cataract extraction was performed. OUTCOMES: The follow-up period was 11 to 15 months. Intraocular pressure was stable in 2 patients treated with surgery, and no cholesterol crystals were observed in the anterior chamber. The intraocular pressure increased in 1 patient treated with laser, and a small amount of cholesterol crystals was still observed in the anterior chamber. LESSONS: Anterior chamber cholesterol crystallization is extremely rare and cannot be treated if it does not cause other lesions. However, glaucoma occurred in all 3 cases in this study, and intraocular pressure increased in 1 case after laser treatment and remained stable in 2 cases after surgical treatment. Therefore, the treatment plan for anterior chamber cholesterol crystallization in glaucoma requires further discussion.

Cellular crosstalk regulates the aqueous humor outflow pathway and provides new targets for glaucoma therapies.

Primary congenital glaucoma (PCG) is a severe disease characterized by developmental defects in the trabecular meshwork (TM) and Schlemm's canal (SC), comprising the conventional aqueous humor outflow pathway of the eye. Recently, heterozygous loss of function variants in TEK and ANGPT1 or compound variants in TEK/SVEP1 were identified in children with PCG. Moreover, common variants in ANGPT1and SVEP1 have been identified as risk alleles for primary open angle glaucoma (POAG) in GWAS studies. Here, we show tissue-specific deletion of Angpt1 or Svep1 from the TM causes PCG in mice with severe defects in the adjacent SC. Single-cell transcriptomic analysis of normal and glaucomatous Angpt1 deficient eyes allowed us to identify distinct TM and SC cell populations and discover additional TM-SC signaling pathways. Furthermore, confirming the importance of angiopoietin signaling in SC, delivery of a recombinant ANGPT1-mimetic promotes developmental SC expansion in healthy and Angpt1 deficient eyes, blunts intraocular pressure (IOP) elevation and RGC loss in a mouse model of PCG and lowers IOP in healthy adult mice. Our data highlight the central role of ANGPT1-TEK signaling and TM-SC crosstalk in IOP homeostasis and provide new candidates for SC-targeted glaucoma therapy.

Altered Ocular Fibrillin Microfibril Composition in Mice With a Glaucoma-Causing Mutation of Adamts10.

Purpose: Previously, we identified a G661R mutation of ADAMTS10 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif 10) as being disease causative in a colony of Beagles with inherited primary open-angle glaucoma (POAG). Mutations in ADAMTS10 are known to cause Weill-Marchesani syndrome (WMS), which is also caused by mutations in the fibrillin-1 gene (FBN1), suggesting functional linkage between ADAMTS10 and fibrillin-1, the principal component of microfibrils. Here, we established a mouse line with the G661R mutation of Adamts10 (Adamts10G661R/G661R) to determine if they develop features of WMS and alterations of ocular fibrillin microfibrils. Methods: Intraocular pressure (IOP) was measured using a TonoLab rebound tonometer. Central cornea thickness (CCT), anterior chamber depth (ACD) and axial length (AL) of the eye were examined by spectral-domain optical coherence tomography. Sagittal eye sections from mice at postnatal day 10 (P10) and at 3 and 24 months of age were stained with antibodies against fibrillin-1, fibrillin-2, and ADAMTS10. Results: IOP was not elevated in Adamts10G661R/G661R mice. Adamts10G661R/G661R mice had smaller bodies, thicker CCT, and shallower ACD compared to wild-type mice but normal AL. Adamts10G661R/G661R mice displayed persistent fibrillin-2 and enhanced fibrillin-1 immunofluorescence in the lens zonules and in the hyaloid vasculature and its remnants in the vitreous. Conclusions: Adamts10G661R/G661R mice recapitulate the short stature and ocular phenotypes of WMS. The altered fibrillin-1 and fibrillin-2 immunoactivity in Adamts10G661R/G661R mice suggests that the G661R mutation of Adamts10 perturbs regulation of the fibrillin isotype composition of microfibrils in the mouse eye.

Brain-derived Neurotrophic Factor in the Aqueous Humor of Glaucoma Patients.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) may be involved in the pathogenesis of glaucoma. BDNF concentrations reported in previous studies have varied widely, and the concentration of BDNF in aqueous humor is unknown. In this study, BDNF concentrations in the aqueous humor of glaucoma patients and control patients were measured with ELISA kits. METHODS: This prospective, observational study examined BDNF levels in aqueous humor in 62 eyes of 43 patients who underwent cataract surgery or trabeculectomy (11 glaucoma patients and 32 non-glaucoma cataract patients as controls). BDNF concentrations were examined by 4 different enzyme-linked immunosorbent assay (ELISA) techniques. RESULTS: The mean ± SD patient age was 72.0 ± 10.1 (range 35 to 87) years. Two of the techniques detected no BDNF in aqueous humor in any samples (n=3 and n=9, respectively); the average value was less than zero. An ultrasensitive ELISA kit did not yield reliable measurements. Finally, in an even more sensitive ELISA (Simoa-HD1), performed by an outside contractor, 25 (54.3%) eyes were below the detection limit, including 20 (55.6%) control and 5 (50%) glaucoma cases. For eyes with detectable BDNF, the overall BDNF concentration was 0.158 pg/mL (n=21): 0.196 pg/mL (n=16) in controls and 0.034 pg/mL (n=5) in glaucoma cases. CONCLUSIONS: BDNF level in aqueous humor varies widely.

Amount of Green Fluorescent Protein in the Anterior Chamber after Intravitreal Injection of Triple-Mutated Self-Complementary AAV2 Vectors is Not Affected by Previous Vitrectomy Surgery.

BACKGROUND: The adeno-associated virus (AAV) vector is a promising vector for ocular gene therapy. Surgical internal limiting membrane peeling before AAV vector administration is useful for efficient retinal transduction. However, no report has investigated localization of AAV vectors after administration into a post-vitrectomy eye. This study investigated the effects of vitrectomy surgery on intravitreal-injected AAV vector-mediated gene expression in the anterior segment and examined the presence of neutralizing antibodies (NAbs) in serum before and after AAV vector administration. METHODS: Of six eyes from three female cynomolgus monkeys, four were vitrectomized (Group VIT) and two were non-vitrectomized (Group IV). All eyes were injected with 50 µL of triple-mutated self-complementary AAV2 vector (1.9 × 1013 v.g./mL) encoding green fluorescent protein (GFP). NAbs in the serum were examined before administration and at 2 and 6 weeks after administration. GFP expression was analyzed at 19 weeks after administration. RESULTS: Immunohistological analysis showed no GFP expression in the trabecular meshwork in any eye. The GFP genome copy in two slices of the anterior segment was 2.417 (vector genome copies/diploid genome) in Group VIT and 4.316 (vector genome copies/diploid genome) in group IV. The NAb titer was 1:15.9 (geometric mean) before administration, 1:310.7 at 2 weeks after administration, and 1:669.4 at 6 weeks after administration. CONCLUSION: Previous vitrectomy surgery did not affect gene expression in the anterior segment after intravitreal injection of AAV vectors.

The Eye as a Transplantation Site to Monitor Pancreatic Islet Cell Plasticity.

The endocrine cells confined in the islets of Langerhans are responsible for the maintenance of blood glucose homeostasis. In particular, beta cells produce and secrete insulin, an essential hormone regulating glucose uptake and metabolism. An insufficient amount of beta cells or defects in the molecular mechanisms leading to glucose-induced insulin secretion trigger the development of diabetes, a severe disease with epidemic spreading throughout the world. A comprehensive appreciation of the diverse adaptive procedures regulating beta cell mass and function is thus of paramount importance for the understanding of diabetes pathogenesis and for the development of effective therapeutic strategies. While significant findings were obtained by the use of islets isolated from the pancreas, in vitro studies are inherently limited since they lack the many factors influencing pancreatic islet cell function in vivo and do not allow for longitudinal monitoring of islet cell plasticity in the living organism. In this respect a number of imaging methodologies have been developed over the years for the study of islets in situ in the pancreas, a challenging task due to the relatively small size of the islets and their location, scattered throughout the organ. To increase imaging resolution and allow for longitudinal studies in individual islets, another strategy is based on the transplantation of islets into other sites that are more accessible for imaging. In this review we present the anterior chamber of the eye as a transplantation and imaging site for the study of pancreatic islet cell plasticity, and summarize the major research outcomes facilitated by this technological platform.

The Development of Ocular Biometric Parameters in Premature Infants without Retinopathy of Prematurity.

PURPOSE: The aim of this study was to investigate the developmental tendencies and distribution of ocular biometric parameters in premature infants without retinopathy of prematurity (ROP). Axial length (AL), anterior chamber depth (ACD), lens thickness (LT) and vitreous length (VL) were measured, and their relationships with birth weight (BW) and postmenstrual age (PMA) were analyzed during their earliest weeks of life. METHODS: This cross-sectional cohort study included 633 premature infants. They were divided into nine groups according to their PMA: 32 weeks, 33 weeks, 34 weeks, and onward to 40 weeks. All participants underwent portable slit-lamp examination, RetCam3 and A-scan ultrasound biometry. The following ocular biometric parameters were recorded: AL, ACD, LT and VL. The t-test, one-way analysis of variance, and the multiple regression analysis model were used to analyze the data. RESULTS: The increases in AL, ACD, LT and VL were 0.14 mm, 0.028 mm, 0.0025 mm and 0.11 mm per week, respectively. AL, ACD, LT and VL were positively correlated with BW (ß = 0.000337, 4.234E-5, 2.697E-5, 0.000278, respectively) and PMA (ß = 0.142, 0.026, 0.011, 0.103, respectively). CONCLUSIONS: With maturation, AL and VL increased and ACD deepened, but there was no significant change in LT. The ocular growth parameters were positively correlated with BW and PMA however the correlations were not strong.

Non-invasive Instrument-Based Tests for Quantifying Anterior Chamber Flare in Uveitis: A Systematic Review.

Purpose: Anterior chamber (AC) flare is a key sign for anterior uveitis. New instrument-based techniques for measuring AC flare can offer automation and objectivity. This review aims to identify objective instrument-based measures for AC flare.Methods: In this systematic review, we identified studies reporting correlation between instrument-based tests versus clinician AC flare grading, and/or aqueous protein concentration, as well as test reliability.Results: Four index tests were identified in 11 studies: laser-flare photometry (LFP), optical coherence tomography, ocular flare analysis meter (OFAM) and the double-pass technique. The correlation between LFP and clinician grading was 0.40-0.93 and 0.87-0.94 for LFP and protein concentration. The double-pass technique showed no correlation with clinician grading and insufficient information was available for OFAM.Conclusion: LFP shows moderate to strong correlation with clinician grading and aqueous protein concentration. LFP could be a superior reference test compared to clinician AC flare grading for validating new index tests.

CPAMD8 loss-of-function underlies non-dominant congenital glaucoma with variable anterior segment dysgenesis and abnormal extracellular matrix.

Abnormal development of the ocular anterior segment may lead to a spectrum of clinical phenotypes ranging from primary congenital glaucoma (PCG) to variable anterior segment dysgenesis (ASD). The main objective of this study was to identify the genetic alterations underlying recessive congenital glaucoma with ASD (CG-ASD). Next-generation DNA sequencing identified rare biallelic CPAMD8 variants in four patients with CG-ASD and in one case with PCG. CPAMD8 is a gene of unknown function and recently associated with ASD. Bioinformatic and in vitro functional evaluation of the variants using quantitative reverse transcription PCR and minigene analysis supported a loss-of-function pathogenic mechanism. Optical and electron microscopy of the trabeculectomy specimen from one of the CG-ASD cases revealed an abnormal anterior chamber angle, with altered extracellular matrix, and apoptotic trabecular meshwork cells. The CPAMD8 protein was immunodetected in adult human ocular fluids and anterior segment tissues involved in glaucoma and ASD (i.e., aqueous humor, non-pigmented ciliary epithelium, and iris muscles), as well as in periocular mesenchyme-like cells of zebrafish embryos. CRISPR/Cas9 disruption of this gene in F0 zebrafish embryos (96 hpf) resulted in varying degrees of gross developmental abnormalities, including microphthalmia, pharyngeal maldevelopment, and pericardial and periocular edemas. Optical and electron microscopy examination of these embryos showed iridocorneal angle hypoplasia (characterized by altered iris stroma cells, reduced anterior chamber, and collagen disorganized corneal stroma extracellular matrix), recapitulating some patients' features. Our data support the notion that CPAMD8 loss-of-function underlies a spectrum of recessive CG-ASD phenotypes associated with extracellular matrix disorganization and provide new insights into the normal and disease roles of this gene.

Islet Transplantation to the Anterior Chamber of the Eye-A Future Treatment Option for Insulin-Deficient Type-2 Diabetics? A Case Report from a Nonhuman Type-2 Diabetic Primate.

Replacement of the insulin-secreting beta cells through transplantation of pancreatic islets to the liver is a promising treatment for type-1 diabetes. However, low oxygen tension, shear stress, and the induction of inflammation lead to significant islet dysfunction and loss. The anterior chamber of the eye (ACE) has gained considerable interest and represents an alternative therapeutic islet transplantation site because of its accessibility, high oxygen tension, and immune-privileged milieu. We have previously demonstrated the feasibility of intraocular islet transplant in mouse and nonhuman primate models of type-1 diabetes and are now assessing its efficacy on glucose homeostasis in a nonhuman primate model of type-2 diabetes. We transplanted allogeneic donor islets (1,500 islet equivalents/kg) into the anterior chamber of one eye in a cynomolgus monkey with high-fat-diet-induced type-2 diabetes. Repeated examinations of the anterior and posterior segments of both eyes were done to monitor the engrafted islets and assess the overall ocular health. Fasting blood glucose level, blood biochemistry, and other metabolic parameters were routinely evaluated to determine the function of the islet graft and diabetes status. The transplanted islets were rapidly engrafted onto the iris and became vascularized 1 month after transplantation. We did not detect changes in intraocular pressure, cataract formation, ophthalmitis, or retinal vessel deformation. A significant lower fasting blood glucose level was observed while the graft was in place, and the transplantation reverts the progression of diabetes. The metabolic markers, hemoglobin A1C and fructosamine, demonstrated improvement following islet transplantation. As a conclusion, intraocular islet transplantation in one eye of a cynomolgus monkey with type-2 diabetes improved its overall plasma glucose homeostasis, as evidenced by short-term measures and long-term metabolic markers. These results further support the future application of the ACE as an alternative site for clinical islet transplants in the context of type-2 diabetes.

The anterior eye chamber: entry of the natural excretion pathway of gadolinium contrast agents?

OBJECTIVE: Previous studies provided evidence that gadolinium can be found in the aqueous chamber (AC) of the eye several hours post injection (p.i.) of gadolinium-based contrast agents (GBCAs). This study aimed to investigate whether gadolinium can be detected promptly after injection of a macrocyclic GBCA on contrast-enhanced T1-weighted MRI in the AC of children. METHODS: This retrospective study encompassed MRI of 200 healthy eyes of children suffering from retinoblastoma of the contralateral eye. MRI was performed with an orbital coil with the children in a state of general anesthesia. Differences of signal intensity ratios (∆SIRs) of the AC to the lens were determined between pre and post contrast-enhanced T1-weighted images (Dotarem®, Guerbet, 0.1 ml/kg body weight, mean (standard deviation) p.i. time = 12:24 (± 2:31) min). RESULTS: A highly significant signal intensity increase was found in the AC of healthy eyes 12 min after GBCA injection (median ∆SIR (interquartile range) = + 0.08 (0.05-0.12), p < 0.0001). In addition, gadolinium enhancement showed a strong negative correlation with children's age in multivariate analysis with adjustment for p.i. time (p < 0.0001). CONCLUSIONS: GBCA leakage into the AC of healthy infantile eyes was found promptly after injection. The negative correlation between patient age and GBCA enhancement might be explained by a maturation process of the blood-aqueous barrier or Schlemm's canal. Future studies should assess the duration and potential diagnostic applications as well as possible safety concerns of gadolinium presence in the AC. KEY POINTS: • Leakage of gadolinium-based contrast agent into the aqueous chamber of infantile eyes was found promptly after intravenous injection (p < 0.0001). • Gadolinium enhancement of the anterior eye chamber was negatively correlated with the children's age (p < 0.0001).

Structural Changes and Astrocyte Response of the Lateral Geniculate Nucleus in a Ferret Model of Ocular Hypertension.

We investigated structural changes and astrocyte responses of the lateral geniculate nucleus (LGN) in a ferret model of ocular hypertension (OH). In 10 ferrets, OH was induced via the injection of cultured conjunctival cells into the anterior chamber of the right eye; six normal ferrets were used as controls. Anterograde axonal tracing with cholera toxin B revealed that atrophic damage was evident in the LGN layers receiving projections from OH eyes. Immunohistochemical analysis with antibodies against NeuN, glial fibrillary acidic protein (GFAP), and Iba-1 was performed to specifically label neurons, astrocytes, and microglia in the LGN. Significantly decreased NeuN immunoreactivity and increased GFAP and Iba-1 immunoreactivities were observed in the LGN layers receiving projections from OH eyes. Interestingly, the changes in the immunoreactivities were significantly different among the LGN layers. The C layers showed more severe damage than the A and A1 layers. Secondary degenerative changes in the LGN were also observed, including neuronal damage and astrocyte reactions in each LGN layer. These results suggest that our ferret model of OH is valuable for investigating damages during the retina-brain transmission of the visual pathway in glaucoma. The vulnerability of the C layers was revealed for the first time.

Effect of Tropicamide on Laser Flare Meter Measurements in Patients with Pseudoexfoliation.

PURPOSE: To investigate the effect of 1% tropicamide on anterior chamber aqueous flare (ACAF) measurements acquired with laser flare meter in patients with pseudoexfoliation. METHODS: Thirty-three eyes of 33 patients with pseudoexfoliation were enrolled. Patients with the history of other ocular diseases, intraocular surgeries, and the presence of severe posterior synechia were excluded. Besides routine ophthalmological examination, ACAF levels were measured by laser flare meter device (Kowa FM 600) before and after instillation of 1% tropicamide. RESULTS: The mean age of 33 patients was 67.3±7.1 (53-85) years. Patients had a mean best corrected visual acuity of 0.25±0.41 (1.80-0.00) logMAR, cup-to-disc ratio of 0.45±0.22 (0.2-1), and IOP of 15.33±2.82 (9-20) mmHg. Although the mean ACAF value increased from 14.68±8.40 (3.4-40.4) photon/ms predilation to 15.41±10.74 (3.8-46.8) photon/ms post-dilation, the difference was not statistically significant (p=0.835). CONCLUSIONS: ACAF values in patients with pseudoexfoliation did not significantly differ after instillation of 1% tropicamide.

Intraocular amyloidosis with multifocal iris and anterior chamber translucent spherules.

Ocular amylodosis, although a rare entity, is known to affect the conjunctiva, extraocular muscles, orbit, lacrimal gland, and skin around the eyes. Intraocular deposition of amyloid mainly confines to the vitreous and cornea. In this report, we describe two cases of intraocular amyloidosis presenting as multiple iris and anterior chamber cysts. Histopathological examination with special stain like Congo Red and Transmission Electron Microscopy confirmed the diagnosis of amyloidosis. Systemic investigations ruled out systemic association confirming the diagnosis of primary ocular amyloidosis.

In Vivo Fluorescence Visualization of Anterior Chamber Injected Human Corneal Endothelial Cells Labeled With Quantum Dots.

Purpose: The injection of cultured human corneal endothelial cells (cHCECs) into the anterior chamber (AC) is a newly developed modality for the successful treatment of corneal endothelium dysfunction. Here, we investigated whether or not cHCECs could be labeled using quantum dots (QDs) composed of semiconductor nanoparticle octa-arginine (R8) to trace injected cHCECs and examined the utility of in vivo fluorescence imaging to analyze the dynamics and accumulation of QD-labeled injected cHCECs in a corneal endothelial dysfunction mouse model. Methods: The cHCECs, either of high quality or with cell-state transition, were labeled by adding a mixture of QDs655 and R8. The labeling efficiency and the unchanging of the cell phenotypes by the labeling was confirmed by flow cytometry. The labeled cHCECs were injected into the AC of either healthy mice or mice with corneal endothelium damaged by cryogenic treatment. The kinetics of the injected cHCECs was traced quantitatively via multiphoton confocal laser microscopy. Results: QD labeling induced no morphologic change in the cHCECs or in the expression of the functional markers of cHCECs (i.e., Na+/K+-ATPase and zonula occludens-1). The injected cHCECs-QDs were quantitatively detected, and the retention of cHCECs-QDs was evident, from 3 to 48 hours post cell injection on the posterior surface in the cryogenically injured corneal endothelium mouse model eyes, yet not in the noninjured healthy control eyes. Conclusions: The findings of this study show that in the field of regenerative medicine, QD labeling of cells presents a convenient and sensitive method of finely monitoring the fate of injected cells in vivo.

Cytokines, chemokines, and flare in the anterior chamber after femtosecond laser-assisted cataract surgery.

PURPOSE: To analyze anterior chamber inflammation after pretreatment with a femtosecond laser platform during cataract surgery and compare the results with those of the manual procedure. SETTING: Eye Clinic, Department of Surgery and Translational Medicine, University of Florence, Italy. DESIGN: Prospective case series. METHODS: Aqueous humor was collected after femtosecond laser pretreatment (femtosecond group) and at the beginning of routine cataract surgery before the primary incision was created (control group). The levels of 14 cytokines and chemokines were measured using a multiplex array system. Surgical parameters (suction time, laser time, effective phacoemulsification time [EPT]) were recorded. Anterior chamber flare was measured by laser photometry preoperatively and 1 day and 7 days postoperatively. RESULTS: Each group comprised 20 eyes. The EPT was significantly lower in the femtosecond group than in the control group. In the femtosecond group, the concentrations of IL (interleukin)-6, IL-8, IL-10, IL-12, vascular endothelial growth factor, and interferon-γ were significantly higher than in the control group. Flare in the anterior chamber measured with flare-cell meter was not significantly different between groups at any timepoint. No correlation was found between cytokine concentrations and age in either group and between cytokine levels and suction or laser time and postoperative flare in the femtosecond group. Also, no correlation was found between postoperative aqueous flare and EPT in either group. CONCLUSIONS: Despite the rise of proinflammatory cytokines in the aqueous humor after femtosecond laser pretreatment, the anterior chamber flare after cataract surgery was similar to that in controls. This might be a result of the lower EPT required after pretreatment.

Expressional and functional involvement of gap junctions in aqueous humor outflow into the ocular trabecular meshwork of the anterior chamber.

Purpose: The ocular trabecular meshwork (TM) responsible for aqueous humor (AH) drainage is crucial for regulating intraocular pressure (IOP) of the eye. An IOP elevation that causes distended TM is involved in the pathogenesis of glaucoma, suggesting intercellular connections important for the TM pathophysiology. The goal of this study was to examine whether gap junction proteins between endothelial cells in the TM are expressional and functional. Methods: The expression levels of the gap junction channels in normal human TM cells were determined with real-time PCR and western blot assays. Immunohistochemistry (IHC) staining was performed to examine the localization of gap junction proteins in normal human TM cells and tissues. IOP and the outflow of AH were measured after intercameral injection of gap junction blockers in C57/BL6 mice. Results: Gap junction proteins GJA1, GJA8, GJB6, and GJC1 were robustly expressed in human TM cells from three individuals. Among the four gap junction channels, GJA1 and GJA8 exhibited the most abundance in the TM. The IHC analysis further confirmed that these proteins were expressed on the membrane between adjacent cells. In the human TM tissues, GJA1, GJA8, GJB6, and GJC1 were also observed along the trabecular beams. Inhibition of gap junctions with intracameral injection of blockers resulted in a statistically significant increase in aqueous humor outflow resistance and IOP elevation in mice. Conclusions: The GJA1 and GJA8 gap junction proteins, in particular, are robustly expressed in human TM cells and tissues. Pharmacological inhibition of gap junction channels causes an increased resistance of AH outflow and an elevation of IOP in mice. The present findings suggest the functional role of gap junction channels for regulation of AH outflow in the TM, and activation of gap junctions might represent a therapeutic strategy for treatment of glaucoma.

Lentiviral Vector-Mediated Expression of Exoenzyme C3 Transferase Lowers Intraocular Pressure in Monkeys.

Primary open-angle glaucoma (POAG) is considered a lifelong disease characterized by optic nerve deterioration and visual field damage. Although the disease progression can usually be controlled by lowering the intraocular pressure (IOP), therapeutic effects of current approaches do not last long. Gene therapy could be a promising method for persistent treatment of the disease. Our previous study demonstrated that gene transfer of exoenzyme C3 transferase (C3) to the trabecular meshwork (TM) to inhibit Rho GTPase (Rho), the upstream signal molecule of Rho-associated kinase (ROCK), resulted in lowered IOP in normal rodent eyes. In the present study, we show that the lentiviral vector (LV)-mediated C3 expression inactivates RhoA in human TM cells by ADP ribosylation, resulting in disruption of the actin cytoskeleton and altered cell morphology. In addition, intracameral delivery of the C3 vector to monkey eyes leads to persistently lowered IOP without obvious signs of inflammation. This is the first report of using a vector to transduce the TM of an alive non-human primate with a gene that alters cellular machinery and physiology. Our results in non-human primates support that LV-mediated C3 expression in the TM may have therapeutic potential for glaucoma, the leading cause of irreversible blindness in humans.