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.

Development of fully automated anterior chamber cell analysis based on image software.

Optical coherence tomography (OCT) is a noninvasive method that can quickly and accurately examine the eye at the cellular level. Several studies have used OCT for analysis of anterior chamber cells. However, these studies have several limitations. This study was performed to supplement existing reports of automated analysis of anterior chamber cell images using spectral domain OCT (SD-OCT) and to compare this method with the Standardization of Uveitis Nomenclature (SUN) grading system. We analyzed 2398 anterior segment SD-OCT images from 34 patients using code written in Python. Cell density, size, and eccentricity were measured automatically. Increases in SUN grade were associated with significant cell density increases at all stages (p < 0.001). Significant differences were observed in eccentricity in uveitis, post-surgical inflammation, and vitreous hemorrhage (p < 0.001). Anterior segment SD-OCT is reliable, fast, and accurate means of anterior chamber cell analysis. This method showed a strong correlation with the SUN grade system. Also, eccentricity could be helpful as a supplementary evaluation tool.

Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber.

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology have been developed and utilized in vivo, they are limited in terms of allowing single cell resolution and continuous longitudinal recordings. Because of the transparency of the cornea, the anterior chamber of the eye (ACE) in mice is well suited to study human and mouse pancreatic islet cell biology. Here is a description of how this approach can be used to perform continuous longitudinal recordings of grafting and revascularization of individual human islet grafts. Human islet grafts are inserted into the ACE, using NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mice as recipients. This allows for the investigation of the expansion of recipient versus donor cells and the contribution of recipient cells in promoting the encapsulation and vascularization of the graft. Further, a step-by-step approach for image analysis and quantification of the islet volume or segmented vasculature and islet capsule forming recipient cells is outlined.

Adipose-derived stem cells integrate into trabecular meshwork with glaucoma treatment potential.

The trabecular meshwork (TM) is an ocular tissue that maintains intraocular pressure (IOP) within a physiologic range. Glaucoma patients have reduced TM cellularity and, frequently, elevated IOP. To establish a stem cell-based approach to restoring TM function and normalizing IOP, human adipose-derived stem cells (ADSCs) were induced to differentiate to TM cells in vitro. These ADSC-TM cells displayed a TM cell-like genotypic profile, became phagocytic, and responded to dexamethasone stimulation, characteristic of TM cells. After transplantation into naive mouse eyes, ADSCs and ADSC-TM cells integrated into the TM tissue, expressed TM cell markers, and maintained normal IOP, outflow facility, and extracellular matrix. Cell migration and affinity results indicated that the chemokine pair CXCR4/SDF1 may play an important role in ADSC-TM cell homing. Our study demonstrates the possibility of applying autologous or allogeneic ADSCs and ADSC-TM cells as a potential treatment to restore TM structure and function in glaucoma.

Magnetic Human Corneal Endothelial Cell Transplant: Delivery, Retention, and Short-Term Efficacy.

Purpose: Corneal endothelial dysfunction leads to corneal edema, pain, and vision loss. Adequate animal models are needed to study the safety and efficacy of novel cell therapies as an alternative to corneal transplantation. Methods: Primary human corneal endothelial cells (HCECs) were isolated from cadaveric donor corneas, expanded in vitro, transduced to express green fluorescent protein (GFP), loaded with superparamagnetic nanoparticles, and injected into the anterior chamber of adult rabbits immediately after endothelial cell or Descemet's membrane stripping. The same volume of balanced salt solution plus (BSS+) was injected in control eyes. We compared different models for inducing corneal edema in rabbits, and examined the ability of transplanted HCECs to reduce corneal edema over time by measuring central corneal thickness and tracking corneal clarity. GFP-positive donor cells were tracked in vivo using optical coherence tomography (OCT) fluorescence angiography module, and the transplanted cells were confirmed by human nuclei immunostaining. Results: Magnetic HCECs integrated onto the recipient corneas with intact Descemet's membrane, and donor identity was confirmed by GFP expression and immunostaining for human nuclei marker. Donor HCECs formed a monolayer on the posterior corneal surface and expressed HCEC functional markers of tight junction formation. No GFP-positive cells were observed in the trabecular meshwork or on the iris, and intraocular pressure remained stable through the length of the study. Conclusions: Our results demonstrate magnetic cell-based therapy efficiently delivers HCECs to restore corneal transparency without detectable toxicity or adverse effect on intraocular pressure. Magnetic delivery of HCECs may enhance corneal function and should be explored further for human therapies.

Distribution of Anterior Chamber Parameters in Normal Chinese Children and the Associated Factors.

PURPOSE: To describe the distribution of anterior chamber depth (ACD), anterior chamber volume (ACV), and anterior chamber angle (ACA) and establish the associated factors in the pediatric population in Shanghai, China. MATERIALS AND METHODS: In this cross-sectional study, children aged 6 to 18 years from 9 primary and middle schools in Shanghai were enrolled. The Pentacam Scheimpflug camera was used to measure anterior eye chamber parameters. The distribution of ACD, ACV, ACA, and their associations with age, sex, body mass index, cycloplegic refractive error, axial length, intraocular pressure, and other parameters were analyzed. RESULTS: A total of 1321 children were included, with a mean age of 9.65±2.95 years. The mean ACA, ACD, and ACV values were 37.95±7.96 degrees, 3.22±0.23 mm and 194.89±28.95 mm, respectively, and were higher in boys than in girls. ACV and ACD had similar growth trend curves with age, whereas ACA was stable. Overall, 5% of the tested children had ACA values ≤24.91 degrees. Greater ACV, deeper ACD, shorter pupil diameter, shorter axial length, and thinner apex corneal thickness were the independent factors associated with wider ACA (R=13.0%, P<0.001). CONCLUSIONS: As one of diagnostic indicators of angle closure, ACA was stable with age. The results of this study should improve the current understanding of the distribution of anterior chamber parameters and the main factors affecting their variation.

Application of polycaprolactone nanofibers as patch graft in ophthalmology.

PURPOSE: The purpose of the study was to evaluate tissue reaction to polycaprolactone (PCL) nanofiber patches in the cornea, conjunctiva, and anterior chamber (AC) in rabbit eyes and to assess their biocompatibility for use as patch grafts. METHODS: Two 100 µ PCL patches were implanted under the conjunctiva and in the corneal stroma of one albino New Zealand rabbit, and pathologic evaluation was done after 3 weeks. In the next step, two PCL patches were implanted; one in the corneal stroma and the other in the AC of two rabbits followed by pathologic evaluation after 3 months. RESULTS: On slit-lamp examination, there was minimum inflammation in all cases. Pathologic examination showed that the contact and probably merging between the host tissue and PCL fibers were achieved with minimal tissue reaction. CONCLUSION: As a biocompatible material, PCL nanofibers seem to be a promising modality for the repair of different tissue defects including melting, thinning, and perforation. They may also be a suitable material for manufacturing keratoprostheses.

Comparison of Efficacy of Difluprednate 0.05% and Loteprednol Gel 0.5% After Cataract Surgery.

PURPOSE: To compare the outcomes and complications of topical difluprednate 0.05% and loteprednol gel 0.5% after routine cataract surgery. METHODS: Subjects received either difluprednate emulsion 0.05% (n=30 eyes) or loteprednol gel 0.5% (n=30 eyes) after routine cataract surgery. Topical steroid drops were initiated 3 days before cataract surgery and continued for 2 weeks postoperatively. Anterior chamber (AC) cell grade, corneal edema, corneal pachymetry, visual acuity, ocular surface quality (Oxford scale), and intraocular pressure (IOP) were evaluated at 1 day, 1 week, and 1 month postoperatively. RESULTS: Patients treated with difluprednate or loteprednol had statistically similar resolution of their AC cell grade and corneal edema at 1 day, 1 week, and 1 month postoperatively (P>0.05 at each study visit). Difluprednate-treated and loteprednol-treated eyes achieved a mean best-corrected visual acuity of at least 20/25 by 1 week postoperatively (0.055 and 0.061 logarithm of the minimum angle of resolution, respectively; P=0.82). The nasal ocular surface quality at 1 week had improved in loteprednol-treated eyes compared with difluprednate-treated eyes (1.0 vs. 1.9 Oxford score, respectively; P<0.001), but similar at all other visits. There was no statistical difference between IOP levels between both treatment groups (P>0.05). In the difluprednate-treated group, one patient developed rebound inflammation and two patients developed cystoid macular edema at their 1-month postoperative visit. CONCLUSIONS: The anti-inflammatory effect, visual recovery, and IOP of patients using topical difluprednate or loteprednol gel after cataract surgery are equivalent. There may be an additional short-term benefit of loteprednol gel in protecting the ocular surface after cataract surgery.

Comparison of the measurements of a novel optical biometry: Nidek AL-Scan with Sirius and a ultrasound biometry.

To investigate the accuracy of the measurements of Nidek AL-Scan by comparing with Sirius (CSO, Florence, Italy), a corneal tomography which also employs the Scheimpflug principle, and a commonly used device, ultrasound biometry (UB) (Aviso A/B, Quantel Medical, MT, USA). Right eyes of 85 healthy volunteers (58 women 27 men) with a mean age of 39.24 ± 14.37 years (range 15-68) were enrolled into this comparative prospective study. Average K 2.4, average K 3.3, CCT (central corneal thickness), WTW (white to white distance), ACD (anterior chamber depth) and AL (axial length) were obtained from the AL-Scan and compared with average SimK, CCT, WTW (horizontal anterior chamber diameter) and ACD obtained from Sirius and also compared with ACD and AL obtained from UB. The statistically significant difference was found between all of the measurements (p < 0.001) except the average keratometry values (K2.4, K3.3, SimK) (p = 0.083). There was a perfect correlation between keratometry, CCT and AL measurements of the devices (ICC = 0.977, 0.954, 0.923, respectively) and there was a strong correlation between the WTW measurements of AL-Scan and Sirius (ICC = 0.865). While ACD parameter of AL-Scan and UB showed a perfect correlation (ICC = 0.977), there was a moderate correlation between AL-Scan and Sirius and also between UB and Sirius (ICC = 0.608 and 0.664, respectively). There was a high correlation between the all measurements, besides ACD, of AL-Scan and Sirius and they can be used interchangeably for average keratometry and WTW confidently. However, ACD and CCT have a broader 95 % LoA (-0.039 to 0.744 and -24.985 to 3.691, respectively). In addition, AL-Scan and UB were in good agreement regarding ACD, while differences in AL measurements of UB and AL-Scan were clinically important (95 % LoA = -0.091 to 0.703). Furthermore, UB and Sirius have a moderate agreement regarding ACD (95 % LoA = -0.047 to 0.680).

Anterior Chamber Angle Morphometry Measurement Changes to Ambient Illumination Scaling in Visante Time Domain Optical Coherence Tomography.

PURPOSE: To test the effect of ambient illumination scaling on the reproducibility and reliability anterior chamber metrics using the Visante time domain optical coherence tomography (TD-OCT) instrument. MATERIALS AND METHODS: The inferior irido-corneal angles of 25 normal, healthy eyes were imaged twice with the Zeiss Visante TD-OCT under five strictly controlled ambient light conditions (foot candles (fc) measured with a light meter at camera/eye interface). Each eye was imaged 10 times totaling 250 assessments. Angle opening distance (AOD500/750), trabecular iris space area (TISA500/750), and scleral spur (SS) angle were graded twice by masked, trained graders at the Doheny Imaging Reading Center using the Visante's intrinsic tools. Lighting effects on measurements, intra-/inter-grader and acquisition analyses, and Bland-Altman plots were computed using Statistical Package for Social Science (SPSS Inc. version 18.0, Armonk, NY). RESULTS: With a near linear relationship of angle metrics to lights levels (R2 = 0.8-0.95), the analysis examines the differences from the brightest to darkest light levels. Decreasing ambient light levels from 1.0 to 0.0 fc decreased the average AOD500 measurement from 407 ± 136 µm to 315 ± 114 µm (mean percent difference (MPD) 29%, p < 0.001), AOD750 from 587 ± 184 µm to 496 ± 155 µm (MPD 18%, p < 0.001), TISA500 from 136 ± 43 µm2 to 101 ± 37 µm2 (MPD 35%, p < 0.001), TISA750 from 269 ± 81 µm2 to 212 ± 68 µm2 (MPD 27%, p < 0.001), and SS angle from 38.3% ± 9% to 32.1% ± 9% (MPD 19%, p < 0.001). Intra-/inter-grader results showed good reproducibility for each grader (MPD = 0.7-3%; coefficient of variation (CV) = 3.2-8.3%; R2 = 0.8-0.95; p < 0.001 for all metrics) and between graders (MPD = 1.4-5.9%; CV = 6.7-14.2%; R2 = 0.81-0.89; Pearson Correlation Coefficient (PCC) = 0.8-0.97 (p<0.001)). Bland-Altman plots did not demonstrate any apparent bias, with similar repeatability and agreement. CONCLUSIONS: The results of this study show the high sensitivity of the anterior chamber to changes in the illumination. The slight decrease in light had a corresponding large decrease in Anterior Chamber Angle (ACA) metrics. With clinical diagnoses and treatments of eye diseases relying on these angle measurements, these findings emphasize the importance of strictly controlling light conditions in order to obtain reproducible measurements of anterior chamber geometry.

Quantitative Assessment of Anterior Segment Inflammation in a Rat Model of Uveitis Using Spectral-Domain Optical Coherence Tomography.

PURPOSE: To develop anterior segment spectral-domain optical coherence tomography (SD-OCT) and quantitative image analysis for use in experimental uveitis in rats. METHODS: Acute anterior uveitis was generated in Lewis rats. A spectral domain anterior segment OCT system was used to image the anterior chamber (AC) and ciliary body at baseline and during peak inflammation 2 days later. Customized MatLab image analysis algorithms were developed to segment the AC, count AC cells, calculate central corneal thickness (CCT), segment the ciliary body and zonules, and quantify the level of ciliary body inflammation with the ciliary body index (CBI). Images obtained at baseline and during peak inflammation were compared. Finally, longitudinal imaging and image analysis was performed over the 2-week course of inflammation. RESULTS: Spectral-domain optical coherence tomography identifies structural features of inflammation. Anterior chamber cell counts at peak inflammation obtained by automated image analysis and human grading were highly correlated (r = 0.961), and correlated well with the histologic score of inflammation (r = 0.895). Inflamed eyes showed a significant increase in average CCT (27 µm, P = 0.02) and an increase in average CBI (P < 0.0001). Longitudinal imaging and quantitative image analysis identified a significant change in AC cell and CBI on day 2 with spontaneous resolution of inflammation by day 14. CONCLUSIONS: Spectral-domain optical coherence tomography provides high-resolution images of the structural changes associated with anterior uveitis in rats. Anterior chamber cell count and CBI determined by semi-automated image analysis strongly correlates with inflammation, and can be used to quantify inflammation longitudinally in single animals.

Noninvasive in vivo imaging of embryonic ß-cell development in the anterior chamber of the eye.

The fetal environment plays a decisive role in modifying the risk for developing diabetes later in life. Developing novel methodology for noninvasive imaging of ß-cell development in vivo under the controlled physiological conditions of the host can serve to understand how this environment affects ß-cell growth and differentiation. A number of culture models have been designed for pancreatic rudiment but none match the complexity of the in utero or even normal physiological environment. Speier et al. recently developed a platform of noninvasive in vivo imaging of pancreatic islets using the anterior chamber of the eye where islets get vascularized, grow and respond to physiological changes. The same methodology was adapted for the study of pancreatic development. E13.0, still undifferentiated rudiments with fluorescent lineage tracing were implanted in the AC of the eye, allowing the longitudinal study of their growth and differentiation. Within 48 h the anlages get vascularized and grow but their mesenchyme displays a selective growth advantage. The resulting imbalance leads to alteration in the differentiation pattern of the progenitors. Reducing the mesenchyme to its bare minimum before implantation allows the restoration of a proper balance and a development that mimics the normal pancreatic development. These groundbreaking observations demonstrate that the anterior chamber of the eye provides a good system for noninvasive in vivo fluorescence imaging of the developing pancreas under the physiology of the host and can have important implications for designing strategies to prevent or reverse the deleterious effects of hyperglycemia on altering ß-cell function later in life.

The anterior chamber of the eye is a transplantation site that supports and enables visualisation of beta cell development in mice.

AIMS/HYPOTHESIS: In vivo imaging of the developing pancreas is challenging due to the inaccessibility of the tissue. To circumvent this, on embryonic day 10.5 (E10.5) we transplanted a mouse developing pancreatic bud into the anterior chamber of the eye (ACE) to determine whether the eye is a useful transplant site to support pancreas development. METHODS: We transplanted an E10.5 dorsal pancreatic bud into the ACE of a syngeneic recipient mouse. Using a mouse insulin promoter-green fluorescent protein (MIP-GFP) mouse as the tissue donor, we non-invasively imaged the pancreatic bud as it develops at single beta cell resolution across time. RESULTS: The transplanted pancreatic bud rapidly engrafts and vascularises when transplanted into the ACE. The pancreatic progenitor cells differentiate into exocrine and endocrine cells, including cells expressing insulin, glucagon and somatostatin. The morphology of the transplanted pancreatic bud resembles that of the native developing pancreas. Beta cells within the transplanted pancreatic bud respond to glucose in a manner similar to that of native fetal beta cells and superior to that of in vitro developed beta cells. Unlike in vitro grown pancreatic explants, pancreatic tissue developing in the ACE is vascularised, providing the developing pancreatic tissue with a milieu resembling the native situation. CONCLUSIONS/INTERPRETATION: Altogether, we show that the ACE is able to support growth, differentiation and function of a developing pancreatic bud across time in vivo.

Species Differences in the Geometry of the Anterior Segment Differentially Affect Anterior Chamber Cell Scoring Systems in Laboratory Animals.

PURPOSE: To determine the impact of anterior segment geometry on ocular scoring systems quantifying anterior chamber (AC) cells in humans and 7 common laboratory species. METHODS: Using normative anterior segment dimensions and novel geometric formulae, ocular section volumes measured by 3 scoring systems; Standardization of Uveitis Nomenclature (SUN), Ocular Services On Demand (OSOD), and OSOD-modified SUN were calculated for each species, respectively. Calculated volumes were applied to each system's AC cell scoring scheme to determine comparative cell density (cells/mm(3)). Cell density values for all laboratory species were normalized to human values and conversion factors derived to create modified scoring schemes, facilitating interspecies comparison with each system, respectively. RESULTS: Differences in anterior segment geometry resulted in marked differences in optical section volume measured. Volumes were smaller in rodents than dogs and cats, but represented a comparatively larger percentage of AC volume. AC cell density (cells/mm(3)) varied between species. Using the SUN and OSOD-modified SUN systems, values in the pig, dog, and cat underestimated human values; values in rodents overestimated human values. Modified normalized scoring systems presented here account for species-related anterior segment geometry and facilitate both intra- and interspecies analysis, as well as translational comparison. CONCLUSIONS: Employment of modified AC cell scoring systems that account for species-specific differences in anterior segment anatomy would harmonize findings across species and may be more predictive for determining ocular toxicological consequences in ocular drug and device development programs.

Physical Factors Affecting Outflow Facility Measurements in Mice.

PURPOSE: Mice are commonly used to study conventional outflow physiology. This study examined how physical factors (hydration, temperature, and anterior chamber [AC] deepening) influence ocular perfusion measurements in mice. METHODS: Outflow facility (C) and pressure-independent outflow (Fu) were assessed by multilevel constant pressure perfusion of enucleated eyes from C57BL/6 mice. To examine the effect of hydration, seven eyes were perfused at room temperature, either immersed to the limbus in saline and covered with wet tissue paper or exposed to room air. Temperature effects were examined in 12 eyes immersed in saline at 20 °C or 35 °C. Anterior chamber deepening was examined in 10 eyes with the cannula tip placed in the anterior versus posterior chamber (PC). Posterior bowing of the iris (AC deepening) was visualized by three-dimensional histology in perfusion-fixed C57BL/6 eyes and by spectral-domain optical coherence tomography in living CD1 mice. RESULTS: Exposure to room air did not significantly affect C, but led to a nonzero Fu that was significantly reduced upon immersion in saline. Increasing temperature from 20 °C to 35 °C increased C by 2.5-fold, more than could be explained by viscosity changes alone (1.4-fold). Perfusion via the AC, but not the PC, led to posterior iris bowing and increased outflow. CONCLUSIONS: Insufficient hydration contributes to the appearance of pressure-independent outflow in enucleated mouse eyes. Despite the large lens, AC deepening may artifactually increase outflow in mice. Temperature-dependent metabolic processes appear to influence conventional outflow regulation. Physical factors should be carefully controlled in any outflow studies involving mice.

Monitoring of strain-dependent responsiveness to TLR activation in the mouse anterior segment using SD-OCT.

PURPOSE: To determine whether spectral-domain optical coherence tomography (SD-OCT) can be used to longitudinally monitor inflammation in the mouse anterior segment and to identify any strain-dependent differences in responsiveness to distinct toll-like receptor (TLR) ligands. METHODS: Corneal inflammation was induced in BALB/c and C57BL/6 mice following central corneal abrasions and topical application of saline, TLR-4 ligand, lipopolysaccharide (LPS), or TLR-9 ligand, CpG-oligodeoxynucleotide (CpG-ODN; CpG). Anterior-segment images were captured using SD-OCT at baseline, 24 hours, and 1 week post treatment. Corneal thickness, stromal haze, and the number of keratic precipitates (KP) and anterior chamber (AC) cells were longitudinally compared to determine differences between mouse strains, time points, and TLR activation. RESULTS: In both mouse strains, treatment with CpG, but not saline or LPS, resulted in a similar number of KPs and AC cells. In C57BL/6 mice, central corneal thickness (CCT) increased in CpG- and LPS-treated eyes at 24 hours, which normalized by 1 week. In BALB/c mice, a significant increase in CCT occurred in eyes treated with CpG at 1 week. Stromal haze peaked in C57BL/6 eyes treated with LPS- or CPG-treatment at 24 hours; however, BALB/c eyes showed persistent and marked increases in corneal haze compared with baseline at 1 week post treatment. CONCLUSIONS: Spectral-domain OCT enables high-resolution, longitudinal, in vivo imaging of anterior segment inflammation in mice and revealed novel strain- and time-dependent differences in response to distinct TLR activation in the cornea.

Identification of adult stem cells in Schwalbe's line region of the primate eye.

PURPOSE: To identify stem cells in the chamber angle of the monkey eye by detection of 5-bromo-2'-deoxyuridine (BrdU) long-term retention. METHODS: Four cynomolgus monkeys were treated with BrdU via subcutaneous pumps for 4 weeks. The eyes of two animals were processed immediately thereafter (group 1) while in the other animals, BrdU treatment was discontinued for 4 weeks to allow identification of cells with long-term BrdU retention (group 2). The number of BrdU-positive nuclei was quantified, and the cells were characterized by immunohistochemistry and transmission electron microscopy (TEM). RESULTS: The number of BrdU-positive cells was higher at Schwalbe's line covering the peripheral end of Descemet's membrane than in Schlemm's canal (SC) endothelium, trabecular meshwork (TM), and scleral spur (SS). Labeling with BrdU in SC, TM, and SS was less intense and the number of labeled cells was smaller in group 2 than in group 1. In contrast, in cells of Schwalbe's line the intensity of BrdU staining and the number of BrdU-positive cells was similar when group 1 and 2 monkeys were compared with each other, indicating long-term BrdU retention. Cells that were BrdU-positive in Schwalbe's line region stained for the stem cell marker OCT4. Details of a stem cell niche in Schwalbe's line region were identified by TEM. CONCLUSIONS: We provide evidence for a niche in the Schwalbe's line region harboring cells with long-term BrdU retention and OCT4 immunoreactivity. The cells likely constitute a population of adult stem cells with the capability to compensate for the loss of TM and/or corneal endothelial cells.

Feedback-controlled constant-pressure anterior chamber perfusion in live mice.

PURPOSE: To describe live mouse, anterior chamber constant-pressure perfusion by an approach using feedback-controlled coupling of pressure and flow to maintain a preset pressure. METHODS: We established a microperfusion system that maintains a constant preset pressure in the anterior chamber of live mice by automatically regulating the microsyringe pump flow rate with a computer-controlled voltage feedback loop. Perfusion was by single-needle cannulation. We characterized the following in C57BL/6 mice aged 3-4 months in vivo: (i) pressure stability, (ii) pressure and flow rate reproducibility, (iii) total outflow facility, and (iv) anterior segment histology after perfusion. RESULTS: Twenty live mice underwent perfusion. Constant pressure was quickly attained and stably maintained. The coefficient of pressure variation over time during perfusion at a preset pressure was <0.001. The average coefficient of variation for repeat pressure and flow rate measurements was 0.0005 and 0.127, respectively. The relationship between flow rate and pressure was linear for perfusions between 15 and 35 mmHg. The total outflow facility was 0.0066 µl/min/mmHg. Perfusion system resistance (0.5 mmHg/min/µl) was negligible relative to the ocular outflow resistance (147 mmHg/min/µl) at physiologically relevant perfusion pressures of 15-35 mmHg. No histological disruption of the drainage tissue was seen following perfusion. CONCLUSIONS: Predetermined pressure was stably maintained during constant-pressure perfusion of live mouse eyes by a method using feedback-controlled coupling of pressure and flow along with single-needle anterior chamber cannulation. Perfusion measurements were reproducible. This approach is potentially useful for exploring aqueous drainage tissue biology, physiology, and pharmacology in live mice.

Anterior chamber cell grading by optical coherence tomography.

PURPOSE: To quantify cells in the ocular anterior chamber (AC) by optical coherence tomography (OCT). METHODS: A time-domain anterior segment OCT system was used to image latex microsphere suspensions in vitro and the AC of uveitis and normal subjects in vivo. The OCT scan pattern, consisting of 2- and 4-mm-diameter concentric circular scans, was divided into central, superior, and inferior regions. A computer algorithm was developed to automatically identify particles in OCT images. A uveitis specialist used slit-lamp biomicroscopy to grade the AC cells on a scale of 0 to 4+. RESULTS: Latex microspheres and ac cells were visualized as reflective spots in oct images. OCT latex microsphere concentration measurements were highly correlated to known particle concentrations (r = 1.000) and had an efficiency of 0.72. in 30 nongranulomatous and 12 granulomatous eyes, the OCT cell counts correlated well with slit-lamp grades in all three regions (Spearman's rho coefficient: >0.63). The average OCT cell count was 3.7 cells/grade in nongranulomatous eyes and 2.0 cells/grade in granulomatous eyes. OCT revealed significant amounts of inferior AC cells in 5 of 16 quiescent uveitis eyes (mean ± SD: 19.9 ± 7.4 cells). OCT captured rare cells in normal eyes (1.1 ± 1.1 cells centrally). CONCLUSIONS: OCT provided quantitative information on AC inflammatory cells. The OCT cell counts correlated well with clinical grading, and particles in the inferior AC that were missed by slit-lamp examination were detected by OCT. OCT could be a valuable tool for the diagnosis and management of anterior uveitis.

An intracameral injection of antigen induces in situ chemokines and cytokines required for the generation of circulating immunoregulatory monocytes.

Anterior Chamber-Associated Immune Deviation (ACAID) induced by an intracameral injection of antigen generates antigen-specific regulatory splenic T cells that suppress specifically cell-mediated immunity specific for the injected antigen. Circulating F4/80(+) cells recovered from mice receiving an intracameral injection of antigen are thought to be ocular in origin and induce the development of thymic and splenic regulatory T cells. We have shown previously that after the intracameral injection of antigen there is a CCR2/CCL2-dependent infiltration of circulating F4/80(+) cells into the anterior chamber associated with the generation of circulating, ACAID-inducing F4/80(+) monocytes. Here we tested the hypothesis that the intracameral injection of antigen induces events in the anterior chamber that are associated with the induction of circulating immunoregulatory monocytes that induce the suppression of cell-mediated immunity. The intracameral injection of antigen resulted in aqueous humor (i) a time- dependent increase of CCL2 and CCL7, (ii) a transient increase in TNF-α, and (iii) an infiltration of CD11b(hi), Gr1(hi) and F4/80(+) as well as F4/80(-) and Gr1(hi) peripheral blood cells into the anterior chamber. Further characterization of these F4/80(+) cells revealed that they are Ly 6C(hi), LY6G(lo) or negative, 7/4 (LY6B)(hi), CD115(+), CD45(+), CD49B(+), and CD62 L(+). Antibody-mediated neutralization of TGF-ß in situ in the anterior chamber prevented the induction of circulating, ACAID-inducing monocytes and ACAID. These cells did not increase in the irides of ACAID-refractory CCR2-/- and CCL2-/- mice that received an intracameral injection of antigen. Our results extend our suggestion that ACAID is initiated as the result of a mild proinflammatory response to intracameral injection that results in the infiltration of a CCR2(+) subset of monocytes into the anterior chamber where there is a TGF-ß-dependent induction of an immunosuppressive phenotype in the infiltrated monocytes that recirculate to induce antigen-specific regulatory T cells.