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.

Breakdown of the blood-eye barrier in choroidal melanoma after proton beam radiotherapy.

PURPOSE: Irradiation of choroidal melanoma is a safe and globe preserving procedure. Chronic inflammatory processes and ischemia are the main reasons for secondary enucleation in the long run. The aim of this study was to determine whether intraocular inflammation and especially inflammatory response after proton beam therapy (PBT) is related to primary tumor characteristics such as height, tumor volume, and initial flare values. METHODS: Twenty-six patients treated for uveal melanoma using PBT were included. All patients were examined for signs of inflammation using laser flare photometry (LFP). Each examination included assessment of the melanoma and fellow eye (which acted as the control) and imaging of the melanoma. RESULTS: Significant differences of flare values between melanoma eyes and control group were found both at baseline (median 17.65 ph/ms (min 4, max 37.10), 7.45 ph/ms (min 0.80, max 16.40), respectively) and during follow-up (median 21.45 ph/ms (min 4.5, max 70.90); 6.05 ph/ms (min 2.40, max 16.40), respectively) (p < 0.001, Wilcoxon test). Flare values in melanoma eyes increased significantly after PBT (p = 0.005, Wilcoxon test) and after a follow-up of 94 days (median, 7-420 days). Flare values of the control group did not change (p = 0.946, Wilcoxon test). The increase of flare values correlated significantly with maximum tumor height and volume (Spearman-Rho 0.633, p = 0.001 and 0.519, p = 0.007, respectively). CONCLUSION: LFP has proven to show significantly higher flare values in melanoma eyes as compared with the control group and provides data on the course of the inflammatory response after treatment. It may ease treatment planning both at baseline and during follow-up.

Application of Laser Flare Photometry in the Quantification of Blood-Aqueous Barrier Breakdown after Micro-incision Vitrectomy.

Purpose: The purpose of the study is to quantify aqueous flare using laser flare photometry (LFM) in patients undergoing 25-G pars plana vitrectomy (PPV) and assess the need for postoperative topical corticosteroid administration . Methods: Prospective evaluation of 50 eyes (50 patients) was performed using LFM until day 30 postoperative. Duration of surgery, indication of PPV, and use of laser and/or cryotherapy were noted. Topical corticosteroids were used if mean LFM values were >50, or if there was anterior chamber fibrin. Results: Mean age of the subjects was 48.62 ± 10.07 years. The preoperative LFM value for 50 subjects was 17.42 ± 25.20. Topical corticosteroids were initiated in only 22 patients. The LFM values of subjects were not different from baseline at 1 month whether or not the subjects received corticosteroids (p > 0.106). Conclusions: With 25-G PPV, there is minimal breakdown of blood-aqueous barrier. LFM helps in monitoring postoperative inflammation, obviating the need for topical corticosteroids in significant number of patients.

Does the disruption of horizontal anterior ciliary vessels affect the blood-aqueous barrier function?

PURPOSE: To investigate the significance of the anterior ciliary vessels (ACVs) preservation during the conventional horizontal strabismus surgery. METHODS: Patients (≥ 8 years) with horizontal strabismus were randomly allocated into group 1 (with ACV preservation) and group 2 (without ACV preservation). The surgical eyes in group 1 were further divided into group A (one rectus muscle operated) and group B (two rectus muscles operated). Similarly, eyes in group 2 were divided into group C (one rectus muscle operated) and group D (two rectus muscles operated). The success rate of ACV preservation was calculated. The anterior chamber flare measurements of each eye by laser flare photometry were recorded on the day prior to and after operation. The flare values between groups and between pre- and post-operation in each group were compared by one-way analysis of variance and a paired t-test respectively. RESULTS: In groups A and B, the success rate of ACV preservation was 82% (27/33) and 70% (28/40)respectively, and the flare values between pre- and post-operation showed no significant differences(4.378 ± 1.527, 4.544 ± 1.452, P = 0.526; 4.625 ± 1.090, 4.989 ± 1.468, P = 0.101 respectively). However, the postoperative values were significantly increased in group C and group D(4.661 ± 1.031, 5.039 ± 1.310, P = 0.025; 4.933 ± 1.691, 5.502 ± 1.430, P = 0.000 respectively). The postoperative flare readings of group D were significantly higher than group B, while group A and group C had no significant variation. CONCLUSION: ACV preservation probably has clinical significance in reducing the undesirable influence on the blood-aqueous barrier.

Correlation between anterior chamber characteristics and laser flare photometry immediately after femtosecond laser treatment before phacoemulsification.

PurposeTo assess the anterior chamber (AC) characteristics and its correlation to laser flare photometry immediately after femtosecond laser-assisted capsulotomy and photodisruption.Patients and methodsThe study included 97 cataract eyes (n=97, mean age 68.6 years) undergoing femtosecond laser-assisted cataract surgery (FLACS). Three cohorts were analysed relating to the flare photometry directly post femtosecond laser treatment (flare <100 n=28, 69.6±7 years; flare 100-249 n=47, 67.7±8 years; flare >249 photon counts per ms cohort n=22, 68.5±10 years). Flare photometry (KOWA FM-700), corneal topography (Oculus Pentacam, Germany: AC depth, volume, angle, pachymetry), axial length, pupil diameter, and endothelial cells were assessed before FLACS, immediately after femtosecond laser treatment and 1 day postoperative (LenSx Alcon, USA). Statistical data were analysed by SPSS v19.0, Inc.ResultsThe AC depth, AC volume, AC angle, central and thinnest corneal thickness showed a significant difference between flare <100 vs flare 100-249 10 min post femtosecond laser procedure (P=0.002, P=0.023, P=0.007, P=0.003, P=0.011, respectively). The AC depth, AC volume, and AC angle were significantly larger (P=0.001, P=0.007, P=0.003, respectively) in the flare <100 vs flare >249 cohort 10 min post femtosecond laser treatment.ConclusionsA flat AC, low AC volume, and a narrow AC angle were parameters associated with higher intraocular inflammation. These criteria could be used for patient selection in FLACS to reduce postoperative intraocular inflammation.

Anterior chamber aqueous flare, pseudoexfoliation syndrome, and glaucoma.

The purpose of this study is to evaluate anterior chamber aqueous flare (ACAF) in Tunisian patients with pseudoexfoliation (PEX) syndrome with or without associated glaucoma. This is a prospective, cross-sectional, comparative study including 53 patients (88 eyes) with PEX syndrome, 48 patients with PEX glaucoma (86 eyes), and 53 healthy sex-and age-matched control subjects (106 eyes). All patients underwent a complete ophthalmic examination and laser flare photometry. Mean ACAF was significantly higher in the PEX syndrome group in comparison with the control group (17.96 ± 10.05 vs 7.06 ± 2.95 ph/ms; p = 10(-4)), in patients with PEX glaucoma compared to PEX syndrome without associated glaucoma (27.99 ± 15.45 vs 17.96 ± 10.05 ph/ms; p = 10(-4)), in the PEX glaucoma group in comparison with control group (27.99 ± 15.45 vs 7.06 ± 2.95 ph/ms; p = 10(-4)), and in patients with unilateral PEX syndrome in comparison with contralateral-unaffected eyes (25.72 ± 14.88 vs 8.58 ± 3.45 ph/ms; p = 0.000). For patients with PEX syndrome, a high ACAF might be a predictor for the development of glaucoma. Further investigations are needed to clarify the role of laser flare photometry in predicting the risk of glaucoma in patients with PEX syndrome.

Transport across Schlemm's canal endothelium and the blood-aqueous barrier.

The majority of trabecular outflow likely crosses Schlemm's canal (SC) endothelium through micron-sized pores, and SC endothelium provides the only continuous cell layer between the anterior chamber and episcleral venous blood. SC endothelium must therefore be sufficiently porous to facilitate outflow, while also being sufficiently restrictive to preserve the blood-aqueous barrier and prevent blood and serum proteins from entering the eye. To understand how SC endothelium satisfies these apparently incompatible functions, we examined how the diameter and density of SC pores affects retrograde diffusion of serum proteins across SC endothelium, i.e. from SC lumen into the juxtacanalicular tissue (JCT). Opposing retrograde diffusion is anterograde bulk flow velocity of aqueous humor passing through pores, estimated to be approximately 5 mm/s. As a result of this relatively large through-pore velocity, a mass transport model predicts that upstream (JCT) concentrations of larger solutes such as albumin are less than 1% of the concentration in SC lumen. However, smaller solutes such as glucose are predicted to have nearly the same concentration in the JCT and SC. In the hypothetical case that, rather than micron-sized pores, SC formed 65 nm fenestrae, as commonly observed in other filtration-active endothelia, the predicted concentration of albumin in the JCT would increase to approximately 50% of that in SC. These results suggest that the size and density of SC pores may have developed to allow SC endothelium to maintain the blood-aqueous barrier while simultaneously facilitating aqueous humor outflow.

A comparative study between clinical grading of anterior chamber flare and flare reading using the Kowa laser flare meter.

To assess the accuracy of standard clinical grading of aqueous flare in uveitis according to the Standardization of Uveitis Nomenclature consensus, and compare the results with the readings of the laser flare meter, Kowa 500. Two examiners clinically graded the flare in 110 eyes. The flare was then measured using the Kowa laser flare meter. Twenty-nine eyes were graded as anterior chamber flare +2; for 18 of these, the clinicians were in agreement, the rest differed by the order of one grade. The range of the laser flare meter for these eyes was 5.2-899.1 photons/ms. The median value was 41.4. Seventy-four eyes were graded with flare +1. Agreement was established in 51 of these eyes. Disagreement for the rest was again by the order of 1, and the flare meter range was 1.1-169.9 photons/ms, median value 18.4. For the clinical measure of flare 0, the clinicians disagreed on three out of five eyes. The flare meter readings ranged from 2.5 to 14.1 photons/ms, median value 9.9. Only two eyes were graded with flare +3 and there was one step disagreement on both of them. We found little evidence of association between the flare readings and intraocular pressure or age. Our findings suggest that clinical evaluation of aqueous flare is subjective. Compared with the Kowa laser flare meter's numeric readings, the discrepancies observed indicate that clinical grading is an approximate science. The laser flare meter provides an accurate, reproducible, non-invasive assessment of aqueous flare that can prove valuable in research and clinical decisions.

The blood-aqueous barrier in health and disease.

The blood-aqueous barrier of the eye is composed by tight junctions in the ciliary process nonpigmented epithelium, the endothelial cells in the iris vasculature, and the inner wall endothelium of Schlemm's canal. Tight junctions are gatekeepers of the paracellular transport limiting the selective diffusion of ions and small solutes through the space between neighboring cells. Tight junctions (ie, junctional adhesion molecules, claudins, occludins, zonula occludens, cingulin) are part of the apical junctional complex that also includes the adherens junctions (ie, cadherin-catenin and nectin-afadin complexes) and the gap junctions (ie, connexins). These junctional complexes respond rapidly to pharmacologic agents and physiological changes. Barrier dysfunction can contribute to the pathophysiology of inflammatory ocular diseases in a passive way by the vascular leakage of blood-borne molecules and inflammatory cells into the anterior segment of the eye.

Impact of P-glycoprotein on blood-retinal barrier permeability: comparison of blood-aqueous humor and blood-brain barrier using mdr1a knockout rats.

PURPOSE: The purpose of this study was to clarify the impact of P-glycoprotein (P-gp) on blood-retinal barrier (BRB) and blood-aqueous humor barrier (BAB) permeability, in contrast to blood-brain barrier (BBB) permeability. METHODS: Permeabilities of six compounds, including P-gp substrates (quinidine, digoxin, and verapamil), were investigated in wild-type and mdr1a knockout rats using retinal, aqueous humor, and brain uptake index (RUI, AHUI, and BUI, respectively) methods and integration plot analysis. RESULTS: In both rat strains, quinidine, digoxin, and verapamil were transported by P-gp across each barrier; however, the impact of P-gp on retinal uptake of quinidine and verapamil was less pronounced than that on brain uptake. The apparent influx permeability clearance (Kin) values of verapamil in retina obtained from wild-type and knockout rats were similar (0.824 ± 0.201 and 0.849 ± 0.980 mL/min·g retina, respectively; mean ± SD; n = 3 rats). The Kin in aqueous humor and brain obtained from knockout rats was, respectively, 3-fold and 12-fold higher than that of wild-type (P < 0.05). In P-gp-deficient conditions, the RUI and AHUI of quinidine, digoxin, and verapamil, as well as the BUI of quinidine and digoxin, were decreased by P-gp inhibitors. However, the BUI of verapamil was not changed by P-gp inhibitors. Results suggest that carrier-mediated influx transporters exist in the blood-ocular barriers and that the function of verapamil influx transporters is markedly different between the retina and brain. CONCLUSIONS: In both rat strains, P-gp operates in the blood-ocular barriers, and the impact of P-gp on BRB permeability to quinidine and verapamil is lower than that on BBB permeability.

Cerebral protection: inflammation, endothelial dysfunction, and postoperative cognitive dysfunction.

PURPOSE OF REVIEW: Postoperative cognitive dysfunction (POCD) is a well recognized perioperative syndrome, with approximately 15% of patients over the age of 60 years displaying objectively measured decrease in cognitive function as a consequence of anesthesia and surgery. The exact cause, however, remains unknown. This review aims to update anesthesiologists on the recent advancements in the understanding of the pathophysiology of POCD. RECENT FINDINGS: Recent evidence suggests that the observed predilection to POCD is likely mediated by a neuro-inflammatory response - with surgery being a major contributing factor. The blood-brain barrier, a highly specialized endothelial layer, is exquisitely sensitive to an inflammatory insult and implicated in the cause of other neurocognitive syndromes also characterized by neuro-inflammation such as cerebral malaria. Inflammatory changes may disrupt the blood-brain barrier and facilitate migration of macrophages into the brain, damaging synapses and neurones and ultimately lead to POCD. This review explores the important question of causality - the potential relationship between inflammation, endothelial dysfunction, and postoperative cognitive decline. SUMMARY: Recent research points to a central role of a neuro-inflammatory cascade in POCD, with endothelial dysfunction potentially aggravating the insult. Investigating the genomic and molecular mechanisms that underlie the intervariation in the inflammatory response to surgery, improving the identification of appropriate endothelial and inflammatory biomarkers, and developing endothelial modulatory and anti-inflammatory (prevention and resolution) strategies are key areas of future translational research. This is important as the elderly, who show increased susceptibility to this and other perioperative illness syndromes, represent an ever-increasing proportion of patients presenting for surgery.

Orchestrated leukocyte recruitment to immune-privileged sites: absolute barriers versus educational gates.

Complex barriers separate immune-privileged tissues from the circulation. Here, we propose that cell entry to immune-privileged sites through barriers composed of tight junction-interconnected endothelium is associated with destructive inflammation, whereas border structures comprised of fenestrated vasculature enveloped by tightly regulated epithelium serve as active and selective immune-skewing gates in the steady state. Based on emerging knowledge of the central nervous system and information from other immune-privileged sites, we propose that these sites are endowed either with absolute endothelial-based barriers and epithelial gates that enable selective and educative transfer of trafficking leukocytes or with selective epithelial gates only.

A contemporary concept of the blood-aqueous barrier.

This review traces the evolution of the concept of the blood-aqueous barrier (BAB) during the past 20 years. The Classical model simply stipulated that the tight junctions of the iris vasculature and ciliary epithelium excluded plasma proteins from the aqueous humor (AH). It failed to reconcile the presence of AH protein levels equal to 1% of that found in plasma. Moreover, models of barrier kinetics assumed that the processes of AH secretion and plasma protein entry were directly linked. Thus, elevations of AH protein levels could only be explained by a pathological breakdown of the BAB. Over the last 20 years it has been shown that the plasma proteins in normal AH by-pass the posterior chamber entirely. Instead, these proteins diffuse from the capillaries of ciliary body stroma, into the iris stroma and then into the anterior chamber. This creates a reservoir of plasma-proteins in the iris stroma that is not derived from the iris vessels. This reservoir is prevented from diffusing posteriorly by tight junctions in the posterior iris epithelium. The one-way valve created by the pupil resting on the anterior lens capsule, combined with the continuous, forward flow of AH through the pupil, prevents protein reflux into the posterior chamber. Importantly, in the new paradigm, secretion of AH and the entry of plasma proteins into AH, are semi-independent events. This opens the possibility that AH protein levels could increase in the absence of breakdown of the BAB. Clinical consequences of this new paradigm of the BAB are discussed.

Pilocarpine-induced flare is physiological rather than pathological.

An elevated aqueous humor protein level (aka flare) has always been considered to represent a pathological breakdown of the blood-aqueous barrier (BAB), regardless of the etiology. Recent studies in humans, using magnetic resonance imaging (MRI) to directly observe BAB kinetics in the posterior chamber of the human eye in-vivo, showed that pilocarpine-induced flare resulting from administration of a single drop of pilocarpine is not the result of breakdown of the BAB in the ciliary body. These MRI studies could not confirm whether pilocarpine caused an increase in iris vascular permeability. In the current studies we completed combined cell-flare meter and intravascular tracer studies, using intravenous horseradish peroxidase (HRP) in rabbits. One hour after receiving 3% pilocarpine in one eye, pupil size significantly decreased and aqueous flare significantly increased in pilocarpine-treated eyes. Light and electron microscopy demonstrated no leakage across either the iris vascular endothelium or the non-pigmented ciliary epithelium in either pilocarpine-treated or control eyes. One animal received HRP directly after pilocarpine to control for a transient increase in permeability before the peak flare response occurred. No leakage was found in the ciliary body or iris of this animal. Additional animals received topical pilocarpine in one eye but after 1 h they were sacrificed without tracer studies. Uveal tissues from these animals were used to assess the distribution of non-HRP protein in the ocular anterior segment and to assess the amount of elutable protein in the iris stromas of both treated and untreated eyes. Immunohistochemistry confirmed the presence of a reservoir of protein in the iris stroma. Analysis of elutable total protein from the iris stroma of pilocarpine-treated and control eyes showed significantly less total elutable protein in pilocarpine-treated eyes. Eyes with the greatest percent change in pupil size (i.e. the strongest miosis) correlated with lowest amounts of residual protein in the iris stroma. The tracer studies confirmed recent MRI studies in humans showing that the source of pilocarpine-induced flare is not disruption of the ciliary epithelial barrier. Extending this work, the current studies also showed no pilocarpine-induced leakage from the iris vasculature. The elutable protein experiments suggested that a primary source of pilocarpine-induced flare was extrusion of a portion of the reservoir of protein in the iris stroma. Taken together, these studies strongly suggest that not all clinically observable flare results from breakdown of the BAB.

In vivo assessment of the permeability of the blood-brain barrier and blood-retinal barrier to fluorescent indoline derivatives in zebrafish.

BACKGROUND: Successful delivery of compounds to the brain and retina is a challenge in the development of therapeutic drugs and imaging agents. This challenge arises because internalization of compounds into the brain and retina is restricted by the blood-brain barrier (BBB) and blood-retinal barrier (BRB), respectively. Simple and reliable in vivo assays are necessary to identify compounds that can easily cross the BBB and BRB. METHODS: We developed six fluorescent indoline derivatives (IDs) and examined their ability to cross the BBB and BRB in zebrafish by in vivo fluorescence imaging. These fluorescent IDs were administered to live zebrafish by immersing the zebrafish larvae at 7-8 days post fertilization in medium containing the ID, or by intracardiac injection. We also examined the effect of multidrug resistance proteins (MRPs) on the permeability of the BBB and BRB to the ID using MK571, a selective inhibitor of MRPs. RESULTS: The permeability of these barriers to fluorescent IDs administered by simple immersion was comparable to when administered by intracardiac injection. Thus, this finding supports the validity of drug administration by simple immersion for the assessment of BBB and BRB permeability to fluorescent IDs. Using this zebrafish model, we demonstrated that the length of the methylene chain in these fluorescent IDs significantly affected their ability to cross the BBB and BRB via MRPs. CONCLUSIONS: We demonstrated that in vivo assessment of the permeability of the BBB and BRB to fluorescent IDs could be simply and reliably performed using zebrafish. The structure of fluorescent IDs can be flexibly modified and, thus, the permeability of the BBB and BRB to a large number of IDs can be assessed using this zebrafish-based assay. The large amount of data acquired might be useful for in silico analysis to elucidate the precise mechanisms underlying the interactions between chemical structure and the efflux transporters at the BBB and BRB. In turn, understanding these mechanisms may lead to the efficient design of compounds targeting the brain and retina.

[Mek1 and Mek2 functions in the formation of the blood placental barrier]. / Le rôle des kinases Mek1 et Mek2 dans la formation de la barrière hématoplacentaire chez la souris.

The ERK/MAPK signaling pathway is involved in several cellular functions. Inactivation in mice of genes encoding members of this pathway is often associated with embryonic death resulting from abnormal placental development. The placenta is essential for nutritional and gaseous exchanges between maternal and embryonic circulations, as well as for the removal of metabolic wastes. These exchanges take place without direct contact between the two circulations. In mice, the hematoplacental barrier consists in a triple layer of trophoblast cells and endothelial cells of the embryo. MEK1 and MEK2 are double specificity serine-threonine/tyrosine kinases responsible for the activation of ERK1 and ERK2. Mek1 inactivation results in placental anomalies due to trophoblast cell proliferation and differentiation defects leading to severe delays in the development of placenta and causing the death of the embryo. Although Mek2(-/-) mutant mice survived without any apparent phenotype, double heterozygous Mek1(+/-)Mek2(+/-) mutants die during gestation from placental malformations. Together, these data emphasize the crucial role of the ERK/MAPK cascade in the formation of extraembryonic structures.

[Glaucoma surgery in childhood]. / Glaukomchirurgie im Kindesalter.

Technical characteristics and a long-term therapeutic strategy due to a long life expectancy play a key role in pediatric glaucoma surgery. The well-established angle surgery (goniotomy and trabeculotomy) achieves successful results in primary childhood glaucoma. Trabeculectomy seems to have been displaced as a secondary approach by glaucoma drainage devices (GDD) in primary childhood glaucoma due to inferior results, especially for children under 3 years of age. Even for secondary childhood glaucoma the results of GDD are encouraging, especially for therapy refractory aphakic glaucoma. In the first 2 years after GDD surgery success rates are about 80% for pediatric glaucoma and the results appear to be independent of the type of glaucoma and implant used. The complications of GDD are balanced to the faster intraocular pressure (IOP) control during the phase of visual acuity development. Cyclodestructive procedures may be applied as a secondary adjuvant approach but they increase the risk of conjunctival scarring and hypotony for subsequent procedures.

[Development of non-invasive clinical examination methods for the anterior segment of the eye and their clinical significance].

1. PURPOSE: Slit-lamp microscopy is a principal ophthalmic clinical method, because it provides microscopic findings of the anterior segment of the eye noninvasively. Its findings, however, are qualitative and there are large inter-observer variations in their evaluation. Furthermore, slit-lamp microscopy provides morphological findings, but a functional evaluation is difficult. We developed two novel methods that establish a qualitative methodology of the slit-lamp microscope and the pathophysiology of the anterior segment of the eye. One is the flare-cell photometer to evaluate flare and cells in the aqueous humor of the eye and the other is an immunohistochemical examination method using tear fluid to evaluate ocular surface disorders. The comprehensive evaluation of these studies is herein overviewed. 2. INNOVATION OF THE FLARE-CELL PHOTOMETER AND ITS CLINICAL SIGNIFICANCE: The breakdown of the blood-aqueous barrier (BAB) causes an increase in protein (flare) and leakage of blood cells (cell) into the aqueous humor of the eye and the severity of BAB breakdown has a positive correlation with the intensity of flare and cells. The flare and cells in the aqueous can be observed qualitatively by slit-lamp microscopy. These findings are primarily distinguished in optics by light scattering. Therefore, detection of the intensity of light scattering due to flare and cells can evaluate the BAB function. The flare-cell photometer comprises 3 novel components: a laser beam system as an incident light, a photomultiplier to detect scattered light intensity and a computer-assisted system to operate the whole system and analyze detected scattered light signals due to flare and cells. The instrument enables us to quantitatively analyze the flare and cells non-invasively and accurately with a wide dynamic measurement range, resulting in a repeated examination of each individual case. It also enables the evaluation of inflammation in the aqueous not only postoperatively but also in endogenous uveitis, evaluation of the effects of anti-inflammatory drugs on BAB and evaluation of aqueous humor dynamics. Furthermore, repeating the examination can minimize inter-individual variations and reduce the number of animals in animal experiments. 3. PATHOPHYSIOLOGICAL EVALUATION METHODS OF OCULAR SURFACE USING TEAR FLUID: Sampling of tears can be performed noninvasively, but the obtainable volume is limited. Therefore, a determination of targeting biomarkers and a development of their micro-volume analysis methods play a crucial role in pathophysiological studies of the ocular surface. Targeting biomarkers should be determined according to the various specified bioactive substances such as eosinophil cationic protein (ECP), cytokines and others. A number of microvolume analysis methods, such as chemiluminescent enzyme immunoassay, immunochromatography, micro-array system and polymerase chain reaction method are used. Objective disorders in the studies include allergic conjunctivitis and infectious diseases such as herpetic keratitis. Quantitative evaluation methods for ECP concentration, antigen-specific secretory IgA in allergic diseases and herpetic keratitis, herpes simplex virus-DNA and cytokine and chemokine profile in tear fluid sampled by filter paper method were investigated. We developed a clinically applicable quantitative immunochemical method for ECP concentration in tear fluid. The results revealed that tear fluid analysis using the above mentioned methods is a clinically useful to investigate the pathophysiology of the ocular surface. 4. CONCLUSION: Laser flare-cell photometer and tear fluid analysis are potent clinical quantitative methods to investigate the pathophysiology of the anterior segment of the eye.

Outflow physiology of the mouse eye: pressure dependence and washout.

PURPOSE: Mice are commonly used in glaucoma research, but relatively little is known about aqueous outflow dynamics in the species. To facilitate future use of the mouse as a model of aqueous humor outflow, several fundamental physiological parameters were measured in the mouse eye. METHODS: Eyes from adult mice of either sex (C57BL/6 background) were enucleated, cannulated with a 33-gauge needle, and perfused at constant pressure while inflow was continuously measured. RESULTS: At 8 mm Hg, total outflow facility (C(total)) was 0.022 ± 0.005 µL/min/mm Hg (all values mean ± SD; n = 21). The flow-pressure relationship was linear up to 35 mm Hg. The conventional outflow facility (C(conv)) was 0.0066 ± 0.0009 µL/min/mm Hg, and the unconventional outflow (F(u)) was 0.114 ± 0.019 µL/min, both measured at room temperature. At 8 mm Hg, 66% of the outflow was via the unconventional pathway. In a more than 2-hour-long perfusion at 8 mm Hg, the rate of facility change was 2.4% ± 5.4% (n = 11) of starting facility per hour. The ocular compliance (0.086 ± 0.017 µL/mm Hg; n = 5) was comparable to the compliance of the perfusion system (0.100 ± 0.004 µL/mm Hg). CONCLUSIONS: Mouse eyes are similar to human eyes, in that they have no detectable washout rate and a linear pressure-flow relationship over a broad range of intraocular pressures. Because of the absence of washout and the apparent presence of a true Schlemm's canal, the mouse is a useful model for studying the physiology of the inner wall of Schlemm's canal and the conventional outflow tissues.

Comparison of tight junction protein expression in the ciliary epithelia of mouse, rabbit, cat and human eyes.

Tight junctions in the nonpigmented epithelium (NPE) of the ciliary processes and the iris vascular endothelium form the ocular blood aqueous barrier that prevents leakage of proteins, immune cells and non-immune cells of blood into the anterior chamber. We attempted to determine whether ultrastructural differences in tight junctions reported in earlier studies are reflected in the expression pattern of tight junction proteins (TJP) and whether the TJP in mice, rabbits and cats resemble those of humans. For immunohistochemistry, 10 µm thick cryosections were rehydrated in PBS and fixed in 50 mM ammonium chloride at room temperature. After rinses in PBS, the sections were incubated twice in 0.1% Triton X-100, 10% goat serum, specific primary antibody or in PBS. After rinses in PBS, the sections were incubated in FITC-conjugated secondary antibody. After rinses in PBS, the sections were mounted with Vectashield mounting medium with propidium iodide, examined and photographed using a confocal microscope. The expression patterns of TJP in ocular ciliary epithelium of human, rabbit, cat and mouse were similar. Occludin immunoreactivity was observed as a sharp line along the junction between pigmented epithelium (PE) and NPE, and along the apico-lateral surfaces of NPE. Very light staining of the ciliary stroma was observed in cat and mouse. Claudin-1 was expressed along the entire boundaries of NPE and was more distinct between PE and NPE in rabbit. The ciliary stroma showed faint staining in cat and mouse. ZO-1 showed staining between PE and NPE, and at the adjacent membrane. Moderate staining was seen in PE in cat and mouse, which suggests that claudin-1, occludin and ZO-1 are expressed along the junction between PE and NPE, and the apico-lateral border of NPE. Lack of major difference in the expression patterns among the different species is important for validating the use of rabbit, mouse and cat in studies of intraocular inflammation in humans.