Protein Concentration In Aqueous Humor Research Articles

The Effect of Intracameral Triamcinolone Acetonide on Controlling Common Complications following Phacoemulsification in Dogs.

The intracameral injection of triamcinolone acetonide (TA) has achieved favorable clinical effects in controlling intraocular inflammatory reactions in humans after cataract surgery. However, the effect of this method remains unclear in veterinary practice. In this paper, 18 dogs with bilateral cataracts were randomly divided into three groups, with 6 dogs in each group. Phacoemulsification and intraocular lens implantation were performed on the 36 eyes of these dogs. A total of 0.1 mL of TA solution was injected into the oculus dexter (OD) anterior chambers. All oculus sinister (OS) anterior chambers of these dogs were used as controls. The results demonstrated that the corneal edema severity scores of the OD (1.5 mg TA) were lower than those of the OS from the 1st to 7th day after surgery, with a significant difference on the 3rd day after surgery (p = 0.033). The corneal edema severity scores in the OD (1.5 mg TA) were significantly lower than those in the OD (0.5 mg TA) on the 3rd day after surgery (p = 0.036). The aqueous humor protein concentration of the OD (1.5 mg TA) had a lower concentration than the OS on the 1st day after surgery (p = 0.004). Furthermore, on the 5th and 10th days, the aqueous humor protein concentration of the OD (1.5 mg TA) was lower than that of the OS (p = 0.038 and p = 0.044, respectively). The aqueous humor PGE2 concentration of the OD (1.5 mg TA) had a lower concentration than the OS on the 1st day after surgery (p = 0.026). The aqueous humor PGE2 concentrations in the OD (1.0 mg TA) and OD (1.5 mg TA) were lower compared to that in the OD (0.5 mg TA) on the 1st day after surgery (p = 0.041 and p = 0.037, respectively). It was demonstrated that TA-based treatment can be safely employed to effectively control common complications after phacoemulsification in dogs.

The Anti-Inflammatory Effects of Angiogenin in an Endotoxin Induced Uveitis in Rats

Angiogenin (ANG) is involved in the innate immune system and inflammatory disease. The aim of this study is to evaluate the anti-inflammatory effects of ANG in an endotoxin induced uveitis (EIU) rat model and the pathways involved. EIU rats were treated with balanced salt solution (BSS), a non-functional mutant ANG (mANG), or wild-type ANG (ANG). The integrity of the blood-aqueous barrier was evaluated by the infiltrating cell and protein concentrations in aqueous humor. Histopathology, Western blot, and real-time qRT-PCR of aqueous humor and ocular tissue were performed to analyze inflammatory cytokines and transcription factors. EIU treated with ANG had decreased inflammatory cells and protein concentrations in the anterior chamber. Compared to BSS and mANG, ANG treatment showed reduced expression of IL-1β, IL-8, TNF-α, and Myd88, while the expression of IL-4 and IL-10 was increased. Western blot of ANG treatment showed decreased expression of IL-6, inducible nitric oxide synthase (iNOS), IL-1β, TNF-α, and phosphorylated NF-κB and increased expression of IL-10. In conclusion, ANG seems to reduce effectively immune mediated inflammation in the EIU rat model by reducing the expression of proinflammatory cytokines, while increasing the expression of anti-inflammatory cytokines through pathways related to NF-κB. Therefore, ANG shows potential for effectively suppressing immune-inflammatory responses in vivo.

Uveitis Therapy With Shark Variable Novel Antigen Receptor Domains Targeting Tumor Necrosis Factor Alpha or Inducible T-Cell Costimulatory Ligand.

PurposeWe assess the efficacy of two next-generation biologic therapies in treating experimental autoimmune uveitis.MethodsVariable binding domains from shark immunoglobulin novel antigen receptors (VNARs) were fused with a mouse IgG2a constant domain (Fc) to generate VNAR-Fc molecules with binding specificity to tumor necrosis factor alpha (TNFα) or inducible T-cell costimulatory ligand (ICOSL). Treatment with VNAR-Fc fusion proteins was compared to treatment with dexamethasone or vehicle in the Lewis rat model of experimental autoimmune uveitis (EAU). Inflammation control was determined by comparing OCT clinical and histologic scores, and aqueous humor protein concentration. The concentration of 27 inflammatory cytokines in the aqueous humor was measured using a multiplex enzyme-linked immunosorbent assay platform.ResultsAdministration of S17-Fc significantly decreased clinical, histologic, and aqueous protein levels when compared to vehicle treatment. Inflammation scores and aqueous protein levels in A5-Fc–treated animals were decreased compared to vehicle treatment, but not significantly. The concentration of vascular endothelial growth factor (VEGF), regulated on activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein 1 alpha (MIP-1α), interleukin (IL)-1β, LPS-induced CXC chemokine (LIX), monocyte chemoattractant protein-1 (MCP-1), and interferon (IFN)-γ were significantly decreased in the eyes of animals treated with dexamethasone. VNAR treatment demonstrated a trend towards decreased cytokine concentrations, but only VEGF and RANTES were significantly decreased by S17-Fc.ConclusionsTreatment with the anti-TNFα VNAR S17-Fc ameliorates EAU as effectively as treatment with corticosteroids.Translational RelevanceVNAR-Fc molecules are a next-generation therapeutic biologic that overcome the limitations of classical biologic monoclonal antibodies, such as complex structure, large size, and limited tissue penetration. This is a novel drug modality that could result in the development of new therapy options for patients with noninfectious uveitis.

Association between corneal endothelial cell densities and elevated cytokine levels in the aqueous humor

Annual reduction rate of corneal endothelial cell density (ECD) varies among etiologies, however, the cause of chronic endothelial cell loss is still unknown. We recently reported the elevation of inflammatory cytokines in the aqueous humor (AqH) in eyes with bullous keratopathy and low ECD. To evaluate the association between ECD and aqueous cytokine levels, we collected a total of 157 AqH samples prospectively. The AqH levels of cytokines were measured and multivariate analyses were conducted to find the correlation between ECD, aqueous cytokine levels and clinical factors, such as number of previous intraocular surgeries and protein concentration in AqH. As a result, ECD was negatively correlated with specific cytokine levels, including IL-1α, IL-4, IL-13, MIP-1β, TNF-α and E-selectin (all P < 0.05). The aqueous cytokine levels showed different correlations with these clinical factors; the number of previous intraocular surgeries was associated with all cytokines except MIP-1α. The AqH protein concentration and the status of intraocular lens showed similar patterns of elevation of IL-1α, IL-4, IL-6, IL-8, IL-10, IL-13, IL-17A, MIP-1β, MCP-1, E-selectin, P-selectin and sICAM-1. In conclusion, elevation of AqH cytokine levels was associated with reduced ECDs. AqH cytokine levels showed significant correlations with clinical factors associated with low ECDs.

An Anti-Interleukin-2 Receptor Drug Attenuates T- Helper 1 Lymphocytes-Mediated Inflammation in an Acute Model of Endotoxin-Induced Uveitis

The aim of the present study was to evaluate the anti-inflammatory efficacy of Daclizumab, an anti-interleukin-2 receptor drug, in an experimental uveitis model upon a subcutaneous injection of lipopolysaccharide into Lewis rats, a valuable model for ocular acute inflammatory processes. The integrity of the blood-aqueous barrier was assessed 24 h after endotoxin-induced uveitis by evaluating two parameters: cell count and protein concentration in aqueous humors. The histopathology of all the ocular structures (cornea, lens, sclera, choroid, retina, uvea, and anterior and posterior chambers) was also considered. Enzyme-linked immunosorbent assays of the aqueous humor samples were performed to quantify the levels of the different chemokine and cytokine proteins. Similarly, a biochemical analysis of oxidative stress-related markers was also assessed. The inflammation observed in the anterior chamber of the eyes when Daclizumab was administered with endotoxin was largely prevented since the aqueous humor protein concentration substantially lowered concomitantly with a significant reduction in the uveal and vitreous histopathological grading. Th1 lymphocytes-related cytokines, such as Interleukin-2 and Interferon-γ, also significantly reduced with related anti-oxidant systems recovery. Daclizumab treatment in endotoxin-induced uveitis reduced Th1 lymphocytes-related cytokines, such as Interleukin-2 and Interferon gamma, by about 60–70% and presented a preventive role in endotoxin-induced oxidative stress. This antioxidant protective effect of Daclizumab may be related to several of the observed Daclizumab effects in our study, including IL-6 cytokine regulatory properties and a substantial concomitant drop in INFγ. Concurrently, Daclizumab treatment triggered a significant reduction in both the uveal histopathological grading and protein concentration in aqueous humors, but not in cellular infiltration.

Altered Antioxidant-Oxidant Status in the Aqueous Humor and Peripheral Blood of Patients with Retinitis Pigmentosa

Retinitis Pigmentosa is a common form of hereditary retinal degeneration constituting the largest Mendelian genetic cause of blindness in the developed world. It has been widely suggested that oxidative stress possibly contributes to its pathogenesis. We measured the levels of total antioxidant capacity, free nitrotyrosine, thiobarbituric acid reactive substances (TBARS) formation, extracellular superoxide dismutase (SOD3) activity, protein, metabolites of the nitric oxide/cyclic GMP pathway, heme oxygenase-I and inducible nitric oxide synthase expression in aqueous humor or/and peripheral blood from fifty-six patients with retinitis pigmentosa and sixty subjects without systemic or ocular oxidative stress-related disease. Multivariate analysis of covariance revealed that retinitis pigmentosa alters ocular antioxidant defence machinery and the redox status in blood. Patients with retinitis pigmentosa present low total antioxidant capacity including reduced SOD3 activity and protein concentration in aqueous humor. Patients also show reduced SOD3 activity, increased TBARS formation and upregulation of the nitric oxide/cyclic GMP pathway in peripheral blood. Together these findings confirmed the hypothesis that patients with retinitis pigmentosa present reduced ocular antioxidant status. Moreover, these patients show changes in some oxidative-nitrosative markers in the peripheral blood. Further studies are needed to clarify the relationship between these peripheral markers and retinitis pigmentosa.

Experimental Uveitis can be Maintained in Rabbits for a Period of Six Weeks After a Safe Sensitization Method

Purpose: New treatments against long-lasting uveitis need to be tested. Our aim was to develop a six-week model of uveitis in rabbits.Methods: Rabbits were presensitized with an s.c. injection of Mycobacterium tuberculosis H37RA emulsified with TiterMax® Gold adjuvant. Uveitis was induced at day 28 and 50, by intravitreal challenges of antigen suspension. Ocular inflammation was assessed till euthanasia at day 71 after s.c. injection of M. tuberculosis H37RA by: (a) the number of inflammatory cells in aqueous humor (AH); (b) the protein concentration in AH; (c) the clinical score (mean of conjunctival hyperaemia, conjunctival chemosis, oedema and secretion); (d) the microscopical score (mean presence of fibrin and synechiae, aqueous cell density and aqueous flare grade, as scored by slit lamp).Results: At the sites of presensitization injection, rabbits presented flat nodules which progressively vanished. The first challenge induced a significant increase in the four parameters (p < 0.05 the Wilcoxon/Kruskal–Wallis test). The AH contained 764 ± 82 cells/µl and 32 ± 0.77 mg protein/ml. During the following days, inflammatory parameters decreased slightly. The second intravitreal challenge increased inflammation (3564 ± 228 cells/µl AH and 31 ± 1 mg protein/ml), which remained at a high level for a longer period of time.Conclusion: We developed a model of long-term uveitis, which could be maintained in rabbits for at least six weeks. Such a model could be used to test the efficacy of either new drugs or various drug delivery systems intended to deliver active agents during a few months.

Expression of promyelocytic leukemia protein and vascular endothelial growth factor in aqueous humor and vitreous fluid in patients with proliferative diabetic retinopathy

AimsTo examine the expression of promyelocytic leukemia protein (PML) in the eye of proliferative diabetic retinopathy (PDR) patients. MethodsPML mRNA levels were measured in proliferative membranes from 12 PDR patients and idiopathic epiretinal membranes from 5 control patients by quantitative RT-PCR. Protein levels of PML and vascular endothelial growth factor (VEGF) in aqueous humor and vitreous fluid samples from 34 PDR patients and 38 control patients were determined using ELISA. ResultsThe PML mRNA expression levels in membrane samples, and the PML protein concentrations in aqueous humor and vitreous fluid samples were significantly lower in PDR patients than control patients. We observed a statistically significant inverse correlation between the concentrations of PML and VEGF in the aqueous humor and vitreous fluid of PDR patients. ConclusionPML may be a good candidate as a diagnostic marker and a therapeutic agent for PDR.

The Effects of Sperminated Pullulans on Cornea Permeability to Puerarin and the Toxicity

To investigate the varied effects of sperminated pullulans (SP) with different amino residues on cornea permeability and its local toxicity. Three groups of rabbits were used: control, low-amino residue content SP (SP-L), and high-amino residue content SP (SP-H). The in vitro and in vivo spreading assays were combined with high performance liquid chromatography (HPLC) to measure the concentration of puerarin in the external medium or aqueous humor when 0% SP, 0.2% SP-L, and 0.2% SP-H were included. The toxicity of SP was determined by corneal hydration values, Draize score, aqueous humor protein concentration, corneal endothelial evaluation, as well as light microscopy and electron microscopy. The application of 0.2% SP-L and 0.2% SP-H to the cornea in vitro increased puerarin apparent permeability coefficient by 1.96-fold (P<0.05) and 2.95-fold (P<0.01), respectively. SP-H showed stronger effect than SP-L (P<0.05). For the in vivo assay, those were 1.81-fold (P<0.05) and 3.71-fold (P<0.01), respectively. With the SP application, the corneal hydration values were <83% and Draize scores were <4, with no apparent changes in histological observations. SP is one potential adjuvant promoting puerarin permeability to the cornea, and the high-content amino residue SP showed stronger effect, without ocular toxicity.

Resolvin D1 reduces the immunoinflammatory response of the rat eye following uveitis.

This study investigated whether the administration of resolvin D1 to rats with endotoxininduced uveitis (EIU) ameliorates the immuno-inflammatory profile of the eye. 24 h after the administration of 200 μg LPS into the footpad of Sprague-Dawley rats, severe changes of the structure of the eye occurred concomitantly with a severe inflammatory and immune response. These latter included strong infiltration of PMN leukocytes CD11b+ T-lymphocytes CD4+ and CD8+ within the eye and a significant release of the cytokines/chemokines TNF-alpha, CXCL8, and RANTES too. Bolus of resolvin D1 (RvD1; 10–100–1000 ng/kg in 200 μL of sterile saline via the tail vein) significantly and dose-dependently (i) reduced the development of the ocular derangement caused by LPS; (ii) reduced the clinical score attributed to EIU; (iii) reduced the protein concentration and myeloperoxidase activity (MPO) in aqueous humor (AqH); and (iv) reduced neutrophils, T-lymphocytes, and cytokines within the eye.

Total protein concentration and T-cell suppression activity of aqueous humour before and after penetrating keratoplasty

We wished to determine whether immune privilege parameters assayed in aqueous humour (AqH) are relevant to the fate of penetrating keratoplasty (PK) in humans. AqH was collected in 28 patients before PK (prospective cohort), in 6 patients with no history of graft rejection undergoing cataract surgery after PK (acceptors), in another 6 patients undergoing treatment of an acute endothelial immune reaction (rejectors), and in 65 controls undergoing uncomplicated cataract extraction. AqH was tested for total protein concentration and the ability to suppress T-cell activation. AqH protein concentrations of acceptors and rejectors post-PK were elevated (2.7 ± 0.8 and 2.7 ± 0.7 mg/ml, respectively) compared with pre-PK AqH level and cataract controls (1.0 ± 0.1 mg/ml, P = 0.01). All AqH samples suppressed T-cell activation, irrespective of source and timing of AqH removal. Assays of immune privilege markers in AqH suggest that PK surgery may result in a sustained loss of integrity of the blood-aqueous barrier. Although trends were evident, values of immune privilege markers determined pre- and post-PK were not statistically significantly different between the study groups. However, further prospective studies determining additional immune privilege markers have to be conducted in order to find out whether these markers might serve as predictive parameters for immune reactions following PK.

Inhibitory Effect of Aminoimidazole Carboxamide Ribonucleotide (AICAR) on Endotoxin-Induced Uveitis in Rats

PURPOSE. To investigate the anti-inflammatory effect of aminoimidazole carboxamide ribonucleotide (AICAR), an analog of adenosine monophosphate (AMP), in endotoxin-induced uveitis (EIU). METHODS. EIU was induced by subcutaneous injection of lipopolysaccharide (LPS) (200 μg) in Lewis rats. AICAR (50 mg/kg, intraperitoneally) was given 6 hours prior and at the same time as LPS injection. Clinical uveitis scores, number of anterior chamber (AC) infiltrating cells, anterior chamber protein concentration, retinal vessel leukocyte adhesion, and protein leakage were measured 24 hours later. Protein levels of C-C chemokine ligand-2 (CCL-2)/monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) in aqueous humor and retina and nuclear translocation of nuclear factor-κB (NF-κB) in the retina were determined by enzyme-linked immunosorbent assay (ELISA). Both mRNA and protein levels of CD14 in peripheral blood mononuclear cells were also measured. RESULTS. AICAR treatment significantly reduced EIU clinical severity as well as inflammatory cell infiltration and protein concentration in aqueous humor. Similarly, the number of retinal vessel-adherent leukocytes and protein leakage were decreased by AICAR treatment. Protein levels of TNF-α, CCL-2/MCP-1, and ICAM-1 in aqueous humor and CCL-2/MCP-1 and ICAM-1 levels in retina were suppressed with AICAR treatment. AICAR also reduced NF-κB translocation and CD14 expression. CONCLUSIONS. AICAR reduces systemic LPS susceptibility and attenuates intraocular inflammation in a rat EIU model by limiting infiltration of leukocytes, suppressing inflammatory mediators, and inhibiting the NF-κB pathway.

A Cannabinoid Ligand, Anandamide, Exacerbates Endotoxin-Induced Uveitis in Rabbits

This study aimed to investigate the effects of anandamide or arachidonylethanolamide (AEA), an endogenous cannabinoid receptor agonist, on intraocular inflammation in an endotoxin-induced uveitis (EIU) model in rabbits. Forty New Zealand albino male rabbits were used (5 groups, 8 animals in each). After establishment of sufficient anesthesia, animals were taken under surgery for intravitreal injections. A maximum amount of 50 μL of solution was injected into the central vitreous with a 30-gauge needle. In the control group, sterile saline was injected into the right eyes of the animals. Likewise, AEA (10(-5) M) in the second group, lipopolysaccharide (LPS; 100 ng) in the third group, and AEA (10(-5) M) and LPS (100 ng) in the fourth group were administered. Fifth group received 0.1 mL subtenon injection of AM251 (10(-5) M), a CB(1)-receptor antagonist, 30 min prior to intravitreal LPS (100 ng) and AEA (10(-5) M) injection. At 24 h after the surgical intervention, clinical evaluation was performed and animals were then euthanized with 100 mg/kg intravenous pentobarbital injections. Immediately after the induction of pentobarbital anesthesia, the anterior chamber of the eyes was quickly punctured using a 30-gauge needle to drain aqueous humor (AH) and obtained specimens were used for cell count, protein measurement, and microbiological contamination tests. After AH collection, enucleation was performed and enucleated material was kept for the pathological evaluation. AEA caused an overall worsening of EIU in studied eyes. It significantly increased the detrimental effects of endotoxin, as assessed by clinical investigation of ocular inflammation, AH leukocyte content, and AH protein concentrations. CB(1)-receptor antagonist AM251 administration reversed some components of this AEA-induced exacerbation to significant extents. AEA exacerbated EIU in rabbit eyes. AM251 has been found beneficial to prevent AEA's aggravating impact on EIU. As AEA is a treatment choice for lowering intraocular pressure in ophthalmology practice, concurrent use of CB(1)-receptor antagonists may be a questionable strategy in cases of secondary glaucoma, to avoid aggravation of the present inflammation.

Model of uveitis in rabbit

Abstract Purpose To develop a long‐term experimental model of uveitis using a new easier method for animals. Methods A suspension of desiccated Mycobacterium tuberculosis H37RA was mixed with TiterMax® Gold Adjuvant to produce a water‐in‐oil emulsion. Pigmented rabbits were pre‐sensitized with a s.c. multisite injection (400 ul) of emulsion. Two challenges were performed by intravitreal injection of 100 ul of M. tuberculosis H37RA suspension, under systemic and topical anesthesia. Clinical examinations and aqueous humors punctures under systemic anesthesia were regularly performed till day 71. Hyperemia, chemosis, edema and secretion were clinically scored from 0 to 3. Ocular inflammation was assessed by: a) the clinical score mean (sum of individual scores divided by 4), b) the number of inflammatory cells in aqueous humor (AH) and c) the protein concentration in AH. Results Rabbits presented a nodule at the sites of pre‐sensitization injection. Clinical score of inflammation reached a maximum 3 days after the first intravitreal challenge. The AH contained 835 cells/ ul and protein concentration in AH was 30 mg /ml. During the following days, inflammatory cell number and protein concentration in AH significantly decreased, whereas the clinical score returned to zero. The second intravitreal challenge induced an inflammation; six days after this challenge, 3800 cells/ul and 30 mg of proteins /ml were counted in AH. Inflammation remained high to moderate over 21 days at least. Conclusion We developed a new model of experimental uveitis, whose schedule was simple. The use of TiterMax® Gold Adjuvant showed advantages over other adjuvants. And uveitis could be maintained during 21 days at least.

A study of aqueous humour proteins in patients of primary open angle glaucoma

Aims: To investigate the changes in the protein composition of aqueous humour in primary open angle glaucoma patients and non-glaucomatous subjects. Patients and Methods: Case control study was conducted at a university hospital to compare the protein profile of aqueous humour obtained from patients of primary open angle glaucoma with those of non glaucomatous subjects. Protein concentration of the aqueous humour samples collected from both glaucomatous and non-glaucomatous patients at the time of surgery was estimated by the Bradford protein assay method. Sodium dodecylsulfate polyacrylamide gel electrophoresis of the samples was also performed. Results: Geometric mean of protein concentration of the samples was 55.73 mg/dl (range: 31-72) in the test group and 46.46 mg/dl (range: 27-65) in the control group and a statistically significant (p ≤= 0.001) difference in protein concentration of aqueous humour between the two groups was observed. Electrophoretic study revealed differences in the aqueous protein composition of the two groups. The glaucomatous group exhibited protein bands of 10 kDa, 20 kDa, 30 kDa, 50 kDa, 60 kDa, 70 kDa, and 90 kDa while the control subjects demonstrated bands of 6 kDa, 10 kDa, 30 kDa, 70 kDa, 90 kDa. Interestingly, almost all the proteins detected in this study corresponded to the molecular weights of heat shock proteins. Conclusions: Primary open angle glaucoma patients differed in protein compositions and had higher concentration of aqueous humour proteins than non glaucomatous patients.

Direct effect of light on 24-h variation of aqueous humor protein concentration in Sprague–Dawley rats

Sprague–Dawley rats 10–12 weeks of age were entrained to a standard light–dark cycle with lights turned on at 6 am and off at 6 pm. Variations of 24-h aqueous humor protein concentration were determined. Samples were taken every 4 h ( N = 10–14) under the standard light–dark condition at 8 pm, midnight, 4 am, 8 am, noon, and 4 pm. Under an acute constant dark condition, when lights were not turned on at 6 am, samples were collected at 8 am, noon, 4 pm, and 8 pm. Aqueous humor protein concentrations under the standard light–dark condition were found in the range of 0.305 ± 0.115 mg/ml (mean ± SD, N = 10) at midnight to 1.505 ± 0.342 mg/ml ( N = 14) at noon. The 3 light-phase protein concentrations were each higher than the 3 dark-phase concentrations. Aqueous humor protein concentrations at 8 am, noon, and 4 pm under the acute constant dark condition were each higher than the concentrations at 8 pm (after both 2 h and 26 h in the dark), midnight, and 4 am, demonstrating an endogenously driven 24-h pattern. At 8 am, noon, and 4 pm, protein concentrations were 56–147% higher when exposed to light. Intraocular pressure (IOP) was monitored using telemetry in separate groups of light–dark entrained rats under the standard light–dark condition and the acute constant dark condition. The 24-h IOP pattern was inverse to the 24-h pattern of aqueous humor protein concentration under the standard light–dark condition, and this IOP pattern was not altered by the acute constant dark condition. In conclusion, an endogenously driven 24-h variation of aqueous humor protein concentration occurred in Sprague–Dawley rats with higher concentrations during the light-phase than the dark-phase. This endogenous pattern of protein concentration was accentuated by a direct effect of light, which was unrelated to the 24-h pattern of IOP.

Inhibitory Effects of Glucosamine on Endotoxin-Induced Uveitis in Lewis Rats

Glucosamine sulfate (GS) is a naturally occurring sugar that exerts immunosuppressive effects in vitro and in vivo. The authors investigated whether GS modulates the inflammatory reaction in endotoxin-induced uveitis (EIU) of rats and the mechanisms by which it exerts its effects. Two-hundred micrograms of lipopolysaccharide (LPS) was injected subcutaneously into Lewis rats to induce EIU. Doses of GS (10, 100, or 1000 mg/kg) were divided into three aliquots and administered intraperitoneally 30 minutes before LPS injection, concurrently with LPS injection, and 30 minutes after LPS injection. Twenty-four hours after LPS injection, aqueous humor was collected for cell counting and measurement of protein concentration. Immunohistochemical staining of the iris-ciliary body was performed to evaluate the effects of GS on intercellular adhesion molecule (ICAM)-1 and nuclear factor (NF)-kappaB activation and to demonstrate macrophage infiltration. The effects of various doses of GS pretreatment were also examined using a mouse macrophage cell line (RAW264.7 cells) and LPS stimulation. Levels of prostaglandin (PG)-E2 and nitric oxide (NO) were determined. Expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 were measured using Western blot analysis. The effect of GS on LPS-induced NF-kappaB activation in RAW cells was also examined. Cell counting and analysis of protein concentration in aqueous humor revealed that GS suppressed EIU in rats treated with a high dose of GS (1000 mg/kg). Immunohistochemistry showed that treatment with GS reduced ICAM-1 expression and suppressed activation of NF-kappaB in the iris-ciliary body. The main inflammatory cells in the iris-ciliary body during EIU were macrophages. In LPS-stimulated macrophage RAW cell culture, GS inhibited the production of NO and PG-E2, the expression of iNOS and COX-2, and the activation of NF-kappaB. GS suppresses EIU in rats by blockading the NF-kappaB-dependent signaling pathway and the subsequent production of ICAM-1 and proinflammatory mediators. This study has extended the authors' previous observation that GS is a potentially important compound for reducing ICAM-1-mediated inflammatory effects in the eye.

New insights into pathophysiology of exfoliation

Abstract Purpose: Pseudoexfoliation (PEX) glaucoma is the most common identifiable cause of open‐angle glaucoma worldwide comprising the majority of glaucoma in some countries. The underlying disorder, PEX syndrome, is a generalized process of the extracellular matrix characterized by the production and progressive accumulation of a fibrillar extracellular material in intra‐ and extracellular tissues. New insights into its molecular pathophysiology have increased the understanding of this disorder and the current concepts of pathogenetic mechanisms of PEX syndrome/glaucoma will be presented in this review. Methods: Basic science, clinical‐pathologic correlations, and review of the literature. Results: Recent molecular biologic data support the pathogenetic concept of PEX syndrome as a type of stress‐induced elastosis, an elastic microfibrillopathy, with TGF‐ß1, increased oxidative stress, a dysbalance of proteolytic enzymes and their inhibitors, an impaired proteasome system, and low grade inflammatory processes being key factors in pathogenesis. Active involvement of trabecular meshwork cells in local production of PEX material in the juxtacanalicular area may be the primary cause of outflow resistance and chronic pressure elevation in PEX patients. Additional pathogenetic factors include marked pigment dispersion, increased aqueous humor protein concentrations, and pressure‐independent risk factors including an impaired ocular and retrobulbar perfusion. Conclusions: PEX syndrome may not only cause severe open‐angle glaucoma but also a spectrum of other complications and imply an increased risk for cardiovascular disease. A thorough awareness of its pathologic features and its effects on ocular tissues is critical for an early recognition and accurate diagnosis.

Concentração de proteínas no humor aquoso de pacientes com glaucoma primário de ângulo aberto em tratamento clínico

To compare total protein concentration in the aqueous humor of primary open-angle glaucoma and non-glaucomatous patients. Aqueous humor samples were obtained from 22 patients just before trabeculectomy for clinically uncontrolled primary open angle glaucoma (POAG group). Aqueous humor (0.1 mL) was aspirated by inserting a 26-gauge needle into the anterior chamber. The same procedure was performed in 22 non-glaucomatous patients just before cataract surgery (control group). Immediately after collection, the aqueous humor was stored at -20 degrees C. Aqueous humor total protein concentration was determined using a colorimetric assay. The geometric mean of total protein concentration of the aqueous humor samples was 32 mg/dL (range: 8-137 mg/dL) in the primary open angle glaucoma group and 16 mg/dL (range: 2-85 mg/dL) in the control group. The ratio of the protein concentration between the two groups was 2.0 (95% confidence interval: 1.3 to 3.2; p=0.003). The total protein concentration in primary open-angle glaucoma aqueous humor was approximately two times higher than that in non-glaucomatous subjects.

Ascorbic acid levels of aqueous humor of dogs after experimental phacoemulsification

Phacoemulsification has been successfully employed in humans and animals for lens extraction. This ultrasonic extracapsular surgical technique induces hydroxyl radical formation in the anterior chamber, which accumulates despite irrigation and aspiration. In this paper we determined the total antioxidant status of aqueous humor after phacoemulsification by measuring aqueous humor ascorbic acid levels. Mixed-breed dogs (n = 11; weighing about 10 kg) with normal eyes as determined by slit-lamp biomicroscopy, applanation tonometry, and indirect ophthalmoscopy had phacoemulsification performed in one eye with the other eye used as a control. Samples of aqueous humor were obtained by anterior chamber paracentesis before surgery and at days 1, 2, 3, 7, and 15 after surgery. Total aqueous humor antioxidant status was inferred from the capacity of aqueous humor to inhibit free radical generation by 2,2-azobis (2-amidopropane) chlorine. Ascorbic acid concentrations were measured by high-pressure liquid chromatography with UV detection. Protein content was determined with the biuret reagent. Statistical analysis was performed by anova followed by the paired t-test. Total antioxidant capacity was reduced from 48 to 27 min during the first 24 h with a gradual increase thereafter, remaining statistically lower than the control eye until 7 days postoperatively. Reduced levels of ascorbic acid followed this reduction in antioxidant capacity (from 211 to 99 microm after 24 h), remaining lower than the control eye until 15 days postoperatively. Protein concentration in aqueous humor increased from 0.62 mg/mL to 30.8 mg/mL 24 h after surgery, remaining statistically lower than the control eye until 15 days postoperatively. Paracentesis alone did not significantly alter the parameters measured. These results indicate that after phacoemulsification, the aqueous humor ascorbic acid levels and antioxidant defenses in aqueous humor are reduced, indirectly corroborating free radical production in the anterior chamber as a result of phacoemulsification. The inflammatory process consequent to the surgical procedure demonstrated by increased protein content in aqueous humor can also contribute to free radical production and ascorbic acid consumption.