BACKGROUNDElevation of intraocular pressure is usually associated with primary open angle glaucoma and caused by increased outflow resistance. A two-color fluorescent tracer technique was developed to investigate the hydrodynamics of aqueous humor outflow with changing intraocular pressure within the same eye, to better understand the relationship between outflow facility and effective filtration area.

METHODSEighteen enucleated bovine eyes were first perfused at 30 mmHg with Dulbecco's phosphate-buffered saline containing 5.5 mmol/L D-glucose. After a stable baseline facility, red fluorescent microspheres (0.5 microm, 0.002% v/v) were exchanged and perfused. Eyes in the one-color control group (n = 6) were immediately perfused with fixative. In the experimental group (n = 6), eyes were perfused with green tracer after intraocular pressure reduced to 7 mmHg, while in the two-color control group (n = 6), eyes were perfused with green tracer with intraocular pressure remaining at 30 mmHg. All 12 eyes were then perfusion-fixed. Outflow facility was continuously recorded in all eyes. Confocal images were taken along the inner wall of the aqueous plexus and the percent of the effective filtration length (PEFL; length of inner wall exhibiting tracer labeling/total length of inner wall) was measured. The relationships between outflow facility and PEFL were analyzed statistically.

RESULTSNo significant differences were found in baseline facilities (microl x min(-1) x mmHg(-1)) among the three groups (the experimental group: 0.93 +/- 0.12; the two-color control group: 0.90 +/- 0.19; the one-color control group: 0.98 +/- 0.13). In the experimental group, the outflow facility was significantly higher at 7 mmHg (4.29 +/- 1.01) than that at 30 mmHg (1.90 +/- 0.67, P < 0.001), which corresponded to a significant increase in the PEFL at 7 mmHg (54.70 +/- 8.42) from that at 30 mmHg ((11.76 +/- 4.56)%, P < 0.001). The PEFL labeled by red fluorescent microspheres in the experimental group ((11.76 +/- 4.56)%) showed no significant difference from that of the one-color control group ((13.39 +/- 2.19)%, P = 0.473) or the two-color control group ((11.49 +/- 4.95)%, P = 0.930). The PEFL labeled by green fluorescent microspheres in the experimental group ((54.70 +/- 8.42)%) was significantly higher than that of the two color control group ((37.34 +/- 8.17)%, P = 0.010). A positive correlation was found between outflow facility and PEFL (r = 0.897, R(2) = 0.804) in the experimental group.

CONCLUSIONSChanges in aqueous humor outflow patterns before and after a change in intraocular pressure can be successfully distinguished within the same eye using our newly developed two-color tracer perfusion technique. The PEFL showed positive correlation with the outflow facility.

Animals ; Aqueous Humor ; physiology ; Cattle ; Intraocular Pressure ; Luminescent Proteins ; metabolism ; Microscopy, Confocal ; Microspheres ; Perfusion ; methods

BACKGROUNDInterleukin (IL)-1beta may effectively decrease introcular pressure (IOP) when administered by subconjunctival injection in normal rabbit. However, IL-1beta is a large molecular agent and an inflammation factor. The aim of this study was to evaluate the penetrability of IL-1beta, and the concentrations of both tumor necrosis factor (TNF)-alpha and IL-6 in the aqueous humor of normal rabbits treated with IL-1beta.

METHODSA total of 170 rabbits were used in the study and were assigned to several different treatment groups as follows: 125 of the rabbits were assigned to two groups. In one group, 33 rabbits were injected subconjunctivally with IL-1beta and 39 were injected with saline alone. In the other group, 27 rabbits were given eye drops containing IL-1beta (400 ng/ml) and 26 were given saline alone. Aqueous humor (AH) was drawn and the concentration of IL-1beta within the fluid measured. The IOP was measured in another six rabbits after administration of eye drops containing IL-1beta (400 ng/ml). A further 20 rabbits were assigned to 3 groups as follows: eight untreated normal controls; six injected subconjunctivally with IL-1beta; and six injected subconjunctivally with saline alone. AH was drawn and the concentration of TNF-alpha in the fluid was measured. Another 19 rabbits were assigned to 3 groups as follows: seven untreated normal controls; and six injected subconjunctivally with IL-1beta; and six injected subconjunctivally with saline alone. AH was drawn and the concentration of IL-6 in the fluid measured. Measurement of cytokine concentration was by radio-immunoassay in all cases.

RESULTSThe IL-1beta concentration in the AH was higher in those animals in which it had been administered subconjunctivally (P < 0.01). The IL-1beta concentration in the AH of the animals given eye drops was almost the same as that in the controls (P > 0.05). The administration of IL-1beta in the form of eye drops had little effect upon IOP reduction. Lower TNF-alpha concentrations were seen in the AH after the subconjunctival administration of IL-1beta, but the concentration of IL-6 was the same as in the normal controls.

CONCLUSIONSIL-1beta shows good corneal penetrability after subconjunctival injection into normal rabbit eyes. The IOP reduction induced by IL-1beta is unlikely be associated with an inflammatory response.

Animals ; Aqueous Humor ; drug effects ; metabolism ; Interleukin-1beta ; pharmacology ; Interleukin-6 ; metabolism ; Rabbits ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo isolate exosomes from rabbit aqueous humor and to investigate their immunosuppression function.

METHODSAqueous humor was collected from 40 New Zealand rabbits and exosomes were isolated by fractional separation and ultracentrifugation methods; the morphology was studied with electron microscopy. The immunosuppressive-related proteins of exosomes were detected with Western blotting; their inhibitory effect on ConA-induced proliferation of T lymphocyte was estimation with CCK-8 cells proliferation assay.

RESULTSEight milliliters of aqueous humor were collected from 40 New Zealand rabbits and 200 μg exosomes was yielded. Under electron microscope, the exosomes had typical structure of lipid bi-layer with a diameter of 50-100 nm. The results of Western blotting showed that these exosomes expressed Hsp70, CD9 and Alix but not Grp94, presenting a typical exosomes protein profile. Moreover, exosomes expressed high level of TGF-β and significantly inhibited the proliferation of T lymphocytes.

CONCLUSIONImmunosuppressive exosomes can be isolated from rabbit aqueous humor, which may be involved in immunotolerance of the eye.

Animals ; Aqueous Humor ; immunology ; Cells, Cultured ; Exosomes ; immunology ; metabolism ; ultrastructure ; Female ; Immune Tolerance ; Male ; Rabbits ; T-Lymphocytes ; immunology

OBJECTIVETo establish a RP-HPLC method for determination of the content of ginsenoside Rg1 in the rabbits aqueous humor, blood and ocular tissues, which were given intragastric administration with the co-xueshuantong soft capsules. The drug concentration in rabbits at different times after oral administration has been determined and the pharmacokinetics characteristics has been researched.

METHODThe compound xueshuantong soft capsules were administrated to the healthy New Zealand rabbits by gavage (10 mg x kg(-1) per rabbit). The concentration of Ginseng Rg1 in aqueous humor, blood and ocular tissues at different time was determined by RP-HPLC.

RESULTRP-HPLC can be established for the determination of ginsenoside Rg1 in the rabbits aqueous humor, blood, ocular tissue. The calibration of curves was linear within the range of 7.60-152.0 mg x L(-1) (r = 0.999 6) for ginsenoside Rg1 in aqueous humor and the calibration of curves were linear within the range of 10.35-103, 50 mg x L(-1) (r = 0.999 8) for ginsenoside Rg1 in blood. Determination of the recovery rate to meet the requirements.

CONCLUSIONThe ginsenosides Rg1 could transmit the blood-ocular barrier into the eyes and reach a certain concentration. The research provides a theoretical and experimental basis for the systemic administration of compound Xueshuantong to treat eye diseases.

Animals ; Aqueous Humor ; metabolism ; Calibration ; Capsules ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; pharmacokinetics ; Female ; Ginsenosides ; pharmacokinetics ; Male ; Rabbits

To elucidate the mechanism of increased intraocular pressure in primary open-angle glaucoma (POAG), the protein profiles of aqueous humor obtained from POAG patients were compared with those of cataract patients as a control group. Aqueous humor proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by the ultrasensitive silver staining technique. In 79% of the samples taken from POAG patients, protein bands of 140,000 or 160,000 daltons were stained, but none were stained from cataract patients. The presence of these protein bands revealed statistically significant differences between the two groups. Protein bands of 140,000 or 160,000 daltons were evenly visible at all ages in POAG patients, and the positivity of bands had no correlation with sex or initial intraocular pressure level. It is possible that the ultrastructural changes of the aqueous outflow pathway in POAG may be related to the changes in the aqueous protein, presence of 140,000 or 160,000 daltons protein bands.
Adult ; Aged ; Aqueous Humor/*metabolism ; Cataract/metabolism ; Electrophoresis, Polyacrylamide Gel ; Eye Proteins/*metabolism ; Female ; Glaucoma, Open-Angle/*metabolism ; Humans ; Male ; Middle Aged ; Molecular Weight

The concentrations of fibronectin in aqueous humor, measured by ELISA which was developed to detect fibronectin, ranged from 5 ng/ml to 100 ng/ml. Aqueous humor was aspirated from human eyes with cataracts and glaucomas using a 26 gauge needle through the peripheral cornea before making the limbal incision into the anterior chamber during surgery. The results of the study show that the average concentration and standard deviation of fibronectin was 0.136 +/- 0.192 microgram/ml in cataract eyes, and 0.962 +/- 0.918 microgram/ml in glaucoma eyes respectively. There was a statistically significant difference between both groups (p = 0.000). However, no significant differences according to age and sex were noted. There was no influence due to preoperative intravenous mannitol injection on fibronectin concentration. The source of aqueous fibronectin is still not clearly known and the mechanism of the higher concentration of fibronectin in glaucoma has not been clearly disclosed, however it is thought that normally present fibronectin is accumulated in the anterior chamber because it can not pass the aqueous outflow pathway, or that fibronectin production may be increased in glaucoma.
Adolescent ; Adult ; Aged ; Aqueous Humor/*metabolism ; Cataract/metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Fibronectins/*analysis ; Glaucoma/metabolism ; Humans ; Male ; Middle Aged

Cyclodialysis and ciliochoroidal detachment were performed in three eyes of three rabbits and in three eyes of another three rabbits, respectively. After aspiration of the aqueous humor, 0.1 ml of 10% sodium fluorescein was injected intracamerally, and the eyeball was enucleated between 30 minutes and one hour after injection and prepared for fluorescence microscopy. Sodium fluorescein concentrations in the supraciliary space were much greater in the group with cyclodialysis or ciliochoroidal detachment than in the normal control group. These results suggest that (1) in the eye with cyclodialysis, the aqueous humor may freely gain access to the supraciliary space through the cleft between the anterior chamber and the supraciliary space and then be removed rapidly and (2) in the eye with ciliochoroidal detachment, the aqueous humor may pass through the uveoscleral outflow pathway.
Animals ; Anterior Chamber/metabolism ; Aqueous Humor/*secretion ; Choroid/metabolism/*surgery ; Ciliary Body/metabolism/*surgery ; Fluorescein ; Fluoresceins/diagnostic use ; Microscopy, Fluorescence ; Rabbits

Primary and secondary corneal endothelial decompensation leads to stromal edema, corneal opacity and loss of visual acuity. The pathogenesis of corneal endothelial decompensation is that adult corneal endothelium in vivo lacks of a robust proliferative response to injury, does not divide sufficiently to replace the lost cells. Previous studies indicate that cell-cell contact inhibition and transforming growth factor-beta2 (TGF-β2) in aqueous humor may be responsible for maintaining human endothelial cells in a non-replicative state in vivo. The results of the experimental investigation by using immunofluorescent staining of the cell cycle-associated proteins and cell proliferation marker Ki67 in corneal endothelium indicate that human corneal endothelial cells in vivo are arrested in the G1-phase and have not exited from the cell cycle. Successful outgrowth in culture of human corneal endothelial cells in vitro and the establishment of the immortalized human endothelial cell line, provide strong evidence that corneal endothelial cells retain proliferative capacity. Experiments with cell culture ex vivo demonstrate that corneal endothelial cells cultured from young donors grow more robustly than those from older donors, and cells cultured from peripheral area of corneas show greater cell density than central regions. Studies have demonstrated that in vitro human corneal endothelia undergo mitotic changes in response to stimulation of growth promoting agents, such as growth factors, EDTA and extracellular matrix. Identification of corneal endothelial stem cells and isolation and culture of human endothelial precursor cells in vitro will be beneficial for further investigation regarding the mechanism of corneal endothelial regeneration as well as corneal endothelial cells in vitro culture.
Aqueous Humor ; metabolism ; Cell Count ; Cell Culture Techniques ; Cell Cycle ; Cell Proliferation ; Cells, Cultured ; Contact Inhibition ; Endothelium, Corneal ; cytology ; Humans ; Stem Cells ; Transforming Growth Factor beta2 ; metabolism

OBJECTIVETo test the immunosuppressive effect of cyclosporine (Cs) in a polymer placed in the anterior chamber of corneal allograft recipients.

METHODSWistar inbred rats with vascularized corneas were recipients of corneal allografts from Sprague-Dawley donor rats. Rats underwent penetrating keratoplasty and were divided randomly into four groups: untreated control animals (UCA); Cs-polymer anterior chamber recipients (CPA); co-polymer subconjunctival recipients (CPS); and Cs-olive oil drop recipients (COO). Grafts were examined by slit lamp every 3 days and clinical conditions were scored. Cs concentration in the aqueous humor was assayed at 1, 2, and 4 weeks. At 1, 2 and 4 weeks after transplantation, the operated eyes were collected for histopathological evaluation of the grafts.

RESULTSThe median survival time of the allografts was 8.2 +/- 1.48 days for the UCA group, 11.4 +/- 2.50 days for the CPS group, and 17.0 +/- 2.00 days for the CPA group. There was a statistically significant difference (P < 0.05) between survival time of the allografts in the animals of the CPA group compared to the other groups of graft recipients. Significantly higher concentrations of Cs were found in the eyes given an anterior chamber implant of Cs-polymer, compared to other treatment groups or untreated rats. A transient inflammatory response in the anterior chamber was observed in the CPA group.

CONCLUSIONSCs-polymer placed in the anterior chamber significantly prolongs corneal allograft survival time in a high risk corneal graft rejection model. This intraocular delivery system may be a valuable adjunct for the suppression of immune graft rejection.

Animals ; Aqueous Humor ; metabolism ; Corneal Transplantation ; Cyclosporine ; administration & dosage ; metabolism ; Drug Delivery Systems ; Graft Survival ; Immunosuppressive Agents ; administration & dosage ; Male ; Rats ; Rats, Wistar ; Transplantation, Homologous

PURPOSE: Transforming growth factor-beta2 is known to be present at elevated levels in the aqueous humor of patients with primary open angle glaucoma (POAG) and diabetes but not in uveitis-related secondary glaucoma. We investigated total TGF-beta2 levels and levels of the active form of TGF-beta2 in the aqueous humor of eyes with different types of glaucoma. METHODS: The concentration of the total and active form of TGF-beta2 was measured in 63 patients with primary open angle glaucoma, neovascular glaucoma complicated with diabetes (NVG), and secondary open angle glaucoma complicated with uveitis (SOAG) using a double antibody 'sandwich-indirect' ELISA method. RESULTS: The levels of total TGF-beta2 in the aqueous samples of POAG, NVG, and SOAG were elevated. The levels of active TGF-beta2 in the aqueous samples of POAG, and NVG were also elevated, whereas the level of active TGF-beta2 was within the normal range in the aqueous samples of SOAG. CONCLUSIONS: These results suggest that the level of TGF-beta2 may play a role in the pathology of various types of glaucoma.
Uveitis/*metabolism ; Transforming Growth Factor beta/*metabolism ; Severity of Illness Index ; Middle Aged ; Humans ; Glaucoma, Open-Angle/*metabolism ; Glaucoma, Angle-Closure/*metabolism ; Enzyme-Linked Immunosorbent Assay ; Diabetic Retinopathy/*metabolism ; Biological Markers/metabolism ; Aqueous Humor/*metabolism ; Aged, 80 and over ; Aged ; Adult