Suppression of VEGF and Decrease in Vascular Leakage by Pyridoxal 5'-Phosphate in Diabetic Rats

PURPOSE: The authors of the present study investigated whether pyridoxal 5'-phosphate (PLP), an active coenzyme of vitamin B6, could inhibit the development of diabetic retinopathy in streptozotocin (STZ)-induced diabetic rats. METHODS: Seven-week-old Spraque-Dawley rats (n = 20) were used in the present study. STZ (70 mg/kg) was injected intraperitoneally to induce diabetes. Blood glucose and body weight were monitored. Intraperitoneal injections of 5 microg and 50 microg PLP were administered every two days from the second week of induced diabetes. During the third week of PLP injections, the concentration level of plasma homocysteine was measured. In addition, functional status was examined by vitreous fluorophotometer and anatomical status by vascular endothelial growth factor (VEGF) staining in the retina. RESULTS: Based on vitreous fluorophotometry examination, the PLP injection group proved to have a lower level of fluorescein concentration in the vitreous. Additionally, immunohistochemical staining revealed down-regulation of VEGF expression in the PLP group. In addition, the PLP group had a lower plasma homocysteine concentration. However, an over-dosage injection of PLP did not appear to have any noticeable impact on the treatment of diabetes. CONCLUSIONS: PLP, an active coenzyme of vitamin B6, proved to have inhibitory effects on VEGF expression and vascular leakage in the diabetic rat retina.

Fluorophotometric measurements of aqueous-humor flow in post-YAG laser iridotomy for acute angle closure

Objective@#This study compared the rates of aqueous-humor flow and trabecular outflow in eyes that had undergone YAG laser iridotomy (LI) for primary-acute-angleclosure (PAC) attack and primary-angle-closure suspect (PACS).@*Methods@#Patients who had PAC attack in one eye and narrow occludable angles (PACS) in the other eye that had undergone YAG LI were recruited. All underwent complete ophthalmologic examination including gonioscopy, ultrasonic pachymetry, A scan, and fluorophotometry to determine the rate of aqueous-humor flow. The Goldmann equation was used to compute the outflow facility using the values of aqueous flow and intraocular pressure (IOP).@*Results@#Fifty eyes of 25 patients were included, 25 of which had PAC attack and 25 were PACS. The central corneal thickness (CCT), anterior-chamber depth, and anterior-chamber volume of the 2 groups were comparable. PAC-attack eyes had significantly higher IOP (18.4 mm Hg) than the PACS (14.12 mm Hg) (p = 0.001). The mean rate of aqueous flow was 2.50 ± 0.94 µL/min and 2.89 ± 1.17 µL/min in the PAC and PACS respectively (p = 0.20). The mean aqueous-outflow facility was 0.29 ± 0.18 µL/min and 0.59 ± 0.37 µL/min respectively (p = 0.0008).@*Conclusion@#A significantly lower aqueous-outflow facility was demonstrated by fluorophotometry among eyes with PAC. Despite the anatomically open angles, they continued to have higher IOPs.

Fuchs' Uveitis: Failure to associate vitritis and disc hyperfluorescence with the disease is the major factor for misdiagnosis and diagnostic delay

Fuchs' uveitis is often diagnosed with substantial delay at the origin of deleterious consequences such as unnecessary treatment. The aim of the study was to analyze the type and frequency of posterior inflammatory and fluorescein angiographic signs in Fuchs' uveitis in conjunction with the other clinical signs and evaluate their respective importane in the diagnosis of the disease. In particular, diagnostic delay and erroneous diagnosis were investigated. Patients seen in our centers between 1995 and 2008 with the diagnosis of Fuchs' uveitis were analyzed. The data collected included age, initial and final visual acuities; clinical findings at presentation, mean diagnostic delay, erroneous diagnosis, laser flare photometry values, fundus and fluorescein angiography manifestations and ocular complications. One hundred and five patients were included. The mean age at diagnosis was 34 years. Twelve patients [11.4%] had bilateral involvement. The mean diagnostic delay was 3.04 +/- 4.30 years. The most frequent clinical signs were vitreous infiltration [97.40%], typical Fuchs' keratic precipitates [94.90%], crystalline lens opacities or cataract [47%], heterochromia [42.60%], ocular hypertension or glaucoma [12.80%]. The mean laser flare photometry value at presentation was 9.85 +/- 6.28 ph/ms. Thirty-nine patients [37.14%] had undergone fluorescein angiography showing disc hyperfluorescence in 97.7% and peripheral retinal vascular leakage in 13.6%. Fuchs' uveitis is significantly underdiagnosed likely because vitreous involvement was previously described but not commonly recognized as an association with Fuchs' uveitis in the clinician's mind and therefore has often been given a different diagnostic label, Moreover, the very frequent inflammatory signs on fluorescein angiography such as disc hyperfluorescence and more rarely peripheral retinal vascular leakage, which has not been typically associated with Fuchs' uveitis, appear to represent an additional factors leading to misdiagnosis. Such clinical findings need to be publicized in order to reduce misdiagnosis, and diagnostic delay

RPE Cell Apoptosis by the Combination Treatment of SAHA and Lactacystin

PURPOSE: To establish a new therapeutic strategy for proliferative vitreoretinopathhy (PVR), we examined the effect of combined treatment with HDAC inhibitor SAHA and proteasome inhibitor lactacystin in human retinal pigment epithelial (RPE) cells, ARPE-19. METHODS: Viability was determined by trypan blue exclusion assay. Mitochondrial membrane potential (MMP) was measured by flow cytometry. Proteasome activity was measured by fluorophotometry. The expression and degradation of apoptosis-related proteins were assesssed by Western blotting. Subcellular location of apoptosis-related factors was monitored by confocal miscroscopy. RESULTS: A single treatment with 5 micro M SAHA or 10 micro M lactacystin did not reduce cell viability. However, combination treatment with 5 micro M SAHA and 10 micro M lactacystin substantially reduced the viability, because the mixture induced the reduction of MMP and nuclear condensation or fragmentation. Moreover, the combination treatment triggered the activation of caspase-3 and the production of PARP cleavage products. These data indicate that the combination treatment efficiently induces apoptosis in ARPE-19 cells. However, co-treatment of SAHA did not augment the proteasome inhibitory activity of lactacystin, nor did co-treatment of lactacystin augment acetylation of histones. It is notable that while p53 and CAD were observed in the mitochondria of cells treated with SAHA, they were translocated into the nucleus after the combination treatment. CONCLUSIONS: These results suggest that the combination treatment of SAHA and lactacystin effectively induced apoptosis in ARPE-19 cells. Further work is warranted to develop this combination therapy as a novel therapeutic strategy for PVR.

A fluorospectrophotometric determination of nitrite in blood / 中国应用生理学杂志

<p><b>AIM</b>To establish a fluorospectrophotometric assay for the measurement of nitrite in blood.</p><p><b>METHODS</b>Interference from hemoglobin and other blood ingredients was removed through sulfuric acid and phosphotungstic acid pretreatment. Fluorescence of 1-[H]-naphthotriazole from the reaction of 2,3-diaminonaphthalene with nitrite was determined with fluorospectrophotometry.</p><p><b>RESULTS</b>The following conditions were proper: Serum or plasma was treated with sulfuric acid and phosphotungstic acid pretreatment for two times, 2,3-diaminonaphthalene of 0.63 mmol x (L(-1)) was used, reaction solution pH and final pH were about 1.60 and 1.70 respectively, solution containing 2,3-diaminonaphthalene and supernatant after pretreatment was water-bathed at 20 degrees C for 15 minutes. The lower limit of detection was 24.27 nmol x L(-1). Nitrite determined in peripheral blood of healthy people was (10.91 +/- 2.38) micromol x L(-1), and its 95% distribution range was (6.24-15.57) micromol x L(-1).</p><p><b>CONCLUSION</b>It's a relatively sensitive, specific, simple method. It's of some value to the study of nitric oxide.</p>

Total radical TRapping antioxidant parameter, calculated / 대한내분비학회지

BACKGROUND: It has been suggested that diabetic patients are under high oxidative stress and plasma MDA concentration is a reliable marker for oxidative stress. However, some studies showed that plasma MDA is not a good marker for oxidative stress. Reeently, the total radical-trapping antioxidant parameter (TRAPc) has been proposed as a marker for the overall antioxidant property of plasma samples. Therefore, in this study, we tried to evaluate whether MDA and TRAPc are reliable markers of the oxidative stress-antioxidant system or not. METHODS: The plasma samples from 67 type 2 diabetic patients and 31 normal subjects were collected. The plasma MDA, protein-bound SH groups, uric acid and vitamin C were determined by fluorophotometry or spectrophotometry. Plasma vitamin E concentration was analyzed by HPLC. Calculated TRAP (TRAPc) were determined by the proposed calculation methods. RESULTS: 1. Diabetic patients had significantly lower TRAPc, compared with normal subjects. 2. SH groups, uric acid, vitamin C and vitamin E were not different between the two groups. 3. MDA and MDA/TG were significantly higher in diabetic subjects. CONCLUSION: From the results of this study, TRAPc seems to be a reliable parameter of overall plasma antioxidant system and the plasma MDA may be used as a marker of oxidative stress, but further long-term logitudinal studies are needed.

Change of Epithelial Barrier Function after Excimer Laser Photorefractive Keratectomy

We used the fluorophotometry to investigate the corneal epithelial barrier function after excimer laser photorefractive keratectomy (PRK). Twenty-five eyes of 21 subjects (13 women, 8 men) underwent PRK to correct rnyopia. Corneal epithelial healing time was measured and corneal epithelial permeability to sodiurn fluorescein was evaluated by fluorophotoinetry at I, 2, and 3 weeks after surgery. The corneal epithelial permeability increased significantly 1 week after surgery and returned to preoperative level 2 weeks after surgery. The permeability differences according to epithelial healing days and corrected diopters were not statistically significant(p>0. 05). These results suggest that PRK delays complete reconstruction of corneal epithelial barrier function. The corneal epithelium regained its functional barrier 2 weeks after PRK in patients, so, at least, during the first 2 weeks, care should be taken to miniinize further epithelial trauma from topical inedication or surgical manipulation.

The Effect of Phacoemulsification on the Corneal Endothelium in Diabetic Patients

We compared the structural and functional corneal endothelial changes between patients with non-proliferative retinopathy and age-matched nondiabetic patients after phacoemulsification with posterior chamber lens implantation. Endothelial cell density was calculated preoperatively, and postoperative 4, 8 weeks by specular microscopy. Fluorophotometry to check corneal endothelial barrier function was underwent preoperatively and at postoperative 4 days. Preoperatively, no significant difference in corneal endothelial cell density and corneal endothelial permeability coefficient was noted between diabetic patients and age-matched non-diabetic patients. But the mean increase of corneal endothelial permeability coefficient at 4 days after surgery was 118.13% in diabetic patients and 61.88% in nondiabetic patients (P<0.05). The mean endothelial cell loss at 4 and 8 weeks after surgery were 10. 29%, 12.55% in diabetic patients and 7.05%, 9.39% in non-diabetic patients.(P<0.05)

The Effect of Choloride Channel on Aqueous Humor Production

Regulating the cell volume is an important factorin secretory function of epithelial cells and regulatory volume decrease (RVD) phenomenon is involved in response to the changes of cell volume and osmolarity. RVD of epithelial cells reflects cellular release of K+ and C1- through channels and K+ and C1- channels were verified in the basal membrane of the non pigmented ciliary epithelial cells. Therefore, we attempted to observe the involvement of C1- channel in aqueous humor production by performing the fluorophotometry after administration of the DIDS(4.4-diisothiocyanatostilbene-2-2-disulfonic acid), the C1 channel blocker. Ten white New Zealand rabbits, 5 for tonometry and 5 for fluorophotometry, were used. One eye was injected 2x10-4M DIDS intravitreally using microsyringe and the other eye was injected normalsaline as a control in each rabbits. Tonometry was performed before the injection and every hour after dosing for 5 hours. Fluorophotometry was performed every 30 minutes for 3 hours starting 2 hours after injection. Wilcoxon`s signed rank test was used for statistical analysis. DIDS decreased intraocular pressure by 12.5%(P<0.05) and reduced aqueous humor flow rate by 28.5%(P0.05). In conclusion, it was observed.

Measurement of Blood Retina Barrier in Branch Retinal Vein Occlusion (BRVO)

Vitreous fluorophotometry was used to measure blood retinal barrier permeability to fluorescein in 15 patients with branch retinal vein occlusion(BRVO). Mean posterior vitreous fluorescein concentration(3mm) was 20.0 +/- 11.3(ng/ml) in affected eyes, and 2.99 +/- 1.22(ng/ml) in unaffected eyes. There was a statistically significant difference between the affected eye and unaffected eye(p<0.05). Also there was a correlation between the hemorrhage area and the posterior vitreous fluorescein concentration(r2=0.819). This study revealed that the permeability of blood retinal barrier was increased in BRVO as compared to the contralateral eye, and the higher permeability values were associated with the extent of area involved.

The effect of subtenon injection of methylprednisolone acetate on the breakdown of blood retinal barrier after cryotherapy

Using computerized vitreous fluorophotometry (VFP, Fluorotron(TM)), we examined the effect of cryotherapy on the blood retinal barrier (BRB) and the effect of subtenon injection of methylprednisolone acetate (Depomedrol(R)). In experiment 1, the right eyes of the 13 pigmented rabbits were treated with heavy cryotherapy after baseline VFP readings. The freezes were applied at 6 places in each quadrant around the equator are in two rows, a total of 24 places circumferentially. The left eyes were reserved as controls. In 6 rabbits (cryo with steroid group), Depomedrol(R) 10 mg of Depomedrol was injected into subtenon space after cryotherapy. The other 7 rabbits were treated with cryotherapy only (cryo only group). The VFP readings were taken 1, 3, 5, and 7 days, 2, 3, 5, and 7 weeks after cryotherapy. Cryotherapy increased the breakdown of BRB significantly. The peak VFP readings were obtained 5 days after cryotherapy in the cryo only group and 7 days after cryotherapy in the cryo with steroid group. In the cryo only group, the severity of the breakdown of BRB was higher than in the cryo with steroid group, and the increased VFP readings could not be normalized until 7 weeks after cryotherapy. In experiment 2, both eyes of the 8 pigmented rabbits were treated with medium cryotherapy after baseline VFP readings. The freezes were applied at 3 places in the superior temporal quadrant and at 3 places in the superior nasal quadrant, a total of 6 places. Depomedrol(R) 10 mg was injected into subtenon space after cryotherapy in the right eyes only. The VFP readings were taken 1, 3, 5, 7, 10, and 14 days after cryotherapy. In this experiment, cryotherapy did not increase the breakdown of BRB. But in the right eye, the severity of the breakdown of BRB was significantly lower than in the left eye 7 and 10 days after cryotherapy. These results suggest that Depomedrol(R) can decrease the severity of the breakdown of BRB after cryotherapy, and may be useful in the prevention of proliferative vitreoretinopathy (PVR).

The Quantitative Analysis of Autofluorescence in the Aging Crystalline Lens

When illuminated by blue light, the human crystalline lens exhibits green autofluorescence. Autofluorescence of the human lens were analyzed quantitatively in vivo as a function of age in 75 healthy individuals{150 eyes) without cataract, ranging in age from 17 to 63 years. Lenses were scanned through the dilated pupil along the optical axis, generating a fluorescence profile consisting of anterior and posterior peaks of lens, using an automated scanning fluorophotometer(Fluorotron master) coupled with a lens system designed for high resolution of the anterior segment structures. Anterior peak autofluorescence increased linearly by 90 ng fluorescein/ml per decade and posterior peak autofluorescence by 70 ng fluorescein/ml per decade. No significant difference between right and left eyes was demonstrated for anterior or posterior peak autofluorescence. We suggest that the quantitative analysis of the autofluorescence of the human lens might be useful to investigate the changes of the aging lens proteins and the pathogenesis of cataract.

Measurement of plasma verapamil levels by high-performance liquid chromatography

1. We have developed an alternative procedure for the measurement of verapamil levels in human plasma by reverse-phase high performance liquid chromatography with fluorimetric detection. 2. Prior to assay, plasma is submitted to a double extraction procedure, using first n-heptane in alkaline medium and then an acid phosphate buffer. Flecainide, a compound not related to verapamil, is used as internal standard. Mean recoveries of 70 and 63 per cent were obtained for verapamil and flecainide, respectively. 3. The sensitivity (5 ng/ml), reproducibility (inter-assay per cent CV = 1.7-8.7; intra-assay per cent CV = 2-4) and high recovery during sample clean-up make this method useful for the quantitation of verapamil in therapeutic monitoring and pharmacokinetic studies. 4. The method is illustrated with the pharmacokinetic results obtained for 14 healthy male volunteers who received a single 240 mg dose of the commercially available tablets of Dilacoron Retard 240 mg. The mean values for the area under the curve from 0 to 24 h (AUC[0-24]), maximum achieved concentration (Cmax) and time to achieve the maximum concentration (Tmax) were 863 ng h-1 ml-1, 112 ng/ml and 4 h, respectively