Gultekin Yucel

Effect of astaxanthin on hepatocellular injury following ischemia/reperfusion

This study investigated the effect of astaxanthin (ASX; 3,3-dihydroxybeta, beta-carotene-4,4-dione), a water-dispersible synthetic carotenoid, on liver ischemia-reperfusion (IR) injury. Astaxanthin (5 mg/kg/day) or olive oil was administered to rats via intragastric intubation for 14 consecutive days before the induction of hepatic IR. On the 15th day, blood vessels supplying the median and left lateral hepatic lobes were occluded with an arterial clamp for 60 min, followed by 60 min reperfusion. At the end of the experimental period, blood samples were obtained from the right ventricule to determine plasma alanine aminotransferase (ALT) and xanthine oxidase (XO) activities and animals were sacrificed to obtain samples of nonischemic and postischemic liver tissue. The effects of ASX on IR injury were evaluated by assessing hepatic ultrastructure via transmission electron microscopy and by histopathological scoring. Hepatic conversion of xanthine dehygrogenase (XDH) to XO, total GSH and protein carbonyl levels were also measured as markers of oxidative stress. Expression of NOS2 was determined by immunohistochemistry and Western blot analysis while nitrate/nitrite levels were measured via spectral analysis. Total histopathological scoring of cellular damage was significantly decreased in hepatic IR injury following ASX treatment. Electron microscopy of postischemic tissue demonstrated parenchymal cell damage, swelling of mitochondria, disarrangement of rough endoplasmatic reticulum which was also partially reduced by ASX treatment. Astaxanthine treatment significantly decreased hepatic conversion of XDH to XO and tissue protein carbonyl levels following IR injury. The current results suggest that the mechanisms of action by which ASX reduces IR damage may include antioxidant protection against oxidative injury.

Effect of hypercholesterolemia on inducible nitric oxide synthase expression in a rat model of elevated intraocular pressure

PURPOSE This study was performed to examine the effect of hypercholesterolemia on inducible nitric oxide synthase (NOS-2) expression and oxidative tissue injury in an experimental rat model of elevated IOP. METHODS Wistar rats were maintained on either regular chow or a high-cholesterol diet for 24 weeks. Intraocular pressure (IOP) was elevated in hypercholesterolemic rats by unilaterally cauterizing three episcleral vessels. Rats were divided into four experimental groups as follows; hypercholesterolemia, hypercholesterolemia+elevated IOP, elevated IOP and control. NOS-2 distribution, lipid peroxidation and retinal nerve fiber layer (RNFL) thickness was evaluated in all experimental groups at the end of 24 weeks. RESULTS Light microscopic evaluation of retinas in hypercholesterolemic rats revealed breaks and discontinuation in focal areas in the outer nuclear layer (ONL). NOS-2 positive staining was observed throughout the outer plexiform layer (OPL), inner plexiform layer (IPL) and ganglion cell layer (GCL) in rats with elevated IOP and/or hypercholesterolemia. Calculated values of RNFL thickness in hypercholesterolemic rats were significantly higher than those in the control and elevated IOP group. Vitreous malondialdehyde (MDA) levels detected in elevated IOP (3.51+/-0.31 nmol/mg protein) and hypercholesterolemia+elevated IOP (5.14+/-1.28 nmol/mg protein) groups were significantly higher than those detected in hypercholesterolemic (1.92+/-1.43 nmol/mg protein) and control (1.89+/-0.24 nmol/mg protein) groups. CONCLUSION The presented data confirms hypercholesterolemia as a risk factor in the development of glaucomatous optic neuropathy (GON) and suggests that increased circulating cholesterol may exacerbate disease progression by inducing NOS-2 expression and elevating oxidant tissue injury.

Nitrotyrosine formation and apoptosis in rat models of ocular injury.

This study was performed to examine inducible nitric oxide synthase (NOS-2) expression, nitrotyrosine formation and apoptosis in rats with elevated intraocular pressure (IOP) and/or ocular inflammation. Ocular inflammation was induced via injection of intra-vitreal lipopolysaccharide (LPS) while IOP was elevated by episcleral vessel cauterization. Animals were randomized to one of the following conditions: elevated IOP, LPS, elevated IOP+LPS, and control. Immunohistochemical staining and western blot analysis of retinal lysates revealed NOS-2 and nitrotyrosine immunoreactivity in all disease groups. NOS-2 expression and protein nitration was significantly greater in rats with elevated IOP+LPS compared to elevated IOP, LPS, and control groups. Nitrite levels in the retina affirmed significantly increased levels of nitric oxide generation in LPS-treated rats with elevated IOP (346 ± 23.8 μM) vs LPS-treated, elevated IOP and control groups (195.6 ± 12.6, 130 ± 2.5 and 76.6 ± 15.6 μM, respectively). Retinal TUNEL staining showed apoptosis in all diseased groups. Percent of apoptotic cells was significantly greater in the elevated IOP+LPS group compared to LPS-treated or elevated IOP groups. Presented data illustrates that both elevated IOP and ocular inflammation augment NOS-2 expression, retinal protein nitration and apoptosis in rats.

Suppressive effect of astaxanthin on retinal injury induced by elevated intraocular pressure.

The aim of this study was to clarify the possible protective effect of astaxanthin (ASX) on the retina in rats with elevated intraocular pressure (EIOP). Rats were randomly divided into two groups which received olive oil or 5mg/kg/day ASX for a period of 8 weeks. Elevated intraocular pressure was induced by unilaterally cauterizing three episcleral vessels and the unoperated eye served as control. At the end of the experimental period, neuroprotective effect of ASX was determined via electrophysiological measurements of visual evoked potentials (VEP) and rats were subsequently sacrificed to obtain enucleated globes which were divided into four groups including control, ASX treated, EIOP, EIOP+ASX treated. Retinoprotective properties of ASX were determined by evaluating retinal apoptosis, protein carbonyl levels and nitric oxide synthase-2 (NOS-2) expression. Latencies of all VEP components were significantly prolonged in EIOP and returned to control levels following ASX administration. When compared to controls, EIOP significantly increased retinal protein oxidation which returned to baseline levels in ASX treated EIOP group. NOS-2 expression determined by Western blot analysis and immunohistochemical staining was significantly greater in rats with EIOP compared to ASX and control groups. Retinal TUNEL staining showed apoptosis in all EIOP groups; however ASX treatment significantly decreased the percent of apoptotic cells when compared to non treated ocular hypertensive controls. The presented data confirm the role of oxidative injury in EIOP and highlight the protective effect of ASX in ocular hypertension.

Transmission electron microscopy and autofluorescence findings in the cornea of diabetic rats treated with aminoguanidine

BACKGROUND The accumulation of advanced glycation end products (AGEs) has been implicated in the pathogenesis of diabetic keratopathy. The present study was aimed to understand if aminoguanidine (AG), an AGE inhibitor, was protective against the development of corneal complications in a diabetic rat model. METHODS Wistar rats were divided into three experimental groups: control, diabetic, and AG-treated diabetic. Diabetes was induced in rats via a single intraperitoneal injection (60 mg/kg) of streptozocin (STZ) and AG was administered in drinking water at a dose of 1 g/L. All animals were sacrificed at the end of 10 weeks and corneas from diabetic and nondiabetic rats were analyzed via transmission electron microscopy (TEM). Corneal autofluorescence measurements were also performed in all experimental groups. RESULTS Electron microscopic evaluation revealed that aminoguanidine treatment in diabetic rats prevented the formation of intracellular spaces between neighbouring cells in the superficial corneal epithelium. Hyperglycemia-induced degeneration of intracellular organelles and formation of cytoplasmic vacuoles in the corneal stroma was also prevented with the treatment of AG. Corneal autofluorescence detected in the diabetic group (5.98 +/- 2.17 Fi/mg protein) was found to be significantly greater than the control (3.92 +/- 0.56 Fi/mg protein) and the AG-treated diabetic group (4.18 +/- 0.59 Fi/mg protein) (p < 0.05). INTERPRETATION The presented data provide evidence that AG is preventive against corneal alterations in experimental diabetes.

Corneal protein nitration in experimental uveitis

Increased expression of inducible nitric oxide synthase (NOS-2) in inflammatory diseases like uveitis suggests that it contributes to the observed pathological state. The aim of this study was to evaluate corneal expression of NOS-2 and corneal protein nitration in a rat model of uveitis. A single injection of intravitreal lipopolysaccharide was used to induce uveitis. Corneal proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by Coomassie blue staining. Expression of NOS-2 and nitrotyrosine (NO2Tyr) formation were determined via immunohistochemistry and Western blot analysis. Total nitrate/nitrite levels in the vitreous were measured by spectral analysis via the Griess reagent. Immunohistochemical analysis revealed increased corneal NOS-2 and NO2Tyr immunoreactivity in rats with uveitis compared with controls. NOS-2 and NO2Tyr immunoreactivity was observed in and around basal cells in the corneal epithelium. Western blot analysis of corneal lysates showed multiple nitrated protein bands in uveitic rats. Spectrophotometric measurement of total nitrate/nitrite levels in the vitreous affirmed significantly increased levels of nitric oxide generation in uveitis (126 ±2.63 μM/mg protein) compared with controls (65 ±6.57 μM/mg protein). The presented data suggests that extensive formation of protein nitration and reactive nitrogen species in the cornea contributes to tissue destruction in uveitis. Hence, selective inhibition of NOS-2 may prevent long-term complications and lead to an improvement in the management of uveitis.

Systemic toxicity of tacrolimus given by various routes and the response to dose reduction

Purpose: To evaluate the long‐term systemic toxicity of tacrolimus (FK‐506) administered by various routes, and to assess the effect of dose reduction on toxicity.

Plasma Homocysteine Levels in Noninsulin-dependent Diabetes Mellitus with Retinopathy and Neovascular Glaucoma

Aim: This study was designed to evaluate plasma homocysteine levels in noninsulin-dependent diabetes mellitus patients (NIDDM) with preproliferative retinopathy and neovascular glaucoma. The experimental goal was to determine the relationship between plasma homocysteine content and the development of microvascular lesions.Methods:Plasma homocysteine levels were assessed in three experimental groups consisting of healthy controls (n=30), NIDDM patients with preproliferative retinopathy (n=20) and NIDDM patients with neovascular glaucoma (n=20). Homocysteine levels were determined via a fluorescence polarization immunoassay method by an Abbot IMX instrument.Results:Plasma homocysteine levels in NIDDM patients with preproliferative retinopathy and neovascular glaucoma (n=40) were found to be significantly higher than those of controls (n=30) (p < 0.01). When statistical analysis was performed separately among the three experimental groups, no significant difference in plasma homocysteine levels were found in patients with preproliferative retinopathy compared to controls. However, homocysteine levels in patients with neovascular glaucoma were found to be significantly higher than the control group (p < 0.001). No significant difference in plasma homocysteine levels could be detected between patients with preproliferative retinopathy and neovascular glaucoma.Conclusions:Hyperhomocysteinemia is a risk factor for the development of microvascular lesions in patients with NIDDM but cannot be used as a marker to assess the progression of lesions observed in neovascular glaucoma.

The role of antioxidants in the prevention of t-butyl hydroperoxide-induced chemiluminescence

An experimental system which assesses the antioxidant potential of ascorbic acid, glutathione, uric acid, and taurine was developed. The system comprised hemoglobin, luminol, t-butyl hydroperoxide, and different concentrations of antioxidants in TRIS-HCl buffer (pH 7.4). Control assays were performed by excluding antioxidants. Chemiluminescence was detected using a liquid scintillation counter in single photon mode. All antioxidants, when applied in the appropriate concentrations, decreased the maximum chemiluminescence values. The minimum concentrations which decreased the chemiluminescence values were defined for each of the antioxidants.

Association between graft function and serum TNF-α, TNFR1 and TNFR2 levels in patients with kidney transplantation

Abstract Introduction: This prospective observational study aimed to assess the relevance of serial postoperative serum TNF-α, TNFR1 and TNFR2 measurements for predicting graft function and acute rejection episodes (AR) after transplantation. Materials and methods: We studied 50 kidney transplant recipients (31 female, 19 male; mean age: 38.36 ± 12.88). Blood samples were collected immediately before and after surgery at day 7, month 1 and month 3. Serum TNF-α, TNFR1 and TNFR2 levels were measured by ELISA using a commercial kit (Invitrogen ELISA). Serum cystatin-C levels were measured by particle-enhanced immunonephelometric method. Glomerular filtration rate (GFR) was estimated by Chronic Kidney Disease-Epidemiology (CKD-EPI) equation. Patients were assigned to their transplant outcomes in terms of acute rejection [AR(+) and AR(−)] and slow (SGF) or immediate graft function (IGF). Results: Among 50 recipients, six had AR(+) and 44 had AR(−), depending on graft function: 17 had SGF and 33 had IGF. Serum creatinine, cystatin-C, TNF-α, TNFR1 and TNFR2 levels demonstrated consistent significantly decreases after transplantation while GFR values had consistent increases (p = 0.001). Pretransplant levels were not statistically different between AR(+) and AR(−) groups (TNF-α: 30.79 ± 5.96 vs. 27.95 ± 2.43 pg/mL, TNFR1: 55.96 ± 21.6 vs. 40.52 ± 7.41 ng/mL, TNFR2: 58.31 ± 8.06 vs. 50.9 ± 3.34 ng/mL, respectively) (p > 0.05). Serum TNF-α, TNFR1 and TNFR2 levels on day 7 and month 1 were also significantly higher in AR(+) group compared to AR(−) (p = 0.012, p = 0.049 for TNF-α, p = 0.001, p = 0.002 for TNFR1, p = 0.001, p = 0.002 for TNFR2). Conclusions: Our preliminary findings suggest that serum TNF-α, TNFR1 and TNFR2 levels might be considered useful markers of evaluating graft function after renal transplantation.