Ayşegül Uysal

Resveratrol attenuates doxorubicin-induced cellular damage by modulating nitric oxide and apoptosis.

Although doxorubicin (DOX) is a commonly used chemotherapeutic agent its clinical use is restricted due to its organ toxicities. The present investigation relates to reducing DOX induced side effects to the liver, kidney and ileum by usage of the antioxidant, anti-inflammatory agent, resveratrol (RES) and to investigate the role of nitric oxide synthase (NOS) in the process. Wistar rats were divided into four groups: control (saline i.p); DOX (20 mg/kg i.p), RES (20 mg/kg i.p) and DOX (20mg/kg i.p)+RES (20 mg/kg i.p). Immunohistochemical activity of both iNOS and eNOS were evaluated after DOX treatment and ultrastructural changes such as cellular damage and mitochondrial degeneration were evaluated. Degenerative ultrastructural changes were demonstrated especially in the DOX treated group. Variations in biochemical marker levels of oxidative stress on ischemia in tissues were not observed. Our data indicate that RES may prevent cellular damage in the early phase of DOX induced toxicity. RES could be used with its beneficial effects during early cellular damage in organ toxicity after DOX treatment in cancer patients.

Assessment of effects of pinealectomy and exogenous melatonin administration on rat sciatic nerve suture repair: an electrophysiological, electron microscopic, and immunohistochemical study

SummaryBackground. Collagen scar formation at the cut end of a nerve, an important problem in clinical practice for neurosurgeons in peripheral nerve surgery, obstructs sprouting of axons into appropriate distal fascicles, and thereby limits nerve regeneration. Researchers attempt to control collagen accumulation in the formation of neuroma by various physical and chemical methods, but these have yielded only limited functional success. This is the first experimental study investigating the effects of melatonin (MLT) on nerve repair and neuronal regeneration in rat sciatic nerve suture repair.Methods. The hypothesis that exogenous MLT administration may inhibit the formation of neuroma in peripheral nerve surgery was investigated in rat sciatic nerve model. In this study, a total of 80 rats were used for control groups (Groups Ia, Ib, IIa, and IId), MLT group (Group Ic), surgical pinealectomy (Px) groups (Groups IIb and IIc), and group of MLT treatment following Px procedure (Group IIe). All animals underwent a surgical intervention consisting of bilateral sciatic nerve section and primary suture repair. At 8 weeks after repair, the animals were killed following completion of recording of nerve action potentials (NAPs). Then, unilateral sciatic nerve specimens including the suture repair region were carefully removed and the excised segments were processed for electron microscopy examination. Afterwards, contralateral sciatic nerve specimens from two animals from each group were removed and stained for immunohistochemical analysis.Results. Results of morphometric analysis revealed that Px procedure caused an elevation of collagen content of the sciatic nerve and macroscopic neuroma formation, and that there was a statistically significant reduction in collagen content of the same region in pinealectomized animals treated with MLT (p<0.001). Accordingly, electrophysiological findings demonstrated that the stimulus intensities required to excite a NAP response were increased in surgical Px group, but the presence of a reduced threshold response was found in the group treated with MLT following Px procedure (p<0.01). Immunohistochemical staining for Type I collagen and Type III collagen was markedly more intense in the epineurium of animals after Px. Virtually no or only weak staining was observed in animals in control groups and the MLT treatment group. Results of immunohistochemical analysis revealed that surgical Px procedure caused a strong immunoreactivity for Type I collagen and Type III collagen in all connective tissue planes of the nerve, especially in the epineurium, and there was a statistically significant reduction in immunoreactivity of the repair region in animals receiving MLT treatment after Px procedure (p<0.001).Conclusion. This study demonstrates that exogenous MLT administration significantly inhibits collagen accumulation in the formation of neuroma in the suture repair site and thereby improves nerve regeneration. From a clinical standpoint, the positive effect of MLT administration on neuroma formation and nerve regeneration seems a particularly attractive treatment option. Therefore, we believe that nerve repair with addition of MLT may be a worthwhile option in addition to other treatment modalities in case of MLT deficiency, such as aging. However, further experimental and clinical studies using functional analysis warranted to confirm this result in future.

Effects of the Calcium Channel Blocker Amlodipine on Serum and Aortic Cholesterol, Lipid Peroxidation, Antioxidant Status and Aortic Histology in Cholesterol-Fed Rabbits

Reactive oxygen metabolites and oxidized fatty acids are proinflammatory and are involved in the pathophysiology of atherosclerosis. Amlodipine, a unique third-generation dihydropyridine-type calcium channel blocker, seems to exert atheroprotective effects through its antioxidant properties related to its chemical structure and independent of its calcium channel-blocking effect. In this study, the interactions of amlodipine with major cellular antioxidants were investigated in order to elucidate the mechanisms underlying its atheroprotective effects. New Zealand white male rabbits were fed regular chow (group 1), chow with 1% cholesterol (group 2), regular chow plus 5 mg/kg/day amlodipine per os (group 3) and 1% cholesterol plus amlodipine (group 4) for 8 weeks. Total cholesterol, malondialdehyde (MDA) and vitamin E concentrations and catalase and superoxide dismutase (SOD) activities were determined in blood drawn before and after the experimental period. Aortic tissue was examined for atherosclerotic changes and aortic total cholesterol, MDA, catalase and SOD were determined. At the end of the 8-week treatment period, serum total cholesterol and plasma MDA were elevated in groups 2 and 4. In group 2, serum vitamin E and plasma SOD diminished (p < 0.05) and catalase increased (p < 0.05). In group 4, SOD activity increased at the end of treatment. MDA levels were lower and plasma SOD activities were higher in group 4 than in group 2. Aortic tissue investigations revealed higher total cholesterol and MDA concentrations and catalase activities in group 2 than in group 4, and the highest tissue SOD activity was recorded in group 4 (p < 0.05 for all comparisons). Morphological examination of aortic tissues exhibited endothelial disarrangement and lipid deposition in group 2. Histopathological alterations related to atherogenesis were less in group 4 than in group 2. Amlodipine seems to exert atheroprotective effects by reducing aortic cholesterol accumulation and blood and aortic lipid peroxidation, enhancing SOD activity both in blood and aortic tissue and suppressing the consumption of vitamin E. On the other hand, the suppression of catalase activity in blood and the aorta interferes with the drugs well-known antioxidant effects.

Expression profiling of stem cell signaling alters with spheroid formation in CD133high/CD44high prostate cancer stem cells

Cancer stem cells (CSC) isolated from multiple tumor types differentiate in vivo and in vitro when cultured in serum; however, the factors responsible for their differentiation have not yet been identified. The first aim of the present study was to identify CD133high/CD44high DU145 prostate CSCs and compare their profiles with non-CSCs as bulk counterparts of the population. Subsequently, the two populations continued to be three-dimensional multicellular spheroids. Differentiation was then investigated with stem cell-related genomic characteristics. Polymerase chain reaction array analyses of cell cycle regulation, embryonic and mesenchymal cell lineage-related markers, and telomerase reverse transcriptase (TERT) and Notch signaling were performed. Immunohistochemistry of CD117, Notch1, Jagged1, Delta1, Sox2, c-Myc, Oct4, KLF4, CD90 and SSEA1 were determined in CSC and non-CSC monolayer and spheroid subcultures. Significant gene alterations were observed in the CD133high/CD44high population when cultured as a monolayer and continued as spheroid. In this group, marked gene upregulation was determined in collagen type 9 α1, Islet1 and cyclin D2. Jagged1, Delta-like 3 and Notch1 were respectively upregulated genes in the Notch signaling pathway. According to immunoreactivity, the staining density of Jagged1, Sox2, Oct4 and Klf-4 increased significantly in CSC spheroids. Isolated CSCs alter their cellular characterization over the course of time and exhibit a differentiation profile while maintaining their former surface antigens at a level of transcription or translation. The current study suggested that this differentiation process may be a mechanism responsible for the malignant process and tumor growth.

Cancer stem cell differentiation: TGFβ1 and versican may trigger molecules for the organization of tumor spheroids.

Cancer stem cells (CSCs) have the ability to self-renew similar to normal stem cells. This process is linked with metastasis and resistance to chemotherapy and radiotherapy. In the present study, we constructed an in vitro differentiation model for CSCs. CSCs isolated and proliferated for one passage were maintained as monolayers or spheroid-forming cells with serum included media for differentiation process. Differentiation of adhesion molecules and cellular ultrastructural properties were investigated and compared in both monolayer and spheroid cultures. CD133+/CD44+ cancer-initiating cells were isolated from DU-145 human prostate cancer cell line monolayer cultures and propagated as tumor spheroids and compared with the remaining heterogeneous cancer cell bulk population. Microarray-based gene expression analysis was applied to determine genes with differential expression and protein expression levels of candidates were analyzed by immunohistochemistry. Electron microscopy showed detailed analysis of morphology. TGFβ1 was found to be significantly upregulated in monolayer CSCs. High expression levels of VCAN, COL7A1, ITGβ3, MMP16, RPL13A, COL4A2 and TIMP1 and low expression levels of THBS1, MMP1 and MMP14 were detected when CSCs were maintained as serum-grown prostate CSC spheroids. Immunohistochemistry supported increased immunoreactivity of TGFβ1 in monolayer cultures and VCAN in spheroids. CSCs were found to possess multipotential differentiation capabilities through upregulation and/or downregulation of their markers. TGFβ1 is a triggering molecule, it stimulates versican, Col7A1, ITGβ3 and, most importantly, the upregulation of versican was only detected in CSCs. Our data support a model where CSCs must be engaged by one or more signaling cascades to differentiate and initiate tumor formation. This mechanism occurs with intracellular and extracellular signals and it is possible that CSCc themselves may be a source for extracellular signaling. These molecules functioning in tumor progression and differentiation may help develop targeted therapy.

The effects of calcium channel antagonist nimodipine on end-plate vascularity of the degenerated intervertebral disc in rats

The vascular channels at the end-plate of the intervertebral disc are very important in maintaining a healthy disc. With age, a reduction of the nutrition of the avascular nucleus pulposus is inevitable. On the other hand the calcium channel antagonist nimodipine has been shown to have a positive effect on blood flow in the region of the vertebral end-plate. To evaluate the effects of nimodipine on the end-plate vascularity in the degenerative discs, we have produced an experimental disc degeneration and evaluated the radiological and histopathological features of the end-plate of the degenerated discs. Adult rats were divided into 3 groups: control (n=5), operated degeneration (n=5), and nimodipine treatment (n=5). Using a posterior approach, a cut was made parallel to the end-plates in the posterior annulus fibrosus in 5 consecutive intervertebral discs between the 5th and 10th vertebral segments of the tails of adult Swiss Albino rats. At 8 weeks, 5 of these animals were treated with nimodipine. In each experimental group, 1 animal was examined using computed tomography (CT) to study the density of the cartilage end-plate of the disc. Then, the animals were sacrificed for subsequent histopathological evaluation. We found that the vascular channel counts and percentage areas from animals treated with nimodipine were higher than from both the non-operative control and operated degeneration groups, although these were not statistically different. Accordingly, the profile of the density histogram in the nimodipine-treated group showed a wide plateau, indicating an increase in the vascularity in this region. From our results, we suggest that nimodipine enhances vascularisation of the cartilage end-plate in the disc. It is possible that the increased proportion of vascular contacts at the end-plate has a beneficial effect in the nutrition of the disc. However, further experimental studies will be needed to determine the validity of this statement in animals or human beings.

The effect of exogenous melatonin administration on trabecular width, ligament thickness and TGF-β1 expression in degenerated intervertebral disk tissue in the rat

Intervertebral disk (IVD) degeneration, a complex pathological condition of varying origins, causes low back pain. Degenerative changes in IVD tissue affect the adjacent vertebral structure, resulting in a decreased vertebral trabecular width. It has been suggested that transforming growth factor-beta 1 (TGF-beta(1)) may have a role in the repair of connective tissue, as it occurs in the IVD degeneration process. In this study, we investigated the effects of exogenous melatonin (MEL) administration on vertebral trabecular width, ligament thickness and TGF-beta(1) expression in degenerated IVD tissue. Fifteen adult male Swiss Albino rats were divided randomly into three groups; nonoperated control, operated degeneration, and MEL treatment groups. In the operated degeneration and MEL treatment groups, cuts were made parallel to the end plates in the posterior annulus fibrosus at the fifth and tenth vertebral segments of the tail to induce IVD degeneration. In each group, TGF-beta(1) immunoreactivity and morphometry of vertebral trabecular width and anterior and posterior ligament thickness were evaluated. Histologically, disorganisation and irregularity of collagen fibres was seen in the degenerated (operated) IVD. Increased TGF-beta(1) expression in multinuclear chondrocytes was also observed as was decreased vertebral trabecular width. Importantly, the reduction of trabecular width observed in the operated degenerated group was reversed after MEL administration (p<0.0001). Similarly, TGF-beta(1) expression in multinuclear chondrocytes was dramatically increased after exogenous MEL application. Thus, there was a regression in histopathological changes after MEL treatment, with disk appearances similar to those of the control group. Based on our findings, we suggest that MEL activates the recovery process in the degenerated IVD tissue, possibly by stimulating TGF-beta(1) activity. This is the first report investigating the involvement of the pineal hormone MEL in the repair of rat IVD.

Comparison of the Effects of Dietary Saturated, Mono-, and n–6 Polyunsaturated Fatty Acids on Blood Lipid Profile, Oxidant Stress, Prostanoid Synthesis and Aortic Histology in Rabbits

Background/Aims: To compare the effects of saturated, monounsaturated and polyunsaturated n–6 fatty acid-enriched diets on the development of atherosclerosis and thrombosis in New Zealand white male rabbits, 3- to 6-month-old animals were supplemented daily (10 g/100 g diet) with butter (n = 8), olive oil (n = 8) or corn oil (n = 8) by oral administration for 7 weeks. Methods: Total cholesterol (TC), HDL- (HDL-C) and LDL-cholesterol (LDL-C), triglycerides (TG), apolipoprotein A-1 (ApoA-1), apolipoprotein B (ApoB), lipid peroxides as thiobarbituric acid-reactive substances (TBARS), thromboxane B2 (TXB2) and 6-ketoprostaglandin F1α (6-ketoPGF1α) concentrations were determined in blood samples drawn before and after each group was fed the different dietary regimens. Histological examination was performed on the aortic tissues. Results: After 7 weeks, TC, ApoB and TXB2 increased significantly (p < 0.05) in the butter-fed animals compared to pre-experimental concentrations. Olive oil administration lead to a significant (p < 0.05) decrease in TC and ApoB levels. The corn oil-enriched diet decreased TC, LDL-C concentrations, TC/HDL-C ratios and 6-ketoPGF1α (stable metabolite of prostacyclin-PGI2; p < 0.05 for all) but increased TBARS levels and TXB2/6-ketoPGF1α ratios. Light microscopic findings were in accordance with these biochemical alterations. Conclusion: Although effective in lipid lowering, corn oil increased oxidant stress as evidenced by increased TBARS and induced endothelial damage which lead to a reduction in PGI2 synthesis and consequently to an increase in the TXB2/6-ketoPGF1α ratio. Olive oil administration did not induce oxidant stress and it had no affect on PGI2 and TXB2 levels which are implicated in platelet aggregation. These findings suggest that oleic acid is more effective than linoleic acid in the protection of endothelial integrity.

Contradictory effects of chlorpromazine on endothelial cells in a rat model of endotoxic shock in association with its actions on serum TNF-α levels and antioxidant enzyme activities

We examined the effects of the phenothiazine derivative, chlorpromazine on thoracic aortic endothelial cell histology (14 h after LPS challenge) in a model of endotoxic shock in rats. Since excessive formation of tumor necrosis factor-alpha (TNF-alpha) and oxygen-derived free radicals contribute to endothelial injury in endotoxemia, we also evaluated the effect of the drug on the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase in liver tissue in this model and tried to find out whether this possible effect was associated with a change in serum TNF-alpha levels (measured 90 min after chlorpromazine administration). Endotoxemia was induced by a single i.p. injection of lipopolysaccharide (LPS) (5 mg kg(-1) in 1.5 ml of saline; LPS from Escherichia coli serotype 055:B5, L-2880, Sigma Chemical Company). Electron microscopic evaluation of the aortas revealed that chlorpromazine (administered 30 min prior to LPS challenge), in smaller doses (3 mg kg(-1)) ameliorated the endothelial cell injury caused by LPS, whereas it caused deterioration of endothelial cell morphology in higher doses (10 and 25 mg kg(-1)). Chlorpromazine administration caused a significant reduction in serum TNF-alpha levels, which was correlated well with an increase in SOD activity in all drug doses (3, 10 and 25 mg kg(-1)). Catalase activity was increased only in the 25 mg kg(-1) chlorpromazine group.

Immunohistochemical profile of transforming growth factor-β1 and basic fibroblast growth factor in sciatic nerve anastomosis following pinealectomy and exogenous melatonin administration in rats

Collagen scar formation at the cut end of a peripheral nerve, an important problem in clinical practice for neurosurgeons, obstructs sprouting of axons into appropriate distal fascicles, and thereby limits the regeneration process. Researchers have attempted to control collagen accumulation and neuroma formation with various physical and chemical methods, but with limited functional success. Recently, it has been demonstrated that transforming growth factor (TGF)-beta and basic fibroblast growth factor (bFGF) play an important role in collagen production by fibroblasts and in Schwann cell activity. In our study, rats were divided into a control group, a melatonin-treated group, a surgical pinealectomy group, and a group treated with melatonin following pinealectomy. They then underwent a surgical sciatic nerve transection and primary suture anastomosis. At 2 months after anastomosis, the animals were sacrificed and unilateral sciatic nerve specimens, including the anastomotic region, were removed and processed for immunohistochemical study from two animals in each group. For each antibody, immunoreactivity was assessed using a semiquantitative scoring system. Strong TGF-beta1 and/or bFGF expression was observed in the epineurium of animals that underwent pinealectomy, but no or weak staining was observed in animals in the control and melatonin treatment groups. Based on these data, we suggest that both TGF-beta1 and bFGF have important roles in control of collagen accumulation and neuroma formation at the anastomotic site, and that the pineal neurohormone melatonin has a beneficial effect on nerve regeneration.