Iloprost and vitamin C attenuates acute myocardial injury induced by suprarenal aortic ischemia-reperfusion in rabbits.

OBJECTIVE: The aim of this study was to evaluate antioxidant and cytoprotective effects of iloprost and Vitamin C in a distant organ after abdominal aorta ischemia-reperfusion injury. MATERIAL AND METHODS: Twenty-eight New Zealand rabbits weighing 2,400-2,800 g were used for this study. The rabbits were divided into four equal groups. These groups are control group, sham group, iloprost group, and iloprost+vitamin C group. Suprarenal aorta was occluded with a vascular clamp. Following 30 minutes of ischemia, the vascular clamp was removed. Rabbits in group 3 received 10 ng/kg/min iloprost and those in group 4 received 10 ng/kg/min iloprost and 10 mg/kg vitamin C. At the end of the reperfusion period, the rabbits were sacrificed by a high intraperitoneal dose of xylazine+ketamine injection. Myocardial tissue samples were taken for electron microscopic analysis. We evaluated SOD, MDA and catalase in myocardial tissue samples. RESULTS: Iloprost and iloprost+vitamin C groups significantly reduced the oxidative stress markers in tissue samples (p<0.05) and significantly decreased the myofibrillar injury and mitochondrial morphology changes in the myocardial tissue as shown with electron microscopy (p<0.05). Myocardial edema was significantly alleviated by iloprost and iloprost+vitamin C administration (p<0.05). CONCLUSIONS: This study clearly showed that myocardial injury and edema occurred after ischemia-reperfusion of abdominal aorta and that groups administered with iloprost and iloprost+vitamin C showed an attenuation of ischemia-reperfusion injury in distant organs (Tab. 3, Fig. 4, Ref. 30).

Effects of melatonin on testis histology, oxidative stress and spermatogenesis after experimental testis ischemia-reperfusion in rats.

BACKGROUND: Testicular torsion due to oxidative stress results in infertility and testicular damage which can be preventable an important health problem worldwide. AIM: The purpose of the present study was to investigate the changes of malondialdehyde (MDA), total antioxidant status (TAS), total oxidant status (TOS) levels; histopathological alterations; morphology, concentration and motilities of the sperm in post ischemic reperfused (I/R) testis tissue. MATERIALS AND METHODS: Forty adult male Wistar rats were carried out and were randomized to five groups; (1) Control group, (2) Ipsilateral left testis ischemia, (3) Melatonin plus ipsilateral left testis ischemia, (4) Contralateral right testis ischemia, 5. Melatonin plus contralateral right testis ischemia. After 1 h ischemia and 24 h perfusion; MDA, TAS and TOS levels were measured, histopathological alterations were determined using by Johnsen's score (JS) and sperm morphology, concentration, motility were examined. RESULTS: MDA, TAS and TOS levels of the testis tissue did not change in all groups (p > 0.05 for all). JS was decreased in I/R group and melatonin treatment reversed histopathological changes and increased JS both in ipsilateral and contralateral testis. Abnormal sperm rate significantly increased in I/R group and melatonin administration changed abnormal sperm rate to normal. CONCLUSIONS: As a result, the present study demonstrated that testicular damage occurs following I/R without an increase of MDA, TAS and TOS levels. Our results also suggested that melatonin is a potent antioxidant agent in preventing testicular I/R injury, as shown by increased JS and changed abnormal sperm rate.

Neuroprotective effect of melatonin on experimental peripheral nerve injury: an electron microscopic and biochemical study.

BACKGROUND AND STUDY AIMS: Melatonin is an important antioxidant agent with a protective role in the prevention of oxidative stress. We designed an experimental study which focused on the potential neuroprotective effect of melatonin on peripheral nerve injury. MATERIALS AND METHODS: Sciatic nerve injury was induced in the mid thigh region of 30 male Wistar rats by clip compression. Melantonin was injected intraperitoneally in 15 of the 30 rats. Electron microscope and biochemical studies were performed to assess the potential beneficial effect of melatonin on peripheral nerve regeneration. Changes to cellular organelles, myelin lamellae and axons were studied. RESULTS: There was a significant difference between the melatonin and nerve injury groups. Rats treated with melatonin demonstrated significant structural protection of the myelin lamellae compared to the nerve injury group. Axonal shrinkage and myelin changes were not prominent histopathologically in melatonin-treated group. Biochemical analysis confirmed the neuroprotective effects of melatonin with significantly lower lipid peroxidation and myeloperoxidase activity measurements in the melatonin-treated group compared to the neural injury group. The results indicate that melatonin can improve neural healing. CONCLUSION: With its neuroprotective effect, as demonstrated in this experimental peripheral nerve injury, melatonin might be used successfully in clinical practice. Further studies on the correct dosage and possible side effects are necessary.

Effect of melatonin and time of administration on irradiation-induced damage to rat testes.

The effect of ionizing irradiation on testes and the protective effects of melatonin were investigated by immunohistochemical and electron microscopic methods. Eighty-two adult male Wistar rats were divided into 10 groups. The rats in the irradiated groups were exposed to a sublethal irradiation dose of 8 Gy, either to the total body or abdominopelvic region using a 60Co source at a focus of 80 cm away from the skin in the morning or evening together with vehicle (20% ethanol) or melatonin administered 24 h before (10 mg/kg), immediately before (20 mg/kg) and 24 h after irradiation (10 mg/kg), all ip. Caspace-3 immunoreactivity was increased in the irradiated group compared to control (P < 0.05). Melatonin-treated groups showed less apoptosis as indicated by a considerable decrease in caspace-3 immunoreactivity (P < 0.05). Electron microscopic examination showed that all spermatogenic cells, especially primary spermatocytes, displayed prominent degeneration in the groups submitted to total body and abdominopelvic irradiation. However, melatonin administration considerably inhibited these degenerative changes, especially in rats who received abdominopelvic irradiation. Total body and abdominopelvic irradiation induced identical apoptosis and testicular damage. Chronobiological assessment revealed that biologic rhythm does not alter the inductive effect of irradiation. These data indicate that melatonin protects against total body and abdominopelvic irradiation. Melatonin was more effective in the evening abdominopelvic irradiation and melatonin-treated group than in the total body irradiation and melatonin-treated group.

Effect of melatonin and time of administration on irradiation-induced damage to rat testes

The effect of ionizing irradiation on testes and the protective effects of melatonin were investigated by immunohistochemical and electron microscopic methods. Eighty-two adult male Wistar rats were divided into 10 groups. The rats in the irradiated groups were exposed to a sublethal irradiation dose of 8 Gy, either to the total body or abdominopelvic region using a 60Co source at a focus of 80 cm away from the skin in the morning or evening together with vehicle (20% ethanol) or melatonin administered 24 h before (10 mg/kg), immediately before (20 mg/kg) and 24 h after irradiation (10 mg/kg), all ip. Caspace-3 immunoreactivity was increased in the irradiated group compared to control (P < 0.05). Melatonin-treated groups showed less apoptosis as indicated by a considerable decrease in caspace-3 immunoreactivity (P < 0.05). Electron microscopic examination showed that all spermatogenic cells, especially primary spermatocytes, displayed prominent degeneration in the groups submitted to total body and abdominopelvic irradiation. However, melatonin administration considerably inhibited these degenerative changes, especially in rats who received abdominopelvic irradiation. Total body and abdominopelvic irradiation induced identical apoptosis and testicular damage. Chronobiological assessment revealed that biologic rhythm does not alter the inductive effect of irradiation. These data indicate that melatonin protects against total body and abdominopelvic irradiation. Melatonin was more effective in the evening abdominopelvic irradiation and melatonin-treated group than in the total body irradiation and melatonin-treated group.

Dose-dependent immunohistochemical changes in rat cornea and retina after oral methylphenidate administration.

Methylphenidate hydrochloride (MPH), more commonly known as Ritalin, is a piperidine derivative and is the drug most often used to treat attention deficit/hyperactivity disorder, one of the most common behavioural disorders of children and young adults. The aim of this study was to investigate dose-dependent immunohistochemical Dopamine 2 receptor (D2) expression and apoptosis in the rat cornea and cornea. In this study, 27 female pre-pubertal Wistar albino rats, divided into three different dose groups (5, 10 and 20 mg/kg) and their control groups, were used. They were treated orally with methylphenidate dissolved in saline solution for 5 days per week during 3 months. At the end of the third month, after perfusion fixation, eye tissue was removed. Paraffin sections were collected for immunohistochemical and terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labelling assay studies. In our study, we observed that the cornea D2 receptor reactivity showed a dose-related increase after MPH treatment, especially in basal cells of the epithelium and a dose-dependent decrease in the retinal ganglion cell which was statistically meaningful. Analysis of the cornea thickness results showed no meaningful difference between groups. Apoptotic cell number showed a meaningful increase in the high dose treated group compared to the other groups of the study. The data suggest that Ritalin has degenerative effect on the important functional part of the eye, such as cornea and retina and its activating dopaminergic mechanism via similar neuronal paths, functionally and structurally, to induce morphological changes. As a result, we believe that this morphological changes negatively effecting functional organization of the affected cornea and retina.

Dose-dependent immunohistochemical and ultrastructural changes after oral methylphenidate administration in rat heart tissue.

Methylphenidate, more commonly known as Ritalin, is a piperidine derivative and is the drug most often used to treat attention deficit/hyperactivity disorder, one of the most common behavioural disorders of children and young adults. Our aims were to investigate dose-dependent immunohistochemical D2 expression and ultrastructural changes of the rat heart tissue, and to demonstrate possible toxicity of the long-term and high dose use of the methylphenidate. In this study, 27 female pre-pubertal Wistar albino rats, divided into three different dose groups (5, 10 and 20 mg/kg) and their control groups, were used. They were treated orally with methylphenidate dissolved in saline solution for 5 days/week during 3 months. At the end of the third month, after perfusion fixation, left ventricle of cardiac tissue was removed. Paraffin, semi-thin and thin sections were collected and immunohistochemical, terminal deoxynucleotidyl transferase-mediated Dig-dUTP nick end labelling assay and ultrastructural studies were performed. In conclusion, we believe that Ritalin is dose-related affecting dopaminergic system to increase heart rhythm and contraction. Thus, this drug may cause degenerative ultrastructural changes in mitochondrial path.

Melatonin prevents inflammation and oxidative stress caused by abdominopelvic and total body irradiation of rat small intestine

We investigated the day-night differences in intestinal oxidative-injury and the inflammatory response following total body (TB) or abdominopelvic (AP) irradiation, and the influence of melatonin administration on tissue injury induced by radiation. Rats (male Wistar, weighing 220-280 g) in the irradiated groups were exposed to a dose of 8 Gy to the TB or AP region in the morning (resting period - 1 h after light onset) or evening (activity span - 13 h after light onset). Vehicle or melatonin was administered immediately before, immediately after and 24 h after irradiation (10, 2.0 and 10 mg/kg, ip, respectively) to the irradiated rats. AP (P < 0.05) and TB (P < 0.05) irradiation applied in the morning caused a significant increase in thiobarbituric acid reactive substance (TBARS) levels. Melatonin treatment in the morning (P < 0.05) or evening (P < 0.05) decreased TBARS levels after TB irradiation. After AP irradiation, melatonin treatment only in the morning caused a significant decrease in TBARS levels (P < 0.05). Although we have confirmed the development of inflammation after radiotherapy by histological findings, neither AP nor TB irradiation caused any marked changes in myeloperoxidase activity in the morning or evening. Our results indicate that oxidative damage is more prominent in rats receiving TB and AP irradiation in the morning and melatonin appears to have beneficial effects on oxidative damage irrespective of the time of administration. Increased neutrophil accumulation indicates that melatonin administration exerts a protective effect on AP irradiation-induced tissue oxidative injury, especially in the morning.

Melatonin prevents inflammation and oxidative stress caused by abdominopelvic and total body irradiation of rat small intestine.

We investigated the day-night differences in intestinal oxidative-injury and the inflammatory response following total body (TB) or abdominopelvic (AP) irradiation, and the influence of melatonin administration on tissue injury induced by radiation. Rats (male Wistar, weighing 220-280 g) in the irradiated groups were exposed to a dose of 8 Gy to the TB or AP region in the morning (resting period - 1 h after light onset) or evening (activity span - 13 h after light onset). Vehicle or melatonin was administered immediately before, immediately after and 24 h after irradiation (10, 2.0 and 10 mg/kg, ip, respectively) to the irradiated rats. AP (P < 0.05) and TB (P < 0.05) irradiation applied in the morning caused a significant increase in thiobarbituric acid reactive substance (TBARS) levels. Melatonin treatment in the morning (P < 0.05) or evening (P < 0.05) decreased TBARS levels after TB irradiation. After AP irradiation, melatonin treatment only in the morning caused a significant decrease in TBARS levels (P < 0.05). Although we have confirmed the development of inflammation after radiotherapy by histological findings, neither AP nor TB irradiation caused any marked changes in myeloperoxidase activity in the morning or evening. Our results indicate that oxidative damage is more prominent in rats receiving TB and AP irradiation in the morning and melatonin appears to have beneficial effects on oxidative damage irrespective of the time of administration. Increased neutrophil accumulation indicates that melatonin administration exerts a protective effect on AP irradiation-induced tissue oxidative injury, especially in the morning.

The effect of tryptophan administration on ileum contractility and oxidant status in mice.

L-tryptophan (TRP) is the precursor amino acid for the synthesis of serotonin (5-HT). 5-HT is effective both on the food intake and gastrointestinal system contractility. The aim of this study was to search the effects of systemic TRP treatment on 5-HT levels of ileum and searching the effect of ileal contractility and oxidant status. Swiss-albino mice were divided into two groups: 1. Control, 2. TRP-treated (100 mg/kg/24 h, i.p., for 7 days). Body weights were recorded at the beginning and at the end of experiments. Acetylcholine-induced contractile responses in the isolated ileum were recorded on polygraph. Ileal tissue malondialdehyde and glutathione levels determined by spectrophotometric and ileal tissue 5-HT levels were measured by immunohistochemical methods. TRP treatment decreased body weight and increased ileal contractile response. In the TRP-treated group, ileum malondialdehyde levels increased and glutathione levels decreased. Immunohistochemical detection showed that ileal 5-HT levels were increased by TRP treatment. There is a relationship between increased oxidative stress and increased contractility in the ileal tissue of the TRP-treated animals. These effects may be related to increased ileal 5-HT synthesis.

Apoptosis and proliferation in gastric epithelium due to Helicobacter pylori: an immunohistochemical and ultrastructural study.

UNLABELLED: The effect of H. pylori infection on gastric epithelial cell apoptosis and proliferation is contradictory. Using immunohistochemistry and electron microscopy, this study sought to demonstrate gastric epithelial changes (ie, apoptosis and proliferation) due to chronic H. pylori infection. METHODS: Eighteen female 6- to 8-week old Swiss Albino mice were inoculated intragastrically with 3 doses of 10(9) CFU/mL H. pylori prepared in a Brucella Broth in 5 days. Nine others served as a control group. At the end of 28 weeks, tissue specimens from the gastric antrum were excised and examined immunohistochemically (epithelial growth factor for regeneration and Caspase-3 for apoptosis) and electron microscopically. Immunohistochemical assessment was performed using the indirect peroxidase-antiperoxidase method. RESULTS: In the H. pylori-infected group, EGF staining in gastric epithelium was found to be decreased significantly compared to that in control group (P < 0.001). Caspase-3 reactivity was commonly observed in surface epithelial cells and glandular epithelial cells in H. pylori-infected group and totally it was statistically significant compared to Caspase-3 staining in control group (P < 0.001). Electron micrograph images demonstrated numerous apoptotic cells with condensed chromatin. CONCLUSION: Chronic H. pylori infection of 28 weeks' duration increases apoptosis in gastric epithelium; however, increased apoptosis does not induce proliferation.

The apoptotic effects of mitomycin C on human endometrial cell cultures and reversal of its effects by beta-carotene and folic acid.

Apoptosis is a complex process involving a variety of mechanisms and it has been shown to be a response of cells to various chemical agents including chemotherapeutic ones. We aimed to induce DNA breaks and apoptosis in cultured endometrial stromal cells by mitomycin C (MMC), a chemotherapeutic agent, and also we aimed to observe the effects of beta-carotene and folic acid on MMC-induced apoptosis. Cultured endometrial stromal cells were exposed to MMC for 48 and 72 hours and in order to reverse MMC effects, we added beta-carotene and folic acid to the cultures. DNA fragmentation was observed in all cells. Apoptotic cell ratios and caspase-3 activity were observed to be dependent on exposure time. Ultrastructural examinations revealed positive effects of beta-carotene and folic acid, however they were not sufficient enough to prevent apoptosis in all cells. Beta-carotene profoundy reduced caspase-3 activity whereas folic acid did not seem to have a similar effect. As apoptosis involves several mechanisms, in a cell in which all these mechanisms are triggered, we think that antioxidants and DNA repair agents alone are not enough to reverse all of them.

Immunohistochemical localization of beta1 and beta4 integrins in mouse endometrium during implantation and early pregnancy.

Implantation presents the remarkable synchronisation between the development of embryo and differentiation of endometrium. Cell-cell adhesion is an important phenomenon taking place during blastocyst implantation in uterine membrane. We think that the investigation of existence and the level of integrins in women can be a guide for treatment of infertility. Our purpose in this study was to show expression beta1 and beta4 integrins on gestational days 4, 6, 12 by immunohistochemical methods and to investigate whether beta4 integrin is a useful marker for receptivity. beta1 and beta4 integrin were exhibited on surface epithelium on gestational day 4. On the other hand, strong beta4 immunoreactivity was detected on surface epithelium and glandular cells on gestational day 12 but no beta1 reactivity was present in the surface epithelium and glandular cells on day 12. In conclusion, both beta1 and beta4 integrins may have a role in implantation process because positive immunoreactivity was seen on apical membrane of surface epithelium on day 4 when implantation occurred. The localization to apical pole of surface epithelium suggest a role for beta1, beta4 integrins in initial embryo and endometrium interaction. It does not seem that beta1 integrin has a role supporting pregnancy since expression of beta4 on surface epithelium and glandular epithelium disappeared on day 12. beta4 integrin expression increasing on day 12 of pregnancy leads us to think a possible functional role supporting pregnancy.

Immunohistochemical localization of epidermal growth factor receptor (EGF-r) and transforming growth factor alpha (TGF-alpha) in developing human ovarian follicles.

In this study, we aimed to detect the distribution of epidermal growth factor receptor (EGF-r) and transforming growth factor alpha in ovarian follicles at different stages. Indirect immunohistochemical methods and EGF-r polyclonal and TGF-alpha monoclonal antibodies were used; tissues were examined with light microscope. While dense collection of both growth factors were observed in primordial follicles, there was a strong reaction especially for EGF-r in follicles. Strong reactivity for EGF-r and moderate reactivity for TGF-alpha were observed in the nearby connective tissue. In examinations of primary follicles for EGF-r presence only, dye uptake was moderate in oocytes and dense in apical and basal cytoplasm of follicle cells. Reactivity was moderate in the nearby connective tissue. In the corpus luteum, there was weak reaction for both growth factors. But in stromal cells, reaction was strong. In degenerated follicle cells and in stroma of atretic follicles, reaction was positive for both growth factors; but EGF-r reactivity was more obvious. While strong staining was observed for both factors especially in granulosa cells surrounding the oocyte in Graafian follicle, moderate TGF-alpha reactivity was determined in oocyte cytoplasm. In conclusion, it is possible that EGF-r and TGF-alpha have ortocrine and paracrine effects on development and regression of human ovarian follicles.

Tryptophan administration increase contractility and change the ultrastructure of mice duodenum.

Serotonin (5-HT) is a metabolite of tryptophan (TRP). 5-HT has been shown to induce contractions in rat duodenum and ileum. We planned to investigate the in vivo effects of TRP administration on duodenal contractility and ultrastructure together. Two equal groups of adult male Swiss-albino mice were used in the experiments. Controls (CONT) and TRP treated (100 mg/kg/24 hr in 0.2 ml. saline solution ip, 7 days). Body weights were recorded at the beginning and at the end of experiments. Duodenum tissues contractility responses to different concentration of KCl and acethycholine (ACh) were recorded on polygraph. The ultrastructural changes in duodenum observed by transmission electron microscopic (TEM) method and 5-HT levels determined by immunohistochemical method. Body weights decreased and duodenal contractile response of ACh increased significantly by TRP treatment. The duodenal ultrastructural changes in TRP group illustrated partially loss of apical surface and fusion in microvilli. Immunohistochemical detection showed that 5-HT increased by TRP treatment. There is a relation between duodenal contractility increased by TRP treatment and changes in the duodenal tissue 5-HT level and ultrastructure.

An investigation on skin wound healing in mice with a taurine-chitosan gel formulation.

The process of wound healing begins immediately following surface lesions or just after exposure to radiation, chemical agents or extreme temperatures. Taurine (2-aminoethane sulfonic acid), an amino acid containing sulfur, is found in almost all tissues in mammals, playing various important physio-logical roles in each organ. Taurine exhibits an antioxidant effect and is also known to have effects on cell proliferation, inflammation and collagenogenesis. Many antioxidants have been used to eliminate the negative effects of oxygen free radicals on wound healing. The objective of the present study was to examine the wound healing effect in mice of taurine-chitosan gel, which releases taurine slowly over a long time period. Fifty mM of taurine in 1.5% chitosan polymer (TAU-GEL) and 1.5% chitosan polymer (CHI-GEL) were applied to full thickness skin wounds of mice once a day for seven days. After seven days of treatment, lipid peroxide formation-malondialdehyde (MDA) and hydroxyproline (HPX) levels and the tensile strength of wound tissues were measured. All results were compared with those of the untreated control group (CONT). The structural alterations in the skin layers were also histologically investigated. It was found that locally administered TAU-GEL form significantly increased wound tensile strength by decreasing the MDA and increasing HPX levels. These results were supported by histological findings. All observations suggest that taurine gel may be effective in wound healing.

The role of taurine added to pulmonary reperfusion solutions in isolated guinea pig lungs.

An experimental comparative study on isolated guinea pig-lungs has been undertaken to determine the probable beneficial effects of adding taurine to pulmonary reperfusion solutions in lung ischemia-reperfusion. 20 guinea pigs were used. The isolated lungs (n = 10 in each group) previously being perfused by oxygenated Krebs-Henseleit solution were put in normothermic ischemic conditions. After 3 hours of normothermic ischemia the lungs were reperfused (with Krebs-Henseleit solution in the control group, Krebs-Henseleit solution plus taurine 10(-2) M in the experiment group) for 20 minutes. Pulmonary artery pressures, tissue malondialdehyde (MDA) and glutathione (GSH) levels were measured before and after the ischemic period and also at the end of reperfusion. Malondialdehyde and glutathione levels of the pefusate were measured before ischemic period and at the end of reperfusion. An electron microscopic analysis was performed on the lung tissues before and after the ischemic period and also at the end of reperfusion. Decreased pulmonary artery pressure, tissue perfusate MDA levels and increased perfusate GSH levels were observed in taurine added group. Electron microscopic evaluation supported our findings indicating preservation of lamellar bodies of type II pneumocytes. It is concluded that taurine may play an important role in protecting tissue against ischemia-reperfusion injury by functioning as an antioxidant.

Immunohistochemical localization of epidermal growth factor receptor (EGF-R) in normal and diseased newborn lung tissues.

The distribution of EGF receptors (EGF-R) was examined in normal, hyaline membrane diseased and pneumonic newborn lung tissues by immunohistochemical methods under the light microscope. The PAP technique with polyclonal antibodies was performed to demonstrate the EGF receptor localisation in these tissues. Strong EGF-R reactivity was observed on bronchiolar epithelium and type I and type II alveolar cells in normal newborn lung tissues; whereas, poor reactivity was observed in alveolar macrophages. On the other hand, strong immunoreactivity was detected in type I alveolar cells and alveolar macrophages in hyaline membrane disease, but no reactivity was present in type II alveolar cells. The strongest immunoreactivity was observed in alveolar macrophages of newborn pneumonic lung tissues. In conclusion, the most meaningful form of reactivity was observed in normal newborn lung tissues of airway track and respiration area. This result is related with the maturation of the lungs after birth.