Meral Baka

Protective Effect of Dietary Supplementation with L-Arginine and L-Carnitine on Hypoxia/Reoxygenation-Induced Necrotizing Enterocolitis in Young Mice

Oxygen-derived free radicals are important components of gastrointestinal injury in necrotizing enterocolitis (NEC). In the present investigation, we examined the protective actions of L-arginine, a nitric oxide synthase substrate, and L-carnitine against hypoxia-reoxygenation (H/R) induced NEC in young mice. Young mice were divided into four groups: group 1 mice were subjected to H/R only; group 2 H/R mice were supplemented with L-arginine in the drinking water (2 g/l) for 7 days; group 3 H/R mice were given L-carnitine solution in water (50 mg/kg p.o.) for 7 days, and group 4 mice served as controls.Hypoxia was induced by placing the mice in a 100% CO2 chamber for 5 min. After hypoxia, the mice were reoxygenated for 10 min with 100% oxygen. We examined the intestinal lesions by light microscopy and measured the intestinal generation of thiobarbituric acid reactive substances (TBARS) and the activities of superoxide dismutase and catalase in the H/R-induced model of NEC. In both L-arginine and L-carnitine groups, the NEC-induced intestinal tissue damage was greatly attenuated, with necrosis limited partially to the mucosa. The tissue TBARS level was significantly higher in group 1 than in any of the other groups (p < 0.001). However, those treated with L-arginine and L-carnitine had TBARS levels similar to those in the control animals. An increased tissue concentration of nitrate, a stable metabolite of nitric oxide, was found in the L-arginine-supplemented group as compared with the control group (p < 0.05). Both superoxide dismutase and catalase activities in the intestine were similar in H/R groups when compared with the intestine of control animals. The present study suggests that oxygen-derived free radicals are involved in the pathogenesis of H/R-induced NEC. This study also shows that dietary supplementation with L-arginine and L-carnitine ameliorates the histological evidence of H/R-induced intestinal injury and significantly decreases lipid peroxidation in H/R-induced bowel injury. Based on these findings, the beneficial effects of L-arginine and L-carnitine in this model may be mediated via mechanisms preventing free radical damage.

Insulin-Like Growth Factor Attenuates Apoptosis and Mucosal Damage in Hypoxia/Reoxygenation-Induced Intestinal Injury

Objective: Necrotizing enterocolitis (NEC) is a potentially lethal disease among premature infants. The aim of the present study was to investigate whether hypoxia-reoxygenation (H/R)-induced intestinal injury was due to increased apoptosis of the intestinal mucosa in young mice and whether pre-treatment of the animals with recombinant human insulin-like growth factor-I (IGF-I), a known anti-apoptotic factor, could protect the intestinal cells from H/R-induced apoptosis or intestinal injury. Study Design: Young mice were divided into three groups: group 1 mice (H/R) were hypoxia-reoxygenation; group 2 mice (H/R + IGF-I) were treated with recombinant human IGF-I by intraperitoneal injection (1 µg/g b.w. once daily) for 7 days, and group 3 mice served as control. Hypoxia was induced by placing young mice in a Plexiglas chamber consisting of 10% oxygen for 60 min. After hypoxia, the young mice were reoxygenated for 10 min with 100% oxygen. Intestinal generation of substances reactive to thiobarbituric acid (TBARS) and active caspase-3 were measured in H/R-induced intestinal injury. Results: Increased numbers of apoptotic cells (apoptotic index) across the villi in young mice subjected to H/R were observed with the TUNEL reaction whereas few apoptotic cells existed in the control animals. In addition, H/R-induced intestinal damage in the H/R + IGF-I group was greatly attenuated, with necrosis limited partially to the mucosa. Tissue-active caspase-3 levels in the H/R group were found to be significantly higher when compared with that of the H/R + IGF-I group of mice and control. However, TBARS concentrations in the intestine were similar in H/R groups when compared to the intestine of control animals. Conclusion: The present study suggests that both necrosis and apoptosis, via mechanisms occurring due to oxygen-derived free radicals and activation of caspase-3, play a role in the pathogenesis of H/R-induced bowel injury. We also show that IGF-I protect intestinal mucosa from necrosis and apoptosis from intestinal H/R injury.

Pinealectomy exaggerates and melatonin treatment suppresses neuroma formation of transected sciatic nerve in rats: gross morphological, histological and stereological analysis

Abstract:  At present, an intensive effort for prevention of neuroma formation following peripheral nerve section continues. It has been recently suggested that surgical pinealectomy (Px) induces elevation of the collagen content in the granulation tissue of a wound, while melatonin application after Px suppresses elevation of the collagen accumulation in the tissue. The aim of the present study was to assess whether melatonin had the ability to suppress collagen production and neuroma formation following peripheral nerve transection. A total of 40 male rats (four groups of 10) were left intact (intact controls) or sham operated (sham group), were Px, or were Px and given melatonin (Px + melatonin group). All animals underwent a surgical intervention consisting of right sciatic nerve neurectomy. After 4 wk, the animals were killed following intracardiac perfusion. Gross morphology of neuroma formation in the proximal nerve segment was examined and proximal neuroma evaluated. Macroscopic and microscopic findings revealed that Px caused a proliferation of connective tissue and large neuroma formation at the proximal ends of transected nerves. Stereological analysis showed that there was a statistically significant reduction in connective tissue content of the same region in Px animals treated with melatonin (P < 0.005). The results achieved in a rodent model of sciatic nerve neuroma formation showed that there was a positive correlation between macroscopic and microscopic observations, and that melatonin enhanced axonal regeneration presumably due to its inhibitory effect on neuroma formation.

Effect of dietary n-3 fatty acids on hypoxia-induced necrotizing enterocolitis in young mice. n-3 fatty acids alter platelet-activating factor and leukotriene B4 production in the intestine.

Necrotizing entercolitis (NEC) is an important neonatal disease with a high mortality rate. Inflammatory mediators, such as mainly platelet-activating factor (PAF), leukotrienes (LT) and tumor necrosis factor play an important role in the genesis of NEC. Diets in Ω–3 (n–3) fatty acids appear to have an antiinflammatory effect, which is thought to be due to decreased active prostaglandins and leukotrienes production after incorporation of these fatty acids into cell membrane phospholipids. We investigated the protective effect of fish oil (source of n–3 fatty acids) on hypoxia-induced model of NEC. Young mice were divided into three groups; group 1 mice were fed standard chow (n–3 fatty acids-free), group 2 was fed a chow supplemented by 10% fish oil for 4 weeks. Group 3 mice served as control. We examined the intestinal lesions by light microscopy and measured intestinal tissue PAF and LB4 levels in hypoxia-induced model of NEC. Significantly increased intestinal PAF and LTB4 levels were found in group 1 mice when compared to group 2 and group 3 mice. The histopathology of the intestinal lesions in group 1 animals was characteristic of ischemic injury. In the n–3 fatty acids-supplemented animals these lesions were milder. The present study shows that endogenously released PAF and LTB4 play an important role in mediating hypoxia-induced intestinal necrosis. The present study also suggests that dietary supplementation with n–3 fatty acids suppress intestinal PAF and LTB4 generation in hypoxia-induced bowel necrosis. The intestinal protective effect of n–3 fatty acids in an experimental model of NEC may open new insight into the treatment and preventation of NEC in neonates.

Effects of Preservatives in Nasal Formulations on the Mucosal Integrity: An Electron Microscopic Study

The preservatives benzalkonium chloride (BZC) and potassium sorbate (PS) are widely used in the formulation of nasal drops and cosmetics. Recently, a number of side effects that resulted from mucosal irritation caused by BZC and PS have been reported. Therefore, this study was performed to investigate the possible clinical and histological alterations induced by in vivo administration of these preservatives to the nasal mucosa of rats. 0.01% BZC and 0.12% PS were administered to the nostrils of male rats for 1 or 4 weeks. Clinical symptoms were recorded during the treatment, and light and electron microscopic examinations were carried out on samples taken from one third central and lower regions of the noses at the end of the treatment periods. Symptomatic changes such as sneezing and nasal rubbing were observed in almost all groups, starting from the 6th day of administration. Light and electron microscopy showed histological changes and nasal lesions induced by the preservatives. The symptomatic and histological changes were more pronounced with prolonged duration of administration. Therefore, it has been concluded that in vivo administration of the preservatives BZC and PS may be irritant to the respiratory epithelium of rats.

The Role of Dietary Supplementation with L-Glutamine in Inflammatory Mediator Release and Intestinal Injury in Hypoxia/Reoxygenation-Induced Experimental Necrotizing Enterocolitis

Background/Aims: Necrotizing enterocolitis (NEC) is a multifactorial syndrome in the neonate. Enteral feeding practices are an important component of gastrointestinal injury in neonatal NEC. In the present study, we examined the protective effect of oral supplementation with L-glutamine, an important specific fuel for the enterocytes, against hypoxia-reoxygenation (H/R)-induced NEC in young mice. Methods: Young mice were divided into four groups: group 1 mice (untreated) underwent H/R; group 2 mice were supplemented with L-glutamine in drinking water (0.5 g/dl) for 3 days, and group 3 mice were supplemented with L-glutamine (3 g/dl) for 10 days. Group 4 mice served as control. Hypoxia was induced by placing the young mice in a 100% CO2 chamber for 5 min. After hypoxia, they were reoxygenated for 10 min with 100% oxygen. We examined the intestinal lesions with light microscopy and measured intestinal generation of PAF and TNF-α in the H/R-induced model of NEC. Results: In group 3 mice, NEC-induced intestinal tissue damage was greatly attenuated with necrosis limited partially to the mucosa. Both intestinal tissue PAF and TNF-α concentrations were significantly higher in the untreated group than in controls (p < 0.001). Group 3 mice (3 g/dl supplemented) showed a significant decrease in intestinal TNF-α concentration compared with young group 1 and group 2 mice (p < 0.05 and p < 0.05, respectively). On the other hand, no significant difference was observed in the intestinal generation of PAF between H/R groups (p > 0.05). Conclusion: The present study suggests that H/R plays an important role in the pathogenesis of NEC and supports the hypothesis that especially PAF and TNF-α are involved in the pathophysiological mechanism of H/R-induced NEC. This study also demonstrates that dietary supplementation with L-glutamine reduces the histologic evidence of H/R-induced intestinal injury. Based on these findings, beneficial effects of L-glutamine in this model of NEC are mediated via mechanisms inhibiting intestinal cytokine release.

Epidural lornoxicam administration – innocent

We aimed to determine the analgesic efficacy and clinical or histopathological neurotoxicity of epidural single-dose lornoxicam. Caudal epidural catheters were inserted into 28 rabbits, divided into four groups, on day 1. Pain latency and degree of motor and sensory loss for each animal for different concentrations of lornoxicam were determined on day 2. All animals were sacrificed on day 3 and laminectomy was performed. Five-mum thick sections of spinal cord, obtained from two segments caudal and two segments rostral from tip of the catheter, were fixed and were stained and evaluated by light microscopy. Lornoxicam produced dose-dependent analgesia (increase in pain latency), brief, mild and reversible motor and sensory block, and histopathological signs of neurotoxicity. Clinical application of epidural lornoxicam should proceed with caution.

Neuroprotective effects of melatonin upon the offspring cerebellar cortex in the rat model of BCNU-induced cortical dysplasia

Cortical dysplasia is a malformation characterized by defects in proliferation, migration and maturation. This study was designed to evaluate the alterations in offspring rat cerebellum induced by maternal exposure to carmustine-[1,3-bis (2-chloroethyl)-1-nitrosoure] (BCNU) and to investigate the effects of exogenous melatonin upon cerebellar BCNU-induced cortical dysplasia, using histological and biochemical analyses. Pregnant Wistar rats were assigned to five groups: intact-control, saline-control, melatonin-treated, BCNU-exposed and BCNU-exposed plus melatonin. Rats were exposed to BCNU on embryonic day 15 and melatonin was given until delivery. Immuno/histochemistry and electron microscopy were carried out on the offspring cerebellum, and levels of malondialdehyde and superoxide dismutase were determined. Histopathologically, typical findings were observed in the cerebella from the control groups, but the findings consistent with early embryonic development were noted in BCNU-exposed cortical dysplasia group. There was a marked increase in the number of TUNEL positive cells and nestin positive cells in BCNU-exposed group, but a decreased immunoreactivity to glial fibrillary acidic protein, synaptophysin and transforming growth factor beta1 was observed, indicating a delayed maturation, and melatonin significantly reversed these changes. Malondialdehyde level in BCNU-exposed group was higher than those in control groups and melatonin decreased malondialdehyde levels in BCNU group (P<0.01), while there were no significant differences in the superoxide dismutase levels between these groups. These data suggest that exposure of animals to BCNU during pregnancy leads to delayed maturation of offspring cerebellum and melatonin protects the cerebellum against the effects of BCNU.

Stereological analysis of sciatic nerve in chickens following neonatal pinealectomy: an experimental study

Background Although the injury to the peripheral nervous system is a common clinical problem, understanding of the role of melatonin in nerve degeneration and regeneration is incomplete. Methods The current study investigated the effects of neonatal pinealectomy on the sciatic nerve microarchitecture in the chicken. The chickens were divided into two equal groups: unpinealectomized controls and pinealectomized chickens. At the end of the study, biochemical examination of 10 sciatic nerve samples from both groups was performed and a quantitative stereological evaluation of 10 animals in each group was performed. The results were compared using Mann-Whitney test. Results In this study, the results of axon number and thickness of the myelin sheath of a nerve fiber in newly hatched pinealectomy group were higher than those in control group. Similarly, surgical pinealectomy group had significantly larger axonal cross-sectional area than the control group (p < 0.05). In addition, the average hydroxyproline content of the nerve tissue in neonatal pinealectomy group was higher than those found in control group. Our results suggest that melatonin may play a role on the morphologic features of the peripheral nerve tissue and that melatonin deficiency might be a pathophysiological mechanism in some degenerative diseases of peripheral nerves. The changes demonstrated by quantitative morphometric methods and biochemical analysis has been interpreted as a reflection of the effects of melatonin upon nerve tissue. Conclusion In the light of these results from present animal study, changes in sciatic nerve morphometry may be indicative of neuroprotective feature of melatonin, but this suggestion need to be validated in the human setting.

Reabsorption of ascites and the factors that affect this process in cirrhosis.

Ascites is one of the main features of liver decompensation in cirrhosis, and it is considered to be a dynamic process. In this study, we aimed to (1) measure the reabsorption rate of ascites; (2) evaluate whether these findings were related to features of ascites, hemodynamics, and serum measurements; and (3) examine morphologic changes in the diaphragm of cirrhotic patients. In all, 42 cirrhotic patients with ascites were enrolled in the study to comprise our study group. Using the dextran 70 test, patient ascites volumes and reabsorption rates were measured. Biopsies from the peritoneal side of the diaphragm were also processed for scanning electron microscopy and lymphatic immunohistochemical studies from the cirrhotic patients and control cadavers. The mean ascites reabsorption rate was 4.5 +/- 4.5 (0.18-14.6) mL/min, which correlated significantly with the calculated ascites volume (r = 0.75, P < 0.001). The mean ascites viscosity was 1.07 +/- 0.07 (0.99-1.17) centipoise, which demonstrated a high degree of negative correlation with the ascites reabsorption rate (r = -0.77, P < 0.001). Patients with a history of spontaneous bacterial peritonitis had significantly lesser ascites reabsorption rates than patients without this particular history. The size of lymphatic stomata in scanning electron microscopy depictions was increased, and lymphatic lacunae were dilated in immunohistochemical studies in the cirrhotic patients with ascites. However, these findings were not uniform in every cirrhotic patient with ascites. The volume and viscosity of ascites seem to influence its reabsorption rate. Additionally, previous episodes of spontaneous bacterial peritonitis may be responsible for the decreased ascites reabsorption rates observed in certain patient populations.