Methyl‐isobutyl amiloride reduces brain Lac/NAA,cell death and microglial activation in a perinatal asphyxia model

Na⁺/H⁺ exchanger (NHE) blockade attenuates the detrimental consequences of ischaemia and reperfusion in myocardium and brain in adult and neonatal animal studies. Our aim was to use magnetic resonance spectroscopy (MRS) biomarkers and immunohistochemistry to investigate the cerebral effects of the NHE inhibitor, methyl isobutyl amiloride (MIA) given after severe perinatal asphyxia in the piglet. Eighteen male piglets (aged < 24 h) underwent transient global cerebral hypoxia‐ischaemia and were randomized to (i) saline placebo; or (ii) 3 mg/kg intravenous MIA administered 10 min post‐insult and 8 hourly thereafter. Serial phosphorus‐31 (³¹P) and proton (¹H) MRS data were acquired before, during and up to 48 h after hypoxia‐ischaemia and metabolite‐ratio time‐series Area under the Curve (AUC) calculated. At 48 h, histological and immunohistochemical assessments quantified regional tissue injury. MIA decreased thalamic lactate/N‐acetylaspartate and lactate/creatine AUCs (both p < 0.05) compared with placebo. Correlating with improved cerebral energy metabolism, transferase mediated biotinylated d‐UTP nick end‐labelling (TUNEL) positive cell density was reduced in the MIA group in cerebral cortex, thalamus and white matter (all p < 0.05) and caspase 3 immunoreactive cells were reduced in pyriform cortex and caudate nucleus (both p < 0.05). Microglial activation was reduced in pyriform and midtemporal cortex (both p < 0.05). Treatment with MIA starting 10 min after hypoxia‐ischaemia was neuroprotective in this perinatal asphyxia model.     

Effects of Metformin and Vanadium on Leptin Secretion from Cultured Rat Adipocytes

Objective: We have reported that glucose utilization regulates leptin expression and secretion from isolated rat adipocytes. In this study, we employed two antidiabetic agents that act to increase glucose uptake by peripheral tissues, metformin and vanadium, as pharmacological tools to examine the effects of altering glucose utilization on leptin secretion in primary cultures of rat adipocytes. Research Methods and Procedures: Isolated adipocytes (100 μL of packed cells per well) were anchored in a defined matrix of basement membrane components (Matrigel) with media containing 5.5 mM glucose and incubated for 96 hours with metformin or vanadium. Leptin secretion, glucose utilization, and lactate production were assessed. Results: Metformin (0.5 and 1.0 mM) increased glucose uptake in the presence of 0.16 nM insulin by 37 ± 10% (p < 0.005) and 62 ± 8% (p < 0.0001) over insulin alone, respectively. Metformin from 0.5 to 5.0 mM increased lactate production by 105 ± 43% (p < 0.025) to 202 ± 52% (p < 0.0025) and at 1.0 and 5.0 mM increased the proportional rate of glucose conversion to lactate by 78 ± 18% (p < 0.005) and 166 ± 41% (p < 0.0025), respectively. At concentrations less than 0.5 mM, metformin did not affect leptin secretion, but at 0.5 mM, the only concentration that significantly increased glucose utilization without increasing glucose conversion to lactate, leptin secretion was modestly stimulated (by 20 ± 9%; p < 0.05). Concentrations from 1.0 to 25 mM inhibited leptin secretion by 25 ± 8% (p < 0.005) to 89 ± 4% (p < 0.0001). Across metformin doses, leptin secretion was inversely related to the percentage of glucose taken up and released as lactate (r = ?0.74; p < 0.0001). Vanadium (5 to 20 μM) increased glucose uptake from 20 ± 7% (p < 0.01) to 34 ± 13% (p < 0.02) and increased lactate production at 5 μM by 17 ± 8% (p < 0.025) and 10 μM by 61 ± 20% (p < 0.02) but did not alter the conversion of glucose to lactate. Vanadium (5 to 50 μM) inhibited leptin secretion by 33 ± 6% (p < 0.0025) to 61 ± 8% (p < 0.0001). Discussion: Both metformin and vanadium increase glucose uptake and inhibit leptin secretion from cultured adipocytes. The inhibition of leptin secretion by metformin is related to an increase in the metabolism of glucose to lactate. The inhibition by vanadium most likely involves direct effects on cellular phosphatases. We hypothesize that the effect of glucose utilization to stimulate leptin production involves the metabolism of glucose to a fate other than anaerobic lactate production, possibly oxidation or lipogenesis.     

Alterations of epithelial layer after ischemic preconditioning of small intestine in rats

Ischemic-reperfusion (IR) injury of the small intestine makes a serious complications associated with various surgical procedures and is related to changes in motility, secretory activity and structural alterations. Preconditioning can reduce range of this damage. The aim of the experimental study was to determine the influence of ischemic preconditioning (IPC) on IR injury on jejunal epithelial layer. Wistar rats (n = 56) were divided in two experimental groups. IR group was subjected to 60 min ischemia of cranial mesenteric artery and followed by reperfusion periods: 1,4,8,24 h (IR1, IR4, IR8, IR24). Group with ischemic preconditioning (IPC+IR) was subjected to two subsequent ischemic attacks (12 min) with 10 min of reperfusion between them, and after 2nd attack ischemia was induced for 60 min followed by relevant reperfusion period. IPC showed the protective impact on the jejunal tissue architecture after 1 h reperfusion, when in IR1 group the highest and significant damage was observed (p < 0.001) in contrast to IPC+IR1 group. Histopathological damage of the intestine in pretreated groups was postponed to 4 h of reperfusion. Protective effect of IPC together with later accumulation of injury signs were confirmed by weaker impact on goblet cell (p < 0.001) and Paneth cell populations (p < 0.05).The increased cells proliferation in preconditioned groups came later, but stronger after 8 h of reperfusion (p < 0.001) and after 24 h of reperfusion still remained at the high activity level (p < 0.001). Our experimental results on the histopathological changes in the jejunum during ischemic preconditioning proved that IPC may have a positive effect on maintaining intestinal barrier function.     

Neuroprotective effects of an alkaloid-free ethyl acetate extract from the root of Sophora flavescens Ait. Against focal cerebral ischemia in rats

Large amounts of brain nitric oxide are produced over several hours after a stroke. This probably causes DNA strand nicks, nitration of cytosolic components of neurons, and ultimately neuronal death. Oxymatrine and matrine are two major alkaloids of the Chinese herb Sophora flavescens Ait. (Leguminosae); they have been demonstrated to inhibit liver injury during warm ischemia and reperfusion and to induce apoptosis, respectively, in vivo and in vitro. However, the neuroprotective efficacy of the EtOAc extract of S. flavescens (ESF) without the alkaloids has not been explored. This study investigated the inhibitory efficacy of ESF, which contain two major flavonoids kurarinone (45.5%) and sophoraflavone G (14.7%), in focal cerebral ischemia. Focal cerebral ischemia was induced using the middle cerebral artery occlusion (MCAO) method. After 1.5 h of MCAO and 24 h of reperfusion, the extent of neurological deficits and the infarct volume were measured in Sprague-Dawley rats. Compared with carnosine (50 mg/kg), as positive control ESF (20 mg/kg) significantly reduced infarct volume and neurological deficits. Treatment of human SH-SY5Y cells with sodium nitroprusside (SNP), a nitric oxide donor, decreased cell viability by causing apoptosis-like cell death. ESF significantly inhibited caspase-3-like enzyme activity and DNA fragmentation. The level of active caspase-3 was maximal 6 h after SNP treatment. However, active caspase-3 and apoptosis were dose-dependently inhibited by ESF treatment. Flow cytometry analysis showed that ESF significantly inhibited cell apoptosis (p<0.05) and reduced the apoptotic index by 79.9% (p<0.01). These results indicate that ESF is neuroprotective in focal cerebral ischemia and the flavonoids in ESF might be responsible for its neuroprotective activity in rats, alone or in part.     

Melatonin renders neuroprotection by protein kinase C mediated aquaporin-4 inhibition in animal model of focal cerebral ischemia

Aim

Aquaporin-4(AQP4) expression in the brain with relation to edema formation following focal cerebral ischemia was investigated. Studies have shown that brain edema is one of the significant factors in worsening stroke outcomes. While many mechanisms may aggravate brain injury, one such potential system may involve AQP4 up regulation in stroke patients that could result in increased edema formation. Post administration of melatonin following ischemic stroke reduces AQP4 mediated brain edema and confers neuroprotection.

Materials and methods

An in-silico approach was undertaken to confirm effective melatonin-AQP4 binding. Rats were treated with 5 mg/kg, i.p. melatonin or placebo at 30 min prior, 60 min post and 120 min post 60 min of middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion. Rats were evaluated for battery of neurological and motor function tests just before sacrifice. Brains were harvested for infarct size estimation, water content measurement, biochemical analysis, apoptosis study and western blot experiments.

Key findings

Melatonin at 60 min post ischemia rendered neuroprotection as evident by reduction in cerebral infarct volume, improvement in motor and neurological deficit and reduction in brain edema. Furthermore, ischemia induced surge in levels of nitrite and malondialdehyde (MDA) were also found to be significantly reduced in ischemic brain regions in treated animals. Melatonin potentiated intrinsic antioxidant status, inhibited acid mediated rise in intracellular calcium levels, decreased apoptotic cell death and also markedly inhibited protein kinase C (PKC) influenced AQP4 expression in the cerebral cortex and dorsal striatum.

Significance

Melatonin confers neuroprotection by protein kinase C mediated AQP4 inhibition in ischemic stroke.     

Delayed Treatment with Carboxy-PTIO Permits a 4-h Therapeutic Window of Opportunity and Prevents Against Ischemia-Induced Energy Depletion Following Permanent Focal Cerebral Ischemia in Mice

We examined whether a nitric oxide scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-l-oxyl-3-oxide (carboxy-PTIO), could offer neuroprotective actions and improve cerebral energy metabolism in a model of stroke. Sixty C57BL/10J mice were given either carboxy-PTIO (0.3–1.2 mg/kg) or vehicle intraperitoneally, 0.5 h after permanent middle cerebral artery occlusion, to evaluate the dose–response effects. An additional 70 animals received carboxy-PTIO (0.6 mg/kg) or vehicle, 2–6 h post-ischemia, for establishing the therapeutic window. Subgroups of animals, treated with carboxy-PTIO (0.6 mg/kg) or vehicle, were used for measuring cerebral bioenergetic metabolites (ATP, ADP, AMP, adenosine). Mice treated with carboxy-PTIO (0.6 mg/kg) had dose-specifically reduced brain infarction, significantly by 27–30% (P < 0.05), even when therapy was delayed up to 4 h after the ischemic insult (P < 0.05). Four hour post-ischemia, ATP depleted in the ischemic hemisphere (P < 0.05). Administration with carboxy-PTIO not only improved the recovery of ATP in the ischemic hemisphere (P < 0.05), but also enhanced adenosine content across the ischemic and non-ischemic hemispheres (P < 0.05). The neuroprotection of carboxy-PTIO may be partly attributed to the beneficial effects of improving cerebral energy metabolism.     

The Protective Effect of Dexanabinol (HU-211) on Nitric Oxide and Cysteine Protease-Mediated Neuronal Death in Focal Cerebral Ischemia

We hypothesized that dexanabinol can prevent neuronal death by protecting neuronal lysosomes from nitric oxide (NO)-mediated toxicity, and in turn, by suppressing the release of cathepsins during cerebral ischemia. Focal cerebral ischemia was induced in two sets of animals by permanent middle cerebral artery occlusion. The first set was used to monitor NO concentration and cathepsin activity, while the second was used for histological examination with hematoxylin and eosin, and TUNEL staining. In post-ischemic brain tissue, NO content and cathepsin B and L activity increased (p < 0.05). Dexanabinol treatment reduced NO concentration and cathepsin activity to the control level (p > 0.05). The number of eosinophilic and apoptotic neurons increased in the post-ischemic cerebral cortex (p < 0.05). However, dexanabinol treatment lowered both of these (p < 0.05). We conclude that dexanabinol might be a useful agent for the treatment of stroke patients.     

Influence of Dietary Copper Proteinate on Performance,Selected Biochemical Parameters,Lipid Peroxidation,Liver, and Egg Copper Content in Laying Hens

This study was performed to determine the effects of copper proteinate on performance, blood chemistry, lipid peroxidation status, and organs as well as copper deposition in the liver and eggs of laying hens. Seventy-two 30-week-old Bovans laying hens were distributed into four groups with three replicates. Animals were fed basal diet containing at least 17% crude protein and 2,800 kcal/kg metabolizable energy supplemented with either 0, 150, 300, or 450 mg/kg copper as copper proteinate. Supplementation of 150 and 300 mg/kg copper increased egg production, whereas 450 mg/kg copper decreased (p < 0.001). Liver copper levels were elevated in 300 and 450 mg/kg copper-supplemented groups (p < 0.001). Egg copper contents increased in all treatment groups (p < 0.01). An increase in glucose (p < 0.001) and decreases in albumin (p < 0.01) and total cholesterol (p < 0.05) levels were determined with 300 and 450 mg/kg copper. Supplementation of 450 mg/kg copper increased alkaline phosphatase and gamma glutamyl transpeptidase activities (p < 0.05), malondialdehyde, and high-density lipoprotein levels (p < 0.01) but decreased alanine aminotransferase and lactate dehydrogenase activities (p < 0.01). No gross and microscopic changes were observed in the liver and kidneys. These results indicated that 150 and 300 mg/kg copper increased egg production without having marked adverse effects, but 450 mg/kg copper altered some blood chemistry variables and reduced egg production in laying hens.     

Ginsenoside Rg1 provides neuroprotection against blood brain barrier disruption and neurological injury in a rat model of cerebral ischemia/reperfusion through downregulation of aquaporin 4 expression

Ginsenoside Rg1 is regarded as one of main bioactive compounds responsible for pharmaceutical actions of ginseng with little toxicity and has been shown to have possibly neuroprotective effects. However, the mechanism of its neuroprotection for acute ischemic stroke is still elusive. The purpose of present study is thus to assess the neuroprotective effects of the ginsenoside Rg1 against blood brain barrier disruption and neurological injury in a rat model of cerebral ischemia/reperfusion, and then to explore the mechanisms for these neuroprotective effects by targeting aquaporin 4. Focal cerebral ischemia was induced by middle cerebral artery occlusion. Neurological examinations were performed by using Longa's 5-point scale. Evans blue dye was used to investigate the effects of ginsenoside Rg1 on blood brain barrier permeability. Immunohistochemical analysis and real-time fluorescence quantitative polymerase chain reaction were used to assess aquaporin 4 expression. As a result, general linear model with repeated measures analysis of variance for neurological scores at 5 repeated measures showed that ginsenoside Rg1-treated group could significantly reduce the changing trend of neurological deficit scores when compared with the middle cerebral artery occlusion model group (p < 0.05). Compared with the middle cerebral artery occlusion model group, ginsenoside Rg1 group has significantly decreased Evans blue content and reduced aquaporin 4 expression at each time point (p < 0.05). In conclusion, ginsenoside Rg1 as a ginsenoside neuroprotective agent could improve neurological injury, attenuate blood brain barrier disruption and downregulate aquaporin 4 expression induced by cerebral ischemia/reperfusion insults in rats.     

Lipid peroxidation reduction and hippocampal and cortical neurons protection against ischemic damage in animal model using Stellaria media

This study was aimed to determine the neuroprotective influence of Stellaria media in terms of restoring normal state of the rat’s hippocampus and cortex after oxidative insult caused by in vitro ischemia and reperfusion. Cell viability and membrane integrity were assessed using MTT and lactate dehydrogenase (LDH) assay, respectively. Ischemic insult was introduced in the rat brain’s hippocampal and cortical slivers by exposing oxygen and glucose deficiency (OGD) for 2 h, followed by 1 h of re-perfusion. Cellular oxidative stress levels were quantified by incorporating 2?,7?-dichlorofluorescein diacetate fluorescent probes. Additionally, the lipid peroxidation was assessed using TBARS assay. Findings revealed significant neuroprotection against OGD-induced mitochondrial impairment at 40 µg/mL of S. media in rat’s hippocampal and cortical slices. The LDH levels were decreased significantly (P < 0.001) during pre-incubation and reoxygenation periods using varied concentrations of S. media extract. Cellular oxidative stress levels results showed significant (P < 0.001) reduction in dichlorofluorescein fluorescence in slices homogenate of hippocampus and cortex using S. media extract. The lipid peroxidation assay results showed decreased (P < 0.01) levels of malondialdehyde in liver tissues of treated rats treated (200 mg/kg body weight) when compared to the ischemic animal. In summary, findings clearly indicated the neuroprotective effects of extract against in vitro ischemia in brain hippocampal and cortex slivers. S. media could undoubtedly be utilized as a healing agent in preventing neuronal cells’ loss during is chemic-reperfusion process.     

Pyrrolidine Dithiocarbamate Activates p38 MAPK and Protects Brain Endothelial Cells From Apoptosis: A Mechanism for the Protective Effect in Stroke?

The antioxidant and inhibitor of nuclear factor κB pyrrolidine dithiocarbamate (PDTC) potently reduces infarct size in various experimental stroke models. In addition, it has been shown to have a favourable safety profile in humans. In this study, we further investigated the mechanistic actions of PDTC on cerebral microvascular endothelial cells as main components of the blood–brain barrier. We propose activation of p38 MAPK by PDTC as an additional protective mechanism. C57/BL6 mice were subjected to transient MCAO for 2 h and treated with PDTC (100 mg/kg) or vehicle i.p. before reperfusion. Infarct size was determined after 24 h. Apoptosis was induced in a cerebral microvascular endothelial cell line and the effect of pretreatment with PDTC and its dependency on p38 MAPK activity was assayed. PDTC administered 2 h after MCAO reduced infarct size by 61% (P < 0.05) and reduces the apoptotic death rate in vivo. In vitro, PDTC reduces the apoptotic death rate of bEnd.3 cells. p38 MAPK was activated by PDTC and its inhibition abrogated the protective effect of PDTC. PDTC reduces infarct size after stroke with a reasonable time window and decreases apoptotic cell death in vivo and in vitro. The attenuation of apoptotic cell death in brain microvascular endothelial cells is dependent on p38 MAPK activity.     

Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative Stress and Neurodegeneration

Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox4 ^−/−) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox4 ^−/− mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy.     

Dietary rapamycin supplementation reverses age‐related vascular dysfunction and oxidative stress,while modulating nutrient‐sensing,cell cycle,and senescence pathways

Inhibition of mammalian target of rapamycin, mTOR, extends lifespan and reduces age‐related disease. It is not known what role mTOR plays in the arterial aging phenotype or if mTOR inhibition by dietary rapamycin ameliorates age‐related arterial dysfunction. To explore this, young (3.8 ± 0.6 months) and old (30.3 ± 0.2 months) male B6D2F1 mice were fed a rapamycin supplemented or control diet for 6–8 weeks. Although there were few other notable changes in animal characteristics after rapamycin treatment, we found that glucose tolerance improved in old mice, but was impaired in young mice, after rapamycin supplementation (both P < 0.05). Aging increased mTOR activation in arteries evidenced by elevated S6K phosphorylation (P < 0.01), and this was reversed after rapamycin treatment in old mice (P < 0.05). Aging was also associated with impaired endothelium‐dependent dilation (EDD) in the carotid artery (P < 0.05). Rapamycin improved EDD in old mice (P < 0.05). Superoxide production and NADPH oxidase expression were higher in arteries from old compared to young mice (P < 0.05), and rapamycin normalized these (P < 0.05) to levels not different from young mice. Scavenging superoxide improved carotid artery EDD in untreated (P < 0.05), but not rapamycin‐treated, old mice. While aging increased large artery stiffness evidenced by increased aortic pulse‐wave velocity (PWV) (P < 0.01), rapamycin treatment reduced aortic PWV (P < 0.05) and collagen content (P < 0.05) in old mice. Aortic adenosine monophosphate‐activated protein kinase (AMPK) phosphorylation and expression of the cell cycle‐related proteins PTEN and p27kip were increased with rapamycin treatment in old mice (all P < 0.05). Lastly, aging resulted in augmentation of the arterial senescence marker, p19 (P < 0.05), and this was ameliorated by rapamycin treatment (P < 0.05). These results demonstrate beneficial effects of rapamycin treatment on arterial function in old mice and suggest these improvements are associated with reduced oxidative stress, AMPK activation and increased expression of proteins involved in the control of the cell cycle.     

Protection against focal cerebral ischemia following exposure to a pulsed electromagnetic field

There is evidence that electromagnetic stimulation may accelerate the healing of tissue damage following ischemia. We undertook this study to investigate the effects of low frequency pulsed electromagnetic field (PEMF) exposure on cerebral injury in a rabbit model of transient focal ischemia (2 h occlusion followed by 4 h of reperfusion). PEMF exposure (280 V, 75 Hz, IGEA Stimulator) was initiated 10 min after the onset of ischemia and continued throughout reperfusion (six exposed, six controls). Magnetic resonance imaging (MRI) and histology were used to measure the degree of ischemic injury. Exposure to pulsed electromagnetic field attenuated cortical ischemic edema on MRI at the most anterior coronal level by 65% (P < 0.001). On histologic examination, PEMF exposure reduced ischemic neuronal damage in this same cortical area by 69% (P < 0.01) and by 43% (P < 0.05) in the striatum. Preliminary data suggest that exposure to a PEMF of short duration may have implications for the treatment of acute stroke. © 1994 Wiley-Liss, Inc.     

Administration of host‐derived probiotics does not affect utilization of soybean meal enriched diets in juvenile turbot (Scophthalmus maximus)

In today's aquaculture, the cost‐intensive and scarce fishmeal is increasingly replaced by plant‐based feedstuff such as soybean meal (SBM). However, SBM contains saponins which can have adverse effects on fish's digestive tract potentially culminating in severe enteritis. In a 60 day feeding trial we studied the use of autochthonous bacteria as probiotics upon SBM supplementation on juvenile turbot. Growth performance, feed conversion, body composition and health status were assessed for five different treatment groups, comprising a fishmeal control (FM ctrl), a SBM control without probiotics (SBM ctrl) and three multi‐species probiotic treatments. For the production of the probiotic treatments a basal diet with a composition identical to the SBM ctrl including 40% SBM of total dry matter likewise was prepared. The basal diet was stepwise top coated with three different probiotic supplementations: (a) three distinct isolates with saponin‐metabolizing ability (SBM + degrad); (b) three distinct isolates inhibitory towards the pathogen, Tenacibaculum maritimum (SBM + anta); and (c) a commercial probiotic application (SBM + com). Individual weight gain was highest in FM ctrl but only SBM + degrad diet showed a significantly lower value (p < 0.05). The feed conversion ratio was lowest in FM ctrl and significantly higher in SBM + degrad (p < 0.01). The protein retention efficiency did only differ significantly between FM ctrl and SBM + degrad (p < 0.05), whereas lipid retention efficiency remained unaffected. Whole body composition and gross energy content were similar in all treatments lacking significant differences. The condition factor was significantly elevated in SBM + degrad compared to FM ctrl (p < 0.05). Hematocrit was highest in FM ctrl and significantly lower in the other treatments (p < 0.01) with SBM + com accounting for the lowest value (p < 0.001). The hepatosomatic index was slightly increased in FM ctrl but no significant difference was detected. Regarding the spleen somatic index SBM + anta treatment revealed the highest and SBM ctrl a significantly lower value (p < 0.05). In conclusion, the growth performance of fish did not benefit from the different probiotic treatments, while body composition and gross energy content remained at an appropriate level. Moreover, the overall health status was on a sufficient level in all treatments which confirms the high dietary tolerability of our putative probiotic isolates by the fish.     

δ-Opioid receptor activation attenuates oxidative injury in the ischemic rat brain

Background  

We have recently shown that δ-opioid receptors (DORs) play an important role in neuroprotection from hypoxic injury via the regulation of extracellular signaling-regulated kinase (ERK) and cytochrome c release. Since ERK and cytochrome c are differentially involved in caspase signaling of oxidative injury that significantly contributes to neuronal damage in ischemia/reperfusion, we considered if DOR activation protects the ischemic brain by attenuating oxidative injury.     

Cornel Iridoid Glycoside Inhibits Inflammation and Apoptosis in Brains of Rats with Focal Cerebral Ischemia

The capacity of cornel iridoid glycoside (CIG) to suppress the manifestations of ischemic stroke was investigated. CIG was administered to rats by the intragastric route once daily for 7 days. Focal cerebral ischemia was induced by 2 h of middle cerebral artery occlusion followed by 24 h of reperfusion. In non-treated rats large infarct areas were observed within 24 h of reperfusion. Examination of the ischemic cerebral cortex revealed microglia and astrocyte activation, increased interleukin-1β (IL-1 β) and tumor necrosis factor-α (TNF-α) concentrations, increased DNA fragmentation in the ischemia penumbra, elevated Bax expression, increased caspase-3 cleavage, and decreased Bcl-2 expression. Pretreatment with CIG decreased the infarct area, DNA fragmentation, IL-1β and TNF-α concentrations, microglia and astrocyte activation, Bax expression, and caspase-3 cleavage while increasing Bcl-2 expression. CIG exerts anti-neuroinflammatory and anti-apoptotic effects which should prove beneficial for prevention or treatment of stroke.     

Protective effect of nicotinamide on neuronal cells under oxygen and glucose deprivation and hypoxia/reoxygenation

Nicotinamide (vitamin B₃) reduces the infarct volume following focal cerebral ischemia in rats; however, its mechanism of action has not been reported. After cerebral ischemia and/or reperfusion, reactive oxygen species (ROS) and reactive nitrogen species may be generated by inflammatory cells through several cellular pathways, which can lead to intracellular calcium influx and cell damage. Therefore, we investigated the mechanisms of action of nicotinamide in neuroprotection under conditions of hypoxia/reoxygenation. Results showed that nicotinamide significantly protected rat primary cortical cells from hypoxia by reducing lactate dehydrogenase release with 1 h of oxygen-glucose deprivation (OGD) stress. ROS production and calcium influx in neuronal cells during OGD were dose-dependently diminished by up to 10 mM nicotinamide (p<0.01). This effect was further examined with OGD/reoxygenation (H/R). Cells were stained with the fluorescent dye 4,6-diamidino-2-phenylindole (DAPI) or antibodies against anti-microtubule-associated protein-2 and cleaved caspase-3. Apoptotic cells were studied using Western blotting of cytochrome c and cleaved caspase-3. Results showed that vitamin B₃ reduced cell injury, caspase-3 cleavage and nuclear condensation (DAPI staining) in neuronal cells under H/R. In addition, nicotinamide diminished c-fos andzif268 immediate-early gene expressions following OGD. Taken together, these results indicate that the neuroprotective effect of nicotinamide might occur through these mechanisms in this in vitro ischemia/reperfusion model.     

Pre‐treatment with the synthetic antioxidant T‐butyl bisphenol protects cerebral tissues from experimental ischemia reperfusion injury

Treatments to inhibit or repair neuronal cell damage sustained during focal ischemia/reperfusion injury in stroke are largely unavailable. We demonstrate that dietary supplementation with the antioxidant di‐tert‐butyl‐bisphenol (BP) before injury decreases infarction and vascular complications in experimental stroke in an animal model. We confirm that BP, a synthetic polyphenol with superior radical‐scavenging activity than vitamin E, crosses the blood–brain barrier and accumulates in rat brain. Supplementation with BP did not affect blood pressure or endogenous vitamin E levels in plasma or cerebral tissue. Pre‐treatment with BP significantly lowered lipid, protein and thiol oxidation and decreased infarct size in animals subjected to middle cerebral artery occlusion (2 h) and reperfusion (24 h) injury. This neuroprotective action was accompanied by down‐regulation of hypoxia inducible factor‐1α and glucose transporter‐1 mRNA levels, maintenance of neuronal tissue ATP concentration and inhibition of pro‐apoptotic factors that together enhanced cerebral tissue viability after injury. That pre‐treatment with BP ameliorates oxidative damage and preserves cerebral tissue during focal ischemic insult indicates that oxidative stress plays at least some causal role in promoting tissue damage in experimental stroke. The data strongly suggest that inhibition of oxidative stress through BP scavenging free radicals in vivo contributes significantly to neuroprotection.