Neuroprotective and Antioxidative Effect of Cactus Polysaccharides In Vivo and In Vitro

Cactus polysaccharides (CP), some of the active components in Opuntia dillenii Haw have been reported to display neuroprotective effects in rat brain slices. In the present study, we investigated the neuroprotective properties of CP and their potential mechanisms on brain ischemia-reperfusion injury in rats, and on oxidative stress-induced damage in PC12 cells. Male Sprague–Dawley rats with ischemia following middle cerebral artery occlusion and reperfusion were investigated. CP (200 mg/kg) significantly decreased the neurological deficit score, reduced infarct volume, decreased neuronal loss in cerebral cortex, and remarkably reduced the protein synthesis of inducible nitric oxide synthase which were induced by ischemia and reperfusion. Otherwise, the protective effect of CP was confirmed in in vitro study. CP protected PC12 cells against hydrogen peroxide (H₂O₂) insult. Pretreatment with CP prior to H₂O₂ exposure significantly elevated cell viability, reduced H₂O₂-induced apoptosis, and decreased both intracellular and total accumulation of reactive oxygen species (ROS) production. Furthermore, CP also reversed the upregulation of Bax/Bcl-2 mRNA ratio, the downstream cascade following ROS. These results suggest that CP may be a candidate compound for the treatment of ischemia and oxidative stress-induced neurodegenerative disease.     

Regulation of Glycogenolysis in Transformed Astrocytes In Vitro

Cultured astrocytes, transformed by Herpesvirus, were used as a model system to study several aspects of the control of glycogenolysis. Adrenergic agonists such as norepinephrine and isoproterenol caused an immediate and dose-dependent increase in the intracellular levels of cyclic AMP. Concomitant with the initial phase of cyclic AMP increase, conversion of phosphorylase b to a and glycogenolysis were observed. The elevation of cyclic AMP, phosphorylase conversion, and glycogenolysis were simultaneously blocked by beta-adrenergic blockers, but not by alpha-adrenergic blocking agents. Repeated administration of norepinephrine caused an attenuated response in both cyclic AMP accumulation and glycogenolysis. Glycogen degradation is also partially regulated by glucose availability. In the presence of glucose, norepinephrine-induced glycogenolysis is blocked, despite elevations in cyclic AMP. The direct role of glucose is postulated, since glucose analogs mimic the effects of glucose.     

Regulation of Glycogen Metabolism in Primary and Transformed Astrocytes In Vitro

Glycogen metabolism was studied in primary and Herpesvirus-transformed cultures of neonatal rat brain astrocytes. A small fraction of the glucose consumed was conserved in glycogen in both the primary and the transformed astrocytic cell cultures. After addition of culture medium containing 5.5 mM glucose, glycogen increased to maximal levels within 2.5 h, the approximate time at which half of the medium glucose was consumed, and rapidly declined thereafter in both the primary and transformed astrocytic cultures. Maximum levels of glycogen were apparently related to the cell density of the Herpesvirus-transformed cultures, but primary cultures did not show this behavior. At any given cell density, maximal levels of glycogen were dependent on the concentration of extracellular glucose. Administration of glucose caused a transient activation of glycogen synthase alpha and a rapid inactivation of glycogen phosphorylase alpha.     

Effect of 6-Phosphogluconate on Phosphoglucose Isomerase in Rat Brain In Vitro and In Vivo

The activity of phosphoglucose isomerase, its kinetic properties, and the effect of 6-phosphogluconate on its activity in the forward (glucose 6-phosphate----fructose 6-phosphate) and the reverse (fructose 6-phosphate----glucose 6-phosphate) reactions were determined in adult rat brain in vitro. The activity of phosphoglucose isomerase (in nmol/min/mg of whole brain protein) was 1,865 +/- 20 in the forward reaction and 1,756 +/- 32 in the reverse reaction at pH 7.5. It was 1,992 +/- 28 and 2,620 +/- 46, respectively, at pH 8.5. The apparent Km and Vmax of phosphoglucose isomerase were 0.593 +/- 0.031 mM and 2,291 +/- 61 nmol/min/mg of protein, respectively, for glucose 6-phosphate and 0.095 +/- 0.013 mM and 2,035 +/- 98 nmol/min/mg of protein, respectively, for fructose 6-phosphate. The activity of phosphoglucose isomerase was inhibited intensely and competitively by 6-phosphogluconate, with an apparent Ki of 0.048 +/- 0.005 mM for glucose 6-phosphate and 0.042 +/- 0.004 mM for fructose 6-phosphate as the substrate. With glucose 6-phosphate as the substrate, at concentrations from 0.05 to 0.5 mM, the activity of the enzyme was inhibited completely in the presence of 0.5-2.0 mM 6-phosphogluconate. With 0.05-0.2 mM fructose 6-phosphate as the substrate, it was inhibited greater than or equal to 85% at the same concentrations of the inhibitor. No significant changes were observed in the values of Km, Vmax, and Ki for phosphoglucose isomerase in the brain of 6-aminonicotinamide-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuroprotective Effect of Hypothermia in Cortical Cultures Exposed to Oxygen-Glucose Deprivation or Excitatory Amino Acids

Abstract: We examined the effect of moderate hypothermia (30°C) on neuronal injury in murine cortical cell cultures. Lowering the temperature during and after a period of oxygen-glucose deprivation reduced both the release of glutamate to the bathing medium and accompanying neuronal degeneration. Hypothermia immediately after brief exposure to high concentrations of NMDA or glutamate also reduced the resulting neuronal degeneration. This protective effect was not eliminated when MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione were added immediately after washout of the exogenously added excitotoxin, suggesting that it was mediated by actions additional to reduction of endogenous late glutamate release. Hypothermia applied only during exposure to NMDA or glutamate, whether brief or prolonged, did not reduce subsequent cytosolic calcium accumulation or neuronal degeneration, suggesting that the postsynaptic induction of NMDA receptor-mediated excitotoxicity is not sensitive to temperature reduction. However, hypothermia during prolonged S -α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate exposure did reduce neuronal degeneration.     

Cell Death Pathways in Astrocytes with a Modified Model of Oxygen-Glucose Deprivation

Traditional oxygen-glucose deprivation (OGD) models do not produce sufficiently stable and continuous deprivation to induce cell death in the ischemic core. Therefore, we modified the OGD model to mimic the observed damage in the ischemic core following stroke and utilized this new model to study cell death pathways in astrocytes. The PO₂ and pH levels in the astrocyte culture medium were compared between a physical OGD group, a chemical OGD group and a mixed OGD group. The mixed OGD group was able to maintain anaerobic conditions in astrocyte culture medium for 6 h, while the physical and the chemical groups failed to maintain such conditions. Astrocyte viability decreased and LDH release into in the medium increased as a function of exposure to OGD. Compared to the control group, the expression of active caspase-3 in the mixed OGD group increased within 2 h after OGD, but decreased after 2 h of OGD. Additionally, porimin mRNA levels did not significantly increase during the first 2 h of OGD, while bcl-2 mRNA levels decreased at 1 h. However, both porimin and bcl-2 mRNA levels increased after 2 h of OGD; interestingly, they both suddenly decreased at 4 h of OGD. Taken together, these results indicate that apoptosis and oncosis are the two cell death pathways responsible for astrocyte death in the ischemic core. However, the main death pathway varies depending on the OGD period.     

In Vitro Growth of Rat Blastocyst under the Effect of Azathioprine

Rat blastocysts were isolated from the uterus on the 5th day after fertilization and set in culture. The effect of azathioprine (Imuran) on the blastocyst's development and on the early trophoblastic differentiation in vitro was investigated. Azathioprine, added to the medium of the blastocyst culture at various concentrations, dose dependently arrested development and had definite cytotoxic effect. In order to study the mechanism of action, a minimal dose of 5 μg/ml, which allowed the survival of about 60% of the blastocysts, was added to the medium after 48 hr of culturing. Under the effect of the substance the area of the spreading blastocyst cells was significantly restricted.
It was found, by autoradiographic methods, that the azathioprine affects the development by restraining DNA synthesis in the throphoblastic cells. Concomitantly RNA synthesis was inhibited and protein synthesis was reduced. The observations indicate, that the impairment of the in vitro differentiation of the blastocysts can be a result of the intracellular inhibitory action of the substance.     

Conditioning Heat Stress Reduces Excitotoxic and Apoptotic Components of Oxygen-Glucose Deprivation-Induced Neuronal Death In Vitro

Abstract: We investigated the effects of sublethal heat stress in murine cortical cell cultures exposed to combined oxygen and glucose deprivation. Pretreatment with sublethal heat stress mildly attenuated the widespread neuronal death induced a day later by 30–60 min of oxygen-glucose deprivation. Heat stress also blunted the increase in extracellular glutamate concentrations induced by the oxygen-glucose deprivation, as well as the neuronal death and ⁴⁵Ca²⁺ uptake induced by exogenous addition of NMDA, although no reduction was seen in neuronal death caused by exogenous kainate or in NMDA-induced whole-cell currents. However, arguing against the idea that the neuroprotective effect of heat stress against neuronal death was exclusively due to reduction of excitotoxicity was the finding that heat stress also reduced the neuronal apoptosis induced by oxygen-glucose deprivation in the presence of glutamate antagonists. This antiapoptotic effect was specific in that heat stress did not reduce neuronal vulnerability to staurosporine-induced apoptosis. Whereas heat stress transiently suppressed protein synthesis, achieving comparable protein synthesis inhibition with cycloheximide did not reproduce the neuroprotective effects of heat stress. These studies suggest that a conditioning heat stress is able to attenuate both the excitotoxic and the apoptotic components of oxygen-glucose deprivation-induced neuronal death in vitro, by mechanisms independent of protein synthesis reduction.     

In Vitro Differences Between Astrocytes of Control and Wobbler Mice Spinal Cord

The Wobbler mouse, a model of amyotrophic lateral sclerosis (ALS), presents motorneuron degeneration and pronounced astrogliosis in the spinal cord. We have studied factors controlling astrocyte proliferation in cultures derived from Wobbler and control mice spinal cord. Basal rate of [³H]thymidine incorporation was 15 times lower in Wobbler astrocytes. While in control cultured cells interleukin-1 (IL-1) and corticosterone (CORT) significantly increased proliferation, both agents were inactive in Wobbler astrocytes. The lack of response to CORT was not due to the absence of glucocorticoid receptors, because similar receptor amounts were found in Wobbler and control astrocytes. In contrast to IL-1 and CORT, transforming growth factor-₁ (TGF-₁) substantially increased proliferation of Wobbler astrocytes but not of control cells. Differences in response to TGF-₁ were also obtained by measuring glial fibrillary acidic protein (GFAP) immunoreaction intensity, which was substantially higher in Wobbler astrocytes. Thus, abnormal responses to different mitogens characterized Wobbler astrocytes in culture. We suggest that TGF-₁ may play a role in the reactive gliosis and GFAP hyperexpression found in the degenerating spinal cord of this model of ALS.     

Effects of Extracellular Potassium on Glycogen Stores of Astrocytes In Vitro

Astrocyte-enriched and meningeal cell cultures of the rat cerebral cortex were prepared, and their glycogen content was measured after 10-90 min under control (2.5 mM) concentrations of potassium after prefeeding with 20 mM glucose. No net change in glycogen level was noted in either culture over this period. Cell cultures were then exposed to increased concentrations of potassium (5, 10, and 15 mM), and their glycogen content was measured after 10-90 min. Both types of cell culture showed complex and variable changes in glycogen content. In general, increased potassium concentrations caused astrocyte glycogen stores to be reduced at physiological increases of potassium levels (from 2.5 to 5 mM and above), although a period of resynthesis was evident at all potassium concentrations. Meningeal cell glycogen levels were highly variable and only affected by high (10 and 15 mM) levels of potassium. These results are discussed with respect to the theory that changes in the external potassium concentration caused by neuronal activity might act as a signal controlling astrocyte glycogen stores.     

In Vitro and In Situ Absorption of Methionine Sulfoxide in Rat Small Intestine

Absorption of methionine and its sulfoxide was investigated in vitro with everted sacs and in situ with circulated loops of rat small intestine. Transmural transport and tissue accumulation of methionine sulfoxide in the everted sacs were in fair agreement with those of methionine. Apparent kinetic parameters for the difference of transmural transport in the absence and presence of 10^?5 m carbonylcyanide m-chlorophenylhydrazone, i.e. for the energy-dependent active transport, were similar for both methionine and its sulfoxide. Methionine was found at a low level in the serosal fluid of the everted sac on incubation with methionine sulfoxide. It was attributed to the methionine leaked out from the tissue but not to that formed by reduction of methionine sulfoxide during the course of intestinal transport. Similar transport was also observed in situ in circulated intestinal loops for methionine and its sulfoxide. The absorption efficiency of methionine sulfoxide in the small intestine is not the reason for the decreased nutritional availability of the most likely oxidation product of methionine.     

Toxicity Evaluation of Chromium Picolinate Nanoparticles In Vivo and In Vitro in Rat

The toxicity of nanoCrpic is still not understood and needs further investigation. Thus, this study investigated the effect of chromium picolinate nanoparticles (nanoCrpic) on the toxicity in vivo and in vitro in rat. In the in vivo study, 36 rats (Wistar, 8-week-old) were randomly divided into the control group (fed basal diet), the low-dose (300 ppb, μg/kg), and high-dose (1,000 ppb) nanoCrpic groups. The trial was conducted for 2 months; at the final stage of the trial, the rats were sacrificed, liver and kidney were examined, and samples of tissues were taken for histological examination. Hepatocytes isolated from 10-week-old Wistar male rats were used for in vitro study to examine the degree of DNA damage following exposure to 0 and 0.294 mM of H₂O₂ for 30 min. Incubation medium was supplemented with 0 (control), 100, and 300 ppb nanoCrpic. In vivo study indicated that no lesions of liver or kidney were detected in 300 and 1,000 ppb nanoCrpic fed rats. The in vitro study evaluated DNA damage according to the percentage and distance of the fragments migration and revealed that there was insignificant difference between the nanoCrpic and control groups (p?>?0.05). This study indicated that nanoCrpic at 300–1,000 ppb in vivo and at 100–300 ppb in vitro showed no signs of toxicity to rats.     

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.     

Effect of Concentrated Fibroblast-Conditioned Media on In Vitro Maintenance of Rat Primary Hepatocyte

The effects of concentrated fibroblast-conditioned media were tested to determine whether hepatocyte function can be maintained without direct contact between hepatocytes and fibroblasts. Primary rat hepatocytes cultured with a concentrated conditioned media of NIH-3T3 J2 cell line (final concentration of 55 mg/ml) showed significantly improved survival and functions (albumin and urea) compared to those of control groups. They also showed higher expression levels of mRNA, albumin and tyrosine aminotransferase compared to hepatocyte monoculture. The results suggest that culture with concentrated fibroblast-conditioned media could be an easy method for in vitro maintenance of primary hepatocytes. They also could be contribute to understand and analyze co-culture condition of hepatocyte with stroma cells.     

体外合成大鼠血清淀粉样A蛋白

 血清淀粉样A蛋白(Serum Amyloid A-SAA)是一个急性期蛋白,在人和小鼠血浆中长时间维持较高的浓度,即会发展为病理学的淀粉样变。但是在大鼠中从未发现这种情况,而对大鼠SAA的研究人们还一无所知。我们使用体外系统研究了大鼠血清淀粉样A蛋白的转录和转译。在体外系统中合成的大鼠SAAmRNA的分子量约为0.5～0.56kb,用合成的mRNA转译的蛋白质分子量为8.2～10kD。用体外系统转录和转译的结果证明,这个体系重复性好,与体内的结果相似。     

Modelling Neuroinflammation In Vitro: A Tool to Test the Potential Neuroprotective Effect of Anti-Inflammatory Agents

Neuron-microglia co-cultures treated with pro-inflammatory agents are a useful tool to study neuroinflammation in vitro, where to test the potential neuroprotective effect of anti-inflammatory compounds. However, a great diversity of experimental conditions can be found in the literature, making difficult to select the working conditions when considering this approach for the first time. We compared the use of neuron-primary microglia and neuron-BV2 cells (a microglial cell line) co-cultures, using different neuron:microglia ratios, treatments and time post-treatment to induce glial activation and derived neurotoxicity. We show that each model requires different experimental conditions, but that both neuron-BV2 and neuron-primary microglia LPS/IFN-γ-treated co-cultures are good to study the potential neuroprotective effect of anti-inflammatory agents. The contribution of different pro-inflammatory parameters in the neurotoxicity induced by reactive microglial cells was determined. IL-10 pre-treatment completely inhibited LPS/IFN-γ-induced TNF-α and IL-6 release, and COX-2 expression both in BV2 and primary microglial cultures, but not NO production and iNOS expression. However, LPS/IFN-γ induced neurotoxicity was not inhibited in IL-10 pre-treated co-cultures. The inhibition of NO production using the specific iNOS inhibitor 1400 W totally abolished the neurotoxic effect of LPS/IFN-γ, suggesting a major role for NO in the neurotoxic effect of activated microglia. Consequently, among the anti-inflammatory agents, special attention should be paid to compounds that inhibit NO production.     

Beta-Amyloid Toxicity in Embryonic Rat Astrocytes

The senile plaques of Alzheimer’s disease contain a high concentration of beta-amyloid (βA) protein, which may affect the glial population in the septal nucleus, an area of increased risk in AD. βA toxicity was measured in septal glia, via a dose-response experiment, by quantifying the effects of three different doses (0.1, 1, and 10 μM) of βA on cell survival. Astrocytes from embryonic day-16 rats were grown in serum-free media in a single layer culture. Cells were treated on day in vitro (DIV)1 and survival was determined on DIV3 to ascertain which concentration was most toxic. In a separate set of experiments, an attempt was made to protect glial cells from the degenerative effects of βA, with treatments of growth factors and estrogen. βA (10 μM) treatment was administered on DIV1, on DIV2 the cells were treated with estrogen (EST, 10 nM), insulin-like growth factors (IGF1 and IGF2, each 10 ng/ml), basic fibroblast growth factor (bFGF, 5 ng/ml) or nerve growth factor (NGF, 100 ng/ml), and on DIV3 the cells were visualized and quantified by fluorescence microscopy with DAPI (4,6-diamidino-2-phenylindole). In addition to dose-response and glial protection, experiments were also conducted to determine whether toxic effects were due to apoptosis. Our results suggest that the survival of glial populations is significantly affected in all three concentrations (0.1, 1.0, and 10 μM) of βA. Glial protection was evident in the presence of NGF, for it showed the significantly highest survival rate relative to the βA treatment alone. Furthermore, toxic effects of βA appear to be due primarily to apoptosis. Significant reversal of βA-induced apoptosis was seen with bFGF and IGF1.     

In Vitro Effect of Tetanus Toxin on GAB A Release from Rat Hippocampal Slices

Abstract Ca²⁺-dependent K⁺-stimulated γ-aminobutyric acid release from rat hippocampal slices was reduced about 30% by pre-incubation of the slices with 10⁴ mouse LD₅₀/ml tetanus toxin for 3 h at 37°C.