Lactate Dehydrogenase Efflux Research Articles

3-Nitropropionic Acid Neurotoxicity in Hippocampal Slice Cultures: Developmental and Regional Vulnerability and Dependency on Glucose

We investigated whether neurotoxic effects of the mitochondrial toxin 3-nitropropionic acid (3-NP) in hippocampal slice cultures are dependent on glucose levels in the culture medium and whether such effects occur via apoptosis or necrosis. In addition, 3-NP toxicity was investigated at two developmental stages of the cultures, prepared from rat brain at postnatal day 5–7 and grown in Neurobasal medium for 1 or 3 weeks. Cultures were exposed to 3-NP in the presence of high (25 mM), normal (5 mM), or low (3 mM) glucose for 48 h, followed by 48 h incubation in medium without 3-NP. Cellular propidium iodide (PI) uptake and lactate dehydrogenase (LDH) efflux into the medium revealed time- and dose-dependent cell death by 3-NP, with EC50 values of about 60 μM in high or normal glucose. Regional vulnerability, as assessed by PI uptake and MAP2 immunostaining, in 3-week-old cultures was as follows: CA1 > CA3 > fascia dentata. In low glucose much lower concentrations of 3-NP (25 μM) triggered neurotoxicity. One-week-old cultures were less susceptible to 3-NP toxicity than 3-week-old cultures, but the dentate granule cells were relatively more affected in the immature cultures. We found no evidence for apoptotic cell death by 3-NP in 3-week-old cultures, but in 1-week-old cultures the putative apoptotic marker c-JUN/AP1 and nuclear fragmentation (Hoechst) were significantly increased in the dentate granule cells.

Glutathione depletion in nigrostriatal slice cultures: GABA loss, dopamine resistance and protection by the tetrahydrobiopterin precursor sepiapterin

Dopaminergic neurons in culture are preferentially resistant to the toxicity of glutathione (GSH) depletion. This effect may be due to high intrinsic levels of tetrahydrobiopterin (BH 4). Here we studied the effects of manipulating GSH and/or BH 4 levels on selective neurotoxicity in organotypic nigrostriatal slice cultures. Following treatments with l-buthionine sulfoximine (BSO, 10–100 μM, 2 days exposure, 2 days recovery), either alone or in combination with the BH 4 precursor l-sepiapterin (SEP, 20 μM), or the BH 4 synthesis inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP, 5 mM), toxic effects were assessed by HPLC analysis of medium and tissues, cellular propidium iodide (PI) uptake, lactate dehydrogenase (LDH) efflux, as well as stereological counting of tyrosine-hydroxylase (TH) positive cells. Thirty micromolar BSO produced 91% GSH and 81% GABA depletion and general cell death, but no significant effect on medium homovanillic acid (HVA) or tissue dopamine (DA) levels. SEP prevented or delayed GABA depletion, PI uptake and LDH efflux by BSO, whereas DAHP in combination with BSO caused (almost) complete loss of medium HVA, tissue DA and TH positive cells. We suggest that under pathological conditions with reduced GSH, impaired synthesis of BH 4 may accelerate nigral cell loss, whereas increasing intracellular BH 4 may provide protection to both DA and GABA neurons.

Effects of S-adenosyl- l-methionine on lipid peroxidation and glutathione levels in rat brain slices exposed to reoxygenation after oxygen-glucose deprivation

We analyzed the effects of S-adenosyl- l-methionine (AdoMet) on tissue oxidative stress in rat brain slices exposed to reoxygenation after oxygen-glucose deprivation. The thiobarbituric acid reactive substances (TBARS), total and oxidized glutathione, and lactate-dehydrogenase efflux (LDH) from tissue to the incubation medium, were measured. Brain slices were incubated without glucose and with N 2, then glucose was added and O 2 was perfused. After the anoxic-reoxygenation period, increase in TBARS, oxidized glutathione and LDH efflux, and decrease in total glutathione levels, were observed. The incubation with AdoMet before the anoxic period reduced TBARS (31–1000 μmol/l), glutathione production was increased (31–1000 μmol/l), LDH efflux decreased 6.41% with 15 μmol/l and 61.5% with 500 μmol/l). In the ex vivo experiments, we administered 50 mg/kg per day p.o., AdoMet for 3 days, then brain slices were collected and the anoxia-reoxygenation experiment was carried out. AdoMet led to the inhibition of brain lipid peroxidation and increased total glutathione production, after 3 h-reoxygenation. The increase of LDH efflux in non-treated rats was reduced by 77%. We conclude that AdoMet exerts citoprotective effects in an experimental model of brain slices reoxygenation after oxygen-glucose deprivation.

Mechanisms of the apoptotic and necrotic actions of trimethyltin in cerebellar granule cells.

In evaluating mechanisms of trimethyltin (TMT)-initiated neuronal damage, the present study focused on involvement of reactive oxygen species, protein kinase C (PKC), and glutamate receptors. Exposure of cerebellar granule cells to TMT (0.01-0.1 microM) produced primarily apoptosis, but higher concentrations were associated with cellular lactate dehydrogenase efflux and necrosis. TMT increased generation of cellular reactive oxygen species, which was inhibited by either L-NAME (inhibitor of nitric oxide synthase, NOS) or catalase, indicating that both NO and H(2)O(2) are formed on TMT exposure. Since chelerythrine (selective PKC inhibitor) also inhibited oxidative species generation, PKC appears to play a significant role in TMT-induced oxidative stress. The metabotropic glutamate receptor antagonist, MCPG, (but not MK-801) prevented oxidative species generation, indicating significant involvement of metabotropic receptors (but not NMDA receptors) in TMT-induced oxidative stress. NOS involvement in the action of TMT was confirmed through measurement of nitrite, which increased concentration dependently. Nitrite accumulation was blocked by L-NAME, chelerythrine, or MCPG, showing that NO is generated by TMT and that associated changes in NOS are regulated by a PKC-mediated mechanism. Oxidative damage by TMT was demonstrated by detection of elevated malondialdehyde levels. It was concluded that low concentrations of TMT (0.01-0.1 microM) cause apoptotic cell death in which oxidative signaling is an important event. Higher concentrations of TMT initiate necrotic death, which involves both an oxidative and a non-oxidative component. TMT-induced necrosis but not apoptosis in granule cells is mediated by glutamate receptors.

Targeted deletion of A(3) adenosine receptors improves tolerance to ischemia-reperfusion injury in mouse myocardium.

A(3) adenosine receptors (A(3)ARs) have been implicated in regulating mast cell function and in cardioprotection during ischemia-reperfusion injury. The physiological role of A(3)ARs is unclear due to the lack of widely available selective antagonists. Therefore, we examined mice with targeted gene deletion of the A(3)AR together with pharmacological studies to determine the role of A(3)ARs in myocardial ischemia-reperfusion injury. We evaluated the functional response to 15-min global ischemia and 30-min reperfusion in isovolumic Langendorff hearts from A(3)AR(-/-) and wild-type (A(3)AR(+/+)) mice. Loss of contractile function during ischemia was unchanged, but recovery of developed pressure in hearts after reperfusion was improved in A(3)AR(-/-) compared with wild-type hearts (80 +/- 3 vs. 51 +/- 3% at 30 min). Tissue viability assessed by efflux of lactate dehydrogenase was also improved in A(3)AR(-/-) hearts (4.5 +/- 1 vs. 7.5 +/- 1 U/g). The adenosine receptor antagonist BW-A1433 (50 microM) decreased functional recovery following ischemia in A(3)AR(-/-) but not in wild-type hearts. We also examined myocardial infarct size using an intact model with 30-min left anterior descending coronary artery occlusion and 24-h reperfusion. Infarct size was reduced by over 60% in A(3)AR(-/-) hearts. In summary, targeted deletion of the A(3)AR improved functional recovery and tissue viability during reperfusion following ischemia. These data suggest that activation of A(3)ARs contributes to myocardial injury in this setting in the rodent. Since A(3)ARs are thought to be present on resident mast cells in the rodent myocardium, we speculate that A(3)ARs may have proinflammatory actions that mediate the deleterious effects of A(3)AR activation during ischemia-reperfusion injury.

Endothelin-1 protects astrocytes from hypoxic/ischemic injury.

Under pathological conditions such as ischemia (I), subarachnoid hemorrhage, and Alzheimer's disease, astrocytes show a large increase in endothelin (ET) -like immunoreactivity. However, it is not clear whether ET is protective or destructive to these cells during brain injury. Using astrocytes from ET-1-deficient mice, we determined the effect of ET-1 on these cells under normal, hypoxic (H), and hypoxic/ischemic (H/I) conditions. Under normal culture conditions, astrocytes from wild-type and ET-1-deficient mice showed no difference in their morphology and cell proliferation rates. ET-3 and ETA receptor mRNAs were up-regulated whereas ETB receptor mRNA was down-regulated in ET-1-deficient astrocytes, suggesting that ET-1 and ET-3 may complement each other's functions and that the expressions of these endothelins and their receptors are regulated by a complex feedback mechanism. Under H and H/I conditions, ET-1 peptide and mRNA were up-regulated in wild-type astrocytes, and the astrocytes without ET-1 died faster than the wild-type astrocytes, as indicated by greater efflux of lactate dehydrogenase. The present study suggests that astrocytes without ET-1 are more vulnerable to H and H/I injuries and that the up-regulation of astrocytic ET-1 is essential for the survival of astrocytes.

Morphologic degeneration of human microvascular endothelial cells induced by iodinated contrast media

The purpose of this study was to characterize the adverse effects of iohexol and ioxaglate on human microvascular endothelial cells, which may result in phlebitis, pain, and thrombosis. The degree of morphologic degeneration and of lactate dehydrogenase (LDH) efflux into the extracellular medium (as an index of cell viability) were determined in endothelial cell culture exposed for 10, 30, or 60 minutes to ioxaglate or iohexol (ionic and nonionic contrast media, respectively) at iodine concentrations of 100 or 150 mg/mL. Ioxaglate induced concentration- and time-dependent morphologic degeneration, including shrinkage and loss of the cell tip in 20%-80% of endothelial cells; iohexol did not. After 60 minutes of exposure, ioxaglate at the higher concentration (150 mg iodine per milliliter) significantly increased the LDH signal (ie, the percentage of LDH released), to 20%. The present findings demonstrate that ioxaglate but not iohexol causes morphologic degeneration of the microvascular endothelial cells. This direct cytotoxic action of ioxaglate probably causes endothelial cell dysfunction, closely associated with the occurrence of phlebitis, pain, and thrombosis.

Effects of Iron Overload and Lindane Intoxication in Relation to Oxidative Stress, Kupffer Cell Function, and Liver Injury in the Rat

Parameters related to liver oxidative stress, Kupffer cell function, and hepatocellular injury were assessed in control rats and in animals subjected to lindane (40 mg/kg; 24 h) and/or iron (200 mg/kg; 4 h) administration. Independently of lindane treatment, iron overload enhanced the levels of iron in serum and liver. Biliary efflux of glutathione disulfide increased by 140, 160, or 335% by lindane, iron, or their combined administration, respectively, and the hepatic content of protein carbonyls was elevated by 5.84-, 2.95-, and 10-fold. Colloidal carbon uptake by perfused livers was not modified by lindane and/or iron, whereas gadolinium chloride (GdCl3) pretreatment diminished uptake by 60–72%. Carbon-induced liver O2 uptake was not altered by lindane, whereas iron produced a 61% increase and the combined treatment led to a 72% decrease over control values. Pretreatment with GdCl3 abolished these effects in all groups. Lindane-treated rats showed acidophilic hepatocytes in periportal areas and some hepatic cells with nuclear pyknosis, whereas iron overload led to moderate hyperplasia and hypertrophy of Kupffer cells and moderate inflammatory cell infiltration. Combined lindane–iron treatment led to hepatocytes with pyknotic nuclei, significant acidophilia, and extensive lymphatic and neutrophil infiltration in the portal space. Hepatic myeloperoxidase activity increased by 1.1-, 2.1-, or 6.7-fold by lindane, iron, or their combined administration, respectively. Liver sinusoidal lactate dehydrogenase efflux increased by 2.2-fold (basal conditions) and 9.7-fold (carbon infusion) in the lindane-iron treated rats, effects that were diminished by 35 and 78% by GdCl3 pretreatment, respectively. These data support the contention that lindane sensitizes the liver to the damaging effects of iron overload by providing an added enhancement to the oxidative stress status in the tissue, and this may contribute to the alteration of the respiratory activity of Kupffer cells and the development of an inflammatory response.

On the regulation of ischaemia-induced glutamate efflux from rat cortex by GABA; in vitro studies with GABA, clomethiazole and pentobarbitone.

Prisms of adult rat cortex were maintained in vitro in either aerobic conditions (control) or conditions simulating an acute ischaemic challenge (hypoxia with no added glucose). Endogenous glutamate efflux increased with time in ischaemic conditions, being 2.7 fold higher than control efflux at 45 min. Returning prisms to control solution after 20 min of simulated ischaemia resulted in glutamate efflux returning to near-control values. Endogenous GABA efflux in ischaemic conditions also increased, being 4.5 fold higher than control efflux at 45 min. Ischaemia-induced glutamate efflux was not accompanied by increased lactate dehydrogenase efflux and was unaltered by omitting calcium from the extra-cellular solution and adding EGTA (0.1 mM). Both GABA and the GABA-mimetic clomethiazole inhibited ischaemia-induced glutamate efflux, with IC(50) values of 26 and 24 microM respectively. The maximum inhibition by either drug was 60 - 70%. Bicuculline (10 microM) abolished the inhibitory effect of GABA (100 microM) but not clomethiazole (100 microM). Picrotoxin (100 microM) abolished the action of both GABA and clomethiazole. Pentobarbitone inhibited glutamate efflux at 100 - 300 microM (maximal inhibition: 39%). Bicuculline (10 microM) abolished this effect. These data suggest that ischaemia-induced glutamate efflux from rat cerebral cortex is calcium-independent and not due to cell damage up to 45 min. The inhibitory effect of GABA, clomethiazole and pentobarbitone on ischaemia-induced glutamate efflux appears to be mediated by GABA(A) receptors. The results suggest that clomethiazole, unlike pentobarbitone, is able to activate the GABAA receptor-linked chloride channel directly and not merely potentiate the effect of endogenous GABA.

Tumor necrosis factor alpha enhances the cytotoxicity induced by nitric oxide in cultured cerebral endothelial cells

It has been shown that independent sources of nitric oxide (NO) and the inflammatory cytokine tumor necrosis factor alpha (TNF α) contribute to the breakdown of the blood-brain barrier (BBB) in the pathogenesis of a number of brain disorders. However, the interaction of NO and TNF α has not been elucidated. The present study was designed to determine whether the toxicity induced by NO is altered by TNF α in brain capillary endothelial cells (BCECs), and if so, whether it is related to the generation of Superoxide. TNF α (50–400 U ml ) did not produce toxicity until at a concentration of 800 U ml . This toxic effect was completely blocked by copper-zinc Superoxide dismutase (SOD)/catalase or N ω-nitro-L-arginine methyl ester (L-NAME) or oxyhemoglobin (HbO 2). Sodium nitroprusside (SNP) reduced with 0.4 mM ascorbate (SNP/Vc) significantly increased Lactate dehydrogenase (LDH) efflux in a concentration-dependent manner. This cytotoxicity of SNP/Vc was also completely inhibited by SOD/catalase or HbO 2. When SNP/Vc used in combination with 400 U ml TNF α, a more remarkable LDH efflux was induced than SNP/Vc alone, even as little as 0.01mM SNP/Vc was toxic, although a dose of 400 U ml TNF α alone had no effect on LDH efflux. In addition, either 0.4 mM SNP/Vc and 800 U ml TNF a alone or 0.4 mM SNP/Vc and 400 U ml TNF α in combination significantly increased malondialdehyde (MDA) content, but nitric oxide synthase (NOS) activity was inhibited only by SNP/Vc and TNF a in combination. These results suggest that TNF α enhances the toxicity of NO in BCECs and that at least part of this enhancement involves the generation of Superoxide.

Antioxidants and gadolinium chloride attenuate hepatic parenchymal and endothelial cell injury induced by low flow ischemia and reperfusion in perfused rat livers

The objective of this study was to determine whether Kupffer cells contribute to parenchymal and endothelial cell damage induced by ischemia-reperfusion in perfused rat livers. Parenchymal and endothelial cell injury were determined by measuring activities of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP), respectively, in the effluent perfusate of livers subjected to 60 min of low flow ischemia followed by 30 min of reperfusion. Upon reperfusion, LDH and PNP activities increased significantly within the first 10 min of reperfusion and remained elevated over control values throughout the duration of reperfusion. Pretreatment with gadolinium chloride, an inhibitor of Kupffer cell function, significantly decreased LDH and PNP efflux during reperfusion by approximately 60% and 50%, respectively. When Kupffer cells were stimulated by vitamin A pretreatment, PNP efflux was doubled during reperfusion. Vitamin E pretreatment attenuated LDH and PNP release by approximately 70% during reperfusion compared to enzyme release in untreated livers. Moreover, the water-soluble antioxidants superoxide dismutase and desferrioxamine reduced reperfusion injury, whereas catalase had no effect on enzyme release. These results demonstrate that superoxide anions released from Kupffer cells are involved in oxidative damage to endothelial cells as well as hepatocytes during the early stages of hepatic reperfusion.

Derangement of Kupffer cell functioning and hepatotoxicity in hyperthyroid rats subjected to acute iron overload

Liver oxidative stress, Kupffer cell functioning, and cell injury were studied in control rats and in animals subjected to L-3,3′,5-tri-iodothyronine (T3) and/or acute iron overload. Thyroid calorigenesis with increased rates of hepatic O2 uptake was not altered by iron treatment, whereas iron enhanced serum and liver iron levels independently of T3. Liver thiobarbituric acid reactants formation increased by 5.8-, 5.7-, or 11.0-fold by T3, iron, or their combined treatment, respectively. Iron enhanced the content of protein carbonyls independently of T3 administration, whereas glutathione levels decreased in T3- and iron-treated rats (54%) and in T3Fe-treated animals (71%). Colloidal carbon infusion into perfused livers elicited a 109% and 68% increase in O2 uptake in T3 and iron-treated rats over controls. This parameter was decreased (78%) by the joint T3Fe administration and abolished by gadolinium chloride (GdCl3) pretreatment in all experimental groups. Hyperthyroidism and iron overload did not modify the sinusoidal efflux of lactate dehydrogenase, whereas T3Fe-treated rats exhibited a 35-fold increase over control values, with a 54% reduction by GdCl3 pretreatment. Histological studies showed a slight increase in the number or size of Kupffer cells in hyperthyroid rats or in iron overloaded animals, respectively. Kupffer cell hypertrophy and hyperplasia with presence of inflammatory cells and increased hepatic myeloperoxidase activity were found in T3Fe-treated rats. It is concluded that hyperthyroidism increases the susceptibility of the liver to the toxic effects of iron, which seems to be related to the development of a severe oxidative stress status in the tissue, thus contributing to the concomitant liver injury and impairment of Kupffer cell phagocytosis and particle-induced respiratory burst activity.

Excitotoxic effects of non-NMDA receptor agonists in organotypic corticostriatal slice cultures

The excitotoxic effects of the glutamate receptor agonists kainic acid (KA) and 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and the corresponding neuroprotective effects of the AMPA/KA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) were examined in corticostriatal slice cultures. The purpose was to examine the feasibility of these cultures for excitotoxic studies, and to demonstrate possible differential excitotoxic effects of KA and AMPA on striatal and cortical neurons. Slices of dorsolateral striatum with overlying neocortex were obtained from neonatal rats and grown on semiporous membranes in serum-free medium for 3–4 weeks before exposure to KA or AMPA for 48 h. The uptake by injured cells of the fluorescent dye propidium iodide (PI) added to the culture medium was used as a quantifiable measure for neuronal degeneration and compared with efflux of the cytosolic enzyme lactate dehydrogenase (LDH) into the culture medium and loss of glutamic acid decarboxylase (GAD) activity in the tissue. Histological sections were also stained by the fluorescent dye Fluoro-Jade (FJ), for degenerating neurons and by immunocytochemical staining for γ-aminobutyric acid (GABA). Digitized images showed a dose (0–24 μM KA, 0–6 μM AMPA) and time (0–48 h) dependent increase in PI uptake in both striatum and cortex. In other cultures exposed to KA (24 μM) or AMPA (6 μM) together with NBQX (0.1–9 μM), NBQX was found to exert a differential neuroprotective effect on striatum and cortex at low doses. NBQX was thus more protective against KA in the cortex than in the striatum, while the opposite was seen in relation to AMPA. Regarding neurodegenerative markers, PI uptake was significantly correlated with (1) LDH release into the culture medium, (2) optical density of Fluoro-Jade staining, (3) loss of GAD-activity in tissue homogenates, and (4) loss of GABA-immunostained neurons. We conclude that both differences between compounds (AMPA vs. KA) and brain areas (striatum vs. cortex) can be demonstrated in corticostriatal slice cultures, which in conjunction with an established set of markers for neuronal cell damage appears to be a feasible model for studies of the neurotoxic and neuroprotective effects of glutamate receptor agonists and antagonists.

Use of brain slices in the study of pathogenic role of inducible nitric oxide synthase in cerebral ischemia-reperfusion

We have recently demonstrated that inducible nitric oxide synthase (iNOS) is expressed in rat forebrain slices exposed to oxygen and glucose deprivation (OGD). Now, we have found that the expression of iNOS after OGD is time-dependent since 20 min of OGD produces the appearance of iNOS at earlier times than 10 min of OGD. OGD also causes neurotoxicity in this model, as revealed by the increase in excitatory amino acid, neuron specific enolase and lactate dehydrogenase (LDH) efflux to the incubation solution. Finally, the administration of the NMDA receptor antagonist MK-801 (100 nM) inhibits both the expression of iNOS and the release of LDH. Our findings demonstrate that this method may be considered an useful in vitro model of ischemia-reperfusion to determine the therapeutic role of neuroprotective tools.

Sex-specific expression of N-methyl d-aspartate receptor (NMDAR) in the preoptic area of neonatal rats

The relationship between the N-methyl- d-aspartate receptor (NMDAR) and the sex-specific neurotoxicity of l-glutamate on the preoptic area (POA) of neonatal rats was studied. The NMDAR were semiquantified by western blot analysis. The kinetic change of intracellular calcium and lactate dehydrogenase (LDH) efflux were monitored as rapid and delayed toxic signals, respectively. The results showed that: (1) the NMDAR expression in POA of male rat is higher than that of females; (2) the l-glutamate (500 μM) induced a more significant elevation of intracellular calcium in neuron derived from male rat than that from female; (3) after glutamate-treatment, the LDH efflux in neuronal culture of male rat is higher than that of females. These results suggest that the quantitative difference in NMDAR between male and female rats may contribute to the sex-specific neurotoxicity of l-glutamate on the POA of neonatal rats.

Stereoselective neuroprotection by novel 2,3-benzodiazepine non-competitive AMPA antagonists against non-NMDA receptor-mediated excitotoxicity in primary rat hippocampal cultures

Glutamate excitotoxicity has been implicated in a variety of acute and chronic neurodegenerative diseases but early phase clinical trials with competitive antagonists at both N-methyl- d-aspartate (NMDA)-receptors and α-amino-3-hydroxy-5-methyl-isoxazolepropionate (AMPA) receptors have been disappointing. A family of atypical 2,3 benzodiazepines, exemplified by GYKI 52466, have been described recently which function as non-competitive AMPA-receptor antagonists. We have investigated the neuroprotective efficacy of LY303070 and LY300164, two analogs of GYKI-52466, in an embryonic rat hippocampal culture model of non-NMDA receptor-mediated excitotoxicity using kainic acid (KA) as an agonist at the AMPA/KA receptor. Overnight treatment with 500 μM KA resulted in prominent neuronal excitotoxicity as assessed by lactate dehydrogenase efflux. LY300164 and LY303070 attenuated KA-excitotoxicity in a dose-dependent manner with IC 50s of 4 and 2 μM, respectively. In contrast, their stereoisomers, LY300165 and LY303071 showed no neuroprotection at concentrations up to 25 μM. In addition, AMPA-mediated excitotoxicity in cyclothiazide pre-treated cultures was also completely blocked by LY303070. Finally, neuroprotection by this class of 2,3 benzodiazepines was not influenced by antagonism of the classical benzodiazepine receptor. LY303070 and LY300164 represent novel non-competitive AMPA-receptor antagonists which may offer unique advantages in the clinic over competitive AMPA-receptor antagonists.

ELEVATED GLUTATHIONE LEVELS ACCOUNT FOR HALF THE PROTECTION SEEN AGAINST GLUCOSE DEPRIVATION WITH OVEREXPRESSION OF BCL-XL IN MOUSE ASTROCYTES

S237 INTRODUCTION: Bcl-2 and bcl-xL are anti-apoptotic genes which are promising candidate genes for gene therapy to ameliorate brain injury associated with stroke. They are thought to have effects on mitochondrial function. Our previous studies showed that overexpression of bcl-2 protects primary mouse astrocytes from glucose deprivation (GD) and hydrogen peroxide (H2 O2) injury [1]. We tested the ability of bcl-xL to protect astrocytes from injury and determined the effect of overexpressing this gene on cellular glutathione, iron and ferritin levels. METHODS: Primary astrocyte cultures were prepared as previously described [2] according to a protocol approved by the Stanford animal care and use committee. The cultures were infected with retroviral vectors to express either bcl-2, bcl-x (L) or the control gene beta-galactosidase. Mock infected controls were also studied. Experiments were performed at least three times on different cultures. Glucose deprivation was performed by washing into a balanced salt solution, combined oxygen-glucose deprivation by washing into anoxic balanced salt solution, or cells were injured by exposure to 400 [micro sign]M H2 O2. Cell injury was quantitated by lactate dehydrogenase efflux. Glutathione was measured using monochlorobimane [1]. Ferritin protein levels were estimated using Western analysis; bands were quantitated by scanning densitometry. Iron levels were quantitated using the colorimetric procedure of Fish [4]. Statistical significance was determined using ANOVA followed by Student Neuman Keuls for multiple comparisons. RESULTS: Astrocytes overexpressing bcl-xL were significantly protected against H2 O2 exposure and glucose deprivation, but not combined oxygen-glucose deprivation. Bcl-xL and bcl-2 overexpressing astrocytes showed higher glutathione levels, and greater iron and ferritin staining than beta-galactosidase infected and mock infected cells. Glutathione levels in bcl-xL overexpressing cells were 20.1 +/- 1 nmol/mg protein, while in beta-galactosidase expressing controls 17.2 +/- 0.8. Higher levels were maintained in bcl-xL overexpressing cells after 7 hr of glucose deprivation. When glutathione levels were reduced in both astrocyte cultures by preincubation with 0.1 mM buthionine sulfoximine, levels were 4.3 +/- 0.8 and 3.9 +/- 0.5 respectively. When these cultures with equivalent glutathione levels were subjected to glucose deprivation the extent of protection by bcl-xL overexpression was reduced to approximately half. DISCUSSION: These results demonstrate that bcl-xL overexpression in astrocytes provides protection against GD and H2 O2 injury, the same spectrum of injuries previously reported to be decreased by bcl-2. These injuries are also the ones in which the greatest loss of glutathione is seen. When glutathione levels were reduced equally in bcl-xL overexpressing and control astrocytes, protection against GD was reduced but not eliminated. This suggests that the protection afforded by this gene is partially due to the elevation of glutathione levels, but not exclusively to this. The elevated ferritin levels observed may be a second component of the protection seen against injury due to overexpression of bcl-xL. Supported in part by a grant from IARS.

Influence of copper-(II) on colloidal carbon-induced kupffer cell-dependent oxygen uptake in rat liver: Relation to hepatotoxicity

Formation of reactive O2 species in biological systems can be accomplished by copper-(II) (Cu2+) catalysis, with the consequent cytotoxic response. We have evaluated the influence of Cu2+ on the respiratory activity of Kupffer cells in the perfused liver after colloidal carbon infusion. Studies were carried out in untreated rats and in animals pretreated with the Kupffer cell inactivator gadolinium chloride (GdCl3) or with the metallothionein (MT) inducing agent zinc sulphate, and results were correlated with changes in liver sinusoidal efflux of lactate dehydrogenase (LDH) as an index of hepatotoxicity. In the concentration range of 0.1–1 μM, Cu2+ did not modify carbon phagocytosis by Kupffer cells, whereas the carbon-induced liver O2 uptake showed a sigmoidal-type kinetics with a half-maximal concentration of 0.23 μM. Carbon-induced O2 uptake occurred concomitantly with an increased LDH efflux, effects that were significantly correlated and abolished by GdCl3 pretreatment or by MT induction. It is hypothesized that Cu2+ increases Kupffer cell-dependent O2 utilization by promotion of the free radical processes related to the respiratory burst of activated liver macrophages, which may contribute to the concomitant development of hepatocellular injury.

Markers for neuronal degeneration in organotypic slice cultures

This protocol describes ways of monitoring spontaneous or induced neuronal degeneration in organotypic brain slice cultures. Hippocampal cultures (4-week-old) are grown in normal serum-free control medium, or exposed to the neurotoxin trimethyltin (TMT) (0.5–100 μM) for 24 h or the excitotoxic glutamate agonist kainic acid (KA) (5–25 μM) for 48 h followed by 24 h or 48 h, respectively, in normal medium. Corticostriatal slice cultures (also 4-week-old) are exposed to KA (6–24 μM) for 48 h and normal medium for control. The resulting neurodegeneration is estimated by (a) propidium iodide (PI) uptake, (b) lactate dehydrogenase (LDH) efflux to the culture medium, (c) ordinary Nissl cell staining, (d) staining by the neurodegenerative marker Fluoro-Jade (FJ), (e) neuronal microtubule degeneration by immunohistochemical staining for microtubule-associated protein 2 (MAP2), and (f) Timm sulphide silver staining for heavy metal alterations. Both hippocampal and corticostriatal slice cultures show a dose- and time-dependent increase in PI uptake and LDH efflux after exposure to TMT and KA. The mean PI uptake and the LDH efflux into the medium correlate well for both types of cultures. Both TMT and KA exposed hippocampal cultures display in vivo patterns of differential neuronal vulnerability as evidenced by PI uptake, FJ staining and MAP2 immunostaining. Corticostriatal slice cultures exposed to a high dose of KA display extensive striatal and cortical degeneration in FJ staining as suggested by a high PI uptake. A change in Timm sulphide silver staining in deep central parts of some control cultures, corresponds to areas with loss of cells in cell staining, loss of MAP2 staining, PI uptake, and FJ staining. We conclude that organotypic brain slice cultures, in combination with appropriate markers in standardized protocols, represent feasible means for studies of excitotoxic and neurotoxic compounds. Themes: Disorders of the nervous system Topics: Neurotoxicity

The guanylate cyclase-coupled natriuretic peptide receptor: a new target for prevention of cold ischemia-reperfusion damage of the rat liver.

The aim of our studies was to investigate hormonal prevention of hepatic preservation damage by the atrial natriuretic peptide (ANP) and the mechanisms involved. Isolated perfusion of rat livers was performed in a nonrecirculating fashion. Twenty minutes of preischemic perfusion was performed with or without different concentrations of ANP, followed by 24-hour storage in cold University of Wisconsin (UW) solution. Two hundred nanomoles of ANP prevented hepatocellular damage during a 2-hour reperfusion period as indicated by a marked attenuation of the sinusoidal efflux of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP), and by reduced Trypan blue uptake. Furthermore, postischemic bile flow as an indicator of liver function was significantly improved by about 60% with 200 nmol/L ANP. No protection was conveyed by 20 nmol/L ANP nor by pretreatment with 200 nmol/L ANP for only 10 minutes. The effects of ANP seemed to be mediated by the guanylate cyclase-coupled A (GC-A) receptor and cyclic guanosine monophosphate (cGMP): whereas expression of both GC-A and GC-B receptors as well as of the GC-C receptor was found, cGMP did protect from ischemia-reperfusion damage, but selective ligands of the B and C receptor did not. To begin to determine the mechanisms of ANP-mediated protection, different parameters were investigated: ANP had no effect on portal pressure as an indicator of hepatic circulation, nor on intracellular energy depletion determined by adenosine nucleotide concentration. However, the marked augmentation of nuclear factor kappaB (NF-kappaB) binding activity during reperfusion was prevented in ANP-pretreated livers. In conclusion, pretreatment with ANP protects the rat liver from cold ischemia-reperfusion damage. This effect is mediated via the GC-A receptor and cGMP, and may be linked to an influence of ANP on NF-kappaB activation. Thus, ANP signaling via the GC-A receptor should be considered as a new pharmacological target to prevent preservation injury of the liver.