Differential mechanisms of neuroprotection by 17 beta-estradiol in apoptotic versus necrotic neurodegeneration.

The major goal of this study was to compare mechanisms of the neuroprotective potential of 17 beta-estradiol in two models for oxidative stress-independent apoptotic neuronal cell death with that in necrotic neuronal cell death in primary neuronal cultures derived from rat hippocampus, septum, or cortex. Neuronal apoptosis was induced either by staurosporine or ethylcholine aziridinium (AF64A), as models for necrotic cell death glutamate exposure or oxygen-glucose deprivation (OGD) were applied. Long-term (20 hr) pretreatment (0.1 microm 17 beta-estradiol) was neuroprotective in apoptotic neuronal cell death induced by AF64A (40 microm) only in hippocampal and septal neuronal cultures and not in cortical cultures. The neuroprotective effect was blocked by the estrogen antagonists ICI 182,780 and tamoxifen and the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002. In glutamate and OGD-induced neuronal damage, long-term pretreatment was not effective. In contrast, short-term (1 hr) pretreatment with 17 beta-estradiol in the dose range of 0.5-1.0 microm significantly reduced the release of lactate dehydrogenase and improved morphology of cortical cultures exposed to glutamate or OGD but was not effective in the AF64A model. Staurosporine-induced apoptosis was not prevented by either long- or short-term pretreatment. The strong expression of the estrogen receptor-alpha and the modulation of Bcl proteins by 17 beta-estradiol in hippocampal and septal but not in cortical cultures indicates that the prevention of apoptotic, but not of necrotic, neuronal cell death by 17 beta-estradiol possibly depends on the induction of Bcl proteins and the density of estrogen receptor-alpha.

Increased formation of reactive oxygen species after permanent and reversible middle cerebral artery occlusion in the rat.

In barbiturate-anesthetized rats, we induced 3 hours of permanent middle cerebral artery occlusion (MCAO) by an intraluminal thread (n = 6), or 1 hour MCAO followed by 2 hours of reperfusion (n = 6). Through a closed cranial window over the parietal cortex, the production of reactive oxygen species (ROS) was measured in the infarct border using online in vivo chemiluminescence (CL) while monitoring the appearance of peri-infarct depolarizations (PID). The borderzone localization of the ROS and direct current (DC) potential measurements was confirmed in additional experiments using laser-Doppler scanning, mapping regional CBF changes through the cranial window after permanent (n = 5) or reversible (n = 5) MCAO. CL measurements revealed a short period (10 to 30 minutes) of reduced ROS formation after vessel occlusion, followed by a significant increase (to 162 +/- 51%; baseline = 100%; P < .05) from 100 minutes of permanent MCAO onward. Reperfusion after a 1-hour period of MCAO led to a burst-like pattern of ROS production (peak: 489 +/- 330%; P < .05). When the experiments were terminated 3 hours after induction of MCAO, CL was still significantly increased above baseline after permanent and reversible MCAO (to 190 +/- 67% and 211 +/- 64%, respectively; P < .05). Simultaneous DC potential recordings detected 6.4 +/- 2.7 PID in the first, 4.7 +/- 2.3 in the second, and 2.8 +/- 2.0 in the third hour after permanent MCAO. In animals with reversible MCAO, PID were abolished from 15-minutes recirculation onward. There was no temporal relationship between ROS production and peri-infarct DC potential shifts. In conclusion, using a high temporal resolution ROS detection technique (CL), we found that permanent MCAO (after an initial decrease) was accompanied by a steady increase of ROS production during the 3-hour observation period, while reperfusion after 1 hour of MCAO produced a burst in ROS formation. Both patterns of ROS production were not related to the occurrence of PID.

Respiratory chain inhibition induces tolerance to focal cerebral ischemia.

The authors show that the inhibitor of the succinate dehydrogenase, 3-nitroproprionic acid (3-NPA), which in high doses and with chronic administration is a neurotoxin, can induce profound tolerance to focal cerebral ischemia in the rat when administered in a single dose (20 mg/kg) 3 days before ischemia. Infarcts were approximately 70% and 35% smaller in the 3-NPA preconditioned groups of permanent and transient focal cerebral ischemia, respectively. This regimen of 3-NPA preconditioning neither induced necrosis, apoptosis, or any other histologically detectable damage to the brain, nor did it affect behavior of the animals. 3-NPA led to an immediate (1-hour) and long-lasting (3-day) decrease in succinate dehydrogenase activity (30% reduction) throughout the brain, whereas only a short metabolic impairment occurred (ATP decrease of 35% within 30 minutes, recovery within 2 hours). The authors found that 3-NPA induces a burst of reactive oxygen species and the free radical scavenger dimethylthiourea, when administered shortly before the 3-NPA stimulus, completely blocked preconditioning. Inhibition of protein synthesis with cycloheximide given at the time of 3-NPA administration completely inhibited preconditioning. The authors were unsuccessful in showing upregulation of mRNA for the manganese superoxide dismutase, and did not detect increased activities of the copper-zinc and manganese superoxide dismutases, prototypical oxygen free radicals scavenging enzymes, after 3-NPA preconditioning. The authors conclude that it is possible to pharmacologically precondition the brain against focal cerebral ischemia, a strategy that may in principal have clinical relevance. The data show the relevance of protein synthesis for tolerance, and suggests that oxygen free radicals may be critical signals in preconditioning.

Acute treatment of hypertension increases infarct sizes in spontaneously hypertensive rats.

We studied the effect of dihydralazine treatment of hypertension in spontaneously hypertensive stroke-prone rats in a model of permanent focal cerebral ischemia (stroke). After occlusion of the middle cerebral artery systemic arterial pressure (SAP) was lowered with a computer controlled infusion device from 163 to 135 or 117 mm Hg for 24h. In the control group SAP was not manipulated. Reduction of SAP to normotension (117 mm Hg) significantly worsened outcome and increased infarct volume measured 7 days after induction of ischemia, whereas a mild reduction of SAP (to 137 mm Hg) had no statistically significant effect on outcome or infarct volume. We conclude that pharmacological treatment of hypertension may negatively affect neurological outcome and infarct volume in a rat stroke model.

Age-related changes of oxygen free radical production in the rat brain slice after hypoxia: on-line measurement using enhanced chemiluminescence.

Superoxide radical production in brain slices of 12-, 24- and 60-day-old rats was measured online during and after 15 min of hypoxia with lucigenin enhanced chemiluminescence. We found a typical radical burst after reoxygenation which developed with aging and is almost nonexistent in the youngest and most prominent in the oldest group. This cannot be explained by a decreasing tissue superoxide dismutase (SOD) concentration in the brain with aging, since the concentration of the enzyme, determined in the same age groups, increased with age.

Rapid Ca2+-dependent NO-production from central nervous system cells in culture measured by NO-nitrite/ozone chemoluminescence.

We have established simple and reliable measurement of constitutive nitric oxide (NO) synthase-dependent nitrite formation in supernatants from primary central nervous system (CNS) cells in culture using NO-ozone chemoluminescence. We found that: (1) astrocytes, endothelial cells and cerebellar granule cells produce NO upon stimulation with the calcium ionophore A23187 (1 microM); (2) application of 100 microM glutamate for 2 min results in NO-production in cerebellar granule cells and cortical neurons. NO-formation upon application of 50 mM KCl was found in cortical neurons; (3) in cultivated cerebral endothelial cells, an inducible form of NO-synthase (iNOS) is found under standard culture conditions. This induction was blocked by dexamethasone applied for at least 48 h and stimulation of constitutive NOS was detectable while iNOS was inhibited. The activity of iNOS was selectively inhibited by application of aminoguanidine for 48 h. Our results suggest that all major CNS cells implied in cerebral blood flow regulation and neurovascular coupling are capable of rapidly producing the vasodilator NO upon intracellular increases of the universal second messenger calcium.

Hyperbaric oxygenation induced tolerance against focal cerebral ischemia in mice is strain dependent.

SV129 or C57BL/6 mice were exposed to hyperbaric oxygenation (HBO, 5 days, 1 h every day, 100% O(2) at 3 atm absolute). One day after the 5th HBO session focal cerebral ischemia was induced. In SV129 mice, HBO induced tolerance against permanent focal cerebral ischemia (n=42, mean infarct volume reduction 27%, P=0.001), but not against transient (30 or 60 min) focal cerebral ischemia. In the C57BL/6 strain of mice, HBO did not induce tolerance against focal cerebral ischemia, even when the duration of ischemia or the HBO protocol were modified. For the first time we demonstrate that HBO can induce tolerance to focal cerebral ischemia, but this effect is strain dependent.

Nitric oxide synthase inhibition does not affect somatosensory evoked potentials in the rat.

Nitric oxide synthase (NOS) inhibition reduces the regional cerebral blood flow (rCBF) responses to somatosensory stimulation. It is controversial whether this is caused by a signalling role of nitric oxide (NO) between neurons and vascular smooth muscle, or by effects of NOS inhibition on neuronal activity. We here report that more than 85% inhibition of NOS activity by topical application of the NOS inhibitor N omega-nitro-L-arginine (L-NNA) for 2 h does not affect somatosensory evoked potentials (SEPs) elicited by vibrissal deflection or electrical forepaw stimulation in choloralose anaesthetised rats equipped with a closed cranial window, whereas cerebral blood flow (CBF) responses due to these stimulation paradigms are reduced by approximately 60%. We conclude that the decrease of the regional vascular response to increased neuronal activity during NOS inhibition is not caused by a suppression of neuronal activity.

Induction of ischemic tolerance in rat cortical neurons by 3-nitropropionic acid: chemical preconditioning.

Sublethal ischemia leads to increased tolerance against subsequent ischemia. We investigated whether tolerance could also be elicited by mild respiratory-chain inhibition (chemical hypoxia) in a rat neuronal-cell enriched culture system. 3-Nitropropionic acid (3-NPA) caused a concentration-dependent inhibition of succinate-dehydrogenase. Two hours preconditioning with 3-NPA 24-48 h before oxygen-glucose deprivation (OGD) reduced neuronal damage morphologically and reduced lactate deydrogenase (LDH) release up to 72% compared to sham-treated sister cultures without 3-NPA. In an attempt to elucidate transcriptional mechanisms, we found no rapid translocation of the hypoxia-sensitive transcription factors N F-KB or hypoxia-inducible factor-I (HIF-I) at 3-NPA concentrations sufficient to trigger tolerance against OGD. In accordance to previous in vivo and brain slice data, we conclude that 3-NPA chemically induces tolerance against oxygen-glucose deprivation in vitro. However, the underlying mechanisms remain elusive.

Cu,Zn SOD in German families with ALS.

We studied the gene for Cu,Zn SOD in 15 German patients with familial ALS and did not find any mutation. Activity of the enzyme and its expression at the protein level was also normal in each patient and in 18 patients suffering from the sporadic form of ALS.

Products of hemolysis in the subarachnoid space inducing spreading ischemia in the cortex and focal necrosis in rats: a model for delayed ischemic neurological deficits after subarachnoid hemorrhage?

OBJECT: The pathogenesis of delayed ischemic neurological deficits after subarachnoid hemorrhage has been related to products of hemolysis. Topical brain superfusion of artificial cerebrospinal fluid (ACSF) containing the hemolysis products K+ and hemoglobin (Hb) was previously shown to induce ischemia in rats. Superimposed on a slow vasospastic reaction, the ischemic events represent spreading depolarizations of the neuronal-glial network that trigger acute vasoconstriction. The purpose of the present study was to investigate whether such spreading ischemias in the cortex lead to brain damage. METHODS: A cranial window was implanted in 31 rats. Cerebral blood flow (CBF) was measured using laser Doppler flowmetry, and direct current (DC) potentials were recorded. The ACSF was superfused topically over the brain. Rats were assigned to five groups representing different ACSF compositions. Analyses included classic histochemical and immunohistochemical studies (glial fibrillary acidic protein and ionized calcium binding adaptor molecule) as well as a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay. Superfusion of ACSF containing Hb combined with either a high concentration of K+ (35 mmol/L, 16 animals) or a low concentration of glucose (0.8 mmol/L, four animals) reduced CBF gradually. Spreading ischemia in the cortex appeared when CBF reached 40 to 70% compared with baseline (which was deemed 100%). This spreading ischemia was characterized by a sharp negative shift in DC, which preceded a steep CBF decrease that was followed by a slow recovery (average duration 60 minutes). In 12 of the surviving 14 animals widespread cortical infarction was observed at the site of the cranial window and neighboring areas in contrast to findings in the three control groups (11 animals). CONCLUSIONS: The authors conclude that subarachnoid Hb combined with either a high K+ or a low glucose concentration leads to widespread necrosis of the cortex.

A miniaturized electrode configuration for isoelectric focusing.

An electrode configuration is described which allows fast isoelectric focusing (IEF) with conventional IEF systems. The equipment, which can be fixed on the cooling plate of a conventional IEF system, consists of a base plate on which flappable electrode holders are fastened. The handling is simple and needs only little time. Graphite rods are used as electrodes, thus avoiding the use of buffer strips. Samples are applied with special applicator strips--permitting the analysis of up to 19 samples on a 50 x 40 mm polyacrylamide gel and up to 44 samples on a 100 x 70 mm gel. Only 30 min are needed for one IEF run.

Application of immunological methods for the visualization of PK after IEF in ultrathin layers.

Immunological methods are recommended for protein visualization after IEF on account of their high sensitivity, good specificity and resolution. We used the indirect immunoassay with peroxidase- and 125I-labelled antibodies to investigate microheterogeneity of pyruvate kinase (PK) from human red blood cells and could show a PK pattern with more than 10 single bands. Different methods were tested as to their applicability for protein fixation and subsequent immunological visualization on the polyacrylamide gel. Beside Western blot, immunofixation and ethanolic fixation can be recommended as fixation techniques especially after isoelectric focusing in ultrathin gels.

Microheterogeneity of human erythrocyte pyruvate kinase: application of immunological visualization techniques after isoelectric focusing in ultrathin gels.

Immunological methods for protein visualization afford high sensitivity, good specificity and resolution. We used the indirect immunoassay with peroxidase- and 125I-labelled antibodies to investigate the microheterogeneity of pyruvate kinase from human red blood cells and can show an enzyme pattern with more than 10 single bands. Different methods were tested as to their applicability for protein fixation and subsequent immunological visualization in polyacrylamide gels. Besides the Western blot, immunofixation and ethanolic fixation can be recommended as fixation techniques, especially after isoelectric focusing in ultrathin gels.

IEF-microheterogeneity of human PK-R: applicability for prenatal diagnosis.

Microheterogeneity of human erythrocyte pyruvate kinase (PK-R) can be obtained by isoelectric focusing (IEF) in ultrathin polyacrylamide gels followed by immunological visualization methods. Using this method to study whether a polymorphismus of PK-R occurs, we could not find any deviations from the normal PK pattern in the case of samples from 100 German donors as well as at a lower number of PK samples from European, African and Asian donors. Furthermore, we compared the IEF-pattern of PK mutants with that of normal new-borns and of foetal PK-samples. Our results show that IEF-microheterogeneity of PK may serve as a tool for prenatal diagnosis in the case of families which are effected by a severe PK deficiency.

[Diagnosis of genetically determined diseases]. / Diagnostik genetisch bedingter Erkrankungen.

Enzymopathies of pyruvate kinase are caused by defects of structural genes forming stable and unstable mutants, respectively. Stable mutants of PK are characterized by high S0,5 PEP and nearly unchanged Vmax. A decrease of PEP affinity can be the result from a very high allosteric constant L0 or from an increased KPEP. From the pattern of PAGE can be concluded that stable PK mutants are tetraheteromers composed of two normal and two shortened polypeptide chains. We suppose that this is the result of a mutation of a codon which stops the polypeptide synthesis of PK earlier. Most PK mutants are unstable. They are characterized by low catalytic activity and high PEP affinity. The kinetic properties of unstable mutants are changed posttranslational by proteolytic modifications. Furthermore low S0,5 PEP values result from a persistence of the isoenzyme PK-K in reticulocytes and erythrocytes, respectively. A prenatal diagnosis of PK enzymopathies can be carried out with a very small blood volume by using the method of isoelectrophoretic focussing.

Mechanical membrane properties of human red blood cells and their change due to metabolic disturbance.

Blood of patients suffering from non-spherocytic chronic haemolytic anemia due to individual mutations of glucose-6-phosphate-dehydrogenase (G-6-PD) and pyruvate kinase (PK) was investigated by biochemical and rheological methods. Both enzymopathies are characterized by a strong increase (by the factor 2 to 4) in the overall RBC-rigidity and the elastic membrane shear modulus of the RBC. There was a positive correlation between the changed elastic shear modulus and the clinical picture in the case of PK deficiency and, in contrast, a negative correlation for patients with G-6-PD enzymopathies. The ability of RBC to change their shape or the static deformability is determined by the excess surface regarding to the enclosed volume, by the rheological properties of the hemoglobin content and by the membrane extension and bending moduli. The dynamic deformability is characterized by the time constant for rapid elastic and plastic deformations and becomes important for entrance and discharge processes in the microcirculation. These rheological relevant properties are subjected to metabolic control. Although for both enzymopathies the mechanism of hemolysis is not understood in detail, it has to be assumed that PK deficient RBC are mostly phagocytized and on the other hand G-6-PD deficient cells are destroyed to a higher extent by intravasal hemolysis. The aim of this study was to investigate whether or not expected changes in the RBC membrane structure due to the decreased ATP production by PK enzymopathies and diminished NADPH production by G-6-PD deficiencies result in abnormal mechanical membrane properties. The answer should give a better understanding of the premature RBC destruction.

Nitric oxide: a modulator, but not a mediator, of neurovascular coupling in rat somatosensory cortex.

We investigated the role of nitric oxide (NO)/cGMP in the coupling of neuronal activation to regional cerebral blood flow (rCBF) in alpha-chloralose-anesthetized rats. Whisker deflection (60 s) increased rCBF by 18 +/- 3%. NO synthase (NOS) inhibition by N(omega)-nitro-L-arginine (L-NNA; topically) reduced the rCBF response to 9 +/- 4% and resting rCBF to 80 +/- 8%. NO donors [S-nitroso-N-acetylpenicillamine (SNAP; 50 microM), 3-morpholinosydnonimine (10 microM)] or 8-bromoguanosine 3', 5'-cyclic-monophosphate (8-BrcGMP; 100 microM)] restored resting rCBF and L-NNA-induced attenuation of the whisker response in the presence of L-NNA, whereas the NO-independent vasodilator papaverine (1 mM) had no effect on the whisker response. Basal cGMP levels were decreased to 35% by L-NNA and restored to 65% of control by subsequent SNAP superfusion. Inhibition of neuronal NOS by 7-nitroindazole (7-NI; 40 mg/kg ip) or soluble guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 100 microM) significantly reduced resting rCBF to 86 +/- 8 and 92 +/- 10% and whisker rCBF response to 7 +/- 4 and 12 +/- 3%, respectively. ODQ reduced tissue cGMP to 54%. 8-BrcGMP restored the whisker response in the presence of 7-NI or ODQ. We conclude that NO, produced by neuronal NOS, is a modulator in the coupling of neuronal activation and rCBF in rat somatosensory cortex and that this effect is mainly mediated by cGMP. L-NNA-induced vasomotion was significantly reduced during increased neuronal activity and after restoration of basal NO levels, but not after restoration of cGMP.

Glucose-6-phosphate dehydrogenase from Plasmodium berghei: kinetic and electrophoretic characterization.

Evidence is given for the existence of a parasite-specific glucose-6-phosphate dehydrogenase in Plasmodium berghei by characterization of its kinetic and electrophoretic properties. After separating the parasites from infected RBC the G6PD was purified by affinity chromatography with 2'5'-ADP-Sepharose 4B. In cellulose acetate electrophoresis malarial G6PD significantly differs from the red cell enzyme. The subunits of the parasite-specific G6PD have a molecular weight of 55 kD in contrast to 59 kD of the RBC enzyme. G6PD from P. berghei shows no cross-reactivity with antibodies against G6PD from rat erythrocytes. The Km-value for G6P of malarial G6PD is increased by one order of magnitude compared with the host cell enzyme.

Evidence that glypican is a receptor mediating beta-amyloid neurotoxicity in PC12 cells.

Docking of beta-amyloid fibrils to neuronal or glial cell membranes may be an early, necessary and intervenable step during the progression of Alzheimer's disease. Formation of neurofibrillary tangles and amyloid plaques as well as neurotoxicity and inflammation may be direct or indirect consequences. In an attempt to find a receptor that mediates those effects, we assessed rat pheochromocytoma PC12 cell 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) reduction after addition of beta-amyloid to the culture medium. Presence of competitive substances in the medium, cell-surface treatment and specific block of cellular synthesis pathways helped to identify the heparan sulphate moiety of a glycosylphosphatidylinositol-anchored protein likely to represent glypican as a possible receptor mediating beta-amyloid neurotoxicity.