Experimental Brain Ischemia: Neuron-Specific Enolase Level in Cerebrospinal Fluid as an Index of Neuronal Damage

Levels of neuron-specific enolase (NSE) were measured in rat CSF following occlusion of the four major arteries to the brain for 10, 20, or 30 min. In the CSF of rats submitted to 30 min of total ischemia, an up to nine-fold increase of NSE level occurred within the first few hours and then slowly diminished. Significant levels were seen for as long as 8 days. Histological observations 3 days after ischemia showed neuronal loss as well as neuronal damage in several forebrain regions such as hippocampus, striatum, and thalamus. Ischemia was followed by transient decreases in exploration behavior and neurological states that were no longer visible 24 h later. After 10 or 20 min ischemia, NSE levels were increased to a lesser degree and fewer damaged neurons were observed. The positive correlation between duration of ischemia and amount of NSE release in CSF indicates that the measurement of NSE in the CSF is a sensitive and reliable index of neuronal lesions.     

Toxicity oft-butylhydroperoxide in hepatocyte monolayers exposed to hypoxia and reoxygenation

Summary Inasmuch as it is known that the toxicity of anesthetic agents is potentiated by hypoxia and that the reductive metabolism of these agents results in the formation of lipid hydroperoxides, we investigated the toxicity of hydroperoxides under low-oxygen concentrations. We found that hypoxia exacerbates the toxicity oft-butyl hydroperoxide, shifting the dose-response curve oft-butyl hydroperoxide vs. lysis of hepatocytes approximately an order of magnitude to the left. Furthermore, although at the end of a 4-h exposure to 0.5% O₂ hepatocyte monolayers seemed normal by three indices (release of⁵¹Cr and serum glutamate transaminase or exclusion of trypan blue), they were completely lysed after an additional 20 h reoxygenation at 20%. O₂. In contrast, monolayers exposed to 2% O₂ for 4 h seemed normal after 20 h reoxygenation. However, cells exposed to both a subtoxic dose of hydroperoxide and 4 h of 2% O₂, although seeming healthy at the end of the hypoxic period, were completely lysed within 20 h after reoxygenation. The study was supported by grant OH 00978 from the National Institutes for Occupational Safety and Health, Atlanta, Georgia.     

Heat Shock RNA Levels in Brain and Other Tissues After Hyperthermia and Transient Ischemia

A number of studies have demonstrated increased synthesis of heat shock proteins in brain following hyperthermia or transient ischemia. In the present experiments we have characterized the time course of heat shock RNA induction in gerbil brain after ischemia, and in several mouse tissues after hyperthermia, using probes for RNAs of the 70-kilodalton heat shock protein (hsp70) family, as well as ubiquitin. A synthetic oligonucleotide selective for inducible hsp70 sequences proved to be the most sensitive indicator of the stress response whereas a related rat cDNA detected both induced RNAs and constitutively expressed sequences that were not strongly inducible in brain. Considerable polymorphism of ubiquitin sequences was evident in the outbred mouse and gerbil strains used in these studies when probed with a chicken ubiquitin cDNA. Brief hyperthermic exposure resulted in striking induction of hsp70 and several-fold increases in ubiquitin RNAs in mouse liver and kidney peaking 3 h after return to room temperature. The oligonucleotide selective for hsp70 showed equivalent induction in brain that was more rapid and transient than observed in liver, whereas minimal induction was seen with the ubiquitin and hsp70-related cDNA probes. Transient ischemia resulted in 5- to 10-fold increases in hsp70 sequences in gerbil brain which peaked at 6 h recirculation and remained above control levels at 24 h, whereas a modest 70% increase in ubiquitin sequences was noted at 6 h. These results demonstrate significant temporal and quantitative differences in heat shock RNA expression between brain and other tissues following hyperthermia in vivo, and indicate that hsp70 provides a more sensitive index of the stress response in brain than does ubiquitin after both hyperthermia and ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thromboxane and Prostacyclin Levels in Fetal Rabbit Brain and Placenta After Intrauterine Partial Ischemic Episodes

The appearance of arachidonic acid (AA) oxidation products in fetal rabbit brain and placenta under normal or partial short-term ischemic episodes induced by placental blood vessel restriction was examined. Intracerebral administration of [3H]AA into close-to-term rabbit fetuses gave rise to radioactively labeled prostaglandin (PG) E2, thromboxane B2, and 6-keto-PGF1 alpha metabolites as detected by HPLC analysis. A significant increase of 20-30% of [3H]AA precursor into eicosanoids was detected in brain of fetuses after 2-h restriction. The thromboxane B2 and 6-keto-PGF1 alpha levels were determined by radioimmunoassay technique over a period of 48 h following ischemic episodes. Thromboxane B2 content in affected animals was higher by five- and twofold at 3 h over control fetal brain and placental tissue values, respectively, and remained significantly higher for 24 h. 6-Keto-PGF1 alpha levels reached a peak value that was greater by 2.5- and 1.5-fold at 6 h for the ischemic brain and placental tissue, respectively, compared with control fetuses. PGE2 levels were less affected, attaining a maximum of 1.9- and 1.1-fold in brain and placenta correspondingly. The thromboxane/prostacyclin ratio reached a maximum in the brain after approximately 3 h, while that in the placenta continued to rise even after 20 h. Persisting high levels of thromboxane are indicative of cerebral vasoconstriction and may suggest possible damaging effects.     

Effects of a New Calcium Channel Blocker, KB-2796, on Protein Synthesis of the CA1 Pyramidal Cell and Delayed Neuronal Death Following Transient Forebrain Ischemia

The effects of a new calcium channel blocker, 1-[bis(4-fluorophenyl)methyl]-4-(2,3,4-trimethoxybenzyl)-piperazine dihydrochloride (KB-2796), on delayed neuronal death (DND) in the hippocampus were examined in gerbils in comparison with those of pentobarbital and flunarizine. The neuronal density in the hippocampal CA1 subfield was counted on the seventh day of recirculation following 5 min of bilateral carotid occlusion, and protein biosynthesis in the brain was also determined at 1, 2, 4, 24, and 72 h following occlusion. The drugs were intraperitoneally administered after recirculation. KB-2796 (10 mg/kg) significantly prevented DND in the CA1 subfield. Pentobarbital (40 mg/kg), but not flunarizine (3 and 10 mg/kg), inhibited DND. Protein synthetic activity in the CA1 subfield was reduced by ischemia and the reduction was not restored even at 72 h after recirculation. KB-2796 did not ameliorate the reduction of protein synthesis in the CA1 subfield by 24 h after recirculation, but in one of three animals restoration of protein synthesis was observed at 72 h of recirculation. Pentobarbital also restored the reduced protein synthesis in two of three animals at 72 h. These results suggest that calcium influx into neurons participates in the pathogenesis of DND, and also that KB-2796 might prevent both morphological and functional cell damage in CA1 neurons induced by transient ischemia.     

Effect of Dichloroacetate on Regional Energy Metabolites and Pyruvate Dehydrogenase Activity During Ischemia and Reperfusion in Gerbil Brain

The objective of this study was to determine whether administration of dichloroacetate (DCA), an activator of pyruvate dehydrogenase (PDH), improves recovery of energy metabolites following transient cerebral ischemia. Gerbils were pretreated with DCA, and cerebral ischemia was produced using bilateral carotid artery occlusion for 20 min, followed by reperfusion up to 4 h. DCA had no effect on the accumulation of lactic acid and the decrease in ATP and phosphocreatine (PCr) during the 20-min insult, nor on the recovery of these metabolites measured at 20 and 60 min reperfusion. However, at 4 h reperfusion, levels of ATP and PCr were significantly higher in DCA-treated animals than in controls, as PCr exhibited a secondary decrease in caudate nucleus of control animals. PDH was markedly inhibited at 20 min reperfusion in both groups, but was reactivated to a greater extent in DCA-treated animals at 60 min and 4 h reperfusion. These results demonstrate that DCA had no effect on the initial recovery of metabolites following transient ischemia. However, later in reperfusion, DCA enhanced the postischemic reactivation of PDH and prevented the secondary failure of energy metabolism in caudate nucleus. Thus, inhibition of PDH may limit the recovery of energy metabolism following cerebral ischemia.     

Noradrenaline Metabolism in Neocortex and Hippocampus Following Transient Forebrain Ischemia in Rats: Relation to Development of Selective Neuronal Necrosis

Noradrenaline (NA) metabolism in the neocortex and hippocampus was examined in rats at 1, 24, and 48 h following 15 min of reversible forebrain ischemia. As assessed by the ratio of accumulated 3,4-dihydroxyphenylalanine (DOPA) to the tissue NA level after inhibition of DOPA decarboxylase, the NA turnover rates were markedly increased (120-148% above the control) at 1 h postischemia in both the neocortex and hippocampal formation (CA1 and CA3 plus dentate gyrus). The DOPA:NA ratio went back to control levels after longer postischemic survival times. The ratio between levels of the deaminated NA metabolite, 3,4-dihydroxyphenylethyleneglycol (DOPEG), and NA, which gives another measure of NA turnover rate, showed similar changes. In the neocortex and the CA3 plus dentate gyrus, the DOPEG:NA ratio was markedly increased (89-118%) 1 h after the ischemia, but this change had disappeared at 24 and 48 h. Thus, both the DOPA accumulation experiments and the NA and DOPEG measurements indicate that following transient forebrain ischemia, there is an increased NA turnover in the hippocampus and cortex only in the early recirculation period and not after longer postischemic survival times. The degree of neuronal necrosis in the CA1 region was examined light microscopically on celestine blue-acid fuchsin-stained sections at 24, 48, and 96 h following the ischemic insult. The neuronal damage in CA1 was sparse after 24 h of recovery, had increased markedly after 48 h, and was very pronounced at 96 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ascorbate-Stimulated Lipid Peroxidation in Human Brain Is Dependent on Iron but Not on Hydroxyl Radical

Abstract: The time dependence of N -acetyl-aspartate (NAA) concentrations relative to lactate and pyruvate in the injured rat spinal cord was investigated. Segments of spinal cord from regions rostral, caudal, and at the epicenter of the injury were analyzed. NAA concentrations were determined by gas chromatography-mass spectrometry and lactate and pyruvate concentrations were determined by UV spectroscopy at 20 min, 60 min, 2 h, 8 h, 24 h, 3 days, and 1 week after injury. NAA levels fell most significantly at the epicenter of the injury, reaching 30% of basal levels within 24 h. In all segments, lactate levels increased significantly shortly after injury, peaking at two to five times normal basal levels between 20 and 60 min after injury. Rostral and caudal to the injury site, lactate elevations and NAA reductions were less dramatic. Pyruvate concentrations were not significantly altered in any of the sections after injury. The temporal and spatial relationships of NAA and lactate changes indicated that ischemic conditions due to injury in the upper thoracic rat spinal cord were distributed asymmetrically. Acute ischemia was more severe caudal to the injury site, and NAA concentrations were more severely impaired in the rostral direction. The results suggest that the extent of neuronal degeneration due to spinal cord injury does not correlate directly with acute ischemic severity as measured by the lactate/pyruvate ratio, and may be more closely related to secondary changes in the neuronal environment.     

Intracellular Calcium Dynamics and Cellular Energetics in Ischemic NG108-15 Cells Studied by Concurrent 31P/19F and 23Na Double-Quantum Filtered NMR Spectroscopy

Abstract: The role of voltage-sensitive Ca²⁺ channels in mediating Ca²⁺ influx during ischemia was investigated in NG108-15 cells, a neuronal cell line that does not express glutamate-sensitive receptor-mediated Ca²⁺ channels. Concurrent ³¹P/¹⁹F and ²³Na double-quantum filtered (DQF) NMR spectra were used to monitor cellular energy status, intracellular [Ca²⁺] ([Ca²⁺]_i), and intracellular Na⁺ content in cells loaded with the calcium indicator 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5FBAPTA) during ischemia and reperfusion. Cells loaded with 5FBAPTA were indistinguishable from unloaded cells except for small immediate decreases in levels of phosphocreatine (PCr) and ATP. Ischemia induced a steady decrease in intracellular pH and PCr and ATP levels, and a steady increase in intracellular Na⁺ content; however, a substantial increase in [Ca²⁺]_i (about threefold) was seen only following marked impairment of cellular energy status, when PCr was undetectable and ATP content was reduced to 55% of control levels. A depolarization-induced increase in [Ca²⁺]_i could be completely blocked by 1 µM nifedipine, whereas up to 20 µM nifedipine had no effect on the increase in [Ca²⁺]_i seen during ischemia. These data demonstrate that voltage-gated Ca²⁺ channels do not mediate significant Ca²⁺ flux during ischemia in this cell line and suggest an important role for Ca²⁺_i stores, the Na⁺/Ca²⁺ antiporter, or other processes linked to cellular energy status in the increase in cytosolic Ca²⁺ level during ischemia.     

丹参酮Ⅱ－A磺酸钠对鼠肝线粒体功能的影响

利用大鼠肝脏线粒体为材料,以琥珀酸为底物,研究了不同浓度的丹参酮Ⅱ－Ａ磺酸钠对线粒体态４、态３呼吸及呼吸控制率,线粒体跨膜电位,线粒体呼吸链复合体（Ⅱ＋Ⅲ）电子传递及质子转移活性的影响。结果证明丹参酮ⅡＡ－磺酸钠是线粒体呼吸链复合体（Ⅱ＋Ⅲ）的有效抑制剂。文中对丹参酮ⅡＡ－磺酸钠在心肌缺血再灌注过程中的保护作用的分子机理进行了讨论。