Studies of the protein synthesis system in the brain cortex during global ischemia and reperfusion.

Previous studies have demonstrated that brain protein synthesis declines after global ischemia and reperfusion. To investigate the role of the translation system in this phenomenon, we examined the ability of partially purified ribosomes, ribosome-bound mRNA and translation cofactors derived from the transiently ischemic cerebral cortex to synthesize protein in vitro. Samples were prepared from canines subjected to 20-min cardiac arrest and after 2 or 8 h of post-resuscitation intensive care. There was no significant decrease in the rate of in vitro protein synthesis as a consequence of either ischemia or reperfusion. Northern hybridization of ribosome-bound RNA revealed a discrete band of mRNA for brain-specific creatine kinase (ck-bb) that was consistent in presence and intensity in all groups. However, mRNA for heat shock 70 protein (hsp-70) was observed only during reperfusion and markedly increased between 2 and 8 h reperfusion. Thus, we conclude that (1) the transcription system is intact during reperfusion and hsp-70 mRNA is made and translocated to the ribosomes during reperfusion, (2) mRNA for ck-bb is not displaced from ribosomes by the appearance of hsp-70 during reperfusion and (3) isolated ribosomes maintain their ability to translate in vitro during the first 8 h of reperfusion after global brain ischemia. Therefore, the early reduction in protein synthesis observed in vivo during post-ischemic brain reperfusion is not due to an intrinsic dysfunction of the ribosomes.

Fluorescent histochemical localization of lipid peroxidation during brain reperfusion following cardiac arrest.

Rats were subjected to cardiac arrest and resuscitation, 90 min of reperfusion, and in situ perfusion fixation. Thiobarbituric acid (TBA) was included in the aldehyde-free perfusion fixative, the TBA reaction was driven in situ by heating, and fluorescence microscopy was utilized to characterize the location of products of the TBA reaction. Absorbance-difference spectra were performed on butanol-extracted brain homogenates to confirm in situ formation of TBA adducts with aldehydic products of lipid peroxidation. Nissl-stained sections revealed good cellular fixation without shrinkage artifacts. Fluorescence was not seen microscopically when TBA was omitted from the perfusion fixative, and little fluorescence was present in normal brains or brains after ischemia only. However, after 90-min reperfusion, intense granular fluorescence was seen in the neuronal perikarya (especially at the base of the apical dendrite) of numerous pyramidal neurons in cortical layers 5 and 6 and in the pyramidal layer of Ammon's horn in the hippocampus. The nuclei of these cells exhibited no fluorescence. Fluorescence was also present in some striatal neurons, but was absent in the adjacent radial bundles. Neither glia nor white matter exhibited similar fluorescence. These observations indicate that neurons in the selectively vulnerable zones of the cortex and hippocampus are early and specific targets of lipid peroxidation during post-ischemic reperfusion.

Assessment of free radical-induced damage in brain proteins after ischemia and reperfusion.

Brain damage initiated during global ischemia has been shown to be exacerbated by iron-dependent lipid peroxidation during early reperfusion. We hypothesized that other cellular components might be involved in similar free radical reactions. In this study we examined three brain protein fractions and ribosomal RNA for evidence of free radical damage during post-ischemic reperfusion. Global brain ischemia was induced by 20-min cardiac arrest. Dogs were divided into four groups: (1) non-ischemic controls; (2) 20-min cardiac arrest without reperfusion; (3) 20-min cardiac arrest and 2 h reperfusion; (4) 20-min cardiac arrest and 8 h reperfusion. Soluble proteins and proteins from ribosomes and synaptosomes were assayed by a dinitrophenylhydrazine method for carbonyl groups, which are characteristic products of protein peroxidation. The ribosomal RNA was also examined by electrophoresis. When proteins from each fraction were peroxidized in vitro by Fenton reagents, carbonyl content increased as [Fe2+] was increased from 0 to 100 microM. However, following reperfusion there was no significant accumulation of carbonyl content in either the soluble (ANOVA P = 0.92) or ribosome (P = 0.10) protein fractions. There was a significant decrease in the carbonyl content of the synaptosome protein fraction after 8 h of reperfusion (P = 0.03). Similarly, although ribosomal RNA fragmentation was observed in ethidium stained agarose gels following in vitro reaction with Fenton reagents, there was no evidence of ribosomal RNA fragmentation or cross-linking following reperfusion. These results suggest that reperfusion free radical reactions do not involve these cellular proteins or ribosomal RNA.

Estrogen protection against atherosclerosis and synthetic estrogen production of cirrhosis in the rabbit.

Two artificial estrogens, ethinyl estradiol and mestranol, were found to cause cirrhosis in the rabbit liver during a study of atherosclerosis. These drugs showed protective effects against atherosclerosis in cholesterol-fed rabbits when administered orally (1 ppm in diet). These drugs and two naturally occurring estrogens, estrone and estradiol, were similarly effective when administered intramuscularly (1.5 mg per rabbit per week) to rabbits fed a diet lower in cholesterol. Reduction by estrogens of plasma cholesterol did not fully account for the reduction of extent of aortic atherosclerosis. Also, no differences in aortic or platelet eicosanoid production from exogenous arachidonic acid were found to explain the effect on atherogenesis. The 2 artificially modified estrogens, ethinyl estradiol and mestranol, caused portal fibrosis with biliary proliferation (even in rabbits not fed cholesterol). Estrone, estradiol, and testosterone were not injurious in this regard at the dosages used. Only the unmodified estrogens reduced atherosclerosis without damaging the rabbit liver.

The effect of EMHP on post-cardiac arrest survival of rats.

1-Ethyl-2-methyl-3-hydroxy-pyrid-4-one (EMHP), a low molecular weight iron chelator that is soluble in hydrocarbon solvents and presumably in lipids, was studied for in vitro inhibition of radical-mediated peroxidation of DNA. We also investigated the acute toxicity of EMHP by administering 40, 100, and 300 mg/kg intravenously to Wistar rats, and we then examined the in vivo effect of the 40 mg/kg dose following a 10-min cardiac arrest and resuscitation in rats. EMHP prevented iron-dependent radical-mediated DNA breaks of the supercoiled plasmid Bluescribe by the Fenton reagent (400 microM iron, 30 microM H2O2) when present at EMHP/Fe ratios of 16:1 and 32:1. The 300-mg/kg dose was lethal in 3 of 5 normal rats, and the 100-mg/kg dose was associated with excessive mortality post-resuscitation. The 40-mg/kg dose was well tolerated post-resuscitation, but it did not improve either 3-day survival or neurologic outcome.

Brain nuclear DNA survives cardiac arrest and reperfusion.

Iron-mediated peroxidation of brain lipids is known to occur during reperfusion following cardiac arrest. Since in vitro damage to DNA is caused by similar iron-dependent peroxidation, we tested whether free radical damage to genomic DNA also develops during reperfusion following cardiac arrest and resuscitation. Genomic DNA was isolated from the cerebral cortex in (i) normal dogs, (ii) dogs subjected to a 20-min cardiac arrest, and (iii) dogs resuscitated from a 20-min cardiac arrest and then allowed to reperfuse for 2 or 8 h. DNA strand nicks were evaluated by in vitro labeling of newly created 3' and 5' termini. DNA base damage was evaluated utilizing reaction with piperidine prior to labeling of 5' termini. The 3' DNA termini were labeled before and after digestion with exonuclease III, and the 5' DNA termini were labeled before and after treatment with piperidine. In vitro experiments with genomic DNA damaged by oxygen radicals verified that these labeling methods identified radical damage. In the experimental animal groups, terminal incorporation and electrophoretic mobility of brain nuclear DNA are not significantly changed either by 20 min of complete brain ischemia or during the first 8 h of reperfusion. We conclude that genomic DNA is not extensively damaged during cardiac arrest and early reperfusion, and therefore such DNA damage does not appear to be an important early aspect of the neurologic injury that accompanies cardiac arrest and resuscitation.

Sign of coma.

The oblique focusing defect of spherical lenses, known as coma, is described by Seidel as the second of five correction terms (third order) to the Gauss theory. The concept is clear for a refracting surface that is free of spherical aberration; however, the impossibility of eliminating spherical aberration from a single lens with spherical surfaces can confuse the understanding of the second aberration, coma. A way of understanding the comatic effect even in the presence of spherical aberration is described. Confusion over the sign of coma, as indicated by mistakes in optics textbooks, is addressed. This paper suggests corrections to some interpretations of coma found in the literature.

Effect of ethinylestradiol on arachidonic acid incorporation, release, and conversion to prostaglandins in rabbit platelets.

Intramuscular administration to female rabbits of 2 mg/kg ethinylestradiol every other day for 10 days increased the uptake and incorporation of [14C]arachidonic acid into platelet lipids, and increased the proportion of [14C]arachidonic acid released from platelets after stimulation by thrombin. The conversion of [14C]arachidonic acid to thromboxane B2 did not differ between the control and ethinylestradiol-treated groups. Thus, the results of this study indicate that the major site in the prostaglandin metabolic pathway influenced by estrogen is the incorporation and release of arachidonic acid in platelet phospholipids.

Effect of testosterone and oxandrolone on thrombocyte aggregation and synthesis of prostaglandins in thrombocytes and aorta of atherosclerosis-susceptible pigeons.

Effect of intramuscular administration (10 mg/kg/week) of testosterone and oxandrolone on a) thrombocyte aggregation and b) synthesis of prostaglandins from [14C]-arachidonic acid in thrombocytes and aorta of atherosclerosis-susceptible White Carneau pigeon was examined. Neither testosterone nor oxandrolone influenced collagen, ADP and arachidonic acid induced aggregation or the synthesis of prostaglandins in thrombocytes. However, both testosterone and oxandrolone stimulated (p less than 0.05) the synthesis of 6-keto-PGF1 alpha (stable product of prostacyclin) und PGE2 in aorta.