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Notes: Abstract: Thirty minutes of insulin-induced reversible hypoglycemic coma (defined in terms of cessation of EEG activity) was produced in anesthetized rats. At the end of the hypoglycemic coma or after recovery for 3, 24, or 72 h induced by glucose infusion, the animals were reanesthetized and their brains frozen in situ. Two control groups were used: untreated controls without prior manipulations, and insulin controls, which received injections of insulin followed by glucose infusion to maintain blood glucose within the physiological range. The brains of these latter animals were frozen 3, 24, or 72 h after glucose infusion. Tissue samples from the cortex, striatum, hippocampus, and thalamus were taken to measure ornithine decarboxylase (ODC) activity, and putrescine and spermidine levels, as well as phosphocreatine (PCr), ATP, glucose, and lactate content. In addition, 20-μm thick coronal sections taken from the striatum and dorsal hippocampus were used for histological evaluation of cell damage and also stained for calcium. Insulin in the absence of hypoglycemia produced a significant increase in ODC activity and putrescine level but had no effect on the profiles of energy metabolites or spermidine. During hypoglycemic coma, brain PCr, ATP, glucose, and lactate levels were sharply reduced, as expected. Energy metabolites normalized after 3 h of recovery. In the striatum, significant secondary decreases in PCr and ATP contents and rises in glucose and lactate levels were observed after 24 h of recovery. ODC activity, and putrescine and spermidine levels were unchanged during hypoglycemic coma. After 3 h of recovery, ODC activity increased markedly throughout the brain, except in the striatum. After 24 h of recovery, ODC activity decreased and approached control values 2 days later. Putrescine levels increased significantly throughout the brain after reversible hypoglycemic coma, the highest values observed after 24 h of recovery (p≤ 0.001, compared with controls). After 72 h of recovery, putrescine levels decreased, but still significantly exceeded control values. Reversible hypoglycemic coma did not produce significant changes in regional spermidine levels except in the striatum, where an approximately 30% increase was observed after 3 and 72 h of recovery (p≤ 0.01 and p≤ 0.05, respectively). Twenty-four hours after hypoglycemic coma, intense calcium staining was apparent in layer III of the cerebral cortex, the lateral striatum, and the crest of the dentate gyrus. After 72 h of recovery, the intense calcium staining included also cortical layer II, the septal nuclei, the subiculum, and the hippocampal CA1-subfield. Changes in polyamine metabolism thus preceded the intense calcium staining in the brain. The results indicate that reversible hypoglycemic coma induces a sharp increase in putrescine level comparable to that observed previously after cerebral ischemia. We, therefore, conclude that the increase in putrescine content is an early biochemical marker of delayed neuronal cell necrosis irrespective of the pathogenesis of this injury. The possible role of polyamines in the manifestation of neuronal necrosis following hypoglycemic coma is discussed.

Notes: Abstract: Biosynthesis and accumulation of the polyamines putrescine, spermidine, and spermine are closely associated with cellular growth processes. We examined polyamine levels and the activity of their first rate-limiting enzyme, ornithine decarboxylase (ODC), in stereotactically induced experimental gliomas of the rat brain 1 and 2 weeks after implantation. Regional ODC activity and polyamine levels were determined in the tumor and in the ipsi- and contralateral striatum, white matter, and cerebral cortex. In the tumor, both ODC activity and polyamine levels markedly increased with progressive tumor growth, as compared to those in the white matter of the opposite hemisphere. In the peritumoral brain tissue, ODC activity did not change, but there was a marked increase of putrescine and, to a lesser degree, of spermidine and spermine almost throughout the whole ipsilateral hemisphere. ODC activity, therefore, seems to be a reliable marker of neoplastic growth in the brain, which may be of use for new clinical concepts of the diagnosis and therapy of brain tumors. The more diffuse distribution of polyamines, however, may be associated with the formation and spreading of edema, which would explain some of the biological effects of tumors on distant brain tissue.

Notes: Abstract: A biochemical method is described for the simultaneous quantitative estimation of unidirectional blood-brain amino acid influx and protein biosynthesis in individual structures of the rat brain. The method involved a double labeling experiment started by the administration of [14C]carboxyl-labeled amino acids and terminated 2 min after infusion of 3H-labeled amino acids, each at tracer quantities, the total labeling period being 45 min. Specific radioactivities of 14C- or 3H-labeled phenyl-alanine, tyrosine, leucine, isoleucine and valine were determined in plasma and in small brain tissue samples for free amino acids, aminoacyl-tRNAs and proteins. Amino acids were converted to their corresponding 5-dimethylamino-naphthalenesulfonyl (Dns, dansyl) derivatives and separated on HPLC C18 reversed-phase columns isocratically according to a newly developed optimizing procedure. The order of influx values between the neutral amino acids in relation to each other was Leu 〉 Tyr 〉 Ile 〉 Phe 〉 Val in every structure examined. Although aminoacylation of tRNAs was found to proceed to a comparable degree for neutral amino acids in all regions investigated, the specific radioactivity of amino acids attached to tRNAs differed substantially from that in the free amino acid pool, especially for leucine and valine. The results indicate the necessity of aminoacyl-tRNA determinations for tracer incorporation studies in protein synthesis analysis. Relative protein synthesis rates in the halothane-anesthetized rat were determined to be 30 and 67–91 pmol total amino acid incorporation/min/mg tissue for white and gray matter, respectively.

Notes: Summary Transient arrest of the cerebral circulation leads to neuronal cell death in selectively vulnerable regions of the central nervous system. It has recently been shown at the light microscopical level that neuronal necrosis is accompanied by a rapid microglial reaction in ischemia (Gehrmann et al. (1992) J. Cereb. Blood Flow Metab. 12:257–269). In the present study we have examined the postischemic microglial reaction in the dorsal rat hippocampus at the ultrastructural level using immuno-electron microscopy. Global ischemia was produced by 30 min of four-vessel occlusion and the microglial reaction then studied after 8, 24 and 72 h. In sham-operated controls microglial cells were not phagocytic; they were randomly distributed throughout the neuropil and occasionally made contacts with other structures such as dendrites in CA1. Ultrastructural signs of activation were observed from 1 day postlesion onward. Reactive microglial cells were consistently seen to phagocytose degenerating neurons particularly in the CA1 stratum pyramidale and in the CA4 sector. They were sometimes interposed between two morphologically distinct types of CA1 neurons, i.e., “dark” (degenerating) and “pale” (surviving) types of neurons. Phagocytic microglial cells also became positive for major histocompatibility complex (MHC) class II antigens at these locations from 1 day after ischemia onward. Furthermore, activated microglial cells were frequent along degenerating dendrites in the stratum radiatum of CA1. After survival times of up to 72 h microglial cells, but not astrocytes, were occasionally observed to undergo mitosis. In addition to their random distribution across the neuropil, microglial cells were frequently observed in a perivascular position under normal conditions. These perivascular microglial cells rapidly expressed MHC class II antigens, extended broad cellular processes and showed signs of phagocytic activity from 1 day onward. These results demonstrate that upon ischemic injury microglial cells proliferate and are rapidly recruited to the site of injury. By virtue of their pronounced cytotoxic potential, microglial cells could be further involved in mediating tissue destruction in ischemia, thus constituting the main immuneffector cell population in this pathological state.

Notes: Abstract The temporospatial relationship between microglial and astrocytic reactions and delayed thalamic cell death was examined 1–7 days following a traumatic cold lesion of the rat sensorimotor cortex using immunocytochemistry in combination with terminal deoxynucleotidyltransferase-mediated biotinylated dUTP nick end labeling (TUNEL) of nuclear DNA fragmentation. No or only occasional TUNEL-positive cells were found in the thalamic relay nuclei up to 3 days after trauma. After 7 days, on the other hand, a considerable number of TUNEL-positive cells were seen in the ventrobasal, the ventrolateral and posterior thalamic nuclei. Already 3 days after trauma, i.e., before cell injury was detectable, many protoplasmic astrocytes, which were reactive for glial fibrillary acidic protein, and ramified microglia, which were positive for complement receptor type 3b (CR3b) but negative for major histocompatibility complex (MHC) class II antigen, were noticed in the thalamus. The number of labeled astro- and microglia further increased after 7 days, when DNA fragmentation became evident. At this time, the morphology of microglia shifted towards bushy and rod-like cells, and microglia became also reactive for MHC class II antigen. Clusters of CR3b- and MHC class II-positive microglia were found in the ventrobasal thalamus. The present findings demonstrate that trauma-induced microglial and astrocytic reactions appear in the thalamus prior the onset of cell damage.

Notes: Summary The incorporation ofl-[3-3H]tyrosine into cat brain proteins was investigated after 1 h of complete ischemia and 7 h of recirculation. Autoradiographs from the cerebral hemispheres, the brain stem and cerebellum revealed that the vast majority of neuronal and glial cells had resumed protein synthesis. Focal reduction of amino acid incorporation was restricted to a few cortical areas within the cerebral hemispheres. A significant number of neurons with no detectable [3H]tyrosine incorporation was only found in the dentate gyrus. The postischemic recovery of protein synthesis supports electrophysiological findings which indicate that nerve cells may survive extended periods of ischemia if the cerebral circulation can be adequately restored.

Notes: Summary A study was made on the effect of ischemia on the vascular permeability to proteins in the cat brain. Evans blue and horseradish peroxidase were used as protein tracers. They were intravenously injected and localized by fluorescence and electron microscopy. Acute complete cerebral ischemia produced by arterial ligations for 15 min to 3h did not induce extravasation of the tracers. Electron microscopical observations on the cortical vessels showed that this was due to a maintained barrier function of the vascular endothelium. Incomplete cerebral ischemia of corresponding duration produced by an arteriovenous shunt (between one common carotid artery and one femoral vein) caused exceptionally extravasation around cortical vessels. Some cats with long shunting time showed signs of increased vascular permeability in the thalamus where the tracers had accumulated in neurons. Severe swelling of capillary endothelial and perivascular glial cells and changes of their cytoplasmic organelles were present in animals without signs of increased vascular permeability to proteins. A discrepancy therefore exists between the maintained impermeability of the capillary endothelium to protein tracers and the ultrastructural changes of the endothelial cell cytoplasm following acute ischemia.

Notes: Summary A study was made on the influence of ischemia on the passage of protein tracers across capillaries in certain blood-brain barrier (BBB) injuries. Total cerebral ischemia was produced by arterial ligation and BBB injury was caused by intracarotid injection of mercuric chloride or by acute hypertension. Changes in the vulnerability of the BBB was correlated with the ischemic impact on the brain as revealed by suppression of electroencephalogram (EEG) or of the pyramidal response (PR) after electrical stimulation of the sensorimotor cortex. In cats not subjected to ischemia both the chemical and the hemodynamic lesions invariably caused extensive extravasation of protein tracers (Evans blue and peroxidase). Episodes of ischemia immediately before the chemical and the hemodynamic lesions, long enough to supress the EEG but not the PR, did not change the pattern of protein extravasation. However, when the duration of ischemia was severe enough to suppress both the EEG and the PR (about 9 min) chemical and hemodynamic insults failed to elicit exudation of proteins. Traumatic lesions caused extravasation regardless of the duration of preceding ischemia. The inhibition of protein extravasation induced by ischemia in the chemical and the hemodynamic lesions is probably related to changes in the vascular endothelium but their exact nature remains obscure.

Notes: Abstract The behaviour of both microflow and evoked potentials was investigated in the right somatomotor cortex of the cat (anaesthetized with chloralose) during electrical stimulation of the contralateral left forepaw. Frequency, amplitude, and time of stimulation were varied. Using the local hydrogen clearance method the changes of microflow were continuously monitored in the same cortical area from which the evoked potentials were recorded. The experiments have shown that activation of the somatomotor cortex by somatic stimulation of the contralateral forepaw results in changes of microflow which clearly correlate to the side and amplitude of the primary evoked potentials. An increase in flow as well as in amplitude of the potentials depends on the stimulation parameters. The changes of microflow are limited to a small area of 1–2 mm in diameter. We conclude that a tight coupling of flow to functional activity exists in the microcirculatory range.