During brain ischemia temperature spontaneously declines. In animal experiments this decline is frequently prevented by stabilizing the temperature at the pre-ischemic level, using an external heat source. The present study examines whether this procedure influences the severity of ischemic injury. Wistar rats were submitted to 30-min four-vessel occlusion followed by 7 days recirculation. During ischemia and the 1st h of recirculation various systemic and electrophysiological variables were recorded. Seven days after the ischemia brains were perfusion-fixed for light microscopical examination. Three brain temperature profiles were compared: spontaneous decline of brain temperature during ischemia from 36 degrees to 31 degrees C (spontaneous hypothermia; n = 5); constant brain temperature of 30 degrees C induced by selective head cooling (induced hypothermia; n = 5); and constant brain temperature of 36 degrees C induced by selective head heating (normothermia; n = 5). Core temperature was maintained constant at 37 degrees C in all groups. In spontaneous hypothermia, 19% of CA1 neurons survived after 30-min ischemia. Induced hypothermia significantly increased this percentage to 69% (P < 0.05); maintenance of brain temperature at normothermia decreased neuronal survival to 1%. Normothermia also led to morphological injury outside the vulnerable regions, an increase in mortality, marked loss of body weight and a prolongation of the electroencephalographic suppression. These findings demonstrate that stabilizing brain temperature at a constant normothermic level by an external heart source introduces an aggravating pathological element that may interfere in an unpredictable way with the manifestation or treatment of ischemic injury.

Regional protein synthesis of brain was measured by quantitative autoradiography in normo- and hypothermic rats submitted to 30 min of four-vessel occlusion. The tracer, [14C]leucine, was applied by controlled intravenous infusion to achieve constant plasma specific activity, and the admixture by proteolysis of unlabeled amino acids to the brain amino acid precursor pool was corrected by measuring the ratio of the labeled-to-unlabeled leucine distribution space in plasma and brain. In normothermic rats preischemic protein synthesis rate was 16.0 +/- 3.2, 9.2 +/- 3.4, 15.5 +/- 2.8, and 15.5 +/- 3.1 nmol of leucine/g/min (mean +/- SD) in the frontal cortex, striatum, hippocampal CA1 sector, and thalamus, respectively. After 30 min of ischemia at a constant brain temperature of 36 degrees C and a recirculation time of 1 h, protein synthesis was reduced in these regions to 6, 9, 8, and 36%, respectively. With ongoing recirculation, protein synthesis gradually returned to normal within 3 days in all areas except in the stratum pyramidale of the hippocampal CA1 sector where inhibition of neuronal protein synthesis was irreversible. Lowering of brain temperature to 30 degrees C during ischemia did not prevent the early global postischemic depression of protein synthesis, but promoted recovery to or above normal within 6 h in all areas including the stratum pyramidale of the CA1 sector. Improvement of protein synthesis in the CA1 sector was associated with improved neuronal survival, which increased from 1% in the normothermic to 69% in the hypothermic animals. These observations suggest that the protective effect of mild hypothermia on ischemic injury of the hippocampal CA1 sector is mediated by the reversal of the postischemic inhibition of protein synthesis.

The effect of single or repeated episodes of cerebral ischemia on protein biosynthesis and neuronal injury was studied in halothane-anesthetized gerbils by autoradiography of [14C]leucine incorporation into brain proteins and light microscopy. For quantification of the protein synthesis rate, the steady-state precursor pool distribution space for labeled and unlabeled free leucine was determined by clamping the specific activity of [14C]leucine in plasma, and by measuring free tissue leucine in samples taken from various parts of the brain. Control values of protein synthesis were 14.6 ± 2.2, 5.8 ± 2.3, 14.2 ± 3.1, and 10.0 ± 3.8 nmol g−1 min−1 (means ± SD) in the frontal cortex, striatum, CA1 sector, and thalamus, respectively. Following a single episode of 5 or 15 min of ischemia, protein synthesis recovered to normal in all brain regions except the CA1 sector, where it returned to only 50% of control after 6 h and to less than 20% after 3 days of recirculation. After three episodes of 5 min of ischemia spaced at 1 h intervals, protein synthesis remained severely suppressed in all brain regions after both 6 h and 3 days of recirculation. Inhibition of protein synthesis after 6 h predicted histological injury after 3 days of recirculation. In animals submitted to a single episode of 5 or 15 min of ischemia, histological damage was restricted to the CA1 sector but injury occurred throughout the brain after three episodes of 5 min of ischemia. These observations demonstrate that persisting inhibition of protein synthesis following cerebral ischemia is an early manifestation of neuronal injury. Prevention of neuronal injury requires restoration of a normal protein synthesis rate.

The rate of leucine incorporation into brain proteins was studied in rats with experimental brain tumors produced by intracerebral transplantation of the glioma clone F98. Incorporation was measured with [14C]leucine using a controlled infusion technique for maintaining constant specific activity of [14C]leucine in plasma, followed by quantitative autoradiography and biochemical tissue analysis. After 45 min the specific activity of free [14C]leucine in plasma was 2.5-3 times higher than in brain and brain tumor, indicating that the precursor pool for protein synthesis was fueled both by exogenous (plasma-derived) and endogenous (proteolysis-derived) amino acids. Endogenous recycling of amino acids amounted to 73% of total free leucine pool in brain tumors and to 60-70% in normal brain. Taking endogenous amino acid recycling into account, leucine incorporation was 78.7 +/- 16.0 nmol/g of tissue/min in brain tumor, and 17.2 +/- 4.2 and 9.7 +/- 3.3 nmol/g/min in normal frontal cortex and striatum, respectively. Leucine incorporation within tumor tissue was markedly heterogeneous, depending on the local pattern of tumor proliferation and necrosis. Our results demonstrate that quantitative measurement of leucine incorporation into brain proteins requires estimation of recycling of amino acids derived from proteolysis and, in consequence, biochemical determination of the free amino acid precursor pool in tissue samples. With the present approach such measurements are possible and provide the quantitative basis for the evaluation of therapeutic interventions.

Reversible cerebral ischemia (of 5 min, 15 min, or 3-times 5 min) was produced in 14 Mongolian gerbils by occluding both common carotid arteries. After 72 h of recirculation, brains were frozen and processed for measuring regional levels of the polyamines putrescine, spermidine and spermine using HPLC and fluorescent detector. Ischemia induced a marked increase in putrescine levels throughout the brain, most pronounced after 3-times 5 min ischemia (P less-than-or-equal-to 0.05 - P less-than-or-equal-to 0.001). Spermine levels were significantly reduced, in the hippocampal CA1-subfield after 5 min of ischemia and, in addition, in the striatum and thalamus after 3-times 5 min ischemia. It is suggested that polyamines are released from necrotic neurons and cleared into the blood. Spermine, released from neurons into the extracellular compartment, may bind to the N-methyl-D-aspartate (NMDA) receptor of cells located in close vicinity and may thus render neurons vulnerable to otherwise subtoxic levels of excitotoxins.

Abstract: Regional [14C]leucine incorporation into brain proteins was studied in gerbils after global ischemia for 5 min and recirculation times of 45 min to 7 days, using a combination of quantitative autoradiography and biochemical analysis. After recirculation for 45 min, incorporated radioactivity was reduced to ∼20–40% of control values in all ischemic brain regions. Specific activity of the tracer, in contrast, was increased, a finding indicating that the reduced incorporation of radioactivity was not due to reduced tracer influx from plasma or a dilution of the tracer by increased proteolysis. After recirculation for 6 h, [14C]leucine incorporation returned to control levels in all regions except the CA1 sector of the hippocampus, where it amounted to <50%. After 1 day, protein synthesis in the CA1 sector returned to ∼70% of control values, followed by a secondary decline to <50% after 3 days and returned to near control values after 7 days. Histological evaluations revealed selective neuronal death in the CA1 sector of the hippocampus after 3 days of recirculation. The complex time course of protein synthesis in the CA1 sector suggests a biphasic mode of injury, which may be related to similar changes of calcium homeostasis. The final return to near normal after CA1 neurons have disappeared is explained by astroglial proliferation and demonstrates that at this time protein synthesis is not a marker of neuronal viability.

Abstract: Recent studies have shown marked increases in brain content of neuropeptide Y (NPY) after seizures induced by intraperitoneal injection of kainic acid and after pentylenetetrazole kindling in the rat. We have now investigated possible changes in the rate of biosynthesis of NPY after kainic acid treatment, by using pulse‐labeling of the peptide and by determining prepro‐NPY mRNA concentrations. For pulse labeling experiments, [3H]tyrosine was injected into the frontal cortex, and the incorporation of the amino acid into NPY was determined after purifying the peptide by gel filtration chromatography, antibody affinity chromatography, and reversed‐phase HPLC. At 2 and 30 days after kainic acid treatment, the rate of tyrosine incorporation was enhanced by ∼380% in the cortex. In addition, concentrations of prepro‐NPY mRNA were determined in four different brain areas by hybridization of Northern blots with a complementary 32P‐labeled RNA probe 2, 10, 30, and 60 days after kainic acid treatment. Marked increases were observed in the frontal cortex (by up to 350% of controls), in the dorsal hippocampus (by 750%), and in the amygdala/pyriform cortex (by 280%) at all intervals investigated. In the striatum only a small, transient increase was observed. The data demonstrate increased expression of prepro‐NPY mRNA and an enhanced rate of in vivo synthesis of NPY as a result of seizures induced by the neurotoxin kainic acid.

Transient forebrain ischemia of the gerbil is a widely used experimental model for the study of the phenomenon of selective vulnerability in the CA1 sector of hippocampus. Despite the fact that detailed studies are available on the time course of various hemodynamic [16], metabolic [5,12,14], and neuropathological alterations [2,6,7], the causal relationship between the pathobiochemical processes and the development of neuronal death is still poorly understood.

Cysteamine and its dimeric form cystamine have been applied to the rat striatum by local injection. Both compounds resulted in a dose-dependent decrease of somatostatin levels. Maximal reduction of somatostatin (by about 50%) was obtained at a dose of 50 micrograms of cysteamine or cystamine after about 6 h. All three molecular weight forms of somatostatin--somatostatin-14, somatostatin-28, and the 13,000 molecular weight form of somatostatin--were reduced, as shown by size exclusion HPLC. Injection of radiolabeled cystamine revealed a fast conversion of the compound to cysteamine, suggesting it is active in the monomeric form. The levels of neuropeptide Y, which is colocalized with somatostatin in striatal neurons, failed to be changed by local or intraperitoneal injection of cysteamine, suggesting that this treatment does not affect vesicles of somatostatin/neuropeptide Y neurons.

Abstract: The origin of afferent somatostatin‐containing fibers terminating in medial and ventral parts of the striatum has been investigated by performing various neuro‐chemical and surgical lesions in the rat. Lesions of the anterior hypothalamus, amygdala, and the hippocampal commissure as well as lesions with 6‐hydroxydopamine and 5,7‐dihydroxytryptamine failed to decrease striatal soma‐tostatin levels. However, thermal coagulation of the globus pallidus or knife‐cut lesions performed ventrally to the striatum resulted in significant decreases in striatal somatostatin content. Analysis of the topographical distribution of somatostatin within the striatum after thermal lesions of the globus pallidus as well as after kainic acid‐induced seizures revealed a preferential loss of the peptide in medial and ventral portions of the striatum, the site of terminating afferent somatostatin nerve fibers. The data suggest that the striatal afferent somatostatin‐containing neurons may originate in the area of the globus pallidus.

The mechanism of somatostatin depletion induced by cysteamine [2-mercaptoethylamine (CySH)] was studied in isolated nerve endings (synaptosomes) from rat brain in vitro. A dose-dependent reduction of somatostatin-like immunoreactivity (SLI) was observed which reached its maximal extent (41%) at a concentration of 300 microM CySH after 1-5 min. There was no release of somatostatin into the incubation medium. CySH at concentrations of up to 10 mM did not interfere in the RIA. Among a variety of compounds, structurally related to CySH 4-aminothiophenol, 2-aminothiophenol and N,N-dimethylaminothiol exhibited the highest efficacy in decreasing somatostatin (60%, 50%, 30%, respectively, at 10 mM and 10 min). The disulfide form of CySH cystamine and dimercaprol resulted in about 15% reduction after 10-min incubation, whereas taurine, alanine, cysteine, and mercaptoethanol were inactive. A saturable, sodium-dependent uptake process was found for the disulfide form of [35S]CySH cystamine [Michaelis-Menten constant (Km) = 18.6 microM, maximum velocity (Vmax) = 2.3 nmol/mg protein X 3 min) which was inhibited by cysteine (87% at 1 mM). [35S]CySH, at concentrations of 20 microM or less, was not stable in buffer solution. It underwent considerable nonenzymatic conversion into its dimeric form (60% at 37 C and 3 min), however it exhibited the same kinetic data for its uptake. Size exclusion HPLC of purified hypothalamic synaptosomes revealed a major SLI peak coeluting with synthetic somatostatin-14 and two minor peaks representing somatostatin-28 and a 13,000 mol wt protein. The three molecular forms of somatostatin were reduced to varied extent by CySH (somatostatin-14 by about 70%, somatostatin-28 by 15%, and the high mol wt form by 30%). Our experiments suggest that high affinity uptake of CySH may precede its action in decreasing somatostatin levels. Increased release or inhibition of synthesis of somatostatin have been excluded as possible mechanisms. It is suggested that SLI is equally affected in nerve endings and in perikarya.

Cysteamine (100 micrograms) markedly reduces the number (by about 60%) and intensity of staining of NADPH diaphorase-reactive neurons 6 h after local injection into the striatum. This effect was reversible (after 24 h) and was only observed when the indirect staining procedure was applied in which NADPH formed by endogenous malate dehydrogenase is used. However, no direct effect of cysteamine on the malate dehydrogenase reaction was found. The decrease in NADPH diaphorase activity parallels the previously reported cysteamine induced decrease in somatostatin contained in the same neurons and may point to a biochemical interrelation of somatostatin and NADPH diaphorase in these neurons.

Pronounced changes in the content of cholecystokinin octapeptide (CCK-8) have been found after limbic seizures induced by i.p. injection of kainic acid. Three hours after injection of the toxin a significant decrease in CCK-8 was observed in the frontal cortex and amygdala/pyriform cortex reflecting an increased release during acute seizures. A persistent decrease in the content of the peptide in the amygdala/pyriform cortex suggests destruction of the respective neurons. In the substantia nigra and in the striatum and, more moderately, in the hippocampus and frontal cortex increases in CCK-8 were observed 10 days after injection of kainic acid suggesting an increased synthesis or decreased release of the peptide in these brain areas subsequently to the acute seizures.

The topographical distribution of the proposed amine transmitters dopamine (DA), serotonin (5-HT), noradrenaline (NA), adrenaline and histamine (HA) and of the metabolites of DA and 5-HT has been investigated in the neostriatum of the rat. DA and, less pronounced, its metabolites 3,4-dihydroxyphenylacetic acid, homovanillic acid and 3-methoxytyramine exhibited gradients with highest levels in dorso-rostal areas and the lowest content in the ventro-caudal part of the striatum. In contrast to this 5-HT, 5-hydroxyindoleacetic acid, and NA exhibited levels which increased from frontal and dorsal areas to the ventro-caudal part of the striatum. The rostral nucleus accumbens and the pallidum were low in DA and high in 5-HT and NA when compared with the dorsal striatum. The turnover rates of DA and 5-HT as judged by the metabolite/amine ratios followed a distribution which was opposite to the respective amine levels. Adrenaline was evenly low in the striatum and only slightly higher in the n. accumbens and pallidum. The levels of HA were considerably lower than those of the other amines. Although HA was also unevenly distributed within the striatum, no clear pattern was found. The topographical distribution of the amines suggests a preferential role of DA in the dorsal striatum and of 5-HT and NA in the ventral part of the striatum including the n. accumbens and the pallidum.

The neuropeptides somatostatin, neurotensin and substance P were investigated in rats during and after limbic seizures induced by systemic injection of kainic acid (10 mg/kg, i.p.). Three hours after injection of the toxin, pronounced decreases (40-50%) in somatostatin-like immunoreactivity in frontal cortex, striatum, dorsal hippocampus and amygdala/pyriform cortex were observed. Concomitantly, neurotensin-like and substance P-like immunoreactivities were also reduced in the frontal cortex and the hippocampus. These early decreases in peptide levels may result from increased release and subsequent inactivation of the peptides during acute seizures. At later time intervals, 3, 10 and 30 days after injection of kainic acid, the initially decreased peptide levels were partially normalized. However, the reduction in somatostatin-like immunoreactivity in amygdala/pyriform cortex and striatum persisted up to 30 days. Neurotensin-like immunoreactivity remained decreased in the frontal cortex. On the other hand, neurotensin- and substance P-like immunoreactivities were increased in the striatum and substantia nigra 10-30 days after injection of kainic acid. These late changes in peptide levels may suggest destruction of peptidergic neurons or adaptive changes induced by the convulsions. Pretreatment of rats with cysteamine (100 mg/kg, i.p.), an agent which decreases brain somatostatin levels, had no effect on the intensity of kainic acid induced convulsions, although a slightly earlier onset of seizures was observed. The changes in peptide levels, especially the marked decreases in somatostatin content after systemic injection of kainic acid, suggest considerable acute and chronic alterations in peptidergic systems caused by limbic convulsions.

The molecular forms of somatostatin contained in the rat striatum were separated by size-exclusion HPLC. Three major peaks of somatostatin-like immunoreactivity (SLI) were resolved. Two peaks cochromatographed with synthetic somatostatin-14 (SS-14) and somatostatin-28 (SS-28), respectively. One peak exhibited a higher molecular weight (about 10,000) and may contain a proform of somatostatin. Local injection of the neurotoxin kainic acid (1 microgram) into the left striatum resulted in a persistent decrease (65-85%) of all three forms of somatostatin. In the contralateral--not injected--striatum a decrease of SLI was also observed which was maximal (45%) after 2 days and was largely abolished after 7 days. This decrease of SLI in the contralateral striatum, however, was due mainly to a decrease of SS-14 and SS-28 but not of the putative proform. Our data suggest that kainic acid causes a destruction of somatostatin-containing perikarya in the injected striatum, whereas in the contralateral striatum increased release with subsequent inactivation of SS-14 and SS-28 takes place. The putative somatostatin proform may serve as neurochemical marker for somatostatin-containing perikarya in the striatum.