Brain fixation for acetylcholine measurements.

Brain fixation using a commercially available microwave oven (power output: 750 W) has been investigated as a means for enzyme inactivation preventing post-mortem changes in brain acetylcholine (ACh) and choline (Ch) levels. Rats and mice were decapitated, and the severed heads immediately irradiated for 5.5 and 3 s, respectively, resulting in a complete inactivation of brain acetylcholine esterase (AChE) and choline acetyltransferase (ChAT). The ACh and Ch contents measured in various brain regions of rat and mouse were: (in rat) striatum 60.5 and 32.4 nmol/g, hippocampus 20.4 and 30.9 nmol/g, cortex 24.2 and 19.6 nmol/g; (in mouse) striatum 70 and 47.2 nmol/g, hippocampus 22. 1 and 30.2 nmol/g, cortex 22.9 and 27.9 nmol/g. These values were found in accordance with those reported in the literature by irradiating whole animals in instruments of higher power capabilities. Thus, the procedure described in the present work may be a simple and valuable means of brain fixation for neurochemical studies of brain ACh in small animal species.

Cerebral ischemia: changes in brain choline, acetylcholine, and other monoamines as related to energy metabolism.

The relationship of cerebral neurotransmitters acetylcholine (ACh), noradrenaline (NA), dopamine (DA), 5-hydroxytryptamine (5HT) to the energy state of the brain was examined in mice at various times following complete ischemia produced by decapitation, in gerbils submitted to transient global ischemia (10 min bilateral carotid artery occlusion, 5 or 30 min recirculation), and in rats 24 hr after irreversible microembolism. Ischemia caused significant reductions in brain monoamine concentrations. The alterations in NA, DA, and 5HT levels persisted during recirculation and were unrelated to energy restoration. They were accompanied by an increase in the concentrations of related metabolites, suggesting that synthesis was unable to compensate for the release of the transmitters at early post-ischemic time periods. As described for the catecholamines and 5HT, ischemia resulted in a significant decrease in ACh level, but recirculation was associated with a rapid increase in ACh concentration. Impaired synthesis and/or increased release of ACh can be responsible for the decrease in ACh concentration during ischemia. Early post-ischemic elevation of ACh may be related to the large increase in brain choline brought about by ischemia.

Lipid metabolism, cerebral metabolic rate, and some related enzyme activities after brain infarction in rats.

Multiple infarcts were produced in cerebral hemispheres of rats by injecting calibrated 50-micron microspheres into the left internal carotid artery, and alterations in lipid and energy metabolism were evaluated 24 hours later in the embolized hemisphere. Total phospholipid content was decreased by 26%, but the different classes of phospholipids were not equally affected. Phosphatidylinositol and phosphatidylserine levels were decreased by about 40% and phosphatidylcholine and phosphatidylethanolamine by 25%, while sphingomyelin level remained unchanged. There was a 3.2-fold increase in total free fatty acid content with a relatively larger rise in polyunsaturated free fatty acids 20:4 and 22:6 (20-fold increase). Determination of enzyme activities showed decreases in Na+,K+-ATPase (-21%) and hexokinase (-14%) but no changes in phosphofructokinase and pyruvate kinase. Study of energy metabolism using the closed system method of Lowry et al showed a significant depression (-36%) of the cerebral metabolic rate. Taken together, these data suggest a relation between lipid alterations and dysfunction of energy metabolism. Phospholipid degradation with subsequent free fatty acid release and alteration in membrane-bound enzymes may have a direct effect on metabolic machinery and may slow cerebral metabolic rate.

[Vascular reactivity to carbon dioxide in the acute stage of cerebral infarction in rats]. / Réactivité vasculaire à l'anhydride carbonique dans la phase aiguë de l'infarctus cérébral chez le Rat.

The effect of CO2 on the cerebral circulation was assessed 24 hours after induction of unilateral brain infarction performed in the rat by injecting radioactive calibrated 50 microns microspheres into the internal carotid artery. The intracerebral distribution of microspheres and regional cerebral blood flow were measured bilaterally in 8 brain regions. In control rats, increase in arterial pCO2 to about 80 mm Hg resulted in 30 to 100% increase in flow according to the area. Cerebral blood flow was also enhanced in the embolized rats, the basal values being multiplied by a factor 1.7 in the embolized hemisphere and by a factor 1.8 in the contralateral hemisphere. These results do not provide evidence for the existence of a "steal" phenomenon between the non infarcted and infarcted hemispheres.

Comparison of the effects of hypertonic glycerol and urea on brain edema, energy metabolism and blood flow following cerebral microembolism in the rat. Deleterious effect of glycerol treatment.

Cerebral microembolism was performed in rats by injecting radioactive calibrated 50 mu microspheres into the left internal carotid artery. The use of radioactive microspheres as embolic agents enabled the number of microspheres to be determined in each cerebral hemisphere. Edema was assessed 24 h after embolization by measuring brain water, sodium, and potassium content. Equiosmolal doses (40 mmol/kg) of glycerol or urea were injected i.p. at various times before sacrifice. Both treatments caused similar changes in water and electrolyte content, brain dehydration being maximal 30 min after urea and 2 h after glycerol injection. Cerebral energy metabolism and regional blood flow were evaluated at the times of maximal brain dehydration. Urea treatment resulted in an improvement of the cerebral circulation whereas glycerol treatment led to a deterioration of cerebral blood flow which cannot be explained by failure to reduce edema and the consequent microcirculatory impairment. Urea treatment had no marked effect on cerebral energy metabolism whereas glycerol injection resulted in an important increase in brain lactate level which may be relevant to the impairment of cerebral reperfusion. These results point out that administration of a metabolized solute like glycerol may exert deleterious effects on the ischemic brain.

[Release and metabolism of brain noradrenaline during experimental ischemia. Effect of glycerol treatment]. / Libération et métabolisme de la noradrénaline cérébrale après réalisation d'une ischémie expérimentale. Influence du traitement par le glycérol.

The release and catabolism of brain NA were studied after labelling of endogenous NA stores with 3H NA in rats submitted to cerebral ischemia by injecting calibrated microspheres into the internal carotid artery. Twenty four hours after embolization, both NA release and catabolism were found to be reduced. They were restored after treatment with hyperosmolar solution of glycerol.

Catecholamine levels and turnover during brain ischemia in the rat.

Unilateral brain ischemia was induced in the rat by injecting radioactive microspheres into the left internal carotid artery. The microspheres were mainly distributed in the left cerebral hemisphere which contained 8 to 10 times more microspheres than the contralateral hemisphere. Embolization caused dopamine (DA) and noradrenaline (NA) depletion only in the left hemisphere. NA levels were already reduced 2 hours after injury while DA was still unaltered after 6 hours. A 30--40% depletion was observed for the two amines after 24 hours. Catecholamine turnover was estimated by measuring the amine depletion after synthesis inhibition with alpha-methyl-p-tyrosine. During the first 2 hours following embolization, DA and NA depletions were slightly increased only in the left hemisphere, indicating an increase in catecholamine efflux. At times 24 hours, an important retardation in amine disappearance after synthesis inhibition was found for DA and NA in the left hemisphere and to a lesser extent for DA in the right hemisphere, suggesting a reduction of the physiological activity of catecholaminergic neurons. These biochemical alterations can be related to the post-stroke behavioural changes of the embolized animals which exhibited an initially increased motor activity followed by a lethargic state.

Influence of various agents on the development of brain edema in the rat following microembolism. Protective effect of gamma-butyrolactone.

Brain edema was induced in rats by injecting 50 mu microspheres, labelled with 85Sr, into the internal carotid artery. The use of radioactive microspheres as embolic agents enabled the number of microspheres to be determined in each cerebral hemisphere. Edema was assessed 12 or 24 h after embolization by measuring brain water content and, in some experiments, sodium and potassium. Pretreatments with dexamethasone, parachlorophenylalanine (an inhibitor of 5-hydroxytryptamine synthesis), mepyramine and metiamide (H1 and H2 histamine receptor antagonists) or aminophylline did not influence significantly the development of brain edema evaluated 24 h after embolization. Aminophylline treatment (100 mg/kg) markedly increased mortality following embolization. Gamma-butyrolactone (300 mg/kg, every 2 h) inhibited significantly the development of brain edema evaluated 12 hours after embolization. Increases in water and sodium in the embolized cerebral hemisphere were reduced by about 50%. This protective effect may be related to the known depressant action on brain metabolism.

Brain edema and blood-brain barrier permeability following quantitative cerebral microembolism.

Cerebral microemboli were formed in rats by injecting 4,000 carbonized microspheres, 50 +/- 10 mu in diameter, labelled with 85Sr, into the internal carotid artery. The use of radioactive microspheres as embolic agents enabled the number of microspheres to be determined in each cerebral hemisphere. The microspheres were mainly distributed in the cerebral hemisphere on the side of the injection. In 61 rats this hemisphere contained 582 +/- 20 microspheres against 99 +/- 9 in the contralateral hemisphere. Brain edema was assessed by measuring brain content of water, sodium and potassium. Blood-brain barrier (BBB) permeability was determined by brain accumulation of 125I-albumin. In the ipsilateral hemisphere brain edema and an increase in BBB permeability appeared 6 hours after embolization and progressed up to 48 hours. Twenty-four hours after embolization, significant correlations were observed between the microsphere content of the cerebral hemispheres and 1) the increases in water and sodium levels, 2) the decrease in potassium level, 3) the increase in BBB permeability. The study of these correlations should make it possible to ignore the poor reproducibility of embolizations and to analyze with increased accuracy the results of various experiments.

Time-dependent changes in the rate of noradrenaline synthesis in various rat brain areas during cold exposure.

The synthesis of noradrenaline (NA) was studied in vivo in the hypothalamus, the brain stem and the "rest of brain" of rats which were exposed to 4 degrees C for 0.5, 2.5 or 24 h. The rate of NA synthesis was estimated 30 min after an i.v. injection of 3H tyrosine (TY) by evaluation of the ratio: 3H-NA specific activity/3H-TY specific activity. Cold exposure did not have the same effect on NA synthesis in the three brain areas. In the hypothalamus, the rate of NA synthesis was increased by a factor of 1.7 and 2 after 0.5 and 2.5 h of cold exposure, respectively and returned to control values after 24 h of cold exposure. In the brain stem, NA synthesis was enhanced by a factor of 1.5 at 2.5 h and returned also to control values at 24 h. In the "rest of brain", cold exposure did not alter NA synthesis at the three intervals studied. These results indicate that the activation of central noradrenergic neurones by cold is only a transient response which is restricted to specific brain areas and which can be interpreted as the consequence of an initial general stress associated with cold exposure.

Evolution in vivo of the synthesis rate of catecholamines in various peripheral organs of the rat during cold exposure.

The synthesis of catecholamines (CA) has been studied in the heart, spleen, submaxillary glands and adrenals of rats exposed to 4 degrees C for 2.5, 24 or 48 h. The synthesis rate has been estimated 30 min after an i.v. injection of 3H tyrosine (TY) by the evaluation of the ratio: 3H-CA specific activity/3H-TY specific activity. In the sub-maxillary glands, cold exposure reduced the noradrenaline (NA) synthesis by 40% at times 24 and 48 h. In the spleen, NA synthesis was multiplied by a factor 1.6 at times 2.5 and 24 h and 2.8 at time 48 h. In the heart, it was increased by a factor 1.3 after 2.5 h, 2.8 after 24 h and 5.5 after 48 h: an important fall in cardiac NA level was observed during the first 24 h of cold exposure indicating that the synthesis capability was unsufficient to compensate the cold-induced NA release. In the adrenals, adrenaline + NA synthesis was not significantly enhanced during the first 24 h of cold exposure and increased by a factor 2.4 at time 48 h. The important increases in CA synthesis which are observed during the 24-48 h interval are likely consecutive to the induction of tyrosine hydroxylase which has been reported in the rat exposed to cold.

[Effect of cold exposure on synthesis of cerebral dopamine (author's transl)]. / Influence de l'exposition au froid sur la synthèse de la dopamine cérébrale

The synthesis of dopamine (DA) has been studied in the striatum and cortex of rats exposed to a temperature of 40C for 2.5 or 24 h. The synthesis rate has been estimated 30 mn after an i.v. injection of 3H tyrosine (TY), by the evaluation of the ratio: 3H-DA specific activity 3H-TY specific activity. Cold exposure modified DA synthesis differently in the two brain areas. In the striatum, DA synthesis was multiplied by a factor of 1.5 after 2.5 h of cold exposure and returned to normal value after 24 h. In the cortex, cold exposure did not significantly change DA synthesis (at any of the two times studied).

[Influence of hypo- and hyperthyroidism on the turnover rate of noradrenaline, dopamine and serotonin in various rat cerebral structures]. / Influence de l'hypo- et de l'hyperthyroidie sur le taux de renouvellement de la noradrénaline, de la dopamine et de la sérotonine dans différentes structures cérébrales du rat

The effect of chronic treatment with tyroxine (T4) or propylthiouracile (PTU) on the turnover of norepinephrine (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT) has been studied in various areas of the rat brain (brain stem, hypothalamus, striatum and "rest of the brain"). The turnover of NE and DA was determined by the decay in endogenous levels after inhibition of tyrosine hydroxylase by alpha-methylparatyrosine and the turnover of 5-HT was evaluated by the initial accumulation of endogenous 5-HT after inhibition of monoamine oxydase by pargyline. T4 treatment accelerated the release of DA from the striatum but had no significant effects on NA release in the various cerebral areas : nevertheless the NE endogenous level was significantly reduced in the brain stem. PTU treatment delayed the release of DA and NA only from the "rest of the brain". Concerning 5-HT, the only significant variation was observed in the hypothalamus of PTU-treated rats and implied increased turnover. The possible relations between the changes in cerebral monoamines turnover and the behavioural alterations which are observed in thyroid disfunction are discussed.