Measurement of local cerebral blood flow with iodo [14C] antipyrine

The autoradiographic diffusible tracer technique for the measurement of local cerebral blood flow was originally designed for use with the radioactive, inert gas 131I-labeled trifluoroiodomethane and is applicable only with tracers that exhibit unrestricted diffusion through the blood-brain barrier. Because of the technical problems associated with the use of gaseous tracers, a suitable nonvolatile tracer has been sought. [14C] Antipyrine has been used previously and found to be unsuitable because of limitations in its diffusion through the blood-brain barrier. An analogue of [14C]antipyrine, iodo [14C]antipyrine, exhibits higher partition coefficients than [14C]antipyrine between nonpolar solvents and water and might, therefore, be expected to diffuse more freely through the barrier. Its use as the tracer in the local blood flow technique leads to values considerably above those obtained with [14C]antipyrine in the rat and cat and essentially the same as those obtained with the gas trifluoro[131I]iodomethane in the cat. Iodo[14C]antipyrine appears, therefore, to be a satisfactory nonvolatile tracer for the measurement of local cerebral blood flow.     

The hydrogen method of measuring local blood flow in subcortical structures of the brain: Including a comparative study with the 14C antipyrine method

Summary A method of measuring local cerebral blood flow, on the basis of inhalation of hydrogen gas and subsequent recording of local clearance curves of hydrogen by means of platinum electrodes, has been tested under different experimental conditions. The values of blood flow recorded from the caudate nucleus in awake cats, during barbiturate anesthesia, and during hypercapnia, are in agreement with previously published results. The preservation of the reactivity of cerebral vessels to CO₂, and to changes in perfusion pressure (autoregulation), indicates that the vessels around the electrode tip maintain their physiological reactions.Further validation of the method is based on a direct comparison with blood flow values in the same structure, measured at short intervals of time by means of the autoradiographic method of Reivich and co-workers. No significant difference has been found in the average blood flow values recorded by the two methods.The hydrogen method therefore is reliable for measuring in acute experiments the local blood flow in subcortical structures of the brain.Supported by US-PHS Grant 2 RO 1 NB 05017 NEUA.     

Interaction of [14C]acetylsalicylic acid with normal human peripheral blood lymphocytes.

Therapeutic concentrations of acetylsalicylic acid (ASA) have strikingly inhibited in vitro and in vivo mitogen- and antigen-induced blastogenesis by human lymphocytes. These observations may be pertinent to the anti-inflammatory actions of ASA. To investigate further the possible effects of ASA on cellular responses, we studied the in vitro interaction of [14C]ASA with lymphocytes. Results indicated that the [14C]ASA association with cells was (a) proportional to ASA concentrations, (b) non-saturable at high concentrations of ASA, (c) dependent on pH, (d) independent of temperature, (e) dependent on cell concentration, (f) not consistently displaced by unlabelled ASA or other drugs, (g) rapid and unchanged over 1 min to 72 hr incubations and (h) reversed by repeated cell washing. These data confirmed that ASA indeed interacted with lymphocytes. The association was rapid, reversible, pH-dependent and not demonstrably specific under these experimental conditions.     

Lymphokine-induced uptake of [14C]glucosamine, [14C]glucose,and [3H]leucine by macrophages

Lymphokine-activated (LK⁺) and control (LK^–) macrophages were cultured for 66 h and then pulsed with [¹⁴C]glucosamine. Uptake of [¹⁴C]glucosamine was greater in LK⁺ than in LK^– cultures. If, after 66 h, the medium was replaced with fresh medium and then pulsed with either [¹⁴C]glucose or [¹⁴C]glucosamine, the uptake of isotope was greatly reduced compared to cultures with no change of medium. However, uptake of both radiolabeled substances was still found to be greater in LK⁺ cultures than in LK^– cultures. Although uptake of both substances was enhanced by lymphokines, the uptake kinetics of each isotope was different. Under similar conditions the uptake of [³H]leucine was not enhanced by lymphokine activation. These data are interpreted to mean that LK⁺ macrophages are metabolically stimulated and utilize more glucose and glucosamine. The difference in kinetics implies a different utilization by macrophages for each substance.     

Autoradiography of [14C]paraquat or [14C]diquat in frogs and mice: accumulation in neuromelanin

The herbicide paraquat has been suggested as a causative agent for Parkinson's disease because of its structural similarity to a metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which may induce a parkinsonism-like condition. MPTP as well as its metabolite 1-methyl-4-phenylpyridine have melanin affinity, and the parkinsonism-inducing potency of MPTP is much stronger in species with melanin in the nerve cells. Autoradiography of [3H]MPTP in experimental animals has shown accumulation in melanin-containing tissues, including pigmented neurons. In the present whole body autoradiographic study accumulation and retention was seen in neuromelanin in frogs after i.p. injection of [14C]paraquat or [14C]diquat. By means of whole body autoradiography of [14C]diquat in mice (a species with no or very limited amounts of neuromelanin) a low, relatively uniformly distributed level of radioactivity was observed in brain tissue. Accumulation of toxic chemical compounds, such as paraquat, in neuromelanin may ultimately cause lesions in the pigmented nerve cells, leading to Parkinson's disease.     

Topography of basal glucose utilization in the hippocampus determined with [1-14C]glucose and [6-14C]glucose

The activity of the pentose phosphate shunt was assessed under basal conditions in subregions of the hippocampus by measuring the uptake and retention of [1-14C]glucose and [6-14C]glucose and their 14C-labelled metabolites. The relative and absolute retention of carbon-14 from each of the two compounds was nearly identical in all regions examined. For each compound, the highest accumulation of 14C occurred in the granule cell layer of the dentate gyrus and in the pyramidal cell layer. Relatively high retention of radioactivity was also found in the molecular layer of dentate gyrus and in the stratum lacunosum-molecular. The stratum radiatum and stratum oriens contained the lowest levels of radioactivity among hippocampal regions. The equal retention of radioactivity from [1-14C]glucose and [6-14C]glucose implies that pentose phosphate shunt activity is very low throughout the hippocampus under the conditions of this study. The uptake and retention of radioactivity was evaluated in different hippocampal regions 10 or 30 min following intravenous injection of [1-14C]glucose. Although there was significantly more radioactivity at 30 min than at 10 min, the same topographic pattern of radioactivity within the hippocampus was observed in rats after both survival periods, indicating that an equal fraction of the [1-14C]glucose utilized in different hippocampal regions is oxidized to 14CO2 under these conditions. Most regions of high glucose utilization in the hippocampus determined with [1-14C]glucose and [6-14C]glucose correspond to regions of intense histochemical staining for cytochrome oxidase reported in the literature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative distribution of rat brain monoamine oxidase A by [14C]clorgyline autoradiography

The distribution of functionally active monoamine oxidase type A (MAO-A) was investigated by in vivo quantitative autoradiography using [¹⁴C]clorgyline in normal, conscious rat brain. [¹⁴C]clorgyline was synthesized by the methylation reaction of N-desmethylclorgyline using [¹⁴C]methyliodide. Sixty minutes after [¹⁴C]clorgyline administration (1.58 MBq/animal i.v.), the brains were removed and prepared for autoradiography by washing the brain sections with 5% trichloroacetic acid solution to remove the nonbinding free tracer. The amount of MAO-A was calculated from the regional acid-insoluble tissue radioactivity and the specific activity of the tracer. The highest amount of MAO-A (5.84 nmol/g tissue) was found in the locus coeruleus. The interpeduncular nucleus, habenular nucleus, fasciculus retroflexus, and solitary tract nucleus possessed over 1.6 nmol/g tissue of MAO-A. Among 23 regions of interest, the lowest amount of MAO-A (0.37 nmol/g tissue) was found in the globus pallidus. The findings of this study suggest that the pattern of MAO-A parallels both in neuroanatomical distribution and in density that of norepinephrine and serotonin innervation. The MAO-A concentration was, however, relatively low in the dopamine-related areas. This corresponded to the previous results obtained by histochemical analysis. In addition, among the white matter structures, a high amount of MAO-A was found specifically in the fasciculus retroflexus.     

The incorporation of [3H] thymidine and [14C] glucosamine into a DNA-polysaccharide complex in normal and scrapie-affected mouse brain

Summary Urea treatment of particulate material sedimented by prolonged centrifugation of brain cell sap has shown the presence of a complex of DNA-polysaccharide into which [³H] thymidine is incorporated into DNA thymidylic acid ten times more rapidly in scrapie than in normal mice. Membrane containing, subcellular, fractions liberate apparently identical material after urea treatment which shows a similarly increased [³H] thymidine incorporation in scrapie mouse brain. The evidence suggests that this material replicates in the cell sap of scrapie mouse brain and subsequently binds to membrane. First approximation calculations indicate that the quantity of DNA involved is of the same order as that which would be expected to be present in the scrapie agent present in mouse brain at peak titre.     

The synthesis of lipids from [1-14C]acetate by isolated rat brain capillaries

Lipid biosynthesis was investigated in isolated cerebral microvessels obtained from adult Sprague-Dawley rats using [1-14C]acetate as precursor. All lipid classes were labelled by [1-14C]acetate. Neutral lipids incorporated about 50% of radiolabelled acetate, among which free fatty acids and triglycerids showed the highest level of incorporation. Moreover, about 4% of radioactivity was found in cholesterol fraction. In phospholipid fraction, phosphatidylcholine and phosphatidylethanolamine were the main radiolabelled phospholipids. [1-14C]acetate was also incorporated into sulphatides and cerebrosides. The presence of bovine serum albumin in incubation medium modified the percentage of incorporation in different lipid fractions.     

Comparison of the rate of absorption and proteolysis of [14C]choleragen and [14C]bovine serum albumin in the rat jejunum.

[14C]choleragen was used to study the rate of disappearance of choleragen enterotoxin from the jejunum of rats. [14C]bovine serum albumin (BSA) was studied in a similar manner. Almost one-third of the labeled toxin had disappeared from the intestine after 6 h. Its rate of disappearance was the same in germfree rats as in conventional rats. The rate of proteolysis of [14C]choleragen and [14C]BSA by intestinal mucodal lysosomal enzymes was also studied. Neither was significantly degraded by neutral proteases; however, heat-inactivated toxin was. They were all degraded by acid proteases; however, the rate of BSA proteolysis was only one-third of that of toxin. Soybean trypsin inhibitor had no effect on the in vivo disappearance of toxin nor on the acid proteases. It did inhibit the neutral protease digestion of heat-treated toxin. Aprotinin and protamine inhibited disappearance in loops of gut but had no effect to inhibit degradation rates. Gangliosides inhibited both rates of disappearance and proolysis of toxin. These agents had some different effects on disappearance rates and proteolysis of BSA. The data indicate that cholera enterotoxin is absorbed by intestinal mucosal cells and is degraded by acid proteases in the cells.     

Studies of cerebrospinal fluid flow and penetration into brain following lateral ventricle and cisterna magna injections of the tracer [14C]inulin in rat

Parasynaptic communication, also termed volume transmission, has been suggested as an important means to mediate information transfer within the central nervous system. The purpose of the present study was to visualize by autoradiography the available channels for fluid movement within the extracellular space following injection of the inert extracellular marker [¹⁴C]inulin into the lateral ventricle or cisterna magna. Bolus injections of 5 μl of 1 μCi of [¹⁴C]inulin were made in awake rats via chronically implanted cannulae. After survival times ranging from 5 min to 4 h, brains were processed for in vivo autoradiography. At 5 min the tracer distributed throughout the ventricles, subarachnoid spaces and cisterns “downstream” of the injection sites. Penetration into the brain from these sites was complex with preferential entry along the ventral side of the brain, especially into the hypothalamus and brainstem. By 4 h virtually the entire brain was labeled irrespective of the site of tracer application. Sustained tracer entry from subarachnoid spaces suggests that some areas act as depots to trap circulating material. This mechanism may contribute to the pattern of deep penetration at later time-points. The spatial and temporal characteristics of fluid movement throughout the brain are instructive in the interpretation of many experimental procedures involving injection of molecules into the cerebrospinal fluid.     

Studies of cerebrospinal fluid flow and penetration into brain following lateral ventricle and cisterna magna injections of the tracer [14C]inulin in rat

Parasynaptic communication, also termed volume transmission, has been suggested as an important means to mediate information transfer within the central nervous system. The purpose of the present study was to visualize by autoradiography the available channels for fluid movement within the extracellular space following injection of the inert extracellular marker [14C]inulin into the lateral ventricle or cisterna magna. Bolus injections of 5 microl of 1 microCi of [14C]inulin were made in awake rats via chronically implanted cannulae. After survival times ranging from 5 min to 4 h, brains were processed for in vivo autoradiography. At 5 min the tracer distributed throughout the ventricles, subarachnoid spaces and cisterns "downstream" of the injection sites. Penetration into the brain from these sites was complex with preferential entry along the ventral side of the brain, especially into the hypothalamus and brainstem. By 4 h virtually the entire brain was labeled irrespective of the site of tracer application. Sustained tracer entry from subarachnoid spaces suggests that some areas act as depots to trap circulating material. This mechanism may contribute to the pattern of deep penetration at later time-points. The spatial and temporal characteristics of fluid movement throughout the brain are instructive in the interpretation of many experimental procedures involving injection of molecules into the cerebrospinal fluid.     

Changes in [14C]deoxyglucose incorporation into rat brain regions during heat-seeking behavior in the cold environment

The central nervous structures involved in behavioral thermoregulatory responses during cold exposure were investigated in conscious rats by means of the 2-deoxy-D-[14C]glucose ([14C]-DG) autoradiographic technique. According to autoradiographs, many brain regions with significant increases or decreases in [14C]-DG incorporation were observed during thermoregulatory behavior. When animals were only exposed to cold, significant increases in [14C]-DG incorporation were observed in the caudate putamen, lateral preoptic area, medial forebrain bundle, ventromedial hypothalamus (VMH), posterior hypothalamus, ventroposteromedial thalamus (VPM), dorsomedial thalamus (MD), substantia nigra (SN) and red nucleus (RN). Lower activities were observed in the hippocampus and the medial habenula. However, when animals performed the behavioral thermoregulation (heat seeking behavior) significant increases were noted in the sulcal prefrontal cortex, sensory-motor cortex, and MD, while decreases were noted in the piriform cortex, VMH, VPM, medial habenula, SN and RN, compared with those in the group without the thermoregulatory behavior.     

Metabolic studies on the uptake of [14C]-histidine and [14C]-histamine and histamine synthesis by guinea-pig basophils, in vitro.

The uptake of [14C]-histidine and [14C]-histamine and the conversion of [14C]-histidine to [14C]-histamine was measured in suspensions of guinea-pig bone marrow cells rich in basophils. When comparable amounts of labelled histidine or histamine were added to equal numbers of basophils, the uptake of histidine was approximately forty-five times greater than that of histamine. Purified eosinophils, neutrophils and mononuclear cells incorporated only a small proportion of [14C]-histidine when compared to the basophil; [14C]-histamine uptake by all these cell types was virtually negligible. Histidine uptake and the amount of histamine formed de novo was directly related to the number of basophils, the time of incubation and the substrate concentration. Histidine uptake was decreased by agents which inhibit glycolysis, oxidative phosphorylation, Na + - K + -dependent ATPase, protein synthesis and RNA synthesis. Inhibition was demonstrable in a dose-dependent fashion and at concentrations which had no apparent effect on cell viability. Inhibitors of DNA synthesis, and of microtubule function, had no influence on histidine uptake. Cytochalasin B, an inhibitor of microfilament function, also decreased histidine uptake but only at concentrations previously showen to affect hexose transport. None of the agents tested affected the uptake of [14C]-histamine or the amounts of new histamine formed from the histidine that had been incorporated. These studies suggest that histidine is preferentially incorporated into the basophil; that the uptake depends on the integrity of a number of metabolic pathways, but that once the histidine is taken up these requirements do not apply to the formation of new histamine. In contrast, histamine appeared to diffuse passively, and in relatively small amounts, into all the cell types tested.     

Metabolism of D-[3-3H]glucose, D-[5-3H]glucose, D-[U-14C]glucose, D-[1-14C]glucose and D-[6-14C]glucose in pancreatic islets in an animal model of type-2 diabetes

This study aims at exploring specific aspects of D-glucose metabolism, so far not yet investigated, in pancreatic islets from adult control rats and animals (STZ rats) injected with streptozotocin during the neonatal period. The latter animals, which represent a current model of type-2 diabetes, displayed a lower body weight, higher plasma D-glucose concentration and lower insulinogenic index than control rats. The protein, DNA and insulin content were all also lower in islets prepared from STZ, rather than control rats. In the presence of 10.0 mM D-glucose, the paired ratio between D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization was also decreased in the islets from STZ rats. No significant difference between control and STZ rats was observed, however, in terms of the ratios between D-[3-3H]glucose and D-[5-3H]glucose utilization, between the generation of radioactive lactate from 14C-labelled D-glucose and tritiated D-glucose utilization and between D-[1-14C]glucose and D-[6-14C]glucose oxidation. These findings reinforce the view that the previously documented preferential impairment of the oxidative modality of glycolysis in islets from STZ rats contrasts with the absence of any major anomaly in other variables of D-glucose catabolism.