Continuous monitoring of glucose with a transcutaneous microdialysis probe.
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Glucose↗
Biomedical subjects
J Korf
Publications and source records attributed to J Korf.
An enzyme-reactor for electrochemical monitoring of choline and acetylcholine: applications in high-performance liquid chromatography, brain tissue, microdialysis and cerebrospinal fluid.
A sandwich-type enzyme reactor in which the enzymes are physically immobilized in a minimal dead space between two cellulose membranes, resulting in improved sensitivity, was developed for the electro-chemical detection of choline (Ch) and acetylcholine (ACh). The reactor contains the enzymes choline oxidase with or without acetylcholine esterase, for the detection of ACh and Ch, respectively. For the HPLC analysis of Ch and ACh the detection system was coupled post column. Levels of Ch and ACh of rat striatum tissue and human cerebrospinal fluid were found to be similar to those determined with published methods. Because of low back pressure--a further advantage of the reactor--the detection system could also be directly coupled to the outlet of a microdialysis device, allowing the on-line real-time measurement of extracellular brain Ch. The versatility of the enzyme reactor for the monitoring of analytes in HPLC eluates, flow injection analysis, with or without prepurification, is emphasized. The usefulness of the reactor-detector system in biomedical applications is illustrated by the measurement of increases of rat striatal extracellular Ch following cardiac arrest.
Acetylcholine↗
Time course and distribution of neuronal degeneration in the dentate gyrus of rat after adrenalectomy: a silver impregnation study.
Recently, Sloviter et al. reported that adrenalectomy (ADX) of young adult rats after 3 months led to a selective loss of granule neurons in the dentate gyrus (DG) and that this loss could be prevented by low doses of corticosterone. In the present study, the ADX-induced neuronal degeneration was investigated in Wistar rats, using a silver impregnation method for degenerating neurons. To examine the time course and distribution of the ADX-induced degeneration, young adult male rats were allowed to survive 2, 3, and 5 days and 1, 2, and 3 weeks after ADX. Argyrophilic neurons were present in the dentate granule cell layer on the second day following ADX. Three days after ADX, the number of argyrophilic granule neurons was much more abundant, and it increased gradually with longer post-ADX survival times. Argyrophilia was specifically confined to dentate granule cells and was accompanied by the occurrence of pyknotic nuclei as observed in adjacent cresyl violet-stained sections. There were significant differences between individual rats in quantity of argyrophilia. About one fifth of the ADX rats showed sporadic or no argyrophilia, in spite of plasma corticosterone levels below the detection limit (10 ng/mL). Sham-operated rats and ADX rats receiving corticosterone (10 mg/L) or dexamethasone (15 mg/L) in their drinking water did not display any argyrophilic neurons in the dentate gyrus. The distribution of the argyrophilia within the DG was highly characteristic with the highest number of degenerating cells in the hidden blade of the middle and the temporal thirds of the DG.(ABSTRACT TRUNCATED AT 250 WORDS)
Adrenalectomy↗
Rat hippocampal lactate efflux during electroconvulsive shock or stress is differently dependent on entorhinal cortex and adrenal integrity.
The role of the entorhinal cortex and the adrenal gland in rat hippocampal lactate formation was assessed during and after a short-lasting immobilization stress and electroconvulsive shock (ECS). Extracellular lactate was measured on-line using microdialysis and enzyme reactions (a technique named lactography); in some rats, unilateral lesions of the entorhinal cortex were made or the bilateral adrenal glands were removed. The stress-evoked increase in hippocampus lactate was not altered either ipsi- or contralateral to an entorhinal cortex lesion. The response to ECS was attenuated only in the hippocampus ipsilateral to the entorhinal cortex lesion. Removal of bilateral adrenal glands caused some delay in the increase in hippocampal lactate after ECS and a major reduction in the stress-evoked lactate response. These results indicate that (1) the entorhinal cortex is activated by ECS, thereby activating hippocampal lactate efflux and presumably metabolism, and (2) the adrenal gland is essential in the response to stress and, to a minor extent, in the ECS-altered hippocampal metabolism.
Adrenal Glands↗
Localization of NMDA and AMPA receptors in rat barrel field.
The aim of this study was to assess the distribution of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-S-methyl-4-isoxazole propionic acid (AMPA) receptors in the barrel field of rat primary somatosensory (SI) cortex using light-microscopic in vitro autoradiography. NMDA receptors were labeled with the competitive antagonist [3H]CGP39653 or with [3H]glycine in the presence of strychnine, and AMPA receptors with [3H]AMPA. In the SI cortex high densities of the NMDA receptor occurred in the supragranular layers and in layer Va. In layer IV high NMDA receptor densities were specifically confined to the barrel hollows. The AMPA binding sites showed less intralaminar variation and no apparent density differences between the barrel hollows and sides in layer IV. It can be concluded that the distribution of NMDA (but not AMPA) receptors in the rat barrel field shows a strong coincidence with the zone of termination of the specific sensory afferents from the ventral posterior nucleus of the dorsal thalamus.
2-Amino-5-phosphonovalerate↗
Automated precolumn fluorescence labelling by carbodiimide activation of N-acetylaspartate and N-acetylaspartylglutamate applied to an HPLC brain tissue analysis.
An automated method is described to couple carboxyl-containing metabolites to the fluorophore 2-aminoanthracene in aqueous solution (containing 75% methanol) in the presence of N,N-dicyclohexylcarbodiimide. The reaction was optimized for N-acetylaspartate (N-Ac-Asp) and N-acetylaspartylglutamate (N-Ac-Asp-Glu). The reactions occurred within 5 min at room temperature in the presence of 0.5-2 mM HCl. At concentrations of electrolytes exceeding 10 mM the coupling reaction became suboptimal. Derivatization was performed in a commercial precolumn derivatization unit. Additional tubing was needed to provide the reagents prior to reversed-phase HPLC and fluorescence detection. The assay is linear over at least three orders of magnitude; as little as 1 pmol could reproducibly be assayed in 100 micrograms wet weight brain tissue extracted with a mixture of methanol and 4 mM HCl (9:1, v/v). N-Ac-Asp and N-Ac-Asp-Glu levels in several brain regions and spinal cord were similar to those so far reported. The compounds could not be detected in peripheral tissue. The advantages, prospects and limitations of the present approach over existing methods to estimate water-soluble carboxylic acids is discussed.
Animals↗
High central D2-dopamine receptor occupancy as assessed with positron emission tomography in medicated but therapy-resistant schizophrenic patients.
We investigated whether the lack of therapeutic response to long-term and adequate neuroleptic treatment was due to a failure to achieve a blockade of cerebral dopamine receptors. Six chronic schizophrenic and medicated patients (DSM-III-R diagnosis, paranoid or disorganized type) were assessed with the Present State Examination and the Brief Psychiatric Rating Scale. According to the Chouinard Rating Scale there were little extrapyramidal symptoms, although no anticholinergic drugs were given. Plasma levels of the neuroleptics were determined and found in the therapeutic range or higher. Dopamine D2-receptor occupancy was determined with positron emission tomography using 11C-methylspiperone as ligand. There was a more than 95% blockade of the D2 receptors in the striatum. These results indicate that the lack of therapeutic response and extra-pyramidal side effects cannot be attributed to an incomplete blockade of cerebral D2 receptors and that the pathogenetic role of these receptors can be questioned in therapy-resistant schizophrenic patients.
Adult↗
Reduction of adenosine A1-receptors in the perforant pathway terminal zone in Alzheimer hippocampus.
The cells of origin of the perforant pathway are destroyed in Alzheimer's disease (AD). In rat the adenosine A1-receptors are specifically localized on the perforant path terminals in the molecular layer of the dentate gyrus. In the present study the density of A1-receptors in the hippocampus of Alzheimer's disease (AD) patients (n = 9) and non-dement controls (n = 3) has been investigated autoradiographically with [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]CPDPX) as the ligand probe. In AD hippocampi binding of [3H]CPDPX was greatly reduced in the outer two thirds of the dentate gyrus molecular layer, likely due to the degeneration of the perforant path. Binding of [3H]CPDPX was not significantly altered in other parts of the AD hippocampus, e.g. the CA1 and the CA3, in spite of a pronounced cellular pathology and reduced N-methyl-D-aspartate (NMDA) receptor densities, assessed as strychnine insensitive [3H]glycine autoradiography. This contrasts with the presumed localization on dendrites of pyramidal neurons of A1 receptors within the CA1 and the CA3.
Adenosine↗
Continuous monitoring of extracellular lactate concentration by microdialysis lactography for the study of rat muscle metabolism in vivo.
A method is described for the measurement and on-line monitoring of muscular extracellular lactate concentration in both anaesthetized and freely moving rats. This method is based on microdialysis sampling and lactic dehydrogenase-catalysed nicotinamide adenine dinucleotide, reduced (NADH)-fluorescence detection techniques. In vivo calibration revealed a resting extracellular lactate concentration of 1.92 +/- 0.13 mmol/l (+/- SEM) in the gastrocnemius muscle of adult male Wistar rats (n = 6), while the average whole-blood lactate level was 0.76 +/- 0.12 mmol/l (+/- SEM). This measured extracellular lactate concentration was 1.73-times higher than that deduced from the arterial lactate concentration. Blocking glycolysis with iodoacetate reduced the extracellular lactate concentration to 52 +/- 6% (+/- SEM, n = 4) of the resting level. The extracellular lactate concentration in rat gastrocnemius muscle had increased to significantly (P less than or equal to 0.05) different levels, 2.4 +/- 0.03 (+/- SEM) or 4.0 +/- 0.55 (+/- SEM) times the control value, 1 h after aortic clamping (n = 3) or cardiac arrest (n = 3), respectively. Stimulation of the sciatic nerve induced elevations of the extracellular lactate concentration in the tibialis anterior muscle which were linearly related to the recorded isometric force-time integral. We also monitored on-line the changes in extracellular lactate concentration in the tibialis anterior muscle of a swimming rat. Our results indicate that microdialysis lactate reflects also intracellular metabolism. Lactography may be a useful alternative to biopsies and nuclear magnetic resonance spectroscopy in clinical medicine and physiology for the monitoring of metabolism in vivo.
Animals↗
The effect of theophylline and immobilization stress on haloperidol-induced catalepsy and on metabolism in the striatum and hippocampus, studied with lactography.
Whether inducing catalepsy in the rat by an intraperitoneal injection of haloperidol (0.5 mg/kg) had an effect on metabolism in the striatum and in the hippocampus, as determined by lactography, and whether reducing the cataleptic state with stress or theophylline (8 mg/kg i.v.) had any impact on metabolism in these two regions of the brain was investigated. Furthermore, whether theophylline reduced catalepsy in rats through the adrenals was investigated. Haloperidol caused a significant increase in the metabolism of lactate, both in the striatum and in the hippocampus. Reducing haloperidol-induced catalepsy with short-term immobilisation stress did not affect the metabolism of lactate, neither in the striatum nor in the hippocampus. Reducing haloperidol-induced catalepsy with theophylline caused a significant rise in the metabolism of lactate in the striatum, while no effect was seen in the hippocampus. Adrenalectomy did not compromise the anti-cataleptic property of theophylline. It is concluded theophylline is a potent antagonist of haloperidol-induced catalepsy, and that this effect is not mediated by the adrenals. Furthermore, it is reported that haloperidol influenced metabolism in regions of the brain not considered to be its primary target. Lactography is considered to be a very useful tool in the study of metabolism during activity.
Adrenalectomy↗
On-line monitoring of extracellular brain glucose using microdialysis and a NADPH-linked enzymatic assay.
A method to monitor extracellular glucose in freely moving rats, based on intracerebral microdialysis coupled to an enzyme reactor is described. The dialysate is continuously mixed with a solution containing the enzymes hexokinase and glucose-6-phosphate dehydrogenase, and the fluorescence of NADPH formed enables the on-line registration of extracellular glucose. The method is applied to monitor changes in extracellular brain glucose during the infusion of glucose, electrically induced seizure, immobilization stress, and repetitive hypoxia. After glucose loading or after seizure, hippocampus dialysate glucose concentration was increased transiently. During immobilization, there was a short-lasting decrease and, thereafter, an increase in the extracellular hippocampus glucose. During repetitive hypoxia in rats with a unilaterally occluded carotid artery, the content of glucose of striatal dialysates followed closely changes in blood pressure. These results illustrate the usefulness of the method in studying changes in brain glucose concentrations under pathological and physiological conditions.
Animals↗
The extracellular concentration of the anti-epileptic drug valproate in the rat brain as determined with microdialysis and an automated HPLC procedure.
The cellular and extracellular brain concentration of valproic acid in freely moving rats has been estimated after intravenous injection of sodium valproate. Some rats were provided with a stereotaxically implanted dialysis probe in the striatum and a cannula in the heart through which the drug was injected and which allowed regular removal of blood. In other rats tissue levels of valproic acid were determined 5 and 90 min after drug injection. Valproic acid was determined by an automated precolumn derivatization procedure followed by HPLC separation and fluorimetric detection. Extracellular concentration was proportional to the blood concentration at every time interval, indicating rapid exchange of the drug between the two compartments. About 50% of the striatal content of valproate was in extracellular space. The experiments demonstrated the usefulness of microdialysis to estimate both the extracellular concentration and the average cellular drug levels, provided a sensitive analysis procedure is available.
Animals↗
Hippocampus pathology in dementia: post-mortem assessed cation shifts and amino acid transmitter contents and its possible neuroimaging in vivo.
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Adult↗
Automated precolumn derivatization of amino acids, small peptides, brain amines and drugs with primary amino groups for reversed-phase high-performance liquid chromatography using naphthalenedialdehyde as the fluorogenic label.
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Aldehydes↗
Improved automated precolumn derivatization reaction of fatty acids with bromomethylmethoxycoumarin as label.
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Animals↗
Lactography as an approach to monitor glucose metabolism on-line in brain and muscle.
1. Thus far metabolic processes in the intact animal (or man) have been studied either by the analysis of body fluids, of biopsies, of tissue obtained post mortem or by techniques, requiring dedicated and expensive equipment (such as positron emission tomography or magnetic resonance spectroscopy). 2. Here we describe a relatively simple and inexpensive technique, that can be applied in vivo to study metabolism in brain regions and muscle in the freely moving rat and in human peripheral tissue. 3. The method is based on microdialysis allowing continuous sampling from the extracellular space, the enzymatic conversion of lactate and the on-line detection of fluorescent NADH. 4. Examples of the application of our technique include the monitoring of lactate efflux from various brain regions of behaving animals under a variety of stress exposures, during ischemia or hypoxia and drug treatments. 5. The results indicate that in brain lactate is not exclusively formed under hypoxia and that neuronal activation leads also to lactate formation, possibly due to the compartmentation of both the involved enzymes and the energy metabolism. 6. The increase of lactate formation in contracting or ischemic muscle or during exercise could also be followed on-line in the rat, suggesting that our approach allows the continuous monitoring of anaerobic metabolism in man e.g. during traumatic or arteriosclerotic limb ischemia or lactic acidosis in shock states. 7. The principle of our approach can easily be adapted to other metabolites, thus enabling to monitor other metabolic pathways in vivo as well.
Animals↗
Graphical method to determine sigmoid ligand binding in vivo at low doses.
In this study, a graphical method is presented to detect sigmoidal binding of in vivo data at low nonsaturating doses. The method can also be applied when the overall binding includes nonspecific binding. Irrespective of the mathematical expression describing the saturation curve, it is shown that a sigmoid curve, in contrast to a nonsigmoid, can be converted into a peak-shaped curve by normalizing the ordinate values with the corresponding value of the abscissa. Computer simulations of in vivo ligand binding demonstrate this principle for in vivo data plots. Experimental results are described to illustrate this approach based on in vivo binding of the neuroleptic drug N-methylspiperone in the rat brain. It is concluded that the method is in particular useful for in vivo applications, e.g., positron emission tomography (PET) studies in human, because low doses are required, and specifically bound, nonspecifically bound, and unbound ligand do not need to be determined separately.
Brain↗
A novel non-perfusion Timm method for human brain tissue.
A simple modification of the Timm sulphide-silver method for unfixed brain tissue is described. Instead of perfusing animals with a sodium sulphide solution, sulphide treatment was performed by exposure to mounted frozen sections to H2S gas. The staining pattern in the rat brain obtained with this modification is similar to that With the Neo-Timm method, primarily yielding staining of the neuropil of telencephalic structures. Because perfusion is not required, the modified Timm method can also be applied to postmortem human tissue. Application of the modified Timm stain to the human tissue is exemplified on the hippocampus with special attention to the non-mossy fiber staining.
Animals↗