Search

Introduction: Total liquid ventilation (TLV) can cool down the entire body within 10-15 min in small animals. Our goal was to determine whether it could also induce ultra-fast and whole-body cooling in large animals using a specifically dedicated liquid ventilator. Cooling efficiency was evaluated under physiological conditions (beating-heart) and during cardiac arrest with automated chest compressions (CC, intra-arrest)., Methods: In a first set of experiments, beating-heart pigs were randomly submitted to conventional mechanical ventilation or hypothermic TLV with perfluoro-N-octane (between 15 and 32 °C). In a second set of experiments, pigs were submitted to ventricular fibrillation and CC. One group underwent continuous CC with asynchronous conventional ventilation (Control group). The other group was switched to TLV while pursuing CC for the investigation of cooling capacities and potential effects on cardiac massage efficiency., Results: Under physiological conditions, TLV significantly decreased the entire body temperatures below 34 °C within only 10 min. As examples, cooling rates averaged 0.54 and 0.94 °C/min in rectum and esophageous, respectively. During cardiac arrest, TLV did not alter CC efficiency and cooled the entire body below 34 °C within 20 min, the low-flow period slowing cooling during CC., Conclusion: Using a specifically designed liquid ventilator, TLV induced a very rapid cooling of the entire body in large animals. This was confirmed in both physiological conditions and during cardiac arrest with CC. TLV could be relevant for ultra-rapid cooling independently of body weight., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

A method for high temporal resolution monitoring of five neurotransmitters, dopamine (DA), noradrenaline (NA), gamma-aminobutyric acid (GABA), glutamate (Glu), l-aspartate (L-Asp), in freely-moving rats using microdialysis and capillary electrophoresis with laser-induced fluorescence detection (CE-LIFD) was developed. An on-line device, including microdialysis and derivatization with naphthalene-2,3-dicarboxaldehyde, mixes the dialysate with derivatization reagents directly in the collection tube, i.e. with no reactor. Thereafter, collected derivatized samples are analyzed off-line with an automated CE system coupled to a LIFD using a 442 nm excitation. The sampling time was limited by the minimal volume required for the analysis by the automated CE system used: neurotransmitters could be determined in 667 nl dialysates (940 nl after derivatization), i.e. in samples collected every 20 s with a flow rate of 2 microl/min. The detection limits at the dialysis probe were 3 x 10(-9), 1 x 10(-9), 1.9 x 10(-8), 4.2 x 10(-7), 2.1 x 10(-7) mol/l for DA, NA, GABA, Glu and L-Asp, respectively. The protocol was validated using in vitro/in vivo tests and the performances--repeatability, linearity, characteristics of the probes--were determined. Finally, the high temporal resolution allowed the simultaneous monitoring of these neurotransmitters in rats with genetic absence epilepsy and revealed, for the first time, increases in GABA concentrations concomitantly with the seizures, detected when our new microdialysis method was combined to electroencephalographic recordings.

Our study was devoted to determine in freely moving rats whether the increase in tissue concentration of tyrosine hydroxylase (TH) elicited by a single administration of RU 24722 could modify the catecholaminergic reactivity of neuronal processes present in the rostrolateral part of the pericerulean area (r-lPCA) in response to tail pinch. Catecholaminergic activity was monitored by measuring in vivo the concentration of dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) using microdialysis coupled to HPLC detection. In this study, the microdialysis probe was implanted at a sufficient distance from the lateral border of rostral nucleus locus ceruleus (LC) to avoid a large contribution of the noradrenergic cell bodies in the measurements performed. We first evidenced that DOPAC measured in the r-lPCA indicated the functional state of catecholaminergic metabolism in neuronal processes (dendrites and fibers) laying in this region. We also showed that the enhancement of TH protein concentration in the r-lPCA following RU 24722 treatment supported an increased in vivo catecholaminergic metabolism in this region. Furthermore, catecholaminergic metabolism response to tail pinch was potentiated in animals with greater TH tissue concentration. Thus, our study reveals that the modulation of both TH concentration and catecholaminergic metabolism in the r-lPCA may be critical in the functioning of cells and neuronal elements present in this region, notably in adaptive responses to noxious stimuli.

These experiments combined extracellular electrophysiological multirecordings from hippocampal organotypic slice cultures with application of drugs to and sampling of extracellular fluid from a restricted region of the slice using a microdialysis probe. Glutamate (Glu) concentrations were monitored in 0.5 or 2 min microdialysis samples, while evoked field potentials responses (EvFPR) in the CA1 region of the hippocampus (stimulation in the CA3 area) were simultaneously recorded using a multi-electrodes array (Physiocard). Glu was assayed by capillary electrophoresis with laser-induced fluorescence detection combined with a continuous flow derivatization of dialysates. The performance of this combined approach was demonstrated by monitoring extracellular Glu concentrations and EvFPR after K+ induced depolarisation, Glu uptake blockade by trans-pyrrolidine-2,4-dicarboxylic acid (PDC), and electrical stimulation. Such an approach allows a global monitoring of the neuronal functioning with a fine time resolution (up to 30 s) on a simple in vitro brain slice model, to be used as a complement to conventional in vivo microdialysis studies.

The measurement of neurotransmitters by capillary electrophoresis (CE) has emerged as a reliable and sensitive method for microdialysis sample analysis. This paper describes a method which employs laser-induced fluorescence detection (LIFD) of catecholamines and excitatory amino acid derivatives formed after reaction with naphthalene-2,3-dicarboxaldehyde. On-line derivatization of very small volumes of microdialysis samples (500 nl) is developed before two off-line analyses (total run time of less than 10 min) are performed to detect derivatives of catecholamines and excitatory amino acids formed in each sample. High microdialysis temporal resolution is reached (2-min fractions) for the simultaneous monitoring of noradrenaline (NA) and glutamate concentrations from rat brain cortex microdialysates. The system performance is evaluated and pharmacological characterization of the determination of NA in cortical dialysates by CE-LIFD is reported.

The effects of a single dexfenfluramine (D-fen) administration on the release of endogenous serotonin (5-hydroxytryptamine, 5-HT), excitatory (glutamate, Glu, aspartate, Asp) and inhibitory (gamma-aminobutyric acid, GABA) amino acids from the frontal cortex were studied by using in vivo microdialysis in freely-moving rats. Extracellular levels of these neurotransmitters were measured with HPLC coupled to electrochemical detection or with capillary electrophoresis coupled to laser-induced fluoresence detection (CE-LIFD). In a first study, single intraperitoneal administration of D-fen (0.5, 1.3, 5 and 10 mg/kg) increased extracellular 5-HT levels in a dose-dependent manner (maximal increase by 982% over baseline for the highest dose) while changes in Glu, Asp or GABA never reached statistical significance. In a second study, 73 nM of D-fen applied locally through the frontocortical dialysis probe, at a flow rate of 1.5 microliters/min in 30 microliters of perfusion fluid for 20 min, increased extracellular 5-HT and Asp levels [the maximal increases were to 1804% and 280% of the respective basal values (100%)] without altering extracellular levels of Glu and GABA. Thus, the order of magnitude of the changes induced by systemic administration or local infusion of D-fen on frontocortical extracellular levels of several neurotransmitters (5-HT > > Asp > GABA = Glu) demonstrate that D-fen, an indirect serotoninergic agonist, mainly increases 5-HT release while producing slight (Asp) or no (Glu, GABA) short-term in vivo variations in amino acid extracellular levels in the rat frontal cortex.

The aim of the present work was to measure, by voltammetry, the catecholaminergic metabolic activity of rat locus coeruleus (LC) neurones in brain slices. For this new experimental approach, we used an optimized protocol of slice preparation intended to prevent neuronal damages due to brain ischaemia. Our results show that the LC neurones exhibit in vitro a stable spontaneous catecholaminergic metabolic activity and that, as in vivo, 3,4-dihydroxyphenylacetic acid (DOPAC) is likely to be the main contributor to the recorded signal. This catecholaminergic metabolic activity can be pharmacologically altered by administering carbachol and clonidine to the superfusion fluid. We also determined the values of bath temperature and superfusion flow rate providing, in our methodological conditions, an optimal catecholaminergic metabolic activity. Finally, we took advantage of both the direct accessibility to the LC and the compactness of this nucleus to determine the spatial resolution of differential normal pulse voltammetry. In conclusion, the study of the subregional mechanisms controling the catecholaminergic metabolism in LC neurones can be performed in brain slices by differential normal pulse voltammetry.

By repeated inbreeding, 2 strains of spontaneously hypertensive and normotensive rats have been simultaneously selected. The activities of tyrosine hydroxylase and phenylethanolamine-N-methyltransferase were determined in various central catecholaminergic nuclei (C1, C2, A6 and A9) and in two peripheral tissues (adrenal glands and superior cervical ganglion). These assays were performed on rats belonging to the normotensive or the hypertensive strain at 3 ages which characterize the development of hypertension (5, 9 and 21 weeks). Except for a decrease in the C1 region of 9-week-old rats, no significant change in tyrosine hydroxylase activity occurred in central or peripheral structures of the spontaneously hypertensive rats when compared to the normotensive rats. In contrast, the activity of the phenylethanolamine-N-methyltransferase (PNMT), was increased in the C2 adrenergic group of the medulla oblongata in young spontaneously hypertensive rats: +43% (P less than 0.001) at 5 weeks of age and +32% (P less than 0.001) in 9-week-old rats. However, there was no significant difference between the 21-week-old rats. No modification of the PNMT activity was found in the C1 adrenergic group of the medulla oblongata. PNMT activity was increased significantly in the adrenal glands of 5-week-old hypertensive rats (+22%, P less than 0.001). By 9 weeks, the difference in PNMT activity in the adrenals was no longer significant. Thus, in young rats of the hypertensive strain, there was an increase in the capacity to synthetize adrenaline in the C2 area of the medulla oblongata and in the adrenal glands. While the enzymatic change present in the adrenals seems to be specific to this new strain of hypertensive rats, the elevation of PNMT activity in a specific region of the medulla oblongata (C2 group) is a characteristic common to at least two independently derived strains of genetically hypertensive rats.

Time-course variations in tyrosine hydroxylase activity were measured in the locus coeruleus of the albino rat after electrolytic coagulation of either the nucleus raphe dorsalis or the nucleus raphe centralis. Highly significant increases were measured at 4 days after lesioning of the raphe dorsalis (30.33%) and the raphe centralis (81.55%) compared with control values, whereas the activity in groups A9 and A10 was unchanged at this time-point. In conjunction with other experimental evidences, an hypothesis is proposed that the catecholaminergic neurons located in the locus coeruleus are directly and/or indirectly controlled by the serotonin-containing neurons located in the anterior raphe system nuclei.

The activities of the three major catecholamines synthesizing enzymes were determined on supernatants from microcubes dissected out from 500 microns thick coronal sections of the rat lower brainstem. The microcubes exhibiting significant or high enzymatic activities represented only one-third of the microcubes analyzed and were generally localized either in the dorso-medial or in the ventro-lateral region of the lower brainstem. In the ventro-lateral region, referred to as the A1-C1 region, the rostro-caudal distribution of the enzymatic activities exhibited a small difference between the maximal TH and DBH activities and the maximum PNMT activity located 500 microns more rostrally. Conversely, in the dorso-medial region, referred to as the A2-C2 region, there was a much larger rostro-caudal difference (about 1500 microns) between the peaks of TH and DBH activities and the more rostral peak of PNMT activity. Such a distribution suggests a separation between the adrenaline (A) and the noradrenaline (NA) containing neurons within the dorso-medial region. According to the results of this study, a preferential microdissection of the A versus NA neurons seems possible in the A2-C2 region of the rat medulla oblongata.

In this study, we sought to determine if there was an interaction between the C2 adrenaline-containing (A) neurons of the rat medulla oblongata and the noradrenaline-containing (NA) cell bodies of the locus coeruleus (LC). For this purpose, the biochemical response of the NA cell bodies of the LC after a lesion of the C2 region was studied by using as markers the in vitro activities of the catecholamine synthesizing enzymes: tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT). An increase in TH activity, not associated with any change in DBH or PNMT activity, was found in the LC (+104%, P less than 0.001) 4 days after a bilateral electrolytic lesion (3 mA for 5 s) of the C2 region. Conversely, the electrolytic lesioning of the neighboring A2 region of NA neurons did not modify the TH activity of the LC. These results suggest the existence of an ascending adrenergic inhibitory control of the NA cell bodies of the LC.

In vivo electrochemical recordings of the metabolism of catecholamines were obtained in the caudal ventrolateral medulla in anesthetized rats submitted to various experimental changes in systemic arterial pressure. Hypertension induced with phenylephrine and reversal of hypovolemia decreased the catechol metabolic activity. In contrast, controlled or hypovolemic hypotension, induced respectively with sodium nitroprusside or blood withdrawal (30% of blood volume), reversibly elicited the opposite pattern. This was suppressed by deafferentation. The changes in catechol metabolic activity in response to hypovolemia were accompanied by similar trends of variations of plasma vasopressin levels. By contrast with the increased catechol metabolic activity secondary to hypotension induced by either prazosin, sodium nitroprusside or hypovolemia, clonidine elicited a decrease in catechol metabolic activity. These data show a dynamic and specific involvement of the metabolism of catecholamines themselves promoted by changes in systemic arterial pressure. This pattern of functioning of catechol metabolism in the caudal ventrolateral medulla appears to be negatively related to systemic arterial pressure changes, a finding which does not fit with the proposed vasodepressor role of the A1-group.

Recent data have shown that 5 days after intraventricular injection of 6-hydroxydopamine (6-OHDA), the tyrosine hydroxylase activity (TH) was increased within the locus coeruleus (LC). We sought to determine if such an alteration occurs within the A1 and A2 noradrenergic (NA) and C1 and C2 adrenergic (A) neurons of the rat medulla oblongata. The TH activity within the cell bodies was significantly increased 2 days after 6-OHDA injection with a maximum at 5 days (LC, + 109%, P less than 0.001; A1-C1, + 40%, P less than 0.01; A2-C2; + 24%, P less than 0.01) while a significant decrease was present 21 days after 6-OHDA. Conversely, dopamine-beta-hydroxylase (DBH) activity exhibited a decrease which was maximal at 21 days (LC,-41%; A1-C1,-33%; A2-C2,-35%, P less than 0.001). Both the TH (-47% at 5 days) and the DBH (-82% at 12 days) activities were decreased within the terminals of the tractus intermediolateralis (TIML). The phenylethanolamine-N-methyltransferase (PNMT) activity was never altered in the cell bodies nor in the terminals analyzed. These data demonstrate that the NA neurons of the rat medulla oblongata and of the LC exhibit a similar pattern of response to the neurotoxin 6-OHDA. Conversely, the lack of change in the PNMT activity confirms the hypothesis of a resistance of the A neurons to 6-OHDA or questions the validity of the PNMT as an A marker.

The concentration of the three major catecholamines (CAs) were determined in 500 micron thick coronal sections of the rat medulla oblongata dissected into microcubes. Noradrenaline (NA) concentrations were always found much higher than the levels of the two other CAs in the same microcube. The highest concentrations of the three CAs were found in the dorso-medial region of the lower brainstem, more exactly in the more caudally located parts of the nucleus tractus solitarii (NTS). In the ventro-lateral region, the CA concentrations were lower and, except for adrenaline (A), did not exhibit any substantial change in their rostro-caudal distribution. Conversely, in the dorso-medial region, there was a clear rostro-caudal pattern of distribution of the three CAs. This distribution was similar for the three amines, since only a small difference (about 500 micron) was found between the maximal NA and A concentrations. Since the three CAs are present in highest concentrations within the same dorso-medial or ventro-lateral groups of microcubes, a microdissection of these two areas seems suitable to study simultaneously the metabolism of the three CAs in the rat lower brainstem. These data also suggest a microdissection procedure to study A metabolism within the C2-C3 A cell bodies and within a region more caudally located, rich in A terminals.