Modification of eicosanoid profile in human blood treated by dual COX/LOX inhibitors.

The arachidonic acid metabolizing enzymes, the cyclooxygenases (COXs) and lipoxygenases (LOXs), have been implicated in the development of a variety of cancers and numerous new therapeutic inhibitors are currently under investigation. However, given the interdependence of the two pathways, the effect of inhibiting one pathway with relatively selective agents can only be appreciated in the in vivo situation. Clearly then, because of their potential beneficial or deleterious effects, it is important to understand the nature and levels of the resulting arachidonic acid metabolites when treating patients with relatively selective inhibitor drugs. In this study, using reference COX-2, 5-LOX and dual COX-2/5-LOX inhibitors, we devised a protocol which permitted the simultaneous quantification of eicosanoid metabolites formed during stimulation of human peripheral venous blood samples with the calcium ionophore, A23187, in the absence and presence of lipopolysaccharide (LPS). Not surprisingly, the end products of both COX and LOX pathways were affected depending on the inhibitor, or combination of inhibitors, used and the concentrations of drug tested. In conclusion, the method described permits the rapid screening of novel compounds for potentially positive and/or negative effects upon the products of arachidonic acid metabolism.

Colchicine poisoning: report of a fatal case with body fluid and post-mortem tissue analysis by high-performance liquid chromatography.

A case involving a suicide by the ingestion of colchicine tablets is presented. Liquid chromatography has been used to measure the drug level in blood and in post-mortem tissues of the patient (a 42-year-old man). Plasma concentration 24 h after ingestion was 4.5 ng/mL. On autopsy, the kidney showed the highest concentration (396 ng/g). High concentrations were also found in the liver (347 ng/g) and heart (334 ng/g). Low concentrations were detected in the lung (58 ng/g), muscle (10 ng/g) and brain (5 ng/g).

Cyclophosphamide cystitis as a model of visceral pain in rats: minor effects at mesodiencephalic levels as revealed by the expression of c-fos, with a note on Krox-24.

The evoked expression of the immediate-early gene-encoded proteins c-Fos and Krox-24 was used to study activation of mesodiencephalic structures as a function of the development of cyclophosphamide (CP) cystitis in behaving rats. This article is the third of a series and completes previously published data obtained at both spinal and hindbrain levels. CP-injected animals received a single dose of 100 mg/kg i.p. under transient volatile anesthesia and survived for 1-4 h in order to cover the entire postinjection period during which the disease develops. Survival times longer than 4 h were not used owing to ethical considerations. Results from CP-injected groups are compared with those from either noninjected controls or saline-injected animals having survived for the same times as CP-injected ones. Quantitative results come from c-fos expression. At mesodiencephalic levels a high and widespread basal c-fos expression was observed in control animals; maximum staining was observed at the midthalamic level. Four groups of nuclei were identified with regard to the density of staining. The first group included nuclei showing clustered, intensely labeled cells; these areas were restricted in extent and related to the maintenance of circadian rythms (intergeniculate leaf, suprachiasmatic nucleus, dorsal parts of either paraventricular thalamic nuclei or central gray), sleep-arousal cycle (supramamillary nucleus), or changes in arterial pressure (laterodorsal tegmental nucleus). The second group included nuclei showing scattered, moderately labeled cells; these areas were widespread at all rostrocaudal levels and related to either autonomic/neuroendocrine regulations (central gray, lateral habenula, hypothalamus) or motor behavior, orienting reflex and oculomotor coordination (unspecific subdivisions of both colliculi and their adjoining mesencephalic regions, zona incerta dorsal). The third group included nuclei with evenly distributed, faintly labeled cells; these areas, which, with few exceptions, covered almost the entire diencephalon, mainly concerned nuclei of multisensory convergence having functions in either discriminative tasks (laterodorsal and lateroposterior thalamic nuclei) or emotional responses (intralaminar and midline thalamic nuclei). The fourth group included nuclei free of labeling; these were areas that received the bulk of unimodal sensory/motor inputs (central inferior colliculus, pretectal optic nuclei, ventral medial geniculate nucleus, ventral anterior pretectal nucleus, dorsal lateral geniculate nucleus, ventrobasal complex; zona incerta ventral, parafascicular thalamic nucleus) and are thus the most discriminative regarding specific modalities. Variations in staining were of the same magnitude in both saline- and CP-injected animals. A sequential study spanning every postinjection hour revealed maximum staining at 1 h postinjection, which was followed by a progressive, time-related decrease. Increases in the number of labeled cells 1 h postinjection were significant in only a restricted number of nuclei showing low basal expression (Edinger-Westphal nucleus and paraventricular, supraoptic, and lateral hypothalamic nuclei); time-related reductions in staining that were correlated to sleep or quiescence behaviors finally resulted in staining equal to or below that seen in control animals. No structures showed significantly increased staining in relation to the full development of cystitis, i.e., with the increase of visceronociceptive inputs. Comparing the present results with those previously obtained at more caudal levels, it appears that subtelencephalic levels primarily driven by visceronociceptive inputs, i.e., those that increase and/or maintain their activity in parallel with the degree of nociception, are confined to brainstem-spinal cord junction levels and only comprise certain subdivisions of the nucleus of the solitary tract (nucleus medialis, nucleus commissuralis, and ventralmost part of area po

Effect of acute stimulation on Fos expression in spinal neurons in the presence of persisting C-fiber activity.

Expression of the inducible transcription factor c-Fos has been examined in the lumbar spinal cord following noxious chemical stimulation (injection of 2% formalin) of the ankles or the ventral skin of the hindpaws of either normal rats, or monoarthritic rats during the chronic phase of the disease. In normal animals the basal expression of c-Fos was low. One day after induction of monoarthritis by an intra-articular injection of killed Mycobacterium butyricum (in complete Freund's adjuvant) there were numerous c-Fos labelled cells in the ipsilateral dorsal horn, and bilaterally in lamina VIII and in other areas of the ventral horn. Four weeks after induction of the arthritis, although marked inflammation of the ankle was still present, all the expression of c-Fos had returned to the basal levels. One hour after formalin stimulation of the ankle or hindpaw skin of normal rats expression of c-Fos was observed throughout the ipsilateral, but not contralateral dorsal horn. Formalin stimulation of the inflamed ankle in four-week arthritic rats induced a 3-to-6 fold increase in c-Fos expression in the ipsilateral dorsal horn compared to formalin stimulation of the ankle in normal rats. In addition, c-Fos expression was induced in the contralateral deep, but not superficial laminae, at a density similar to that produced ipsilaterally by formalin stimulation of the ankle of normal rats. Formalin stimulation of the contralateral ankle in monoarthritic rats (i.e. the non-inflamed ankle) induced an ipsilateral expression of c-Fos which was similar to that observed after stimulation of the arthritic ankle. This stimulation of the normal ankle also resulted in an expression of c-Fos in the contralateral deep, but not superficial laminae, that was similar to that induced ipsilaterally by stimulation of the arthritic ankle. Finally, formalin stimulation of the hindpaw skin (which was not inflamed) of the arthritic limb induced the same number of c-Fos labelled cells in the superficial laminae as did formalin stimulation of the skin of normal rats; but in the deep laminae there was a 1.6-fold increase in the number of labelled cells. These different observations show that the down-regulation of c-Fos expression observed in chronic monoarthritis is in fact associated with a sensitization and an extension of the field of its expression in response to an acute nociceptive stimulation.

Cyclophosphamide cystitis as a model of visceral pain in rats. A survey of hindbrain structures involved in visceroception and nociception using the expression of c-Fos and Krox-24 proteins.

The evoked expression of the immediate early gene-encoded proteins c-Fos and Krox-24 was used to study activation of hindbrain neurons as a function of the development of cyclophosphamide (CP) cystitis in behaving rats. CP-injected animals received a single dose of 100 mg/kg i.p. under transient volatile anesthesia and survived for 1 to 4 h in order to cover the whole postinjection period during which the disease develops. CP-injected groups included: (1) animals with minor simple chorionic edema, an early characteristic of inflammation (1 h postinjection); (2) animals with well-developed simple chorionic edema (2 h postinjection); (3) animals with mild inflammation (chorionic edema accompanied by epithelial cleavage; 3 h postinjection); and (4) animals with complete inflammation (4 h postinjection). In addition to onset of chorionic edema, the earliest postinjection period also included the general aspects of the nervous reaction consecutive to the injection process (handling, transient volatile anesthesia and postanesthesia awakening, abdominal pinprick, CP-blood circulating effects). Controls included both noninjected animals and saline-injected animals surviving for the same times as CP-injected ones. Quantitative results come from c-Fos expression. It has been shown that: (1) saline injection is a significant stimulus for only nucleus O and central gray pars alpha and nucleus medialis of the dorsal vagal complex; (2) all structures driven by CP injection (nucleus O and central gray pars alpha, locus coeruleus, Barrington's nucleus and parabrachial area mostly in its ventral and lateral subdivisions, dorsal vagal complex, ventrocaudal portion of lateral bulbar reticular formation) responded vigorously shortly after injection, but only two (dorsal vagal complex, ventrocaudal portion of lateral bulbar reticular formation) showed increased or renewed activity when cystitis completely developed, i.e., when noxious visceral inputs reached highest levels. Regarding the sequential activation of these structures in relation to postinjection time, evidence is given that: (1) a large variety of hindbrain structures are differentially involved in either the general reaction consecutive to the injection process or to various degrees of cystitis; (2) these structures extend from the brain-spinal cord to the pons-mesencephalon transitional junction levels; (3) the two structures most powerfully driven by visceronociceptive inputs are also the most caudal ones, being located at the brain-spinal cord junction level; and (4) the dorsal vagal complex could be the main hindbrain visceral pain center, with three particular subdivisions, the nucleus medialis, nucleus commissuralis, and ventralmost part of area postrema, being involved.

Antioxidant properties of hydroxy-flavones.

The antioxidant properties of 24 hydroxy-flavones were evaluated. Results show that 2',3',4'-OH substitution on the B ring plays a crucial role in radical scavenger activity in the DPPH assay and in the inhibitory effect on pereoxydation of tissue lipids in the MDA test. The formation of stable radicals for this type of compounds has been studied by ESR. In addition, it has been found that 7-hydroxy-flavones are potent competitive inhibitors of xanthine oxidase. It is proposed that the C-7 OH of flavones may take the place of the C-2 or C-6 OH of xanthine in the active site of the enzyme. A C-4' OH or C-4' OMe substitution on the 7-hydroxy flavones is not favourable to a fit in the active site. The 2',3',4'-trihydroxy-flavones inhibited XO by another process, which remains to be determined. In summary, this study provides evidence that hydroxy-flavones exhibit interesting antioxidant properties expressed either by the capacity to scavenge free radicals (for 2',3',4'-trihydroxy-flavones) or to competitively inhibit xanthine oxidase (for 7-hydroxy-flavones). These compounds may be drug candidates for treating pathologies related to free radical oxidation.

[Erythrocyte ketone reductase activity, total plasma haloperidol and acute psychoses]. / Activité cétone réductase érythrocytaire, taux plasmatiques d'halopéridol et psychoses aiguës.

Haloperidol (HAL) is a widely used and clinically effective neuroleptic. Its metabolism differs in various animal species. In humans, reduced haloperidol (RHAL), a hydroxy metabolite of HAL, is produced by a cytosolic ketone reductase. Interconversion is known to occur whereby HAL is found in the plasma after administration of RHAL in vivo. Interconversion of HAL and RHAL has been observed in man. However, the capacity for reductive HAL is greater than its oxidation back from RHAL. RHAL, the resulting metabolite of HAL, is reported to be about 10-25% as active as HAL in an animal model. Large intersubject variation has been observed in the pharmacokinetics of HAL and RHAL. A wide variation in reductive drug-metabolizing has been observed in schizophrenic patients treated with HAL. Both high and low RHAL/HAL ratios or RHAL levels were reported to be linked to poor response in HAL-treated patients and might be correlated with the therapeutic window effect of HAL treatment. It is conceivable, therefore, that subjects with high reductive capacity relative to oxidative capacity may have less therapeutic response from the same dose of HAL than those with a low reductive capacity relative to oxidative capacity. This aim of this study was to investigate the HAL reduction among a sample of HAL-treated schizophrenic patients. Because ketone reductases are generally not tissue specific, we investigate the reductase activity in Red blood cells (as described by Inaba), before and during the treatment. Steady-state plasma drug levels during 2 weeks of treatment were quantified. We examined the relationships between fixed doses of HAL treatment, Red blood cells ketone reductase activity, plasma HAL and RHAL levels and the percentage of change of the Positive and Negative Syndrome Scale for Schizophrenia. The participants in this study were 15 inpatients consecutively being treated in the adult psychiatric wards of the University of Lille. All subjects met DSM III-R criteria for schizophrenia (paranoid form). Upon induction subjects were evaluated clinically by trained raters using the Positive and Negative Syndrome Scale for Schizophrenia (PANSS). Subjects were required to score 40 or higher on the general psychopathology subscale of the PANSS to continue participation. All subjects were drug free. Haloperidol was administered orally at three times daily dose. Patients were randomized to treatment at three orally fixed doses: 6 mg per day, 10 mg per day and 15 mg per day. Patients were treated for 2 periods of one week. At the end of each period, dosages could be modified according to the clinic evolution of the patient. PANSS was repeated by the same raters blinded to the haloperidol dosage, plasma concentration and Rbc haloperidol ketone reductase activity, at the beginning and at the end of each period. Blood samples were collected on the same day that clinical assessment were made. Multiple regression analysis (forward stepwise) revealed that Red blood cells reductase activity at D0 is an important variable predicting haloperidol plasma levels at week 2. Similarly Red blood cells reductase activity at D0 and D7 predicted Reduced haloperidol plasma concentrations at week 2. In this sample, no parameter was found to be consistency predicted the percentage change in the PANSS positive subscale from baseline, at week 2. Nevertheless, Red blood cells reductase activity at D0, Reduced haloperidol/haloperidol ratio at week 2, haloperidol plasma levels at week 2 and the dose of haloperidol at week 1 were important variables predicting the percentage change in the PANSS general subscale from baseline at week 2. These results suggest that the knowledge of reductase activity could predict the treatment response in acute schizophrenic patients.

[Favourable effect of zolpidem on catatonia]. / Effet favorable du zolpidem sur un état catatonique.

According to the literature, electroconvulsive therapy and benzodiazepines, especially lorazepam, are recommended for the treatment of catatonia. We report the case of a 56-year-old woman with catatonia resistant to electroconvulsive therapy and benzodiazepines. Treatment with zolpidem led to durable improvement. This case suggests that zolpidem should be tested in catatonia since side effects are minimal.

Cyclophosphamide cystitis as a model of visceral pain in rats: model elaboration and spinal structures involved as revealed by the expression of c-Fos and Krox-24 proteins.

The evoked expression of the immediate early gene (IEG)-encoded proteins c-Fos and Krox-24 was used to monitor spinal visceronociceptive processing that results from cyclophosphamide cystitis in behaving rats. Animals received a single dose of 100 mg/kg i.p. of cyclophosphamide and survived for 30 min to 5 h. Longer survival times were not considered because of ethical considerations. Cyclophosphamide-injected animals developed characteristic behavioral signs in parallel with development of bladder lesions and spinal evoked expression of IEG-encoded proteins. Histological examination of the urinary bladder was used to evaluate the degree of cystitis and as a criterion for selection of groups of animals to be quantitatively analyzed. Controls consisted of freely behaving animals including control (un-injected), sham (saline-injected) or diuretic (furosemide-injected) animals. Behavioral modifications consisted of lacrimation, piloerection, assumption of a peculiar "rounded-back" posture, which was accompanied by head immobility and various brief "crises" (tail hyperextension, abdominal retractions, licking of the lower abdomen, backward withdrawal movements). Abnormal behaviors, which first appeared (lacrimation, piloerection) at the end of postinjection hour 1, progressively increased in severity (rounded-back posture) over the following 90 min to reach a plateau at about postinjection hour 2; the rounded-back posture was maintained up to time of death. Histological modifications of bladder tissue were assessed using a 4-grade scale in a blind setting. The 1st grade consisted of control or sham animals with no bladder lesion; 2nd grade, animals with simple chorionic edema; 3rd grade, animals with chorionic edema associated with mucosal abrasion, fibrin deposit, and onset of polymorphonuclear leukocyte infiltration; 4th grade, animals with complete cystitis corresponding to an increase in severity and spread of all the signs of cystitis described above plus petechial hemorrhage. Simple chorionic edema was observed from 30 min to 3 h postinjection, but with a progressive increase in severity over time. Edema accompanied by epithelial abrasion was observed for animals that survived 3-4 h postinjection; complete inflammation was observed in animals that survived 4-5 h postinjection. The study of c-Fos- and Krox-24-encoded protein expression demonstrated that few lumbosacral spinal areas were specifically involved in the processing of visceral inputs in response to bladder stimulation. These areas were the parasympathetic column (SPN), the dorsal gray commissure (DGC as the caudal extent of lamina X), and superficial layers of the dorsal horn.(ABSTRACT TRUNCATED AT 400 WORDS)

Antioxidant activities of Crataegus monogyna extracts.

Interesting antioxidant activities of extracts from different vegetative and reproductive organs of Crataegus monogyna harvested at different stages of growth have been determined by the malondialdehyde-thiobarbituric acid (MDA) test on hepatic microsomal preparations and compared to the contents in total phenolics, proanthocyanidins, catechins, flavonoids, and phenolic acids. The best correlations were established with total phenols while activities in leaves seem to be influenced by flavonoids and in flowers and fruits by proanthocyanidins and catechins.

High-performance liquid chromatographic determination of cyclic 3',5'-AMP with fluorescence detection. Vasoactive intestinal peptide-induced modification of its concentration in neuroblastoma cells.

The efficiency of ion-pair reversed-phase HPLC on a Vydac C18 column with 50 mM ammonium acetate (pH 4.75)-methanol-acetonitrile (88:9:3, v/v/v) as the mobile phase with isocratic separation and fluorescence detection for the determination of cAMP in cellular extracts was evaluated. This method was compared with a radioimmunoassay technique in terms of linearity, reproducibility and sensitivity. No interactions with other nucleotides such as AMP, ADP, ATP and cGMP were observed. Application to the measurement of cAMP modifications was studied in a neuroblastoma cell line: LA-N-2 cells stimulated by a neuropeptide, vasoactive intestinal peptide.

Correlation of clinical response (PANSS) and plasma levels of haloperidol and reduced haloperidol in schizophrenia.

1. The authors attempted to correlate plasma concentrations in H/rH and clinical efficacy from 8 schizophrenic patients (DSM IIIR) on H. 2. No significant correlations were found between H, rH plasma levels and positive and negative subscale for each patient. 3. The authors observed an opposite evolution concerning the mean results between plasma concentrations and PANSS total score.

[Value of the determination of erythrocyte haloperidol ketone reductase activity in the evaluation of haloperidol therapy]. / Intérêt du dosage de l'activité halopéridol cétone réductase érythrocytaire dans l'optimisation d'un traitement par halopéridol.

Serum levels of haloperidol and reduced haloperidol as well as the reduced haloperidol/haloperidol ratios were determined in nine acute schizophrenics on oral haloperidol medication and correlated over 21 days with psycho pathology and extra-pyramidal symptom scores. We have investigated red blood cells haloperidol reductase activity in the group of patients. Significant correlations were found between haloperidol plasma levels and positive sub scale for each patient (r = 0.86 and p < 0.01; r = 0.70 and p < 0.05). We found a correlation between red blood cells reductase activity and the improvement of the psychotic anxiety scale (r = 0.64/and p < 0.05; r = 0.67 and p < 0.05), but not with reduced haloperidol/haloperidol ratios in plasma. The knowledge of reductase activity could predict the treatment response in acute schizophrenic patients. We suggest that the reported inter individual and inter ethnic differences in haloperidol and reduced haloperidol and in clinical response and adverse effects may be a reflection of genetic control of the two oxidative pathways mediated by cytochrome P450 isozyme and/or the reductase pathway mediated by haloperidol reductase in individual subject.

Hindbrain structures involved in pain processing as revealed by the expression of c-Fos and other immediate early gene proteins.

We have used the evoked expression of the immediate early gene-encoded proteins (c-Fos, Fos B, Jun B, Jun D, c-Jun and Krox-24) to monitor sensory processing in the hindbrain structures of rats undergoing somatic inflammation. Experiments were performed on freely moving animals that did not experience constraints other than those imposed by the disease itself. Local injections of chemicals were used to cause subcutaneous inflammation of the plantar foot or monoarthritis by intracapsular injection. Labelling was studied at survival times that corresponded either to the time points of maximum labelling in the spinal cord (4 h for the subcutaneous model, 24 h and two weeks for the monoarthritis model) or at survival times that corresponded to the chronic phase of monoarthritis evolution (six, nine and 15 weeks). Controls consisted of freely moving, unstimulated animals. Basal expression was observed for all immediate early genes and in a variety of structures, but always remained moderate. All immediate early gene-encoded protein expressions except c-Jun were evoked, but except for c-Fos, and to a lesser extent Jun D, intensities of staining always remained faint. The following results will be mainly based on c-Fos expression, as this protein proved to be the most effective marker for all the survival times studied. Somatic pain evoked c-Fos expression in a subset of discrete subregions of both the caudal medulla oblongata and transitional areas of the pontomesencephalic junction. In the caudal medulla oblongata, structures involved were the caudal intermediate reticular nucleus, the subnucleus reticularis dorsalis, the ventrolateral reticular formation and the lateral paragigantocellular nucleus. Structures involved at the pontomesencephalic junction level mostly included the superior and dorsal lateral subnuclei of the parabrachial area, the nucleus cuneiformis and the most caudal portions of the lateral central gray, also including the laterodorsal tegmental nucleus; labelling in other lateral subnuclei of the parabrachial area always remained moderate. Staining in the caudal reticular areas was evident only at short survival times (4 and 24 h survival times in subcutaneous and monoarthritis models, respectively). Staining in nuclei of the pontomesencephalic junction was evident in all cases except for the very long survival periods (six to 15 weeks) of monoarthritis. In all cases staining was bilateral with contralateral predominance with regard to the stimulated limb. The present work demonstrates that hindbrain structures involved in somatic pain processing can be effectively identified in behaving animals and that c-Fos is the most reliable activity marker in this case.(ABSTRACT TRUNCATED AT 400 WORDS)

Spinal and hindbrain structures involved in visceroception and visceronociception as revealed by the expression of Fos, Jun and Krox-24 proteins.

We have used the evoked expression of the immediate early gene-encoded proteins (Krox-24, c-Fos, Fos B, Jun D, Jun B, c-Jun) to monitor visceral processing in both the spinal cord and hindbrain structures of rats undergoing either mechanical colorectal or chemical intraperitoneal stimulation. Experiments were conducted under controlled volatile anaesthesia to suppress affective reactions that visceral stimulations may induce. The results refer to the effects of anaesthesia alone, and of both innocuous and noxious stimulations. Non-nociceptive and nociceptive stimulation but not anaesthesia were effective in evoking c-Fos, c-Jun, Jun B and Krox-24 expressions in the spinal cord. Intraperitoneal injections labelled cells mostly at the thoracolumbar junction levels, while colorectal distension labelled cells mostly at the lumbrosacral junction levels. Labelling was widely distributed throughout the gray matter including superficial layers, deep dorsal horn, lamina X and sacral parasympathetic columns. Krox-24- and, to a lesser degree, c-Jun-labelled cells were quite numerous in the superficial layers of the dorsal horn; Jun B, and especially c-Fos, were very effective in demonstrating inputs to all parts of the spinal cord. Both anaesthesia and noxious visceral stimulation were effective in evoking c-Fos, Krox-24 and Jun B expressions in discrete hindbrain subregions. The structures which are primarily labelled under anaesthesia are the rostral ventrolateral medulla, the external medial and lateral nuclei of the parabrachial area, the medial and dorsal subnuclei of the nucleus of the solitary tract, the area postrema, the central gray including pars alpha and nucleus O, the nucleus beta of the inferior olive, the locus coeruleus, and the inferior colliculi and adjacent parts of central gray. The structures which are primarily labelled following noxious visceral stimulation are the caudal intermediate reticular nucleus as part of the caudalmost ventrolateral medulla and the superior lateral nucleus of the rostrolateral parabrachial area. Labelling in the caudal intermediate reticular nucleus was maximal for colorectal distension. Labelling in the superior lateral nucleus was specific to peritoneal inflammation. The Edinger-Westphal nucleus is a structure in which noxious-evoked labelling was superposed onto the anaesthesia-evoked labelling. Nociception-evoked overexpression in this nucleus was maximal for intraperitoneal inflammation. The present work demonstrates that the central effects induced by either anaesthesia or visceroception including pain can be effectively monitored through the induction of an array of immediate early genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential time course and spatial expression of Fos, Jun, and Krox-24 proteins in spinal cord of rats undergoing subacute or chronic somatic inflammation.

We have used the evoked expression of both immediate early gene (IEG)-encoded proteins (Krox-24, c-Fos, Fos B, Jun D, Jun B, c-Jun), and dynorphin to monitor sensory processing in the spinal cords of rats undergoing subacute or chronic somatic inflammation (i.e., subcutaneous inflammation of the plantar foot and monoarthritis, respectively). Behavioral and immunocytochemical approaches were conducted in parallel up to 15 weeks postinjection in order to detect possible relationships between clinical evolution and spatiotemporal pattern of IEG-encoded protein expression. Each disease had specific characteristics both in terms of their clinical evolution and pattern of evoked protein expression. All IEG proteins were expressed in both cases. Most of the staining was observed in both the superficial layers of the dorsal horn and deep dorsal horn (laminae V-VII and X). Monoarthritis was distinguished by a high level of total protein expression. Staining was especially dense in the deep dorsal horn. More labelled cells were observed at 1-2 days and at 2 weeks postinjection, corresponding to the initiation and progressive phases of the disease, respectively. Subcutaneous inflammation was characterized by a moderate level of total IEG expression. More labelled cells were observed in the first day following injection. It is the relative degree of expression of each IEG-encoded protein with regard to the others that characterized the progression of the diseases. Early stages of the diseases coincided with the expression of all Fos and Jun proteins, while late stages showed an increase in Jun D and Fos B involvement; Krox-24 was induced mostly during the early phases and/or periods of paroxysm of the diseases. Persistent stimulation was characterized by a predominant expression in deep versus superficial layers of the dorsal horn. Evoked expression of c-Jun in motoneurons was only observed in monoarthritis. The peak of dynorphin expression was late in regard to both the induction of inflammation and period of maximal IEG-encoded protein expression. The present work indicates that the neural processing that takes place during progression of these diseases can be monitored well at the spinal cord level by using the expression of an array of IEG-encoded proteins. Study of long term evolutive diseases and especially those that evolve into chronicity can largely benefit from such an approach.

Atrazine in plasma and tissue following atrazine-aminotriazole-ethylene glycol-formaldehyde poisoning.

A high performance liquid chromatography method has been used to study the plasma kinetics of atrazine in a human fatality after ingestion of a herbicide mix containing atrazine, aminotriazole, ethylene glycol and formaldehyde. A hemodialysis was performed in an effort to eliminate these toxic substances. The mean atrazine clearance over 4 h was 250 mL/min and the dialysance of atrazine was calculated as 76%. On autopsy, the kidney showed the highest concentration of atrazine (97.62 micrograms/g-1 wet tissue) with lesser concentrations in the lung, small intestine and liver, and the lowest concentration in the heart.

Calbindin-D28K (CaBP28k)-like Immunoreactivity in Ascending Projections.

This study concerns the involvement of calbindin-D28K (CaBP28k)-containing neurons in the efferent projections of both the trigeminal nucleus caudalis and the dorsal vagal complex (nucleus of the solitary tract and area postrema) in rats. Recent evidence has shown that these projections are particularly important for the processing of visceroception and/or nociception at central levels. The trigeminal nucleus caudalis has dense projections to both the nucleus of the solitary tract and the parabrachial area; the dorsal vagal complex is intimately connected to the parabrachial area. CaBP28k is a calcium-binding protein the function of which could be a determining factor in controlling the excitability of cells by acting on intrinsic calcium metabolism. CaBP28k content of projections was ascertained using a double labelling approach that combined the retrograde transport of a protein - gold complex to identify projection cells and immunocytochemistry to identify CaBP28k-positive cells. The trigeminal nucleus caudalis is rich in both CaBP28k-immunoreactive cells and cells projecting to the parabrachial area or the nucleus of the solitary tract. Cells containing both the protein and the retrograde tracer, however, were mostly restricted to the superficial layers (laminae I and outer II) and to their rostral extensions, the dorsal paramarginal and paratrigeminal nuclei. These trigeminal subdivisions are targets for nociceptive, visceroceptive and thermal inputs of peripheral origins. The dorsal vagal complex is rich in CaBP28k. Dense populations of immunoreactive cells are observed in the ventrolateral part of the area postrema and all of the three main subdivisions of the nucleus of the solitary tract (rostral gustatory, ventrolateral respiratory and medial cardiovascular subregions). The subnucleus commissuralis, subnucleus centralis and dorsal subnuclei are particularly densely stained. The subnucleus centralis, which is involved in regulating food and water intake, does not project to the parabrachial area. The area postrema, subnucleus commissuralis and dorsal subnuclei, which are implicated in cardiovascular and/or ingestive behaviours, have dense projections to the parabrachial area, many of which contain CaBP28k. The present results demonstrate that CaBP28k-containing cells form a major part of the solitary and trigeminal projection systems, including subregions that are involved in visceroception and/or nociception processing. The location of solitary nucleus projection cells overlaps those of some neuropeptidergic projecting populations, suggesting colocalization. Consequently, certain neuropeptidergic actions may be CaBP28k-dependent.

Calbindin-D28K (CaBP28k)-like Immunoreactivity in Ascending Projections.

This study concerns the involvement of calbindin-D28K (CaBP28k)-containing neurons in ascending spinal projections to the brainstem (nucleus of the solitary tract, lateral reticular nucleus area), pontine (parabrachial area) and mesencephalic (periaqueductal grey) structures. All these central structures are important in the processing of visceroception and visceronociception and all are targets for spinal efferents from similar areas. CaBP28k controls the excitability of cells by acting on intrinsic calcium metabolism. Results refer to the caudal spinal areas where the visceroceptive regions are concentrated. Experiments were performed through a double labelling approach that combined the retrograde transport of a protein - gold complex to identify the projection cells and immunohistochemistry to identify the CaBP28k-positive cells. The caudal spinal cord is rich in both CaBP28k-containing and projection cells. Cells colocalizing the protein and the retrograde tracer were quite numerous, with a particularly high concentration in the superficial layers of the dorsal horn (laminae I and outer II) and the lateral spinal nucleus. The other spinal areas containing immunoreactive projection cells were the reticular part of the neck of the dorsal horn, the medial laminae VII and VIII, lamina X and the sacral parasympathetic nucleus. The superficial layers and the neck of the dorsal horn are targets for nociceptive, visceroceptive and thermal inputs; the sacral parasympathetic column and lamina X are involved in visceroceptive integration. A functional role for the lateral spinal nucleus has not yet been established. Quite similar results were obtained for each of the ascending pathways under study. The high incidence of CaBP28k in spinal pathways suggests that calbindin has a major role in controlling the excitability of spinal cells subserving the processing of visceroception and/or visceronociception information to supraspinal levels. The participation of CaBP28k-immunoreactive cells in spinal ascending tract cells largely outnumbers those previously reported for various neuropeptides (Leah et al., Neuroscience, 24, 195 - 207, 1988)

High-performance liquid chromatographic assay for amiodarone N-deethylation in microsomes of rat liver.

A reversed-phase high-performance liquid chromatographic assay using ultraviolet detection is described for determining the production of the major N-dealkylated metabolite of amiodarone in rat liver microsomes. The principal advantages of this method are its simple sample preparation (protein precipitation by acetonitrile), low detection limit for N-desethylamiodarone (0.05 mumol/l) and relatively short analysis time (16 min). Its analytical applicability is demonstrated by the comparison of the kinetic parameters (maximum velocity and Michaelis-Menten constant) between Sprague-Dawley and Dark-Agouti rats.