Hippocampal Deformations and Entorhinal Cortex Atrophy as an Anatomical Signature of Long-Term Cognitive Impairment: from the MCAO Rat Model to the Stroke Patient.

Stroke patients have an elevated risk of developing long-term cognitive disorders or dementia. The latter is often associated with atrophy of the medial temporal lobe. However, it is not clear whether hippocampal and entorhinal cortex atrophy is the sole predictor of long-term post-stroke dementia. We hypothesized that hippocampal deformation (rather than atrophy) is a predictive marker of long-term post-stroke dementia on a rat model and tested this hypothesis in a prospective cohort of stroke patients.Male Wistar rats were subjected to transient middle cerebral artery occlusion and assessed 6 months later. Ninety initially dementia-free patients having suffered a first-ever ischemic stroke were prospectively included in a clinical study. In the rat model, significant impairments in hippocampus-dependent memories were observed. MRI studies did not reveal significant atrophy of the hippocampus volume, but significant deformations were indeed observed-particularly on the ipsilateral side. There, the neuronal surface area was significantly lower in ischemic rats and was associated with a lower tissue density and a markedly thinner entorhinal cortex. At 6 months post-stroke, 49 of the 90 patients displayed cognitive impairment (males 55.10%). Shape analysis revealed marked deformations of their left hippocampus, a significantly lower entorhinal cortex surface area, and a wider rhinal sulcus but no hippocampal atrophy. Hence, hippocampal deformations and entorhinal cortex atrophy were associated with long-term impaired cognitive abilities in a stroke rat model and in stroke patients. When combined with existing biomarkers, these markers might constitute sensitive new tools for the early prediction of post-stroke dementia.

Stobadine-induced hastening of sensorimotor recovery after focal ischemia/reperfusion is associated with cerebrovascular protection.

In a model of 1 hour-intraluminal occlusion of rat middle cerebral artery (MCA), we investigated the spontaneous recovery of vascular functions and functional deficit together with ischemia volume evolution at 24 h, 3 days and 7 days of reperfusion. Infarct cerebral volumes and edema were quantified with histological methods. Endothelium-dependent and smooth muscle potassium inward rectifier current (Kir2.x)-dependent relaxing responses of MCA were tested using Halpern arteriograph and Kir2.x current density evaluated on MCA myocytes with whole-cell patch-clamp technique. Sensorimotor recovery was estimated according to performances obtained with adhesive removal test and prehensile traction test. A time-dependent improvement of smooth muscle K(+)-dependent vasorelaxation and Kir2.x current density is observed at 7 days of reperfusion while endothelium-dependent relaxation is still impaired. In parallel a significant reduction of functional deficit is observed at 7 days of reperfusion together with a time-matched reduction of striatal infarct and edema volumes. Administration of an antioxidant agent, stobadine, at time of reperfusion and 5 h later allowed: (i) a neuroprotective effect with a significant reduction of infarct size compared to vehicle-treated rats; (ii) a prevention of endothelial-dependent relaxation and Kir2.x current density reductions of MCA ipsilateral to occlusion; (iii) a hastening of the functional recovery. The beneficial effect of stobadine underlines a link between vascular protection, neuronal protection and sensorimotor recovery that could become a promising pharmacological target in the treatment of cerebral ischemia.

Neurogliovascular unit after cerebral ischemia: is the vascular wall a pharmacological target.

Ischemic stroke induces drastic alterations of the functions of the neurogliovascular unit with dramatic consequences on the well-being of the patients in terms of cognitive and motor handicap. Nowadays, only very few therapeutics are available as a treatment of ischemic stroke. Ischemia is a multifactorial pathology involving different cerebral cellular components such as neurons, astrocytes and vessels working as a functional unit. Recent experimental strategy investigation involving different agents with antioxidant properties (dt-BC, stobadine) or pleiotropic effects (lipopolysaccharide, LPS) has been developed to evaluate whether the vascular wall could be considered as a potential target in neuroprotection concept.

PPAR: a new pharmacological target for neuroprotection in stroke and neurodegenerative diseases.

PPARs (peroxisome-proliferator-activated receptors) are ligand-activated transcriptional factor receptors belonging to the so-called nuclear receptor family. The three isoforms of PPAR (alpha, beta/delta and gamma) are involved in regulation of lipid or glucose metabolism. Beyond metabolic effects, PPARalpha and PPARgamma activation also induces anti-inflammatory and antioxidant effects in different organs. These pleiotropic effects explain why PPARalpha or PPARgamma activation has been tested as a neuroprotective agent in cerebral ischaemia. Fibrates and other non-fibrate PPARalpha activators as well as thiazolidinediones and other non-thiazolidinedione PPARgamma agonists have been demonstrated to induce both preventive and acute neuroprotection. This neuroprotective effect involves both cerebral and vascular mechanisms. PPAR activation induces a decrease in neuronal death by prevention of oxidative or inflammatory mechanisms implicated in cerebral injury. PPARalpha activation induces also a vascular protection as demonstrated by prevention of post-ischaemic endothelial dysfunction. These vascular effects result from a decrease in oxidative stress and prevention of adhesion proteins, such as vascular cell adhesion molecule 1 or intercellular cell-adhesion molecule 1. Moreover, PPAR activation might be able to induce neurorepair and endothelium regeneration. Beyond neuroprotection in cerebral ischaemia, PPARs are also pertinent pharmacological targets to induce neuroprotection in chronic neurodegenerative diseases.

"Use-dependent" effects of cisapride on postrest action potentials in rabbit ventricular myocardium.

Repercussions of cisapride-induced blocking effects on repolarisation of K(+) channels in open and inactivated states investigated in rabbit ventricular myocardium during rest and under stimulation were compared with effects of K(+)-blocking drugs (4-aminopyridine, dofetilide, terikalant). Major lengthening in the first postrest action potential indicates affinity for closed channels. Gradual lengthening during stimulation implies affinity for open channels. Four (control, add-in, steady-state, washout) 20-min rest periods were alternated with regular stimulation (0.5 Hz). Each drug was added during add-in and steady-state periods. Similarly to dofetilide (10 nM) and terikalant (0.3 microM), cisapride (1 microM) increasingly lengthened action potentials during stimulation, whereas 4-aminopyridine (1 mM) prolonged mostly the first postrest action potential. Our results indicate that cisapride induced use-dependent lengthening of repolarisation, compatible with an affinity for open K(+) channels. We also found that in isolated rabbit ventricular myocytes, cisapride (1-10 microM) decreased the inward rectifier K(+) current, an effect contributing to the proarrhythmic potential.

Sequence and analysis of chromosome 5 of the plant Arabidopsis thaliana.

The genome of the model plant Arabidopsis thaliana has been sequenced by an international collaboration, The Arabidopsis Genome Initiative. Here we report the complete sequence of chromosome 5. This chromosome is 26 megabases long; it is the second largest Arabidopsis chromosome and represents 21% of the sequenced regions of the genome. The sequence of chromosomes 2 and 4 have been reported previously and that of chromosomes 1 and 3, together with an analysis of the complete genome sequence, are reported in this issue. Analysis of the sequence of chromosome 5 yields further insights into centromere structure and the sequence determinants of heterochromatin condensation. The 5,874 genes encoded on chromosome 5 reveal several new functions in plants, and the patterns of gene organization provide insights into the mechanisms and extent of genome evolution in plants.

Increase in endogenous brain superoxide dismutase as a potential mechanism of lipopolysaccharide-induced brain ischemic tolerance.

A low dose (0.5 mg/kg) of lipopolysaccharide (LPS), administered 72 hours before 60-minute middle cerebral artery occlusion, induced a delayed neuroprotection proven by the significant decrease (-35%) of brain infarct volume in comparison with control, whereas infarct volumes remained unchanged in rats treated 12, 24, or 168 hours before ischemia. This delayed neuroprotective effect of LPS was induced only with low doses (0.25 to 1 mg/kg), whereas this effect disappeared with a higher dose (2 mg/kg). The delayed neuroprotection of LPS was induced in the cortical part of the infarcted zone, not in the subcortical part. The beneficial effect of LPS on consequences of middle cerebral artery occlusion was suppressed by dexamethasone (3 mg/kg) and indomethacin (3 mg/ kg) administered 1 hour before LPS, whereas both drugs had no direct effect on infarct volume by themselves, suggesting that activation of inflammatory pathway is involved in the development of LPS-induced brain ischemic tolerance. Preadministration of cycloheximide, an inhibitor of protein synthesis, also blocked LPS-induced brain ischemic tolerance suggesting that a protein synthesis is also necessary as a mediating mechanism. Superoxide dismutase (SOD) could be one of the synthesized proteins because lipopolysaccharide increased SOD brain activity 72 hours, but not 12 hours, after its administration, which paralleled the development of brain ischemic tolerance. In contrast, catalase brain activity remained unchanged after LPS administration. The LPS-induced delayed increase in SOD brain content was suppressed by a previous administration of indomethacin. These data suggest that the delayed neuroprotective effect of low doses of LPS is mediated by an increased synthesis of brain SOD that could be triggered by activation of inflammatory pathway.

Differential role of nitric oxide pathway and heat shock protein in preconditioning and lipopolysaccharide-induced brain ischemic tolerance.

The purposes of this study were to investigate the role of nitric oxide (NO), nitric oxide synthase (NOS), and 70 kDa heat shock protein in brain ischemic tolerance induced by ischemic preconditioning and lipopolysaccharide. Focal cerebral ischemia was induced in rats by intraluminal middle cerebral artery occlusion. Infarct volume was significantly reduced (1) in rats subjected to 3 min ischemia 72 h prior to 60 min ischemia; (2) in rats administered lipopolysaccharide (0.5 mg/kg; i.p.) 72 h prior to 60 min ischemia compared with controls. The beneficial effect of ischemic preconditioning was unchanged despite prior administration of nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor. Conversely, the protective effect of lipopolysaccharide was nullified by L-NAME. Using immunohistochemical techniques, we observed that (1) ischemic preconditioning but not lipopolysaccharide induces the expression of 70 kDa heat shock protein in cerebral cortex and (2) lipopolysaccharide induces early increased expression of endothelial NOS in cerebral blood vessels. The results suggest that (1) endothelium-derived NO plays a role of a trigger in the brain tolerance induced by lipopolysaccharide, and (2) 70 kDa heat shock protein is involved in the protection afforded by ischemic preconditioning but not by lipopolysaccharide.

Relationship between inward rectifier potassium current impairment and brain injury after cerebral ischemia/reperfusion.

Functional alterations of barium-sensitive potassium inward rectifier (KIR) current, which is involved in the vasodilation of middle cerebral arteries (MCA) in rat brain, have been described during brain ischemia/reperfusion (I/R). The authors investigate the effects of I/R on KIR current recorded in isolated myocytes from MCA of control rats and from contralateral and ipsilateral MCA of ischemic rats by the whole-cell patch-clamp technique, and the relationship between its alteration and the severity of brain injury. The vascular smooth muscle cells exhibited similar morphologic features in all conditions, and the KIR was present in the three groups of myocytes, exhibiting a characteristic inward rectification and a normal external potassium dependence. The KIR density was significantly reduced in cell of MCA ipsilateral to occlusion with a maximum at -135 mV, whereas there was no difference between control and contralateral cells. This alteration in KIR density in occluded MCA was significantly correlated with severity of brain injury and brain edema. These results suggest that the alteration of KIR density in MCA myocytes after I/R and the consecutive impaired dilation of MCA may contribute to aggravation of the brain injury.

Simple and sensitive method for determination of metronidazole in human serum by high-performance liquid chromatography.

A simple and sensitive HPLC method for determination of metronidazole in human plasma has been developed. A step of freezing the protein precipitate allowed an efficient separation of aqueous and organic phases minimizing the noise level and improved therefore the limit of quantitation (10 ng ml(-1) using 1 ml of plasma sample). The separation of compounds was performed on a RP 18 column with acetonitrile-aqueous 0.01 M phosphate solution (15:85, v/v) as mobile phase. Detection was performed by UV absorbance at 318 nm. Metronidazole was well resolved from the plasma constituents and internal standard. An excellent linearity was observed between peak-height ratios plasma concentrations over a concentration range of 0.01 to 10 microg ml(-1). Within-day and between-day precision (expressed by relative standard deviation) and accuracy (mean error in per cent) did not exceed 4% between 1 and 10 microg ml(-1) and 8.3 and 7.2% respectively for the limit of quantitation. The method is suitable for bioavailability and pharmacokinetic studies in humans.

A communication process: a new paradigm applied to high-dilution effects on the living body.

Living beings communicate with their world nonverbally, whether on a somatic or a psychological level. This paradigm of signifiers or sense takes place in the framework of the logic of analogy. The signifier is the semantic object that materially designates information to be transmitted and dealt with; a homeopathic remedy is the mimetic representation of the disease. Differential levels of information organize the spread of signifiers; each level is the result of regulation and integration of the previous level. The living self is the never-ending process whereby levels of information are synthesized in the face of the informing environment. Such representations meet one another in the communication between the patient and the physician-remedy system. The medical device must reinform the patient and make the patient's signs and symptoms move toward a higher level of integration. The dilution of the remedy permits us to receive and treat it as information about disease. Signs and symptoms can be recognized as an erroneous adaptation; the organism is engaged in a process of paradoxical negation. The action of the remedies consists of a dynamic analogy between pieces of information. The paradigm of signifiers offers a new possibility for the exploration of informative therapeutics.

Murine macrophage elastolytic activity induced by Aspergillus fumigatusstrains in vitro: evidence of the expression of two macrophage-induced protease genes.

The interaction between Aspergillus fumigatus conidia and murine macrophages of various origins was investigated. Cocultures were carried out between A. fumigatus strains and freshly isolated murine pulmonary alveolar macrophages or two murine macrophage cell-lines: murine alveolar cell-line MALU and murine astrocytoma cell-line J774. By measuring the variation of elastolytic activity in the coculture supernatants with two elastin substrates, we demonstrated that either viable or fixed A. fumigatus or C. albicans yeasts or nonspecific particles induced significant macrophage elastolytic activity. The effect of A. fumigatus supernatant or the purified A. fumigatus galactomannan suggested also the possible involvement of this polysaccharide in macrophage-protease gene expression, release, and activity in invasive aspergillosis. The effect of inhibitory compounds demonstrated the potential implication of a macrophagic metalloprotease and a macrophagic cysteine protease. RNA analysis allowed us to demonstrate the induction of expression of two macrophagic protease genes in stimulated macrophages. Two distinctive mechanisms appeared to be implicated in macrophage protease induction: nonspecific phagocytosis in the earliest times of the coculture and (or) specific galactomannan recognition after its gradual release by the mycelium.

cGMP-stimulated cyclic nucleotide phosphodiesterase regulates the basal calcium current in human atrial myocytes.

EHNA (Erythro-9-[2-hydroxy-3-nonyl]adenine) is a wellknown inhibitor of adenosine deaminase. Recently, EHNA was shown to block the activity of purified soluble cGMPstimulated phosphodiesterase (PDE2) from frog, human, and porcine heart with an apparent Ki value of approximately 1 microM and with negligible effects on Ca2+/calmodulin PDE (PDE1), cGMP-inhibited PDE (PDE3), and low Km cAMP-specific PDE (PDE4) (Méry, P.F., C. Pavoine, F. Pecker, and R. Fischmeister. 1995. Mol. Pharmacol. 48:121-130; Podzuweit, T., P. Nennstiel, and A. Muller. 1995. Cell. Signalling. 7:733- 738). To investigate the role of PDE2 in the regulation of cardiac L-type Ca2+ current (ICa), we have examined the effect of EHNA on ICa in freshly isolated human atrial myocytes. Extracellular application of 0.1-10 microM EHNA induced an increase in the amplitude of basal ICa ( approximately 80% at 1 microM) without modification of the current-voltage or inactivation curves. The maximal stimulatory effect of EHNA on ICa was comparable in amplitude with the maximal effect of isoprenaline (1 microM), and the two effects were not additive. The effect of EHNA was not a result of adenosine deaminase inhibition, since 2'-deoxycoformycin (1-30 microM), another adenosine deaminase inhibitor with no effect on PDE2, or adenosine (1-10 microM) did not increase ICa. In the absence of intracellular GTP, the substrate of guanylyl cyclase, EHNA did not increase ICa. However, under similar conditions, intracellular perfusion with 0.5 microM cGMP produced an 80% increase in ICa. As opposed to human cardiomyocytes, EHNA (1-10 microM) did not modify ICa in isolated rat ventricular and atrial myocytes. We conclude that basal ICa is controlled by PDE2 activity in human atrial myocytes. Both PDE2 and PDE3 may contribute to keep the cyclic nucleotides concentrations at minimum in the absence of adenylyl and/or guanylyl cyclase stimulation.

Biological effects of continuous exposure of embryos and young chickens to electromagnetic fields emitted by video display units.

The effects of continuous exposure of embryos and young chickens to electromagnetic fields (EMFs) emitted by video display units (VDUs) were investigated. Embryos and brood were continuously exposed during embryonic and postembryonic phases to EMFs emitted by two types of VDU (TV or computer). Embryonic mortality was evaluated in three independent experiments. Young chickens were immunized three times by porcine thyroglobulin (Tg). Blood samples were assayed after each immunization for specific anti-Tg antibodies (IgG), plasma corticosterone (CORT), and plasma melatonin (MLT). In the sham-exposed samples, embryonic death (10-33%) was restricted to the perinatal period and the IgG, CORT, and MLT responses of young chickens crested after the second immunization. Constant EMF exposure was accompanied by significantly increased fetal loss (47-68%) and markedly depressed levels of circulating anti-Tg IgG, CORT, and MLT. Collectively, these findings indicate that continuous exposure to EMFs, issuing from VDUs, adversely affects embryos and young chickens.

A role for bursa fabricii and bursin in the ontogeny of the pineal biosynthetic activity in the chicken.

The tripeptide bursin (Lys-His-Gly-NH2) is a B cell differentiation hormone derived from the bursa fabricii. The latter is a cloacal diverticulum and the site of B lymphocyte differentiation and selection in aves; also the bursa fabricii is involved in endocrine functions. Herein we demonstrate that in the chicken, the bursa fabricii and bursin are crucial to the ontogeny of both the pineal response to antigenic challenge and pineal circadian synthetic activity. In early embryonically bursectomized chickens, the plasma melatonin response to immunization by porcine thyroglobulin (Tg) was abolished. Also, the amplitudes of both plasma melatonin and pineal N-acetyltransferase (NAT) circadian rhythms were reduced by 50%, whereas the activity of hydroxyindole-O-methyltransferase (HIOMT) remained unchanged. Conversely, administration of either minute amounts (100 pg, 100 fg) or highly dilute (5 x 10(-27) g) bursin, with the exception of a highest dose (100 micrograms), to bursaless embryos induced recovery of normal antigen-induced melatonin response and normal amplitudes of melatonin and NAT rhythms. These findings establish that early in embryonic life, the bursa fabricii and its derived signal (bursin) are essential for normal development of pineal synthetic activity and underline the efficacy of very dilute bursin as an informative signal.

Effect of calcitonin gene-related peptide and vasoactive intestinal peptide on murine CD4 and CD8 T cell proliferation.

The effects of alpha calcitonin gene-related peptide (alpha CGRP) and vasoactive intestinal peptide (VIP) on the proliferation of CD4 and CD8 T-murine lymphocytes were investigated. When stimulated by a combination of phorbol 12-myristate-13-acetate (PMA) and calcium ionophore (A23187), both neuropeptides in a range of 10(-7)-10(-10) M had an inhibitory effect on the proliferative response of unfractionated splenocytes as well as of purified CD4 and CD8 T lymphocytes. The inhibitory effect of these two neuropeptides was completely or partially blocked by the antagonists of CGRP and VIP receptors. CGRP8-37 and (p-Cl-D-Phe6, Leu17VIP, respectively. The inhibitory effects of each neuropeptide on purified T cells were observed within 4 h after PMA/A23187 activation and their inhibitory actions were correlated with a decrease of IL-2 production. In addition, the two neuropeptides in a range of 10(-7)-10(-10) M induced a rapid and dose-dependent increase in intracellular cAMP in CD4 and CD8 T cells. This suggests the involvement of this second messenger in the inhibitory effects of these two neuropeptides. Taken together these results show that CD4 and CD8 spleen cells represent at least two of the cellular targets for CGRP and VIP inhibition of proliferation mediated by the same type of mechanism.

[4-Arylpiperazine derivatives: a seedbed of drugs for various therapeutic indications]. / Dérivés 4-arylpipéraziniques: une pépinière de médicaments aux indications thérapeutiques vari s?

The main focus of this review is the pharmacology and the therapeutic use of chemical related agents, the arylpiperazine derivatives. These compounds produce a variety of behavioural responses and pharmacological effects which directly and principally result from activation of serotonin systems. However, minor modifications in the chemical structure of these products involve important changes in affinity and selectivity for 5-HT receptors since it can also display significant affinity for dopaminergic, adrenergic or histaminergic receptors. The different arylpiperazine drugs therapeutically used are described as well as some compounds presently under investigation.

Idiotypic restriction of murine monoclonal antibodies to a defined antigenic region of human thyroglobulin.

In previous studies, we demonstrated that anti-human thyroglobulin (hTg) autoantibodies in patients with thyroid disorders exhibit a restricted epitopic specificity towards antigenic region II defined by its reactivity with four murine monoclonal antibodies (mAb 3, 6, 10, 15). To analyze the relationships between epitopic specificity and idiotypic expression of these mAb, two polyclonal anti-idiotypic sera were generated in rabbits by immunization with F(ab')2 fragments of mAb 3 and mAb 10. These anti-idiotypic preparations (AI 3 and AI 10) were tested against a panel of hTg-mAb produced in different strains of mice (HR BIOZZI and BALB/c). The idiotypic analysis showed that AI 3 and AI 10 specifically recognized framework-associated idiotopes as well as paratope-associated idiotopes shared by region II mAb. These results demonstrate that specificity for region II was strongly associated with a restricted idiotype suggesting a high sequence homology between V regions. In addition, naïve BALB/c mice immunized with AI 3 or AI 10 produced anti-hTg (Ab3) antibodies that recognize region II epitopes. These latter findings reveal that anti-Id contain a population of Ab2 beta carrying the internal image of region II epitopes.

Nitric oxide regulates the calcium current in isolated human atrial myocytes.

Cardiac Ca2+ current (ICa) was shown to be regulated by cGMP in a number of different species. Recently, we found that the NO-donor SIN-1 (3-morpholino-sydnonimine) exerts a dual regulation of ICa in frog ventricular myocytes via an accumulation of cGMP. To examine whether NO also regulates Ca2+ channels in human heart, we investigated the effects of SIN-1 on ICa in isolated human atrial myocytes. An extracellular application of SIN-1 produced a profound stimulatory effect on basal ICa at concentrations > 1 pM. Indeed, 10 pM SIN-1 induced a approximately 35% increase in ICa. The stimulatory effect of SIN-1 was maximal at 1 nM (approximately 2-fold increase in ICa) and was comparable with the effect of a saturating concentration (1 microM) of isoprenaline, a beta-adrenergic agonist. Increasing the concentration of SIN-1 to 1-100 microM reduced the stimulatory effect in two thirds of the cells. The stimulatory effect of SIN-1 was not mimicked by SIN-1C, the cleavage product of SIN-1 produced after liberation of NO. This suggests that NO mediates the effects of SIN-1 on ICa. Because, in frog heart, the stimulatory effect of SIN-1 on ICa was found to be due to cGMP-induced inhibition of cGMP-inhibited phosphodiesterase (cGI-PDE), we compared the effects of SIN-1 and milrinone, a cGI-PDE selective inhibitor, on ICa in human. Milrinone (10 microM) induced a strong stimulation of ICa (approximately 150%), demonstrating that cGI-PDE controls the amplitude of basal ICa in this tissue. In the presence of milrinone, SIN-1 (0.1-1 nM) had no stimulatory effect on ICa, suggesting that the effects of SIN-1 and MIL were not additive. We conclude that NO may stimulate ICa in human atrial myocytes via inhibition of the cGI-PDE.