333 resultados para EPILEPSY-PRONE RAT
Resumo:
The cellular localisation of neurofilament triplet subunits was investigated in the rat neocortex. A subset of mainly pyramidal neurons showed colocalisation of subunit immunolabelling throughout the neocortex, including labelling with the antibody SMI32, which has been used extensively in other studies of the primate cortex as a selective cellular marker. Neurofilament-labelled neurons were principally localised to two or three cell layers in most cortical regions, but dramatically reduced labelling was present in areas such as the perirhinal cortex, anterior cingulate and a strip of cortex extending from caudal motor regions through the medial parietal region to secondary visual areas. However, quantitative analysis demonstrated a similar proportion (10-20%) of cells with neurofilament triplet labelling in regions of high or low labelling. Combining retrograde tracing with immunolabelling showed that cellular content of the neurofilament proteins was not correlated with the length of projection. Double labelling immunohistochemistry demonstrated that neurofilament content in axons was closely associated with myelination. Analysis of SMI32 labelling in development indicated that content of this epitope within cell bodies was associated with relatively late maturation, between postnatal days 14 and 21. This study is further evidence of a cell type-specific regulation of neurofilament proteins within neocortical neurons. Neurofilament triplet content may be more closely related to the degree of myelination, rather than the absolute length, of the projecting axon.
Resumo:
PURPOSE: To evaluate whether anti-vascular endothelial growth factor (VEGF) neutralizing antibodies injected in the vitreous of rat eyes influence retinal microglia and macrophage activation. To dissociate the effect of anti-VEGF on microglia and macrophages subsequent to its antiangiogenic effect, we chose a model of acute intraocular inflammation. METHODS: Lewis rats were challenged with systemic lipopolysaccharide (LPS) injection and concomitantly received 5 µl of rat anti-VEGF-neutralizing antibody (1.5 mg/ml) in the vitreous. Rat immunoglobulin G (IgG) isotype was used as the control. The effect of anti-VEGF was evaluated at 24 and 48 h clinically (uveitis scores), biologically (cytokine multiplex analysis in ocular media), and histologically (inflammatory cell counts on eye sections). Microglia and macrophages were immunodetected with ionized calcium-binding adaptor molecule 1 (IBA1) staining and counted based on their differential shapes (round amoeboid or ramified dendritiform) on sections and flatmounted retinas using confocal imaging and automatic quantification. Activation of microglia was also evaluated with inducible nitric oxide synthase (iNOS) and IBA1 coimmunostaining. Coimmunolocalization of VEGF receptor 1 and 2 (VEGF-R1 and R2) with IBA1 was performed on eye sections with or without anti-VEGF treatment. RESULTS: Neutralizing rat anti-VEGF antibodies significantly decreased ocular VEGF levels but did not decrease the endotoxin-induced uveitis (EIU) clinical score or the number of infiltrating cells and cytokines in ocular media (interleukin [IL]-1β, IL-6, tumor necrosis factor [TNF]-α, and monocyte chemoattractant protein [MCP]-1). Eyes treated with anti-VEGF showed a significantly decreased number of activated microglia and macrophages in the retina and the choroid and decreased iNOS-positive microglia. IBA1-positive cells expressed VEGF-R1 and R2 in the inflamed retina. CONCLUSIONS: Microglia and macrophages expressed VEGF receptors, and intravitreous anti-VEGF influenced the microglia and macrophage activation state. Taking into account that anti-VEGF drugs are repeatedly injected in the vitreous of patients with retinal diseases, part of their effects could result from unsuspected modulation of the microglia activation state. This should be further studied in other ocular pathogenic conditions and human pathology.
Resumo:
Serum-free aggregating cell cultures of fetal rat telencephalon were examined by biochemical and immunocytochemical methods for their development-dependent expression of several cytoskeletal proteins, including the heavy- and medium-sized neurofilament subunits (H-NF and M-NF, respectively); brain spectrin; synapsin I; beta-tubulin; and the microtubule-associated proteins (MAPs) 1, 2, and 5 and tau protein. It was found that with time in culture the levels of most of these cytoskeletal proteins increased greatly, with the exceptions of the particular beta-tubulin form studied, which remained unchanged, and MAP 5, which greatly decreased. Among the neurofilament proteins, expression of M-NF preceded that of H-NF, with the latter being detectable only after approximately 3 weeks in culture. Furthermore, MAP 2 and tau protein showed a development-dependent change in expression from the juvenile toward the adult form. The comparison of these developmental changes in cytoskeletal protein levels with those observed in rat brain tissue revealed that protein expression in aggregate cultures is nearly identical to that in vivo during maturation of the neuronal cytoskeleton. Aggregate cultures deprived of glial cells, i.e., neuron-enriched cultures prepared by treating early cultures with the antimitotic drug cytosine arabinoside, exhibited pronounced deficits in M-NF, H-NF, MAP 2, MAP 1, synapsin I, and brain spectrin, with increased levels of a 145-kDa brain spectrin breakdown product. These adverse effects of glial cell deprivation could be reversed by the maintenance of neuron-enriched cultures at elevated concentrations of KCl (30 mM). This chronic treatment had to be started at an early developmental stage to be effective, a finding suggesting that sustained depolarization by KCl is able to enhance the developmental expression and maturation of the neuronal cytoskeleton.
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The rat adrenal gland contains ganglion cells able to synthesize nitric oxide (NO). This messenger molecule controls and modulates adrenal secretory activity and blood flow. The present study analyzed the number, size, and distribution of NO-producing adrenal neurons in adulthood and during postnatal development by means of beta-nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. This method reliably visualizes the enzyme responsible for NO generation. The reactive neurons per adrenal gland were 350-400 in both male and female adult rats. The positive nerve cell bodies were mostly located in the medulla, few being detected within the cortex and the subcapsular region. Dual labeling with anti-microtubule-associated protein 2 antibody, specific for neuronal elements, confirmed this distribution. Anti-microtubule-associated protein 1b antibody identified a subset of NADPH-d-positive neurons, displaying different degrees of maturation according to their position within the adrenal gland. At birth, there were about 220 NADPH-d-labeled neurons per adrenal gland in both sexes. As confirmed by dual immunocytochemical labeling, their great majority was evenly distributed between the cortex and the subcapsular region, the medulla being practically devoid of stained neurons. After birth, the number of adrenal NADPH-d-positive ganglion cells displayed a strong postnatal increase and reached the adult-like distribution after 1-2 months. During the period of increase, there was a transient difference in the numbers of these cells in the two sexes. Thus we present here evidence of plasticity in the number, size, and distribution of NADPH-d-positive adrenal neurons between birth and adulthood; in addition, we describe transient sex-related differences in their number and distribution during the 2nd postnatal week, which are possibly related to the epigenetic action of gonadal hormones during this period.
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AIMS/HYPOTHESIS: Pro-atherogenic and pro-oxidant, oxidised LDL trigger adverse effects on pancreatic beta cells, possibly contributing to diabetes progression. Because oxidised LDL diminish the expression of genes regulated by the inducible cAMP early repressor (ICER), we investigated the involvement of this transcription factor and of oxidative stress in beta cell failure elicited by oxidised LDL. METHODS: Isolated human and rat islets, and insulin-secreting cells were cultured with human native or oxidised LDL or with hydrogen peroxide. The expression of genes was determined by quantitative real-time PCR and western blotting. Insulin secretion was monitored by EIA kit. Cell apoptosis was determined by scoring cells displaying pycnotic nuclei. RESULTS: Exposure of beta cell lines and islets to oxidised LDL, but not to native LDL raised the abundance of ICER. Induction of this repressor by the modified LDL compromised the expression of important beta cell genes, including insulin and anti-apoptotic islet brain 1, as well as of genes coding for key components of the secretory machinery. This led to hampering of insulin production and secretion, and of cell survival. Silencing of this transcription factor by RNA interference restored the expression of its target genes and alleviated beta cell dysfunction and death triggered by oxidised LDL. Induction of ICER was stimulated by oxidative stress, whereas antioxidant treatment with N-acetylcysteine or HDL prevented the rise of ICER elicited by oxidised LDL and restored beta cell functions. CONCLUSIONS/INTERPRETATION: Induction of ICER links oxidative stress to beta cell failure caused by oxidised LDL and can be effectively abrogated by antioxidant treatment.
Resumo:
Pre-operative assessment and surgical management of patients with non-lesional extratemporal epilepsy remain challenging due to a lack of precise localisation of the epileptic zone. In most cases, invasive recording with depth or subdural electrodes is required. Here, we describe the case of 6.5-year-old girl who underwent comprehensive non-invasive phase I video-EEG investigation for drug-resistant epilepsy, including electric source and nuclear imaging. Left operculo-insular epilepsy was diagnosed. Post-operatively, she developed aphasia which resolved within one year, corroborating the notion of enhanced language plasticity in children. The patient remained seizure-free for more than three years.
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This study was conducted to identify enzyme systems eventually catalysing a local cerebral metabolism of citalopram, a widely used antidepressant of the selective serotonin reuptake inhibitor type. The metabolism of citalopram, of its enantiomers and demethylated metabolites was investigated in rat brain microsomes and in rat and human brain mitochondria. No cytochrome P-450 mediated transformation was observed in rat brain. By analysing H2O2 formation, monoamine oxidase A activity in rat brain mitochondria could be measured. In rat whole brain and in human frontal cortex, putamen, cerebellum and white matter of five brains monoamine oxidase activity was determined by the stereoselective measurement of the production of citalopram propionate. All substrates were metabolised by both forms of MAO, except in rat brain, where monoamine oxidase B activity could not be detected. Apparent Km and Vmax of S-citalopram biotransformation in human frontal cortex by monoamine oxidase B were found to be 266 microM and 6.0 pmol min(-1) mg(-1) protein and by monoamine oxidase A 856 microM and 6.4 pmol min(-1) mg(-1) protein, respectively. These Km values are in the same range as those for serotonin and dopamine metabolism by monoamine oxidases. Thus, the biotransformation of citalopram in the rat and human brain occurs mainly through monoamine oxidases and not, as in the liver, through cytochrome P-450.
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Extremely preterm infants commonly show brain injury with long-term structural and functional consequences. Three-day-old (P3) rat pups share some similarities in terms of cerebral development with the very preterm infant (born at 24-28 weeks of gestation). The aim of this study was to assess longitudinally the cerebral structural and metabolic changes resulting from a moderate neonatal hypoxic ischemic injury in the P3 rat pup using high-field (9.4 T) MRI and localized (1) H magnetic resonance spectroscopy techniques. The rats were scanned longitudinally at P3, P4, P11, and P25. Volumetric measurements showed that the percentage of cortical loss in the long term correlated with size of damage 6 h after hypoxia-ischemia, male pups being more affected than female. The neurochemical profiles revealed an acute decrease of most of metabolite concentrations and an increase in lactate 24 h after hypoxia-ischemia, followed by a recovery phase leading to minor metabolic changes at P25 in spite of an abnormal brain development. Further, the increase of lactate concentration at P4 correlated with the cortical loss at P25, giving insight into the early prediction of long-term cerebral alterations following a moderate hypoxia-ischemia insult that could be of interest in clinical practice.
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To study inflammatory reactions occurring in relation to demyelination, aggregating rat brain cell cultures were subjected to three different demyelinating insults, i.e., (i) lysophosphatidylcholine (LPC), (ii) interferon-gamma combined with lipopolysaccharide (IFN-gamma+LPS), and (iii) anti-MOG antibodies plus complement (alpha-MOG+C). Demyelination was assessed by measuring the expression of myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG), and the activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP). The accompanying inflammatory reactions were examined by the quantification of microglia-specific staining, by immunostaining for glial fibrillary acidic protein (GFAP), and by measuring the mRNA expression of a panel of inflammation-related genes. It was found that all three demyelinating insults decreased the expression of MBP and MOG, and induced microglial reactivity. LPC and alpha-MOG+C, but not IFN-gamma+LPS, decreased CNP activity; they also caused the appearance of macrophagic microglia, and increased GFAP staining indicating astrogliosis. LPC affected also the integrity of neurons and astrocytes. LPC and IFN-gamma+LPS upregulated the expression of the inflammation-related genes IL-6, TNF-alpha, Ccl5, Cxcl1, and iNOS, although to different degrees. Other inflammatory markers were upregulated by only one of the three insults, e.g., Cxcl2 by LPC; IL-1beta and IL-15 by IFN-gamma+LPS; and IFN-gamma by alpha-MOG+C. These findings indicate that each of the three demyelinating insults caused distinct patterns of demyelination and inflammatory reactivity, and that of the demyelinating agents tested only LPC exhibited general toxicity.
Lipoprotein lipase and leptin are accumulated in different secretory compartments in rat adipocytes.
Resumo:
Adipose cells produce and secrete several physiologically important proteins, such as lipoprotein lipase (LPL), leptin, adipsin, Acrp30, etc. However, secretory pathways in adipocytes have not been characterized, and vesicular carriers responsible for the accumulation and transport of secreted proteins have not been identified. We have compared the intracellular localization of two proteins secreted from adipose cells: leptin and LPL. Adipocytes accumulate large amounts of both proteins, suggesting that neither of them is targeted to the constitutive secretory pathway. By means of velocity centrifugation in sucrose gradients, equilibrium density centrifugation in iodixanol gradients, and immunofluorescence confocal microscopy, we determined that LPL and leptin were localized in different membrane structures. LPL was found mainly in the endoplasmic reticulum with a small pool being present in low density membrane vesicles that may represent a secretory compartment in adipose cells. Virtually all intracellular leptin was localized in these low density secretory vesicles. Insulin-sensitive Glut4 vesicles did not contain either LPL or leptin. Thus, secretion from adipose cells is controlled both at the exit from the endoplasmic reticulum as well as at the level of "downstream" secretory vesicles.
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The biodistribution of transgene expression in the CNS after localized stereotaxic vector delivery is an important issue for safety of gene therapy for neurological diseases. The cellular specificity of transgene expression from rAAV2/1 vectors using the tetON expression cassette in comparison with the CMV promoter was investigated in the rat nigrostriatal pathway. After intrastriatal injection, although GFP was mainly expressed into neurons with both vectors, the relative proportions of DARPP-32+ projection neurons and parvalbumin+ interneurons were respectively 13:1 and 2:1 for the CMV and tetON vectors. DARP32+ neurons projecting to the globus pallidus were strongly GFP+ with both vectors, whereas those projecting to the substantia nigra pars reticulata (SNpr) were efficiently labeled by the CMV but poorly by the tetON vector. Numerous GFP+ cells were evidenced in the subventricular zone with both vectors. However, in the olfactory bulb (OB), GFP+ neurons were observed with the CMV but not the tetON vector. We conclude that the absence of significant amounts of transgene product in distant regions (SN and OB) constitutes a safety advantage of the AAV2/1-tetON vector for striatal gene therapy. Midbrain injections resulted in selective GFP expression in tyrosine hydroxylase+ neurons by the tetON vector whereas with the CMV vector, GFP+ cells covered a widespread area of the midbrain. The biodistribution of GFP protein corresponded to that of the transcripts and not of the viral genomes. We conclude that the rAAV2/1-tetON vector constitutes an interesting tool for specific transgene expression in midbrain dopaminergic neurons.
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Knowledge of T(1) relaxation times can be important for accurate relative and absolute quantification of brain metabolites, for sensitivity optimizations, for characterizing molecular dynamics, and for studying changes induced by various pathological conditions. (1)H T(1) relaxation times of a series of brain metabolites, including J-coupled ones, were determined using a progressive saturation (PS) technique that was validated with an adiabatic inversion-recovery (IR) method. The (1)H T(1) relaxation times of 16 functional groups of the neurochemical profile were measured at 14.1T and 9.4T. Overall, the T(1) relaxation times found at 14.1T were, within the experimental error, identical to those at 9.4T. The T(1)s of some coupled spin resonances of the neurochemical profile were measured for the first time (e.g., those of gamma-aminobutyrate [GABA], aspartate [Asp], alanine [Ala], phosphoethanolamine [PE], glutathione [GSH], N-acetylaspartylglutamate [NAAG], and glutamine [Gln]). Our results suggest that T(1) does not increase substantially beyond 9.4T. Furthermore, the similarity of T(1) among the metabolites (approximately 1.5 s) suggests that T(1) relaxation time corrections for metabolite quantification are likely to be similar when using rapid pulsing conditions. We therefore conclude that the putative T(1) increase of metabolites has a minimal impact on sensitivity when increasing B(0) beyond 9.4T.
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The acute effects of various vasopressor agents on the diameter of the common carotid artery were studied in halothane-anesthetized normotensive rats. The animals were infused intravenously for 60 min with equipressor doses of angiotensin II (10 ng/min), the alpha1-stimulant methoxamine (5 microg/min), lysine vasopressin (5 mU/min), or vehicle. The arterial diameter was measured by using a high-resolution ultrasonic echo-tracking device. The three vasoconstrictors increased the carotid artery diameter, but this effect was significantly more pronounced with lysine vasopressin. Even a nonpressor dose of lysine vasopressin (1 mU/min) caused a significant increase in the arterial diameter. The lysine vasopressin-induced vasodilatation could be prevented by the administration of d(CH2)5Tyr(Me)AVP (10 microg, i.v.), a selective V1-vasopressinergic receptor antagonist. These data therefore suggest that a short-term increase in blood pressure induces in rats a distention of the carotid artery. The increase in arterial diameter seems to involve an active mechanism with lysine vasopressin caused by the stimulation of V1-vasopressinergic receptors.
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The authors report three children who suffered temporary oromotor or speech disturbances as focal epileptic manifestations within the frame of benign partial epilepsy of childhood with rolandic spikes and review similar cases described in the literature. The deficit can occur as an initial symptom of the disorder without visible epileptic seizures and interferes in a variable way with simple voluntary oromotor functions or complex movements including speech production, depending on the exact location and spread of the discharging epileptic focus around the perisylvian region. The most severe deficit produces the anterior operculum syndrome. More subtle non-linguistic deficits such as intermittent drooling, oromotor apraxia or dysfluency, as well as linguistic ones involving phonologic production, can occur. The rapidity of onset, progression and recovery of the deficit is very variable as well as its duration and presumably reflects the degree of epileptic activity. In some cases, rapid improvement with antiepileptic medication occurs and coincidence between the paroxysmal EEG activity (which is usually bilateral) and the functional deficit is seen. The clinical and EEG profile of the seizures disorder and the dynamic of the deficit in these cases bear a strong resemblance to what is seen in the acquired epilepsy-aphasia syndrome (Landau and Kleffner). The variations in clinical symptoms appear more related to the main site, local extension and bilaterality of the epileptic foci rather than a basic difference in physiopathology.