Molecular and chemical neuropathology最新文献

The mechanism by which aluminum induces formation of perikaryal neurofilament (NF) inclusions remains unclear. Aluminum treatment inhibits: 1. The incorporation of newly synthesized NF subunits into Triton-insoluble cytoskeleton of axonal neurites; 2. Their degradation and dephosphorylation; 3. Their translocation into axonal neurites. It also fosters the accumulation of phosphorylated NFs within perikarya. In the present study, we addressed the relationship among these effects. Aluminum reduced the assembly of newly synthesized NF subunits into NFs. During examination of those subunits that did assemble in the presence of aluminum, it was revealed that aluminum also interfered with transport of newly assembled NFs into axonal neurites. Similarly, a delay in axonal transport of microinjected biotinylated NF-H was observed in aluminum-treated cells. Aluminum also inhibited the incorporation of newly synthesized and microinjected subunits into the Triton-insoluble cytoskeleton within both perikarya and neurites. Once incorporated into Triton-insoluble cytoskeletons, however, biotinylated subunits were retained within perikarya of aluminum-treated cells to a greater extent than within untreated cells. Notably, these subunits were depleted in the presence and absence of aluminum within 48 h, despite the persistence of the aluminum-induced perikaryal accumulation itself, suggesting that individual NF subunits undergo turnover even within aluminum-induced perikaryal accumulations. These findings demonstrate that aluminum interferes with multiple aspects of neurofilament dynamics and furthermore leaves open the possibility that aluminum-induced perikaryal NF whorls may not represent permanent structures, but rather may require continued recruitment of cytoskeletal constituents.

L-Glutamate, a major excitatory amino acid, plays an important role in learning and memory. L-Glutamate uptake into synaptic vesicles is an ATP-dependent process. Exposure of neurons to high, sustained extracellular concentrations of glutamate results in excitotoxicity. Elevated levels of phosphomonoesters (PMEs), phosphodiesters (PDEs), and phosphocreatine (PCr) have been reported in Alzheimer disease (AD). In this article, the effects of selected PMEs, PDEs, and PCr on vesicular L-[3H]glutamate uptake into isolated bovine synaptic vesicles are investigated. D-myo-Inositol-1-monophosphate (I1P), D-myo-inositol-2-monophosphate (I2P), sn-glycero-3-phosphate, (alpha-GP) and PCr significantly stimulated L-[3H]glutamate uptake into synaptic vesicles. Phosphoethanolamine (PE), phosphocholine (PC), L-phosphoserine (L-PS) sn-glycero-3-phosphocholine (GPC), and sn-glycero-3-phosphoethanolamine (GPE) had little or no effect on vesicular L-glutamate uptake. These observations suggested that the vesicular uptake of glutamate can be regulated by endogenous PMEs and PCr. The mechanism of activation by I1P, I2P, and alpha-GP appears to be stimulation of Mg(2+)-ATPase activity. These effects on vesicular glutamate uptake may be important in diseases in which the levels of these metabolites are altered, as they are in AD.

It has already been shown that the administration of convulsant 3-mercaptopropionic acid at 150 mg/kg enhances binding affinity of muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB) to certain rat CNS membranes without affecting site number. Herein we employed a 100 mg/kg dose and tested [3H]QNB binding to cerebellar, hippocampal, and striatal membranes obtained from rats killed at preseizure, seizure, and postseizure stages. In cerebellum, binding increased 24, 65, and 19% a1 preseizure, seizure, and postseizure stages, respectively; in hippocampus, values were 12 and 20% higher at pre- and seizure stages, but failed to differ from controls at postseizure; in striatum, increases of 10 and 18% were recorded at seizure and postseizure, with no changes at preseizure. Neither a subconvulsant dose (20 mg/kg) nor in vitro drug addition had any effect on binding. Results indicate a differential response to the convulsant, with reversible changes in cerebellum and hippocampus, and a delayed response in striatum, supporting the concept of area-dependent neuronal plasticity.

The expression of mRNA for connexin 43, a gap junction protein putatively found in astrocytes, is studied in two experimental models of epilepsy: the electrically kindled rat and the tetanus-toxin-injected rat. Rats were kindled by electrical stimulation of the amygdala to Racine class 5 seizures and divided into cohorts of three to undergo 3, 6, or 10 such events, respectively. Another two cohorts of rats received injections of tetanus toxin at strengths of 3 and 9 MLD50, respectively, into the amygdala. Features of epileptogenicity were identified electrographically in both cohorts during the first 4 wk following toxin injection with spontaneous ictal events recorded in the latter cohort. All rats were sacrificed 4 wk after electrode or cannula implantation, except for two toxin-injected cohorts that were sacrificed at wk 8 or 10. The epileptogeonic area in the region of the amygdala was harvested and pooled by cohort for Northern blot analysis. These were compared with control nonimplanted tissues. In the tetanus-toxin-injected animals, at time-points of 4, 8, and 10 wk, connexin 43 mRNA expression in epileptogenic tissues is found to be decreased or unchanged relative to control cases. Kindled rats demonstrated reductions of connexin mRNA with a trend toward normalizing levels with increasing numbers of stimulations when compared to control animals. Connexin 43 immunostained sections of the basolateral amygdala showed a similar trend in protein expression. Both experimental models of epilepsy show no connexin 43 mRNA upregulation despite varying degrees of epileptogenicity. This study therefore does not support the hypothesis that an increase in transcription is the basis for any proposed increase in gap junction communication involving connexin 43 in the context of epileptogenicity or as a reaction to increased neuronal excitability.

Serotonin (5HT) is one of the classical neurotransmitters expressed earlier in the embryonic rat brain, and it was proposed as a developmental signal in the central nervous system. In the adult brain, 5HT seems to be involved in neuronal plasticity. It was postulated that S-100 protein, a glial neurotrophic factor, could be modulated by 5HT probably through the glial 5HT1A receptors. In a model of chronic inhibition of endogenous 5HT synthesis produced by the daily administration of parachlorophenylalanine (PCPA) for 2 wk, we have studied by immunohistochemical methods and digital morphometric analysis the expression of two proteins present in rat brain astrocytes: glial fibrillary acidic protein (GFAP) and S-100 protein. The effectiveness of the PCPA treatment was tested by the use of specific anti-5HT antibodies that showed absence of 5HT fibers in 5HT innervation areas like frontal cortex and hippocampus. Different effects of PCPA treatment on serotoninergic raphe nuclei were observed: dorsal raphe nucleus (DRN) seemed to be more sensitive to the PCPA's action than ventral raphe nucleus (VRN). In DRN and in the two 5HT innervation areas studied, glial cells responded to the 5HT depletion induced by PCPA showing astrocytes with large and tortuous processes. Astrocytes from 5HT-depleted regions showed higher immunostaining for S-100 protein than controls. There was not any modification in optical density of S-100 protein immunostaining in VRN, the area less sensitive to PCPA treatment. These observations indicated that astrocytes are sensitive to the 5HT level, and in presence of low 5HT concentration in the intercellular space, astrocytes could react by synthesizing glial proteins like GFAP and S-100 protein.

Aluminum is a neurotoxic metal that may be involved in the progression of neurodegenerative diseases, including Alzheimer disease and amyotrophic lateral sclerosis (ALS). Although the mechanism of action is not known, aluminum has been shown to alter Ca2+ flux and homeostasis, and facilitate peroxidation of membrane lipids. Since abnormal increases of intracellular Ca2+ and oxygen free radicals have both been implicated in pathways leading to neurodegeneration, we examined the effect of aluminum on these parameters in vitro using primary cultures of cerebellar granule cells. Exposure to glutamate (1-300 microM) caused a concentration-dependent uptake of 45Ca in granule cells to a maximum of 280% of basal. Pretreatment with AlCl3 (1-1000 microM) had no effect on 45Ca accumulation, but increased the uptake induced by glutamate. Similarly, AlCl3 had no effect on intracellular free Ca2+ levels measured using fluorescent probe fura-2, but potentiated the increase induced by glutamate. The production of reactive oxygen species (ROS) was examined using the fluorescent probe dichlorofluorescin. By itself, AlCl3 had little effect on ROS production. However, AlCl3 pretreatment potentiated the ROS production induced by 50 microM Fe2+. These results suggest that aluminum may facilitate increases in intracellular Ca2+ and ROS, and potentially contribute to neurotoxicity induced by other neurotoxicants.

Hepatic encephalopathy (HE) is characterized by symptoms pointing at disturbances in glutamatergic neurotransmission in the brain, particularly in the striatum. The binding parameters of ligands specific for different recognition sites in the N-methyl-D-aspartate (NMDA) receptor complex and the distribution of the receptor subunit mRNAs (NR1, NR2A-D) were assessed in rats with acute HE induced with a hepatotoxin, thioacetamide (TAA). The binding of: 1. L-[3H]glutamate (NMDA-displaceable); 2. [3H]dizocilpine and N-(1-[2-thienyl]-cyclohexyl) [3H]piperidine ([3H]TCP); and 3. The coactivator site agonist [3H]glycine was assayed in purified membranes of the cerebral cortex, hippocampus, and striatum. In HE rats, Bmax of NMDA-displaceable glutamate binding was increased in the cerebral cortex and hippocampus, but slightly decreased in the striatum. In this region, the binding affinity was also slightly increased. In HE, Bmax of [3H]dizocilpine binding was unchanged in the striatum and cerebral cortex, but substantially decreased in the hippocampus. Pretreatment with phorbol ester enhanced the binding of dizocilpine more in HE than in control rats. Bmax of [3H]TCP binding was decreased in the cerebral cortex and striatum, but increased in the hippocampus. The different responses of these two phencyclidine site antagonists to HE may be indicative of a conformational change within the ion channel and/or the presence of microdomains reacting differently to extrinsic factors. HE did not affect glycine binding, but potentiated the maximal stimulation of [3H]dizocilpine binding by glycine in the cerebral cortex. The results emphasize the brain region and domain specificity of the responses of the NMDA receptor complex to HE.

Biotinyl derivatives of several lectins were used to study the localization of glycoconjugates in the cerebral microcapillaries and various brains of rats given at 24-h intervals two i.p. administrations of a hepatotoxin-thioacetamide (TAA) and examined 21 d posttreatment. At this time, the rats were asymptomatic with regard to hepatic encephalopathy but showed specific and selective changes in the blood-brain-barrier (BBB) transport of basic amino acid, but no BBB damage, and region-specific neuronal injury in the hippocampus and neocortex. The lectins tested recognized the following sugar residues: beta-D-galactosyl (Ricinus communis agglutinin [RCA-1]); N-acetyl-glucosaminyl and N-acetyl-neuraminic acid (wheat-germ agglutinin [WGA]); N-acetyl-D-galactosaminyl (Helix pomatia agglutinin [HPA]); beta-D-galactosyl and D-galactosyl neuraminic acid (peanut agglutinin [PNA]), and alpha-D-galactosyl and alpha-D-mannosyl (concanavalin A [Con A]). The treatment markedly decreased the binding to the cerebromicrovascular network of the hippocampus and neocortex of RCA-1 and WGA. The binding of these two lectins to their complementary monosaccharide residues appears to reflect subtle changes in BBB function, with a detection threshold below the conventional BBB permeability tests. The changes in the binding of the other two lectins: an increase of HPA binding and a decrease of Con A binding, confined to neocortical neurons and pyramidal cells of hippocampus injured by TAA treatment.

We examined the change of regional cerebral blood flow (rCBF) and local cerebral glucose utilization (LCGU) in the middle cerebral artery (MCA) occlusion or recirculation model of rats, and tested anti-ischemic effects of a free radical scavenger, 3-methyl-1-phenyl-pyrazolon-5-one (MCI-186). A remarkable increase in LCGU was observed in the cortex supplied by the anterior cerebral artery after recirculation. This hypermetabolism of glucose was at least partly caused by the postischemic oxidative injury, since MCI-186 ameliorated the high LCGU in this area. These results suggested the usefulness of this type of free radical scavenger for inhibiting the postischemic injury.

After acute trimethyltin (TMT) intoxication (21 d after a single i.p. injection at a dose of 8 mg/kg) the histological, immunohistochemical, and immunochemical investigation of adult rat hippocampus showed a distinct pattern of neuronal loss, and an increase in both glial fibrillary acidic protein- (GFAP) immunoreactive cells and GFAP concentration, as expected. S-100-immunoreactive cells also increased markedly, whereas the concentration of S-100 increased even more than that of GFAP. The data show that S-100 is an index of glial reaction to damage after TMT intoxication and suggest the potential usefulness of exploring the possibility that it may play a role in induced neurodegenerative processes.