Molecular and chemical neuropathology最新文献

Mice infected with LP-BM5 develop a severe immunodeficiency accompanied by learning and memory deficits, gliosis, and neurotransmitter abnormalities. The neurochemical alterations are consistent with elevated excitotoxin levels, suggesting that infected mice may incur neuronal damage. Although the number of neocortical neurons was unchanged in mice 12 wk after LP-BM5 infection, the expression of cytoskeletal proteins declined, particularly in the frontal and parietal cortex as indicated by MAP2, NF-200, and synaptophysin immunoreactivity. In contrast, the number of striatal neurons decreased 19%. The remaining neurons were smaller, with fewer synaptic boutons, and showed decreased synaptophysin and NF-200, immunoreactivity. Immunoblots of cortex and striatum confirmed decreases in MAP2, NF-200 and synaptophysin expression. Finally, although NCAM expression decreased in the striatum, it increased in the cortex. These results indicate that LP-BM5-infected mice sustain significant neuronal damage, which may contribute to their behavioral deficits. Moreover, the increase in cortical NCAM expression suggests active synaptic remodeling to compensate for the persistent excitotoxic environment in these mice, whereas striatal neurons degenerate. These concurrent degenerative and compensatory processes may also occur in the brains of patients with AIDS dementia complex (ADC), who suffer extensive degeneration of the basal ganglia and cerebral cortex.

The distribution of the metabotropic glutamate receptor mGluR1a was studied in the normal and kainate-lesioned rat hippocampus using a monoclonal (MAb) and a polyclonal antibody to mGluR1a. Many labeled nonpyramidal neurons were observed in the stratum oriens of CA1 in sections incubated with MAb. In comparison, fewer labeled neurons were observed in this layer in sections incubated with polyclonal antibody. Many nonpyramidal neurons were observed in the stratum lucidum of CA3 and the hilus of the dentate gyrus, with both antibodies. The cell bodies of pyramidal neurons were unlabeled. A dense network of labeled processes was observed in the neuropil of the CA fields at electron microscopy. Some dendrites were very densely labeled and did not contain dendritic spines. These were identified as dendrites of nonpyramidal neurons. Other dendrites contained lightly labeled dendritic shafts, but densely labeled dendritic spines, and were identified as dendrites of pyramidal neurons. Intravenous kainate injections resulted in destruction of pyramidal neurons and a massive decrease in mGluR1a immunoreactivity in the CA fields. This decrease was obvious even at 1-5 d postinjection, when the nonpyramidal neurons in the stratum oriens remained densely labeled, suggesting that pyramidal neurons contributed significantly to mGluR1a staining in the CA fields. We conclude that the dendritic spines of hippocampal pyramidal neurons contain mGluR1a, even though little staining is observed in their parent dendritic shafts or cell bodies.

The activities of the enzymes related to glutathione synthesis, degradation, and functions as well as reactive oxygen scavenging enzymes were analyzed in different brain regions, such as cerebral hemisphere, cerebellum, brainstem, thalamus, and hypothalamus after 1 and 3 mo of streptozotocin-induced diabetes in rats. Parallel studies were also made in age-matched control rats and insulin-treated diabetic rats. The content of glutathione (GSH) and its synthesizing enzyme gamma-glutamylcystein synthetase and also superoxide dismutase (SOD) and catalase activities (reactive oxygen scavenging enzymes) were significantly decreased from almost all the brain regions studied. However, glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), gamma-glutamyl transpeptidase (gamma-GTP), and glutamine synthetase (GS) activities were increased in the diabetic rat brain. Insulin treatment to the diabetic rats resulted in partial to full recovery in these enzymes activities. The present results emphasize the potentially serious alterations of brain free radical scavenger system in uncontrolled Type I diabetes.

The biological roles of nitric oxide (NO) and cGMP as inter- and intracellular messengers have been intensively investigated during the last decade. NO and cGMP both mediate physiological effects in the cardiovascular, endocrinological, and immunological systems as well as in central nervous system (CNS). In the CNS, activation of the N-methyl-D-aspartic acid (NMDA) type of glutamatergic receptor induces Ca(2+)-dependent NOS and NO release, which then activates soluble guanylate cyclase for the synthesis of cGMP. Both compounds appear to be important mediators in long-term potentiation and long-term depression, and thus may play important roles in the mechanisms of learning and memory. Aging and the accumulation of amyloid beta (A beta) peptides are important risk factors for the impairment of memory and development of dementia. In these studies, the mechanism of basal- and NMDA receptor-mediated cGMP formation in different parts of adult and aged brains was evaluated. The relative activity of the NO cascade was determined by assay of NOS and guanylate cyclase activities. In addition, the effect of the neurotoxic fragment 25-35 of A beta (A beta) peptide on basal and NMDA receptor-mediated NOS activity was investigated. The studies were carried out using slices of hippocampus, brain cortex, and cerebellum from 3- and 28-mo-old rats. Aging coincided with a decrease in the basal level of cGMP as a consequence of a more active degradation of cGMP by a phosphodiesterase in the aged brain as compared to the adult brain. Moreover, a loss of the NMDA receptor-stimulated enhancement of the cGMP level determined in the presence of cGMP-phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) was observed in hippocampus and cerebellum of aged rats. However, this NMDA receptor response was preserved in aged brain cerebral cortex. A significant enhancement of the basal activity of NOS by about 175 and 160% in hippocampus and cerebellum, respectively, of aged brain may be involved in the alteration of the NMDA receptor response. The neurotoxic fragment of A beta, peptide 25-35, decreased significantly the NMDA receptor-mediated calcium, and calmodulim-dependent NO synthesis that may then be responsible for disturbances of the NO and cGMP signaling pathway. We concluded that cGMP-dependent signal transduction in hippocampus and cerebellum may become insufficient in senescent brain and may have functional consequences in disturbances of learning and memory processes. A beta peptide accumulated during brain aging and in Alzheimer disease may be an important factor in decreasing the NO-dependent signal transduction mediated by NMDA receptors.

The distributions of constitutive and inducible 70-kDa heatshock proteins (Hsc70 and Hsp70, respectively) and ubiquitin (Ub) were investigated in autopsy specimens from 24 adult human brains. The objectives were to verify that the milder fixation and celloidin embedding applied to those specimens preserved protein immunoreactivity in the tissue sections, even with extended intervals between death and fixation, and to determine the typical pattern of distribution of the proteins in aged human cerebellum and caudate nucleus. To achieve these objectives, the patterns of immunoreactivity in human specimens were compared with those in normal rat brain after three methods of immersion fixation: 1. 1% Formalin; 2. 10% Formalin; 3. Methacarn (a modification of Carnoy's solution). Additionally, some rats were left refrigerated, but unfixed for up to 24 h to mimic the postmortem interval that commonly occurs prior to fixation of human autopsy material. Tissues were embedded in celloidin, sectioned at 100 microns, and the celloidin dissolved to permit immunostaining. Immunoreactivity for all antigens was greatly diminished in the rat brain by fixation in 10% formalin compared to 1% formalin or methacarn. Rat and human brain tissues fixed in the latter two solutions showed similar patterns of low levels of Hsp70 immunostaining in gray matter and other areas where neuronal somata were concentrated, whereas Hsc70 immunostaining was much greater in those same areas. Little Hsc70 or Hsp70 immunoreactivity was detected in the white matter from either source, but immunoblots of human gray and white matter suggested that white matter contained more Hsc70 and Hsp70 than apparent by tissue section immunoreactivity. Ubiquitin immunostaining in rat and human brain showed the same high levels as Hsc70 in gray matter, but unlike Hsc70, was also visible in white matter. These patterns remained the same in rat brains even if fixation was delayed for 24 h. In three human brain specimens, elevated Hsc70 staining, but not Hsp70 or Ub, was found in a ring pattern similar to that described as the ischemic penumbra in experimentally induced brain ischemia. These results indicated that dilute formalin preserved Hsc/Hsp70 and Ub antigenicity well, and that the proteins had similar distributions in human and rat brains, despite the extended postmortem delay in fixation of the former. They also suggested that evidence of premortem, localized cellular metabolic stress may be preserved in the postmortem human brain by an alteration in the typical distribution of Hsc70.

Hypothermia diminishes the ischemia-induced protein kinase C (PKC) translocation and inhibition, and also reduces transmitter release during ischemia. To study the role of PKC in the mechanism of glutamate release during ischemia, we measured extracellular glutamate levels in the striatum with the microdialysis technique, in the presence and absence in the dialysate of the PKC activator 4 beta-phorbol-12,13-dibutyrate (PDBu) and the protein kinase inhibitor staurosporine. We confirm that hypothermia attenuates the elevation of extracellular levels of glutamate in the striatum during ischemia. In the presence of PDBu, the glutamate levels in the dialysate increased from 0.3 mumol/L to an end ischemic level of 4.8 mumol/L during hypothermic ischemia (33 degrees C). These levels were significantly higher than in hypothermic ischemia (33 degrees C) without added PDBu. Staurosporine significantly mitigated the glutamate levels during normothermic ischemia. Our data suggest that PKC is involved in the temperature-dependent elevations of extracellular glutamate levels in the striatum during ischemia, and we propose that compounds preventing PKC activation may mimic the hypothermic protective action against ischemic brain damage.

L-Deprenyl is a complex drug, and number of mechanisms have been proposed to explain its effects. These include blockade of dopamine metabolism, amplification of dopamine responses, induction of superoxide dismutase or delaying apoptosis. Using in situ hybridization techniques, we have shown that L-deprenyl (5-10 mg/kg intraperitoneally, killed after 24 h) increases aromatic L-amino acid decarboxylase (AADC) mRNA levels in rat substantia nigraventral tegmental area. In human brain tissue, AADC is present at low levels, suggesting a possible rate-limiting role in monoamine synthesis. This is particularly important in parkinsonian patients, since the therapeutic efficacy of L-DOPA is attributed to its enzymatic decarboxylation to dopamine. The present findings support that one of the effects of L-deprenyl may be to facilitate the decarboxylation of L-DOPA by increasing the availability of AADC.

Changes in the concentration of membrane-bound phospholipids following single (25-min) spinal cord ischemia and 3 h of reperfusion were determined. These were compared with the changes following brief repeated (8-, 8-, and 9-min) ischemia followed each time by reperfusion for 1 h, or the same periods of ischemia followed by 8 h, 8 h, and 24 h of reperfusion, respectively. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and sphingomyelin (SM) were assayed in regions of the spinal cord of the rabbit, including gray matter, white matter, dorsal horns, intermediate zone, and ventral horns. The brief repeated ischemia with 1-h reperfusions produced more extensive degradation of phospholipids in almost all regions compared with the equivalent time of ischemia (25 min) in a single period. After a lengthy reperfusion after repeated ischemia, the phospholipids were resynthesized with the exception of the phosphatidylinositol in the gray matter. The resynthesis was most pronounced in the dorsal horns and in the white matter.

The messenger ribonucleic acid (mRNA) of gap junction protein connexin 43 was quantified in the tetanus toxin rat model of focal epilepsy following injection of toxin into the left amygdala. Animals were monitored electrographically at weekly intervals with bilateral amygdala electrodes. Cohorts of 3 rats were sacrificed at weeks 1, 2, 3, 4, 6, 8, and 10, and bilateral regions containing the amygdala and posterior cerebral cortex were sampled, frozen, and later pooled for northern blot analysis. Spike generation was manifest in all animals during the first 4 wk followed by variable attenuation and cessation by 10 wk. Electrode implantation alone was shown by regression analysis to cause significant (p < 0.05) elevation of connexin mRNA in weeks 1-4. Injection of toxin diminished connexin mRNA expression in the amygdala when compared to electrode implantation alone. No trend in connexin mRNA expression was established over time in either amygdala or cerebral cortex in the acute epileptic or chronic postepileptic phase. No association between connexin 43 mRNA expression and the development of epileptogenicity was found in the context of a self-limiting animal model of focal epilepsy.

Twelve 4-urea-5,7-dichlorokynurenic acid derivatives were synthesized by reacting the 4-tosylimino derivative of 5,7-dichlorokynurenate methyl ester first with triphosgene and then with a secondary amine. Compounds were screened in mice for anticonvulsant activity using maximal electroshock (MES), subcutaneous pentylenetetrazole (Met), and threshold tonic extension (TTE) tests. A rotorod test was used to determine neurotoxicity. Seven of the derivatives had anticonvulsant activity in TTE testing at 100 mg/kg. One compound, 2-methyl carboxylate-5,7-dichloro-4-([¿diphenylamino¿-carbonyl]amino)-quino line, had an ED50 value of 134 mg/kg (95% conf. int.: low-78.5, high-205.7; slope 1.9, SE = 0.44) in TTE testing. Two derivatives had MES activity. Only one compound, an N,N-diethylamino derivative, was neurotoxic in the rotorod test. Compounds were screened at a 10-microM concentration for activity in displacing 5,7-dichlorokynurenic acid from synaptosomal membrane fragments. Since 9 of the 12 compounds synthesized and tested have demonstrated anticonvulsant activity, this class of chemicals offers promise for the production of useful therapeutic agents.