Molecular and chemical neuropathology最新文献

Contradictory results have been reported on the downregulation and role of the brain-specific protein metallothionein-III (MT-III, GIF) in Alzheimer disease (AD). In this article, the importance of MT-III downregulation in AD brain was re-evaluated in temporal and frontal cortex, hippocampus, and cerebellum of 11 AD patients and two groups of five and six control subjects, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify the levels of MT-III mRNA relative to the levels of three constitutive RNAs: beta-actin, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), and ribosomal RNA 18S (rRNA 18S). The distribution of MT-III was similar to that of each of the three constitutive RNAs. The relative levels of each of these RNAs was high in brain regions examined in both AD patients and control subjects. Our findings do not support a downregulation of MT-III mRNA in the frontal cortex as well as the temporal cortex and hippocampus of AD patients. However, the level of MT-III mRNA was not constant in the investigated samples, suggesting that MT-III mRNA regulation could be controlled by factors other than AD pathology. Brain-derived neurotrophic factor (BDNF) mRNA levels were hardly detectable by RT-PCR in human brain tissue; a trend for a decrease was apparent in the temporal cortex of AD patients. In conclusion, the content of MT-III mRNA in the brain of AD patients was not detectably impaired, whereas BDNF mRNA may be affected.

Quinolinic acid (QUIN) is an endogenous excitotoxic agonist of the N-methyl-D-aspartate (NMDA) type of glutamate receptor, which causes slowly progressing degeneration of vulnerable neurons in some brain regions. Using changes in the activity of membrane-bound gamma-glutamyl transpeptidase (GGT) as a marker of cell damage, we found a significant decrease of this enzyme activity, which was preferentially located in the ipsilateral hippocampal formation and entorhinal cortex, 4 d after the unilateral intracerebroventricular (icv) injection of 0.5 mumol QUIN. The dose of QUIN divided into two half-doses injected bilaterally led to a symmetrical decline of GGT activity in hippocampal areas. The lesion was characterized by a suppression of GGT activity in hippocampal and entorhinal capillaries, corresponding to 60 and 81% of their initial value, respectively, but no significant changes were ascertained in synaptosomal membranes. The changes in the activity of capillary GGT were associated with the decrease of apparent maximal velocity Vmaxapp, whereas apparent Michaelis constant K(m)app (0.69-0.79 mM) remained unaffected. In the nonlesioned brain, concanavalin A (Con A) affinity chromatography revealed five glycoforms of synaptosomal GGT in contrast to only one found in hippocampal and entorhinal capillaries. The results document that neither the saccharide moiety of GGT nor the value of enzyme K(m)app is significantly affected by the QUIN-induced lesion of the rat brain. However, the suppression of GGT activity, which is accompanied by a decrease in the value of Vmaxapp in brain microvessels, may suggest dysfunction of the blood-brain barrier (BBB) in the QUIN-injured rat brain.

In this work, the tertiary butylhydroperoxide- (t-BuOOH) treated mouse was used as a model to study the oxidative stress that is associated with various neurodegenerative diseases. DNA was found to be an early target of t-BuOOH attack. Necrosis was associated with extensive DNA fragmentation that occurred in almost all regions of the brain within 20 min following intracerebroventricular (icv) injection of 109.7 mg/kg t-BuOOH. Apoptosis was associated with high levels of DNA fragmentation that was observed at 48 h after icv administration of 21.9 mg/kg t-BuOOH. Susceptibility to DNA damage was found to be age-dependent, since 24-mo-old mice exhibited consistently higher and more pervasive DNA damage than 8 mo-old-mice. Extensive DNA damage was seen in various brain regions in patients with Alzheimer disease (AD) and with both Alzheimer and Parkinson disease (AD-PD). These results directly implicate DNA damage in neurodegeneration. The DNA fragmentation ob-served can lead to both apoptosis and necrosis, as suggested by gel electrophoresis. Nicotinamide, a precursor of NAD in the brain, was able to prevent DNA fragmentation induced by low-dose t-BuOOH, when coadministered with the toxin.

Activated astrocytes, intrinsic components of both local and remote (axonal target regions) central nervous system injury responses, are now recognized as active metabolic and regulatory mediators in many neurological disorders. To further define these responses, we devised a new ventral surgical approach to unilaterally lesion the inferior olivary nuclear complex, which has a single predominant remote target, the cerebellum. Activated astrocyte number, volume, and density, as well as the total volume of brainstem involved in the astrocytic response, all peaked at postlesion day (pld) 4, returning toward, but not to, unoperated control values at pld 24 (p < 0.05). In contrast, the peak astrocyte response in the cerebellum was delayed, being greatest at pld 6 (p < 0.05 compared to control or pld 2). These responses were associated with increases in overexpression of S100 beta, an astrocyte-derived neurite growth factor, and with an increase in cerebellar steady-state levels of a neuronal injury response protein, the beta-amyloid precursor protein (beta-APP). This is similar to correlated increases in these two proteins that are found in epilepsy and Alzheimer disease. Our studies defining remote astrocytic and neuronal responses may be important for understanding glial-neuronal mechanisms underlying the spread of neuropathological changes in conditions such as Alzheimer disease.

Perinatal asphyxia (PA) produces changes in nitric oxide synthase (NOS) activity in neuronal and endothelial cells of the striatum and neocortex. The changes were examined using a histochemical NADPH-diaphorase (NADPH-d) staining method. Newborn rats were exposed to severe PA at 37 degrees C and other groups were subjected to severe PA under hypothermic condition (15 degrees C) for 20 or 100 min, respectively. Quantitative image analysis was performed on the striatum and neocortex in order to count cell number of reactive neurons and to compare the pattern of staining between the different groups of animals. Severe asphyctic pups showed an important neuronal loss in striatum and neocortex that was reduced by hypothermia. NADPH-d(+) neurons with reactive processes were found in the lateral zone of the striatum and neocortex in asphyctic pups. Controls and hypothermic striatum showed rounded cells without reactive process, while no cells were stained in cortex. There was also an increase in NADPH-d activity in endothelial cells in severe asphyctic pups in striatum and neocortex vs control and hypothermically treated animals. Our data evidenced that an inappropriate activation of NOS in neuronal and endothelial cells induced by PA is related to neuronal injury. Hypothermia inhibits neuronal injury and may be a valuable neuroprotective agent.

The effect of cyclic adenosine 3',5'-monophosphate (cAMP) on epileptiform activity in rat hippocampal slices was investigated. Bath-applied cAMP reversibly decreased the frequency of extracellularly recorded discharges in the CA3 subfield induced by bethanechol- or theophylline-containing solutions. Because cAMP was presumed to be relatively membrane impermeant, we developed and tested the hypothesis that this cAMP-mediated effect occurred extracellularly through the catabolic conversion of cAMP to 5'-AMP and, in turn, to adenosine, a known inhibitory neuromodulator. Three predictions derived from this catabolic hypothesis were tested. First, blockers of the enzymes involved were predicted to antagonize this effect of cAMP. In contrast, the coapplication of a cAMP-phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), or a 5'-nucleotidase inhibitor, adenosine 5'-[alpha, beta-methylene] diphosphate (AMP-CP), enhanced the cAMP-induced suppressive effect. Second, the nonhydrolyzable cAMP analogs, dibutyryl- and 8-bromo-cAMP, were predicted to be ineffective. Low concentrations (5-40 microM) of these two derivatives, however, also suppressed bethanechol-induced discharges, while, at a higher concentration (100 microM), both analogs increased discharge frequencies. Third, enzymatic catabolism of adenosine was predicted to antagonize cAMP's effect, but coapplying adenosine deaminase (10 U/mL) did not diminish this action. Because these data did not support the catabolic hypothesis, other, as yet undefined, mechanisms must be responsible for the discharge-suppressant effect of cAMP.

The aim of this paper was to determine whether prolonged drinking of lead acetate-containing water by adult rats, which imitates environmental exposure to lead (Pb), affects some morphological and biochemical properties of rat brain microvessels. We noted a significant increase of lead level in capillaries and synaptosomes obtained from brains of rats under chronic toxicity conditions. Intravenously injected horseradish peroxidase (HRP) was used to evaluate the functional state of the blood-brain barrier (BBB). The results indicate that, systematically administered at low doses, lead induces BBB dysfunction. The changes, revealed in light microscopy and confirmed by electron microscopic studies, are typical for "leaky" microvessels, reported for variety of neuropathological conditions associated with BBB damage. Enhanced pinocytotic activity of the endothelial cells and the opening of interendothelial tight junctions, together with enormous phagocytizing action of the pericytes, are the most characteristic ultrastructural features noted. The presence of specific type of perivascular cells containing droplets of lipids in the cytoplasm, together with changes in phospholipid profile in brain capillaries, suggest that altered lipid composition of membranes may, at least in part, be responsible for changes in observed membrane permeability.

Conventionally, assessment of the neurotoxicity of environmental pollutants relies on high-dosage treatment and nonspecific end points. In the present study, the early temporal and regional alterations in the mRNAs of neurotrophins were investigated following subtoxic doses of methylmercury (MeHg) in adult Sprague-Dawley rats using in situ hybridization histochemistry and phosphoimaging evaluation. Decreases in brain-derived neurotrophic (BDNF) mRNA labeling intensities were seen in the dentate gyrus (DG; 44% of controls), and in the CA1 (72% of controls) and CA3c (70% of controls) cell layers of hippocampus after 8 mg MeHg/kg (ip) at 4 h, and at 1 h only in the DG. The decrease in BDNF mRNA expression in the DG was dose-dependent. At 3 d, regional levels had recovered. No significant changes could be detected in mRNA levels of the BDNF high-affinity receptor trkB or neurotrophin-3 mRNA at either 1 h, 4 h, or 3 d. Cresyl violet staining and GFAP immunohistochemistry did not reveal any major neuropathology in hippocampus at 2 wk. Thus, MeHg causes specific downregulation of BDNF mRNA, unlike many other perturbations of central nervous system homeostasis that have been shown to lead to upregulation of this mRNA.

Qualitative and quantitative evaluations of cytoskeletal proteins are critical for understanding physiological and pathological processes affecting the nervous system. Most of such studies on human samples have only used immunohistochemical techniques. We describe a complementary immunoblotting approach, for the assessment of neuronal cytoskeletal proteins, which employs fresh frozen postmortem tissues. We found that cytosolic fractions are suitable for qualitative and quantitative evaluations of four major dendritic cytoskeletal proteins: microtubule-associated protein (MAP)-2, MAP-5, and high- and medium-molecular-weight nonphosphorylated neurofilaments. The enhanced chemiluminescence (ECL) technique revealed consistent and distinctive immunoblotting patterns for all four proteins in both monkey (no postmortem delay) and human (17-34 h postmortem interval) samples, some of which differed from those found in rodents. Quantitations of blots, by tissue protein-optical density curves that demonstrated linearity of the measurements in the 0- to 100-microgram range, support the feasibility of these immunoassays for the study of neurologic disorders.

Using mice genetically deficient in the complement (C)-system component C5, this study explored a potential novel role of the C-system in Ca(2+)-mediated control of glutamate AMPA receptor functions. We found that Ca2+ preincubation of frozen brain tissue sections enhances AMPA binding capacity more dynamically in C5 deficient (C5-) than congenic C5 sufficient (C5+) mice. The Ca(2+)-mediated response was mostly localized to the CA3 and CA1 subdivisions of the pyramidal layers of the hippocampal formation. In C5- mice, kainic acid (KA) excitotoxicity that models hippocampal neurodegeneration abolished the Ca(2+)-mediated induction of hippocampal AMPA binding. The changes in AMPA binding preceded temporally and overlapped anatomically the appearance of apoptotic features in the same hippocampal neuron layers. C5- mice showed greater hippocampal neurodegeneration then C5+ mice. NMDA binding controlled for specificity of glutamate-mediated changes and found no C5 genotypic influences. The study gives further credence to the role of the C-system in modifying the intensity and outcome during response to conditions leading to hippocampal neurodegeneration.