Molecular and chemical neuropathology最新文献

A fluororeceptor assay (FRA) has been developed for the determination of antibodies against acetylcholine receptor (AChR), employing an antifluorescein serum and fluorescein-labeled AChR. Antiserum raised against rat muscle AChR in rabbit and the labeled AChR are incubated with antifluorescein serum at room temperature. At high levels of anti-AChR, binding of the labeled AChR prevented subsequent binding of the fluorescein groups by antifluorescein, resulting in little change in the signals of the label. Conversely, at low levels of anti-AChR, the free fraction of the labeled AChR is available to be bound by antifluorescein, which markedly reduced fluorescence intensity of the label. Thus, the fluorescence intensity of the assay mixture directly reflects the amount of anti-AChR antibodies in the serum. It is concluded that the availability of fluorescein-labeled AChR and the antibody directed against it permit measurement of anti-AChR antibodies in human myasthenia. The quality of the assay and its preliminary clinical application have been evaluated.

The effects of cadmium, copper, and zinc on beta APP metabolism were investigated in COS-7 and PC12 cells. Cadmium chloride (CdCl2) increased beta APP steady-state protein levels and decreased beta APP posttranslational processing. These changes were not accompanied by alterations in beta APP mRNA levels or splicing. In addition, cytosolic alpha-actin and G3PDH levels were not affected. Further, neither zinc (ZnCl2) nor copper (CuSO4) altered beta APP levels or affected its normal processing. Pulse-chase studies revealed that the rate of beta APP maturation decreased twofold in the presence of 25 microM CdCl2 compared to untreated controls. beta APP secretion from the cell also dramatically slowed. These two factors result in the accumulation of partially processed beta APP inside cells. The presence of CdCl2 also decreased the amount of an 8-kDa beta APP C-terminal fragment, indicating that the cellular compartment in which beta APP accumulates is not accessible to alpha-secretase. Studies using Brefeldin A suggest that this compartment may be the cis or medial Golgi. However, A beta production was proportionately increased. These data show that CdCl2 can modulate the beta APP cleavage to favor A beta. Finally, beta APP mis- metabolism was shown to be unrelated to the hsp70 induction elicited by CdCl2; both heat shock and CuSO4 induced hsp70 but had no effect on steady-state levels of beta APP, although heat shock did slow beta APP maturation. These data indicate that hsp70 alone cannot chaperone beta APP through an alternate processing pathway leading to A beta production.

Oxidative damage on biological molecules has been proposed as a major cause of alterations observed in aging brain as well as in neurodegenerative diseases. In this study, we measured membrane fluidity in mitochondria extracted from three cerebral regions and cerebellum of Alzheimer disease (AD) patients and age-matched controls by means of fluorescence polarization technique. A significant reduction of mitochondrial membrane fluidity was found in AD, except in cerebellum. In controls, a decrease of membrane fluidity was observed along with age, and it was also related to the content of the oxidized nucleoside 8-hydroxy-2'-deoxyguanosine (OH8dG) in mitochondrial DNA (mtDNA). Alteration in membrane fluidity seems to be a result of lipid peroxidation, since it dramatically decreased when mitochondria were exposed to FeCl2 and H2O2. The parallel increase of viscosity in mitochondrial membrane and the amount of OH8dG in mtDNA is suggestive of a relationship between these biological markers of oxidative stress. These results provide further evidence that oxidative stress may play a role in the pathogenesis of AD.

This in vivo study compared the pharmacokinetics of intracerebroventricularly administered tertiary butylhydroperoxide (tBuOOH) (109.7 mg/kg) among six different brain regions in two age groups of mice (2- and 8-mo-old mice). Brains were dissected at 11 time-points ranging from 0.5-60 min. Pharmacokinetics parameters did not differ between the two age groups. This demonstrates that previously reported age-related differences in tBuOOH toxicity may not be owing to pharmacokinetic differences between the two age groups. Differences were found when comparing the pharmacokinetics of tBuOOH among the various brain regions. Area under the curve (AUC) values were highest in the striatum and thalamus, and lowest in the cerebellum. The half-life of tBuOOH varied widely among the regions with the longest half-lives in the cortex and hippocampus, and the shortest in the striatum and cerebellum. The oxidation of glutathione and the induction of DNA damage are critical aspects of tBuOOH toxicity. These data show that region-dependent differences in toxicity reported previously may result from factors, such as tBuOOH-induced glutathione oxidation and DNA damage.

The occurrence and distribution of myelin-associated glycoprotein (MAG)-like immunoreactivity was investigated in the rat using a polyclonal antibody to MAG purified from rat brain. In the nervous system, MAG immunoreactivity was found in the periaxonal portion of the myelinated fibers and in a small number of oligodendroglia in the cortex, hippocampus, and the spinal cord. The sheath of Schwann cells in unmyelinated fibers and satellite cells in the spinal ganglia were also immunoreactive for MAG. In the endocrine system, the noradrenaline-containing cells in the adrenal medulla and some endocrine cells in the duodenum showed MAG immunoreactivity. In the immune system, numerous reticular cells with slender cytoplasmic processes, which formed a dense network, were immunopositive for MAG within the germinal center in the lymph nodes and spleen. In the thymus, a number of epithelial reticular cells within the medulla showed variation in staining intensity. These findings provide new information on the wide distribution of MAG immunoreactivity in the nervous, endocrine, and immune systems, and may contribute to the further understanding of the biological roles of this protein.

Anesthetic agent, arterial pCO2 level, and opioid peptides have all been implicated in the pathophysiology of experimental stroke models. The effects of halothane, alpha-chloralose, and differing concentrations of arterial pCO2 on injury volume and CSF beta-endorphin levels were studied in a feline model of experimental focal cerebral ischemia. The type of anesthetic agent used had no effect on injury volume following 6 h of focal cerebral ischemia. Over a 6-h period, beta-endorphin levels significantly increased from 10.1 +/- 5.0 fmol/mL at zero time to 14.4 +/- 7.2 fmol/mL at 6 h under halothane anesthesia (p < 0.05), whereas they did not significantly change (10.1 +/- 6.7 to 7.8 +/- 4.7 fmol/mL) under alpha-chloralose anesthesia. In contrast, hypercapnia had no effect on beta-endorphin levels, but significantly increased injury volume from 30.6 +/- 5.7% of the ipsilateral hemisphere under normocapnic conditions to 37.1 +/- 5.9% under hypercapnic conditions (p < 0.05). These results suggest that hypercapnia increases injury volume in a feline model of focal cerebral ischemia, and pCO2 should be controlled in experimental focal cerebral ischemia models.

Comparison of astroglial immunoreactivity in mesencephalon, cerebellum, and hippocampus of 25-d-old rat pups exposed to 2,4-dichlorophenoxyacetic acid (2,4-D) through the mother's milk was made using a quantitative immunohistochemical analysis. A glial reaction was detected at the level of serotonergic nuclei and extreme astrogliosis in the hippocampus and cerebellum. A quantitative analysis of reactive astrocytes was performed by using GFAP and S-100 protein as specific markers. The study showed a significant increase in their number, size, number of processes, and density of immunostaining in 2,4-D-exposed animals. Exposure to 2,4-dichlorophenoxyacetic acid on the first days of life modifies the astroglial cytoarchitecture in parallel to previously described neuronal changes.

We investigated the influence of ion compositions on the membrane potential in LA-N-1 human neuroblastoma cells using bisoxonol as a potential-sensitive fluorescent dye. The ability of K+, ouabain, veratridine, and maitotoxin to induce membrane depolarization was evaluated. Increasing concentrations of K+ ions from 10 to 50 mM caused a dose-dependent increase of bisoxonol fluorescence, which was completely independent on Na+ and Ca2+. Ouabain (5 mM), an inhibitor of the Na+, K(+)-ATPase, failed to induce membrane depolarization. Veratridine (40 and 100 microM), a Na+ channel activator, only in the presence of 10 micrograms of Leiurus scorpion venom reduced the membrane potential. Maitotoxin (MTX) from 3 to 10 ng/mL depolarized LA-N-1 cells in a dose-dependent manner, and produced a rapid and sustained increase of intracellular free calcium monitored by means of fluorescent probe fura-2. The MTX-induced depolarization and the increase in cytosolic free calcium concentration were dependent on extracellular Ca2+ ions. On the other hand, Na+ ions also seem to be, although only partially, implicated in the MTX effects, since both the blockade of tetrodotoxin (TTX)-sensitive voltage-operated Na+ channels and the removal of Na+ ions were able to reduce the depolarization. In conclusion, our data indicate that the depolarizing action of MTX on LA-N-1 cells is Ca(2+)- and Na(+)-dependent, although the latter only partially, and that this effect is dependent on Ca2+ influx into the cells likely through a voltage-insensitive calcium-entry system.

Breakdown or disruption of the cytoskeleton has been implicated in the neurodegenerative processes of a variety of diseases, including Alzheimer disease (AD) and stroke. Studies of such diseases in the human involve the use of postmortem brain tissue. Postmortem delay may vary considerably from a few hours to a few days, and within this period, a degree of cytoskeletal breakdown may occur. It is therefore crucial to examine alterations occurring in the cytoskeleton as a result of postmortem delay and subtract these from those caused by the disease. In this study, the distribution of tau, MAP2, and MAP5 immunohistochemistry was examined following postmortem intervals of 0-72 h in the rat cerebral cortex, corpus callosum, caudate nucleus, and hippocampus. Each microtubule-associated protein (MAP) underwent unique changes that were dependent both on postmortem interval and the brain region examined. Following long postmortem delays, some of the changes in these proteins were similar to those seen in rodent models of cerebral ischemia. These results demonstrate that MAPs are not stable during postmortem delay in the rat. Therefore, caution must be exercised when interpreting changes in MAPs in human postmortem tissue, especially in cases where ischemic injury may be involved. Examination of control tissue carefully matched for postmortem delay is therefore essential to allow meaningful interpretation of cytoskeletal abnormalities in human neurodegenerative disease.

The relationship between the behavioral effects and motor activity induced by N-methyl-D-aspartate (NMDA) (150 mg/kg, ip) and brain polyamine concentration was studied in male Wistar rats. Motor activity was evaluated by an automated subtraction analysis system to measure the duration and vigor of any kind of movement. The behavioral modifications exhibited by the nonconvulsant NMDA-treated rats were evaluated according to the composition and sequence of behavioral components as: hypoactivity (pattern A), partially stereotyped activity (pattern B), and generalized stereotyped activity (pattern C). The concentration of polyamines in the frontal cortex and hippocampus was measured 8 and 24 h after drug injection. A relationship was found between the concentration of putrescine in both regions and the motor activity. In addition, the concentrations of putrescine also correlated with the vigor of the movements performed. Moreover, the putrescine concentration in the frontal cortex and hippocampus paralleled the behavioral patterns. The histological examination of the frontocortical and hippocampal areas did not reveal any evidence of damage. In conclusion, partially or generalized stereotyped activity elicited by systemic NMDA administration induces an increase in putrescine in the brain not linked to histological damage.