Molecular and chemical neuropathology最新文献

Interleukin-1 alpha (IL-1 alpha), IL-1 beta, interleukin-1 receptor type I (IL-1RI, signaling receptor), and IL-1 receptor antagonist (IL-1Ra, endogenous inhibitor) are pivotal components of the IL-1 system. IL-1 and other cytokines induced by IL-1, such as TGF-beta 1, may participate in the growth of various tumor cells. In children, primary nervous system tumors represent the most common solid malignancy. We investigated the levels of IL-1 alpha, IL-1 beta, IL-1RI, IL-1Ra, and TGF-beta 1 mRNAs in pediatric astrocytomas (n = 19), ependymomas (n = 13), and primitive neuroectodermal tumors (n = 22) using sensitive and specific RNase protection assays. The data show a significant distinct cytokine mRNA profile among brain tumor types. Pilocytic, nonpilocytic, and anaplastic astrocytomas have significant increased levels of IL-1 beta, IL-1RI, and TGF-beta 1 mRNAs, but low levels of IL-1Ra mRNA; this may have implications for an IL-1 beta feedback system and IL-1 beta<-->TGF-beta 1 interactions in astrocytomas. Ependymomas show increased levels of IL-1 alpha and IL-1 beta mRNAs associated with low levels of IL-1Ra mRNA; primitive neuroectodermal tumors do not exhibit increased levels of any cytokine component examined. The data also suggest that a dysregulation of the balance between stimulatory and inhibitory cytokines may be involved in the growth and development of brain tumors via autocrine/paracrine mechanisms.

Effects of prenatal ethanol exposure on extracellular glutamate accumulation stimulated by glutamate receptor agonists were studied in rat cerebellar granule cell cultures. The prenatal exposure to ethanol was achieved via maternal consumption of a Sustacal liquid diet containing either 5% ethanol or isocaloric sucrose (pair-fed) substituted for ethanol from gestation d 11 until the day of parturition. Neither the basal level of extracellular glutamate nor the increased accumulation of glutamate stimulated by KCl (40 mM) or by ionotropic glutamate receptor agonists, N-methyl-D-aspartate (NMDA) or kainate (KA) (100 microM each), in cells prepared from the ethanol-fed group was significantly different from that in cells prepared from the pair-fed group. Glutamate accumulation stimulated by quisqualate (QA, 100 microM) or by trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD, 250 microM) in the ethanol-fed group was higher than that in the pair-fed group by 116 and 36%, respectively. In the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 100 microM), an ionotropic QA receptor antagonist, the QA-induced accumulation of glutamate in the ethanol-fed group was still higher than that in the pair-fed group. In the presence of MK-801 (5 microM), an antagonist of the NMDA receptor, the enhanced accumulation of glutamate stimulated by either QA or t-ACPD was still observable in the ethanol-fed group as compared to the pair-fed group. Addition of (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG, 500 microM), a selective antagonist of the metabotropic glutamate receptor, abolished the enhanced accumulation of glutamate stimulated by either QA or t-ACPD in the ethanol-fed group. Although immunoblotting of mGluR1 and mGluR2/3 did not show apparent differences between the pair-fed and the ethanol-fed groups, the overall results suggest that the effect of prenatal ethanol exposure was selectively through a pathway mediated by the metabotropic glutamate receptor.

Oxidative stress in the central nervous system (CNS) may cause oxidation of lipoprotein particles. The oxidized lipoproteins may damage cellular and subcellular membranes, leading to tissue injury and cell death. Human low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) are oxidized by transition metal ions, such as Cu2+. Using PC 12 cells, we tested the cytotoxicity of oxidized LDL and VLDL. Cell death was increased in a dose-dependent manner. Antioxidants added to the incubation medium, such as vitamins E or C, or resveratrol showed some protection. Results indicated that oxidized lipoproteins may serve as an oxidative stressor, which may initiate the neuronal cell death leading to the manifestation of Alzheimer disease (AD).

Acetylcholinesterase (AChE) is the enzyme responsible for the hydrolysis of the neurotransmitter acetylcholine in the central nervous system. Recently, we have found that AChE promotes the assembly of amyloid-beta peptides (A beta) into Alzheimer fibrils. The action of AChE on the state of aggregation of the A beta peptide supposes a near neighbor relationship between these two molecules. In the present work, we have studied A beta-AChE interactions using the crosslinker reagent disuccinimidyl suberate (DSS), in the presence of [125I]-A beta peptide. The A beta-AChE complexes formed by crosslinkage were then analyzed by SDS-PAGE and autoradiography. We observed the formation of [125I] A beta-labeled complexes of 70, 160, 250, and 300 kDa corresponding to monomers, dimers, tetramers, and oligomers of AChE, respectively crosslinked with the A beta peptide. Our results suggest that AChE and the A beta peptide may be involved in physiologically relevant interactions, related to the pathogenesis of Alzheimer disease (AD).

To investigate the relationship between cerebral amyloid angiopathy (CAA) and cystatin C, we studied five CAA patients on whose cerebral blood vessels colocalization of cystatin C and beta-protein was recognized immunohistochemically. One patient was suspected as familial CAA and the other patients were sporadic cases. Two patients had low concentration of cystatin C in their cerebrospinal fluid (CSF) as we have previously reported in CAA patients. Enzyme-linked immunosorbent assay (ELISA) revealed that cystatin C and beta-protein have been included at the ratio of about 1:100 in the crude amyloid fibrils of one patient. Using a monoclonal antibody (MAb) against cystatin C, we performed affinity chromatography and immunoblotting on her amyloid fibril fraction. Eluate showed a band with a mol wt of 14,000 and the N-terminal 14 amino acid residues of 14-kDa protein were identical with that of cystatin C. This molecular weight is not identical to that of the truncated form of cystatin C deposited in hereditary cerebral hemorrhage with amyloidosis in Iceland (HCHWA-I), but that of normal cystatin C. DNA sequence analysis of five patients showed no point mutations in the cystatin C gene. Cystatin C and beta-protein colocalization, which was recognized in amyloid lesions of CAA, suggests that cystatin C deposition may be related to beta-protein deposition. We hypothesize that cystatin C deposition in sporadic cerebral amyloid angiopathy with cystatin C deposition (SCCAA) involves a different mechanism from that in HCHWA-I, which may be related to low CSF concentration of cystatin C without amino acid substitutions.

Lipid peroxidation has been suggested to be a potential cause of neuronal damage in neurodegenerative diseases. Changes in several parameters of lipid peroxidation, including basal (unstimulated) lipid peroxidation, stimulated lipid peroxidation, tissue iron concentrations, and the concentrations of some oxygen radical scavengers, have been reported in neurodegenerative diseases. However, the in vitro interaction of oxygen radical scavengers and stimulated lipid peroxidation in neurodegenerative disease has been less well-studied. The purpose of the present study was to determine the effects of oxygen radical scavengers on ascorbate-stimulated lipid peroxidation in Alzheimer disease (AD). We have found that some parameters of ascorbate-stimulated lipid peroxidation are altered in AD and that the effects of superoxide dismutase (SOD) on ascorbate-stimulated lipid peroxidation are significantly different in AD as compared to aged.

Turnover of [3H]phosphoinositides (PI) was examined in brain slices from the hippocampus of rats undergoing soman-induced seizure activity. Hydrolysis of PI was determined by measuring the accumulation of [3H]inositol-1-phosphate (IP1). Incubation of hippocampal slices in the presence of carbachol or norepinephrine (NE) increased PI hydrolysis. Stimulated hydrolysis by NE, but not carbachol was significantly reduced in slices from soman-challenged rats undergoing convulsive activity. NE-stimulated PI hydrolysis was not reduced in slices from animals exposed to soman that did not exhibit convulsive activity. In rats surviving for 24 h, the response to NE was not different from control rats. In control slices, NE-stimulated hydrolysis of PI was potentiated by GABA. No potentiation by GABA was seen in slices from animals undergoing seizures. Uptake and incorporation of myo-[2-3H]inositol into phospholipids was reduced in slices from rats undergoing convulsions. Reduced IP1 production appeared to be owing, in part, to decreased synthesis of inositol lipids. These observations suggest that during soman-induced seizure activity, there is an apparent decrease in the response of the PI second messenger system to NE stimulation, and that this may contribute to the severity and duration of convulsions and brain damage resulting from exposure to soman and other anticholinesterase compounds.

The content and composition of gangliosides were examined in an experimental mouse brain tumor, EPEN, that was grown subcutaneously in the flank of the syngeneic C57BL/6J (B6) host and in the B6 severe combined immunodeficiency (SCID) host. SCID mice lack functional T- and B-lymphocytes, but have a normal complement of macrophages. The content and distribution of the brain tumor gangliosides were similar whether the tumor was grown in the immunocompetent B6 host or in the B6-SCID host. N-acetylneuraminic acid- (NeuAc) containing GM3 was the major ganglioside in the subcutaneous tumors and in the cultured EPEN cells. Significant amounts of N-glycolylneuraminic acid- (NeuGc) containing gangliosides were found in the tumor grown in both mouse hosts. NeuGc-containing gangliosides are not expressed in normal mouse brain, but are present in macrophages and serum. An extremely complex pattern of minor gangliosides was found in the subcutaneous tumors on two-dimensional, high-performance thin-layer chromatograms. Most of the minor gangliosides comigrated with those found in mouse macrophages. The results show that the absence of functional T- and B-lymphocytes does not markedly affect brain tumor ganglioside composition and suggest that NeuGc-containing gangliosides in the EPEN can be derived from tumor infiltrating host cells (mostly macrophages) and from the extracellular milieu (serum).

Effects of tacrine (1,2,3,4-tetrahydro-9-aminocridine) on memory deficits in rats treated with ethylcholine aziridinium ion (AF64A) were studied using active avoidance test in the two-way shuttle box. Neurotoxin AF64A injected at a dose of 6 nmol (i.c.v., bilaterally) causes nonspecific tissue damage in hippocampal fields CA2 and CA3. Two weeks after treatment with 6 nmol, AF64A active avoidance performance of toxin-treated rats was significantly deteriorated compared to vehicle-treated animals estimated in learning test (68 +/- 3.5 and 83 +/- 3.2% of correct responses, respectively; p < 0.01) and in retention test (53 +/- 5 and 76 +/- 3.6%, respectively; p < 0.01). Under these conditions, chronic treatment with tacrine at a daily dose of 1 mg/kg for 12-14 d reverses the effect of AF64A on the active avoidance performance both in learning (78 +/- 3.2%) and retention (72 +/- 4%) tests. It is supposed that behavioral effects of tacrine considerably depend on a severity of neurodegeneration in the hippocampus.

Disruption of the blood-brain barrier (BBB) and neuronal cytoskeletal damage were evaluated in two commonly used rat models of traumatic brain injury. Adult rats received a lateral cortical impact (CI) or lateral fluid percussion (FP) injury of mild or moderate severity or a sham procedure. Six hours after trauma, the brains were removed and analyzed with immunocytochemical techniques for alterations in the serum protein, IgG, and the cytoskeletal protein, microtubule-associated protein 2 (MAP2). Both models induced profound alterations in these proteins in the ipsilateral cortex and hippocampus compared to sham-injured controls. Following an injury of moderate severity, the CI injury resulted in greater IgG extravasation in the cortex and hippocampus than the FP injury. Conversely, after a mild injury, IgG extravasation in the hippocampus was greater for FP than CI. All of the animals in the CI group and most of the FP group showed a loss of MAP2 in the hippocampus. The specific subregions within the cortex and hippocampus that were affected by the injury varied between models, despite having identical impact sites. These data demonstrate that there are both similarities and differences between a CI and FP injury on vascular and neuronal cystoskeletal integrity, which should be considered when utilizing these animal models to study selected features of human head injury.