Biomedical subjects

N Y Calingasan

Publications and source records attributed to N Y Calingasan.

At least 19 recordsLinked to original sources

PubMed2004

Evidence that infiltrating neutrophils do not release reactive oxygen species in the site of spinal cord injury.

The release of reactive oxygen species (ROS) by neutrophils, which infiltrate the region of damage following spinal cord injury (SCI), was investigated to determine if such release is significant following spinal cord injury. The relationship of extracellular levels of hydroxyl radicals and hydrogen peroxide obtained by microdialysis sampling and oxidized protein levels in tissue to neutrophil infiltration following spinal cord injury was examined. Neither of the reactive oxygen species were elevated in the site of spinal cord injury relative to their concentrations in normal tissue at a time (24 h) when the numbers of neutrophils were maximum in the site of injury. Surprisingly, ablation with a neutrophil antiserum actually increased the level of oxidized proteins in Western blots. Thus, our findings are (1) that neutrophils, which infiltrate the site of damage following a spinal cord injury, do not release detectable quantities of reactive oxygen species; and (2) that the presence of neutrophils reduces the concentrations of oxidized proteins in the site of spinal cord injury. Therefore, release of reactive oxygen species by neutrophils does not contribute significantly to secondary damage following spinal cord injury. Reduced levels of oxidized proteins in the presence of neutrophils may reflect removal of damaged tissue by neutrophils.

Animals↗

PubMed2000

Dietary restriction attenuates the neuronal loss, induction of heme oxygenase-1 and blood-brain barrier breakdown induced by impaired oxidative metabolism.

Experimental thiamine deficiency (TD) is a model of impaired oxidative metabolism associated with region-selective neuronal loss in the brain. Oxidative stress is a prominent feature of TD neuropathology, as evidenced by the accumulation of heme oxygenase-1 (HO-1), ferritin, reactive iron and superoxide dismutase in microglia, nitrotyrosine and 4-hydroxynonenal in neurons, as well as induction of endothelial nitric oxide synthase within the vulnerable areas. Dietary restriction (DR) reduces oxidative stress in several organ systems including the brain. DR increases lifespan and reduces neurodegeneration in a variety of models of neuronal injury. The possibility that DR can protect vulnerable neurons against TD-induced oxidative insults has not been tested. The current studies tested whether approximately 3 months of DR (60% of ad libitum intake) altered the response to TD. Six month-old ad libitum-fed or dietary restricted C57BL/6 mice received a thiamine-deficient diet either ad libitum, or under a DR regimen respectively for eleven days. The TD mice also received daily injections of the thiamine antagonist pyrithiamine. Control ad libitum-fed or DR mice received an unlimited amount, or 60% of ad libitum intake, respectively, of thiamine-supplemented diet. As in past studies, TD produced region-selective neuronal loss (-60%), HO-1 induction, and IgG extravasation in the thalamus of ad libitum-fed mice. DR attenuated the TD-induced neuronal loss (-30%), HO-1 induction and IgG extravasation in the thalamus. These studies suggest that oxidative damage is critical to the pathogenesis of TD, and that DR modulates the extent of free radical damage in the brain. Thus, TD is an important model for studying the relationship between aging, oxidative stress and nutrition.

Aging↗

PubMed2000

Quantitative alpha-ketoglutarate dehydrogenase activity staining in brain sections and in cultured cells.

The activity of a key mitochondrial enzyme, the alpha-ketoglutarate dehydrogenase complex (KGDHC), declines in the brains of patients with neurodegenerative diseases such as Alzheimer's disease, as well as in thiamine-deficient (TD) animals. The decreased activity often occurs without a reduction in enzyme protein, which negates the use of immunocytochemistry to study cellular or regional changes in enzyme activity within the brain. To overcome this limitation, an activity staining method using nitroblue tetrazolium was developed. The histochemical activity staining was standardized in cultured cells. The assay was linear with time and was highly specific for KGDHC. The dark-blue reaction product (formazan) formed a pattern that was consistent with mitochondrial localization. Treatment of the cultured cells with both reversible and irreversible inhibitors decreased formazan production, whereas conventional enzyme assays on cell lysates only revealed loss of KGDHC activity with irreversible inhibitors. The activity staining was also linear with time and highly specific for KGDHC activity in mouse brain sections. Staining occurred throughout the brain, and discrete neuronal populations exhibited particularly intense staining. The pattern of staining differed markedly from the distribution of KGDHC protein by immunocytochemistry. Generalized decreases in the intensity of activity staining that occurred in the TD brains compared to controls were comparable with the loss of KGDHC activity by conventional enzyme assay. Thus, the present study introduces a new histochemical method to measure KGDHC activity at the cellular and regional level, which will be useful to determine changes of in situ enzyme activity.

Animals↗

PubMed2000

The alpha-ketoglutarate dehydrogenase complex in neurodegeneration.

Altered energy metabolism is characteristic of many neurodegenerative disorders. Reductions in the key mitochondrial enzyme complex, the alpha-ketoglutarate dehydrogenase complex (KGDHC), occur in a number of neurodegenerative disorders including Alzheimer's Disease (AD). The reductions in KGDHC activity may be responsible for the decreases in brain metabolism, which occur in these disorders. KGDHC can be inactivated by several mechanisms, including the actions of free radicals (Reactive Oxygen Species, ROS). Other studies have associated specific forms of one of the genes encoding KGDHC (namely the DLST gene) with AD, Parkinson's disease, as well as other neurodegenerative diseases. Reductions in KGDHC activity can be plausibly linked to several aspects of brain dysfunction and neuropathology in a number of neurodegenerative diseases. Further studies are needed to assess mechanisms underlying the sensitivity of KGDHC to oxidative stress and the relation of KGDHC deficiency to selective vulnerability in neurodegenerative diseases.

Brain↗

PubMed2000

Metabolic impairment elicits brain cell type-selective changes in oxidative stress and cell death in culture.

Abnormalities in oxidative metabolism and inflammation accompany many neurodegenerative diseases. Thiamine deficiency (TD) is an animal model in which chronic oxidative stress and inflammation lead to selective neuronal death, whereas other cell types show an inflammatory response. Therefore, the current studies determined the response of different brain cell types to TD and/or inflammation in vitro and tested whether their responses reflect inherent properties of the cells. The cells that have been implicated in TD-induced neurotoxicity, including neurons, microglia, astrocytes, and brain endothelial cells, as well as neuroblastoma and BV-2 microglial cell lines, were cultured in either thiamine-depleted media or in normal culture media with amprolium, a thiamine transport inhibitor. The activity levels of a key mitochondrial enzyme, alpha-ketoglutarate dehydrogenase complex (KGDHC), were uniquely distributed among different cell types: The highest activity was in the endothelial cells, and the lowest was in primary microglia and neurons. The unique distribution of the activity did not account for the selective response to TD. TD slightly inhibited general cellular dehydrogenases in all cell types, whereas it significantly reduced the activity of KGDHC exclusively in primary neurons and neuroblastoma cells. Among the cell types tested, only in neurons did TD induce apoptosis and cause the accumulation of 4-hydroxy-2-nonenal, a lipid peroxidation product. On the other hand, chronic lipopolysaccharide-induced inflammation significantly inhibited cellular dehydrogenase and KGDHC activities in microglia and astrocytes but not in neurons or endothelial cells. The results demonstrate that the selective cell changes during TD in vivo reflect inherent properties of the different brain cell types.

Aldehydes↗

PubMed2000

Vascular factors are critical in selective neuronal loss in an animal model of impaired oxidative metabolism.

Thiamine deficiency (TD) models the cellular and molecular mechanisms by which chronic oxidative deficits lead to death of select neurons in brain. Region- and cell-specific oxidative stress and vascular changes accompany the TD-induced neurodegeneration. The current studies analyzed the role of oxidative stress in initiating these events by testing the role of intercellular adhesion molecule-1 (ICAM-1) and endothelial nitric oxide synthase (eNOS) in the selective neuronal loss that begins in the submedial thalamic nucleus of mice. Oxidative stress to microvessels is known to induce eNOS and ICAM-1. TD increased ICAM-1 immunoreactivity in microvessels within the submedial nucleus and adjacent regions 1 day prior to the onset of neuronal loss. On subsequent days, the pattern of ICAM-1 induction overlapped that of neuronal loss, and of induction of the oxidative stress marker heme oxygenase-1 (HO-1). The intensity and extent of ICAM-1 and HO-1 induction progressively spread in parallel with the neuronal death in the thalamus. Targeted disruption of ICAM-1 or eNOS gene, but not the neuronal NOS gene, attenuated the TD-induced neurodegeneration and HO-1 induction. TD induced ICAM-1 in eNOS knockout mice, but did not induce eNOS in mice lacking ICAM-1. These results demonstrate that in TD, an ICAM-1-dependent pathway of eNOS induction leads to oxidative stress-mediated death of metabolically compromised neurons. Thus, TD provides a useful model to help elucidate the role of ICAM-1 and eNOS in the selective neuronal death in diseases in which oxidative stress is implicated.

Animals↗

PubMed2000

Vascular endothelium is a site of free radical production and inflammation in areas of neuronal loss in thiamine-deficient brain.

Free radical production in vascular endothelial cells and inflammatory responses in perivascular microglia accompany the selective neuronal death induced by TD. Lipid peroxidation and tyrosine nitration occur in neurons within susceptible areas. Thus, region- and cell-specific oxidative stress contributes to selective neurodegeneration during TD. These data are consistent with the hypothesis that in TD, vascular factors constitute a critical part of a cascade of events leading to increases in blood-brain barrier permeability to nonneuronal proteins and iron, leading to inflammation and oxidative stress. Inflammatory cells may release deleterious compounds or cytokines that exacerbate the oxidative damage to metabolically compromised neurons. Similar mechanisms may operate in the pathophysiology of neurodegenerative diseases in which vascular factors, inflammation and oxidative stress are implicated including AD.

Alzheimer Disease↗

PubMed1999

Cultures of astrocytes and microglia express interleukin 18.

Interleukin 18 (IL-18 or interferon-gamma inducing factor) is a recently discovered pro-inflammatory cytokine and powerful stimulator of the cell-mediated immune response. IL-18 is produced by several sources including monocytes/macrophages, keratinocytes and the zona reticularis and zona fasciculata of the adrenal cortex. IL-18 occurs in brain but its cellular source in the CNS has never been investigated. The presence of IL-18 and its response to stimulation in the brain was tested with primary cultures of microglia, astrocytes and hippocampal neurons. IL-18 mRNA was present in astrocytes and microglia, but not in neurons. The endotoxin lipopolysaccharide (LPS) did not affect IL-18 in astrocytes, but LPS robustly increased IL-18 mRNA in microglia. IL-18 protein was constitutively expressed in astrocytes and induced in microglia by LPS. The levels of interleukin-1beta converting enzyme (ICE), an activating enzyme, and caspase 3 (CPP32), an inactivating enzyme, were assessed to investigate the presence of the appropriate processing enzymes in the cultured cells. ICE was present at constitutive levels in microglia and astrocytes suggesting that these cell types may produce and secrete matured IL-18. Active forms of CPP32 were not detectable in either cell type indicating the absence of a degradative pathway of IL-18. The present results demonstrate that microglia and astrocytes are sources of brain IL-18 and add a new member to the family of cytokines produced in the brain.

Animals↗

PubMed1999

Protein-bound acrolein: a novel marker of oxidative stress in Alzheimer's disease.

Several lines of evidence support the role of oxidative stress, including increased lipid peroxidation, in the pathogenesis of Alzheimer's disease (AD). Lipid peroxidation generates various reactive aldehydes, such as 4-hydroxynonenal (HNE), which have been detected immunochemically in AD, particularly in neurofibrillary tangles, one of the major diagnostic lesions in AD brains. A recent study demonstrated that acrolein, the most reactive among the alpha,beta-unsaturated aldehyde products of lipid peroxidation, could be rapidly incorporated into proteins, generating a carbonyl derivative, a marker of oxidative stress to proteins. The current studies used an antibody raised against acrolein-modified keyhole limpet hemocyanin (KLH) to test whether acrolein modification of proteins occurs in AD. Double immunofluorescence revealed strong acrolein-KLH immunoreactivity in more than half of all paired helical filament (PHF)-1-labeled neurofibrillary tangles in AD cases. Acrolein-KLH immunoreactivity was also evident in a few neurons lacking PHF-1-positive neurofibrillary tangles. Light acrolein-KLH immunoreactivity occurred in dystrophic neurites surrounding the amyloid-beta core, which itself lacked acrolein-KLH staining. The pattern of acrolein-KLH immunostaining was similar to that of HNE. Control brains did not contain any acrolein-KLH-immunoreactive structures. The current results suggest that protein-bound acrolein is a powerful marker of oxidative damage to protein and support the hypothesis that lipid peroxidation and oxidative damage to protein may play a crucial role in the formation of neurofibrillary tangles and to neuronal death in AD.

Acrolein↗

PubMed1999

Metabolic impairment induces oxidative stress, compromises inflammatory responses, and inactivates a key mitochondrial enzyme in microglia.

Microglial activation, oxidative stress, and dysfunctions in mitochondria, including the reduction of cytochrome oxidase activity, have been implicated in neurodegeneration. The current experiments tested the effects of reducing cytochrome oxidase activity on the ability of microglia to respond to inflammatory insults. Inhibition of cytochrome oxidase by azide reduced oxygen consumption and increased reactive oxygen species (ROS) production but did not affect cell viability. Azide also attenuated microglial activation, as measured by nitric oxide (NO.) production in response to lipopolysaccharide (LPS). It is surprising that the inhibition of cytochrome oxidase also diminished the activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), a Krebs cycle enzyme. This reduction was exaggerated when the azide-treated microglia were also treated with LPS. The combination of the azide-stimulated ROS and LPS-induced NO. would likely cause peroxynitrite formation in microglia. Thus, the possibility that KGDHC was inactivated by peroxynitrite was tested. Peroxynitrite inhibited the activity of isolated KGDHC, nitrated tyrosine residues of all three KGDHC subunits, and reduced immunoreactivity to antibodies against two KGDHC components. Thus, our data suggest that inhibition of the mitochondrial respiratory chain diminishes aerobic energy metabolism, interferes with microglial inflammatory responses, and compromises mitochondrial function, including KGDHC activity, which is vulnerable to NO. and peroxynitrite that result from microglial activation. Thus, activation of metabolically compromised microglia can further diminish their oxidative capacity, creating a deleterious spiral that may contribute to neurodegeneration.

Animals↗

PubMed1999

Oxidative stress is associated with region-specific neuronal death during thiamine deficiency.

Thiamine deficiency (TD) is a model of chronic impairment of oxidative metabolism and selective neuronal loss. TD leads to region-specific neuronal death and elevation of inducible nitric oxide synthase (iNOS) in macrophages/microglia in mouse brain. Identification of the initial site of neuronal death in the submedial thalamic nucleus allowed us to test the role of iNOS and oxidative stress in TD-induced neuronal death. The pattern of neuronal loss, which begins after 9 days of TD, overlapped with induction of the oxidative stress marker heme oxygenase-1 (HO-1) in microglia. Neuronal death and microglial HO-1 induction spread to engulf the whole thalamus after 11 days of TD. As in past studies, reactive iron and ferritin accumulated in microglia beginning on day 10. The lipid peroxidation product, 4-hydroxynonenal (HNE) accumulated in the remaining thalamic neurons only after 11 days of TD. These responses were not likely mediated by iNOS because HO-1 induction and HNE accumulation were comparable in iNOS knockout mice and wild-type controls. These results show that region and cell specific oxidative stress is associated with selective neurodegeneration during TD. Thus, TD is a useful model to help elucidate neuron-microglial interaction in neurodegenerative diseases associated with oxidative stress.

Aldehydes↗

PubMed1999

Oxidative stress and a key metabolic enzyme in Alzheimer brains, cultured cells, and an animal model of chronic oxidative deficits.

Oxidative stress and diminished metabolism occur in several neurodegenerative disorders. Brains from Alzheimer's disease (AD) patients exhibit several indicators of oxidative stress and have reduced activities of the alpha-ketoglutarate dehydrogenase complex (KGDHC), a key mitochondrial enzyme. Whether these abnormalities are secondary to neurodegenerative processes or are inherent properties of the cells cannot be determined in autopsy brain. Studies in cultured fibroblasts suggest that AD-related differences in oxidative stress and KGDHC reflect inherent properties of AD cells. KGDHC is sensitive to oxidative stress whether the enzyme is studied in cells, in purified mitochondria, or as an isolated protein. Reductions of brain KGDHC in living rodents lead to oxidative stress and selective cell death. The results suggest that KGDHC participates in a deleterious cascade of events related to oxidative stress that are critical in selective neuronal loss in neurodegenerative diseases.

Alzheimer Disease↗

PubMed1998

Induction of nitric oxide synthase and microglial responses precede selective cell death induced by chronic impairment of oxidative metabolism.

Abnormal oxidative processes including a reduction in thiamine-dependent enzymes accompany many neurodegenerative diseases. Thiamine deficiency (TD) models the cellular and molecular mechanisms by which chronic oxidative aberrations associated with thiamine-dependent enzyme deficits cause selective neurodegeneration. The mechanisms underlying selective cell death in TD are unknown. In rodent TD, the earliest region-specific pathological change is breakdown of the blood-brain barrier (BBB). The current studies tested whether nitric oxide and microglia are important in the initial events that couple BBB breakdown to selective neuronal loss. Enhanced expression of endothelial nitric oxide synthase and nicotinamide adenine dinucleotide phosphate diaphorase reactivity in microvessels, as well as the presence of numerous inducible nitric oxide synthase-immunoreactive microglia, accompanied the increases in BBB permeability. Nitric oxide synthase induction appears critical to TD pathology, because immunoreactivity for nitrotyrosine, a specific nitration product of peroxynitrite, also increased in axons of susceptible regions. In addition, TD elevated iron and the antioxidant protein ferritin in microvessels and in activated microglia, suggesting that these cells are responding to an oxidative challenge. All of these changes occurred in selectively vulnerable regions, preceding neuronal death. These findings are consistent with the hypothesis that the free radical-mediated BBB alterations permit entry of iron and extraneuronal proteins that set in motion a cascade of inflammatory responses culminating in selective neuronal loss. Thus, the TD model should help elucidate the relationship between oxidative deficits, BBB abnormalities, the inflammatory response, ferritin and iron elevation, and selective neurodegeneration.

Animals↗

PubMed1998

Immunochemical characterization of the deficiency of the alpha-ketoglutarate dehydrogenase complex in thiamine-deficient rat brain.

Mitochondrial dysfunction is a common feature of many neurodegenerative disorders. The metabolic encephalopathy caused by thiamine deficiency (TD) is a classic example in which an impairment of cerebral oxidative metabolism leads to selective cell death. In experimental TD in rodents, a reduction in the activity of the thiamine diphosphate-dependent, mitochondrial enzyme alpha-ketoglutarate dehydrogenase complex (KGDHC) occurs before the onset of pathologic lesions and is among the earliest biochemical deficits found. To understand the molecular basis and the significance of the deficiency of KGDHC in TD-induced brain damage, the enzyme activity and protein levels of KGDHC were analyzed. The effect of TD on the subregional/cellular distribution of KGDHC and the anatomic relation of KGDHC with selective cell death were also tested by immunocytochemistry. Consistent with several previous studies, TD dramatically reduced KGDHC activity in both anatomically damaged (thalamus and inferior colliculus) and spared (cerebral cortex) regions. Immunocytochemistry revealed no apparent correlation of regional KGDHC immunoreactivity or its response to TD with affected regions in TD. The basis of the enzymatic and immunocytochemical behavior of KGDHC was further assessed by quantitative immunoblots, using antibodies specific for each of the three KGDHC components. Despite the marked decrease of KGDHC activity in TD, no reduction of any of the three KGDHC protein levels was found. Thus, TD impairs the efficacy of the KGDHC catalytic machinery, whereas the concentration of protein molecules persists. The generalized decline of KGDHC activity with no apparent anatomic selectivity is consistent with the notion that the compromised mitochondrial oxidation sensitizes the brain cells to various other insults that precipitate the cell death. The current TD model provides a relevant experimental system to understand the molecular basis of many neurodegenerative conditions in which mitochondrial dysfunction and KGDHC deficiency are prominent features.

Alcohol Amnestic Disorder↗

PubMed1998

Disturbances of the blood-brain barrier without expression of amyloid precursor protein- containing neuritic clusters or neuronal loss during late stages of thiamine deficiency in guinea pigs.

Generalized oxidative deficits associated with experimental thiamine deficiency (TD) lead to selective neurodegeneration. In mouse brain, TD produces region-specific breach of the blood-brain barrier (BBB), neuronal loss and an accumulation of amyloid precursor protein (APP) in abnormal neurites. The APP-laden abnormal neurites within the damaged areas of mouse brain aggregate into neuritic clusters which strikingly resemble the neuritic component of Alzheimer amyloid plaques. However, amyloid beta-peptide (Abeta) immunoreactivity has not been demonstrated in these neuritic clusters, possibly because the Abeta region of APP in mice contains three amino acid substitutions as compared with the amino acid sequence of human Abeta. In contrast, the guinea pig nucleic acid sequence is more related to the human sequence and the Abeta region is identical in sequence to that of human APP. Thus, the current studies tested whether the presence of an authentic Abeta fragment of APP (i.e., identical to that of man) might make guinea pigs more vulnerable to the development of Abeta-containing neuritic clusters following TD. During late stages of TD, BBB abnormalities, manifested by immunoglobulin G (IgG) extravasation and increased NADPH diaphorase reactivity in microvessels, occurred in brain areas known to be damaged by TD in mice. However, despite the prolonged thiamine deprivation and the advanced neurological symptoms of guinea pigs, no significant neuronal loss or altered APP/Abeta immunostaining occurred in any brain region. Microglial activation, another early marker of damage in mice, was not evident in thiamine-deficient guinea pig brain. Ferritin immunoreactivity and iron deposition in oligodendrocytes within areas of BBB abnormalities were either slightly enhanced or unchanged as compared to controls. This is the first report of brain abnormalities in the guinea pig model of dietary and pyrithiamine-induced TD. The results demonstrate species differences in the response to TD-induced damage, and further support the role of BBB and nitric oxide in the initial events in TD pathology.

Amyloid beta-Peptides↗

PubMed1997

Importance of vascular changes in selective neurodegeneration with thiamine deficiency.

These results demonstrate that early alterations in the BBB may underlie selective vulnerability in this model of chronic reduced oxidative metabolism. Changes in the BBB (IgG extravasation) precede alterations in APP processing and cell death. Since thiamine-dependent enzymes are also reduced in the brain in Alzheimer's disease, similar processes may be important in the pathophysiology of the disease.

Amyloid beta-Protein Precursor↗

PubMed1997

Thiamine deficiency alters APP but not presenilin-1 immunoreactivity in vulnerable brain regions.

Presenilin-1 (PS-1) and amyloid precursor protein (APP) have been linked to the pathogenesis of Alzheimer's disease. While APP accumulation is well documented in several models of brain injury, the role of PS-1 levels in neurodegeneration, if any, remains to be elucidated. The current studies examined PS-1 and APP expression in brain following thiamine deficiency (TD), a nutritional model associated with impaired oxidation and selective neurodegeneration. TD did not alter PS-1 immunoreactivity in any region of rodent brain before or after cell loss. In contrast, APP immunoreactivity accumulated in swollen neurites within, or around lesions in rats, or in abnormal clusters in mice. Thus, alterations in APP but not PS-1 levels are involved in TD-induced neurodegeneration.

Amyloid beta-Protein Precursor↗

PubMed1996

Absence of neuronal and glial proteins in human and rat leptomeninges in situ.

Human leptomeningeal (arachnoid and pia mater) cells in culture have been demonstrated in replicated studies to express typical neuronal proteins such as neurofilament protein and neuron-specific enolase. In addition, they can express glial fibrillary acidic protein. The present study examines the possibility that neuronal and glial proteins might be present in rat and human leptomeningeal cells in situ. The neuronal proteins 160 kDa and 200 kDa neurofilaments, neuron-specific enolase and microtubule-associated protein 2 were, however, not immunolocalized in either the pia mater or arachnoid. Glial fibrillary acidic protein and galactocerebroside were also not detected, while fibronectin and vimentin immunoreactivities were robust in all layers of the leptomeninges. Together with the previously reported expression of some neuronal and astroglial markers in cultured human leptomeninges, these observations suggest that culture alters the properties of leptomeningeal cells.

Animals↗