Sequential and concerted gene expression changes in a chronic in vitro model of parkinsonism.

Many mechanisms of neurodegeneration have been implicated in Parkinson's disease, but which ones are most important and potential interactions among them are unclear. To provide a broader perspective on the parkinsonian neurodegenerative process, we have performed a global analysis of gene expression changes caused by chronic, low-level exposure of neuroblastoma cells to the mitochondrial complex I inhibitor and parkinsonian neurotoxin rotenone. Undifferentiated SK-N-MC human neuroblastoma cells were grown in the presence of rotenone (5 nM), and RNA was extracted at three different time points (baseline, 1 week, and 4 weeks) for labeling and hybridization to Affymetrix Human U133 Plus 2.0 GeneChips. Our results show that rotenone induces concerted alterations in gene expression that change over time. Particularly, alterations in transcripts related to DNA damage, energy metabolism, and protein metabolism are prominent during chronic complex I inhibition. These data suggest that early augmentation of capacity for energy production in response to mitochondrial inhibition might be deleterious to cellular function and survival. These experiments provide the first transcriptional analysis of a rotenone model of Parkinson's disease and insight into which mechanisms of neurodegeneration may be targeted for therapeutic intervention.

Constructing a standard climacteric scale.

Issues relating to the design of scales and their psychometric properties are discussed in the context of constructing a standard measure of core climacteric symptoms. Seven factor analytic studies of climacteric symptoms are examined to determine whether or not there is sufficient consensus across studies to permit agreement on the symptom content and the structure of such a scale. It is argued that these factor analytic studies do indeed contain sufficient consensus on the basis of which a standard climacteric scale can be constructed. Such a scale is described.

Has psychosocial research on the menopause any clinical relevance?

The relevance of the findings of longitudinal studies, and other types of psychosocial studies, of general population samples of climacteric women to day-to-day clinical practice is examined. It is shown that the findings of such studies can act as a useful guide to the assessment and treatment of menopausal women in a clinical setting. However, it is argued that the relevance of these studies to clinical practice could be even greater if certain clinically derived considerations were taken into account in the methodology and design of such studies, and therefore provide a more clinically relevant evidence base for the overall management of women at the time of the menopause.

In vivo labeling of mitochondrial complex I (NADH:ubiquinone oxidoreductase) in rat brain using [(3)H]dihydrorotenone.

Defects in mitochondrial energy metabolism have been implicated in several neurodegenerative disorders. Defective complex I (NADH:ubiquinone oxidoreductase) activity plays a key role in Leber's hereditary optic neuropathy and, possibly, Parkinson's disease, but there is no way to assess this enzyme in the living brain. We previously described an in vitro quantitative autoradiographic assay using [(3)H]dihydrorotenone ([(3)H]DHR) binding to complex I. We have now developed an in vivo autoradiographic assay for complex I using [(3)H]DHR binding after intravenous administration. In vivo [(3)H]DHR binding was regionally heterogeneous, and brain uptake was rapid. Binding was enriched in neurons compared with glia, and white matter had the lowest levels of binding. In vivo [(3)H]DHR binding was markedly reduced by local and systemic infusion of rotenone and was enhanced by local NADH administration. There was an excellent correlation between regional levels of in vivo [(3)H]DHR binding and the in vitro activities of complex II (succinate dehydrogenase) and complex IV (cytochrome oxidase), suggesting that the stoichiometry of these components of the electron transport chain is relatively constant across brain regions. The ability to assay complex I in vivo should provide a valuable tool to investigate the status of this mitochondrial enzyme in the living brain and suggests potential imaging techniques for complex I in humans.

Constructing a standard climacteric scale.

Issues relating to the design of scales and their psychometric properties are discussed in the context of constructing a standard measure of core climacteric symptoms. Seven factor analytic studies of climacteric symptoms are examined to determine whether or not there is sufficient consensus across studies to permit agreement on the symptom content and the structure of such a scale. It is argued that these factor analytic studies do indeed contain sufficient consensus on the basis of which a standard climacteric scale can be constructed. Such a scale is described.

3-Nitropropionic acid exacerbates N-methyl-D-aspartate toxicity in striatal culture by multiple mechanisms.

We examined the effects of 3-nitropropionic acid-induced succinate dehydrogenase inhibition on neuronal ATP content, N-methyl-D-aspartate-induced neuronal death, resting membrane potential, and N-methyl-D-aspartate-induced changes in cytosolic calcium concentration ([Ca2+]c) in cultured rat striatal neurons. Exposure of cultures to 3 mM 3-nitropropionic acid for 3 h did not cause overt toxicity, but reduced ATP content by 35%. Treatment with 3-nitropropionic, or removal of Mg2+ from the medium, enhanced subsequent N-methyl-D-aspartate toxicity, reducing the LC50 from 250 microM to 12 microM or 30 microM, respectively. Even after Mg2+ removal, enhancement of N-methyl-D-aspartate toxicity by 3-nitropropionic acid remained pronounced, with the LC50 further decreasing to 3 microM. The mean resting membrane potential of neurons treated with 3-nitropropionic acid was -37 mV, while that in control neurons was -61 mV. Treatment with 3-nitropropionic did not affect baseline [Ca2+]c as determined by fura-2 microfluorimetry. N-methyl-D-aspartate (30 microM) caused a rapid rise in [Ca2+]c, the initial magnitude of which was not affected by 3-nitropropionic acid. However, after a 1-h treatment, [Ca2+]c was dramatically higher in 3-nitropropionic acid-treated neurons. This increased calcium load was washed out slowly and only partially, although calcium in control neurons washed out rapidly and almost completely. These results suggest that in striatal neurons, the enhancement of N-methyl-D-aspartate toxicity caused by succinate dehydrogenase inhibition may be due to synergism between partial relief of the Mg2+ blockade of the N-methyl-D-aspartate receptor and other mechanisms, including disruption of neuronal calcium regulation. This synergism may be relevant to the neuronal death observed in neurodegenerative disorders.

A prospective randomized study of inpatient iv. antibiotics for community-acquired pneumonia. The optimal duration of therapy.

STUDY OBJECTIVE: To compare therapeutic outcome and perform a cost-benefit analysis of inpatients with community-acquired pneumonia (CAP) treated with a shortened course of i.v. antibiotic therapy. DESIGN: A prospective, randomized, parallel group study with a follow-up period of 28 days. SETTING: Bronx Veterans Affairs Medical Center (VAMC) and the Castle Point VAMC; university-affiliated VAMC general medical wards from September 1993 to March 1995. PATIENTS: Seventy-two male veterans and 1 female veteran with 75 episodes of CAP defined by a new infiltrate on chest radiograph and either history or physical findings consistent with pneumonia. Study population was 42%(31) black, 33%(24) white, and 25%(18) Hispanic. INTERVENTIONS: Patients were randomized (1:1:1) to 1 of 3 treatment groups: group 1 received 2 days of i.v. and 8 days of oral therapy; group 2 received 5 days of i.v. and 5 days of oral therapy; and group 3 received 10 days of i.v. therapy. Antibiotics consisted of cefuroxime, 750 mg every 8 h for the i.v. course, and cefuroxime axetil, 500 mg every 12 h for the oral therapy. MEASUREMENTS AND RESULTS: No differences were found in the clinical course, cure rates, or resolution of chest radiograph abnormalities among the three groups. A significant difference was found in the length of stay (LOS) among the three groups. The mean +/- SD LOS was 6 +/- 3 days in group 1, 8 +/- 2 days in group 2, and 11 +/- 1 days in group 3. The shortened LOS could potentially save $95.5 million for the Department of Veterans Affairs and $2.9 billion for the US private sector. CONCLUSIONS: Adult patients hospitalized for CAP who are not severely ill can be successfully treated with an abbreviated (2-day) course of i.v. antibiotics and then switched to oral therapy. A longer course of i.v. therapy prolongs hospital stay and cost, without improving the therapeutic cure rate.

Bioenergetics and glutamate excitotoxicity.

Bioenergetic defects and abnormalities in glutamate neurotransmission have both been proposed to play important roles in neurological diseases of varying chronology, etiology and pathology. Recent experimental evidence suggests an intimate relationship between these two systems. Metabolic inhibition predisposes neurons to glutamate-mediated "excitotoxic" damage. The exact mechanism of this increased susceptibility is yet to be defined, but may involve, singly or in combination, decreased voltage-dependent Mg2+ blockade of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor, abnormalities in cellular Ca2+ homeostasis, or elevated production of reactive oxygen species. It is believed that enhancement of excitotoxicity by impaired metabolism may be a ubiquitous mechanism of neuronal death in neurological disease. Further elucidation of the exact mechanism of this enhancement may lead to the discovery of new targets for therapeutic intervention.

Manipulation of membrane potential modulates malonate-induced striatal excitotoxicity in vivo.

Malonate is a reversible inhibitor of succinate dehydrogenase (SDH) that produces neurotoxicity by an N-methyl-D-aspartate (NMDA) receptor-dependent mechanism. We have examined the influence of pharmacological manipulation of membrane potential on striatal malonate toxicity in rats in vivo by analysis of lesion volume. Depolarization caused by coinjection of the Na+,K(+)-ATPase inhibitor ouabain or a high concentration of potassium greatly exacerbated malonate toxicity; this combined toxicity was blocked by the noncompetitive NMDA antagonist MK-801. The toxicity of NMDA was also exacerbated by ouabain. The overt toxicity of a high dose of ouabain (1 nmol) was largely prevented by MK-801. Coinjection of the K+ channel activator minoxidil (4 nmol) to reduce depolarization attenuated the toxicity of 1 mumol of malonate by approximately 60% without affecting malonate-induced ATP depletion. These results indicate that membrane depolarization exacerbates malonate neurotoxicity and that membrane hyperpolarization protects against malonate-induced neuronal damage. We hypothesize that the effects of membrane potential on malonate toxicity are mediated through the NMDA receptor as a result of its combined agonist- and voltage-dependent properties.

ARL-15896, a novel N-methyl-D-aspartate receptor ion channel antagonist: neuroprotection against mitochondrial metabolic toxicity and regional pharmacology.

ARL-15896 [(+)-alpha-phenyl-2-pyridineethanamine], formerly known as FPL-15896, is a novel N-methyl-D-aspartate (NMDA) receptor ion channel antagonist. Using quantitative receptor autoradiography, we examined the regional binding characteristics of ARL-15896 and compared them to those of MK-801, the prototypical NMDA receptor channel blocker. The affinity of ARL-15896 was much lower (3000-fold) than that of MK-801 in all brain regions examined. In addition, in contrast to MK-801, which has a higher affinity in the forebrain than in the cerebellum (IC50 of 10 nM vs 24 nM), ARL-15896 had a higher affinity in the cerebellum than in the forebrain (IC50 of 17 microM vs 45 microM). The neuroprotective potential of ARL-15896 was investigated in a rat model of excitotoxicity, the intrastriatal injection of malonate. Malonate is a competitive inhibitor of succinate dehydrogenase, and its toxicity has been shown to be mediated largely by the NMDA receptor. Administration of ARL-15896 either intrastriatally (200 nmol) or subcutaneously (9.0 mg/kg) reduced the volume of the lesion produced by 1 mumol of malonate by 80%, a degree similar to that reported for MK-801. ARL-15896 was also protective when administered after the malonate injection. Furthermore, in contrast to MK-801, administration of ARL-15896 was not associated with any apparent behavioral side effects. This report is consistent with previous studies suggesting that drugs with regional pharmacological profiles similar to that of ARL-15896 have better clinical tolerability; it also indicates that ARL-15896 is an effective neuroprotective agent.

Pulmonary scar carcinoma: report of three cases and review of the literature.

Pulmonary scar carcinoma was described as a distinct clinicopathological entity over 50 years ago. There are many theories on the formation of this entity. We present three cases of pulmonary scar carcinoma with a high ratio of adenocarcinoma. One patient had a favorable postoperative course despite a 14-month delay in treatment. Necropsy specimen of another patient showed two primary scar carcinomas unrelated to each other. Literature review and discussion of etiology, diagnosis, and treatment modalities of pulmonary scar carcinoma were done. Pathogenesis and prognosis of the neoplasms associated with apical scars are not clearly understood.

Intrastriatal injections of the succinate dehydrogenase inhibitor, malonate, cause a rise in extracellular amino acids that is blocked by MK-801.

The effects of intrastriatal injections of a reversible inhibitor of succinate dehydrogenase, malonate, on the extracellular concentrations of amino acid neurotransmitters were examined using a microdialysis probe that was positioned a fixed distance from an injection cannula. Malonate (2 mumol) caused a 23 +/- 5-fold increase in extracellular glutamate (Glu), a 18 +/- 6-fold increase extracellular gamma-aminobutyric acid (GABA) and a modest increase in extracellular aspartate (Asp, 2.9 +/- 0.8-fold increase). Administration of the NMDA receptor antagonist MK-801 (5 mg/kg) prior to injection of malonate almost completely blocked these increases. This study provides direct evidence that inhibition of succinate dehydrogenase causes an increase in extracellular amino acid neurotransmitters and further evidence that bioenergetic defects may contribute to the pathogenesis of chronic neurodegenerative diseases through an excitotoxic mechanism.

Exacerbation of NMDA, AMPA, and L-glutamate excitotoxicity by the succinate dehydrogenase inhibitor malonate.

We report that a subtoxic dose of the succinate dehydrogenase (SDH) inhibitor malonate greatly enhances the neurotoxicity of three different excitatory amino acid agonists: N-methyl-D-aspartate (NMDA), S-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (S-AMPA), and L-glutamate. In male Sprague-Dawley rats, intrastriatal stereotaxic injection of malonate alone (0.6 mumol), NMDA alone (15 nmol), S-AMPA alone (1 nmol), or glutamate alone (0.6 mumol) produced negligible toxicity as assessed by measurement of lesion volume. Coinjection of subtoxic malonate with NMDA produced a large lesion (15.2 +/- 1.4 mm3), as did coinjection of malonate with S-AMPA (11.0 +/- 1.0 mm3) or glutamate (12.8 +/- 0.7 mm3). Administration of the noncompetitive NMDA antagonist MK-801 (5 mg/kg i.p.) completely blocked the toxicity of malonate plus NMDA (0.5 +/- 0.3 mm3). This dose of MK-801 had little effect on the lesion produced by malonate plus S-AMPA (9.0 +/- 0.7 mm3), but it attenuated the toxicity of malonate plus glutamate by approximately 40% (7.5 +/- 0.9 mm3). Coinjection of the AMPA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)-quinoxaline (NBQX; 2 nmol) had no effect on malonate plus NMDA or malonate plus glutamate toxicity (12.3 +/- 1.8 and 14.0 +/- 0.9 mm3, respectively) but greatly attenuated malonate plus S-AMPA toxicity (1.5 +/- 0.9 mm3). Combination of the two antagonists conferred no additional neuroprotection in any paradigm. These results indicate that metabolic inhibition exacerbates both NMDA receptor- and non-NMDA receptor-mediated excitotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the excitotoxic potential of the reversible succinate dehydrogenase inhibitor malonate.

Although the mechanism of neuronal death in neurodegenerative diseases remains unknown, it has been hypothesized that relatively minor metabolic defects may predispose neurons to N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxic damage in these disorders. To further investigate this possibility, we have characterized the excitotoxic potential of the reversible succinate dehydrogenase (SDH) inhibitor malonate. After its intrastriatal stereotaxic injection into male Sprague-Dawley rats, malonate produced a dose-dependent lesion when assessed 3 days after surgery using cytochrome oxidase histochemistry. This lesion was attenuated by coadministration of excess succinate, indicating that it was caused by specific inhibition of SDH. The lesion was also prevented by administration of the noncompetitive NMDA antagonist MK-801. MK-801 did not induce hypothermia, and hypothermia itself was not neuroprotective, suggesting that the neuroprotective effect of MK-801 was due to blockade of the NMDA receptor ion channel and not to any nonspecific effect. The competitive NMDA antagonist LY274614 and the glycine site antagonist 7-chlorokynurenate also profoundly attenuated malonate neurotoxicity, further indicating an NMDA receptor-mediated event. Finally, the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) antagonist NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)-quinoxaline) was ineffective at preventing malonate toxicity at a dose that effectively reduced S-AMPA toxicity, indicating that non-NMDA receptors are involved minimally, if at all, in the production of the malonate lesion. We conclude that inhibition of SDH by malonate results in NMDA receptor-mediated excitotoxic neuronal death. If this mechanism of "secondary" or "weak" excitotoxicity plays a role in neurodegenerative disease, NMDA antagonists and other "antiexcitotoxic" strategies may have therapeutic potential for these diseases.

Polysynaptic regulation of glutamate receptors and mitochondrial enzyme activities in the basal ganglia of rats with unilateral dopamine depletion.

After nigrostriatal dopaminergic denervation, the output nuclei of the basal ganglia, the medial globus pallidus and substantia nigra pars reticulata (Snr), become overactive, in part, because of increased activity of excitatory afferents from the subthalamic nucleus (STN). Because STN uses glutamate as a transmitter, we examined whether there are regulatory changes in glutamate receptor binding in the basal ganglia. Rats received unilateral 6-hydroxydopamine lesions of the medial forebrain bundle and substantia nigra pars compacta that were confirmed by apomorphine-induced rotation and 3H-GBR-12935 binding. As an indirect index of relative synaptic activity, succinate dehydrogenase and cytochrome oxidase activities were assayed histochemically in sections adjacent to those used for receptor binding. There were increases in enzymatic activity in entopeduncular nucleus (EP; the rodent homolog of medial globus pallidus), SNr, and globus pallidus (GP, the rodent homolog of lateral globus pallidus) in the lesioned hemisphere, suggesting increased synaptic activity, perhaps due to increased firing of the STN. Ipsilateral to the lesion, and postsynaptic to the STN, there were profound decreases in the binding of 3H-AMPA (alpha-amino-3-hydroxy-5-methylisoxazole propionic acid) in EP and SNr (45% and 30%, respectively); there were no alterations in the striatum, globus pallidus, or STN, and binding throughout the unlesioned hemisphere was equivalent to that in unlesioned control animals. In contrast, 3H-MK-801 binding to the NMDA receptor ion channel was not reduced in SNr, and was too low to be measured reliably in EP and STN. 3H-MK-801 binding was reduced by 6% in striatum and 39% in globus pallidus.(ABSTRACT TRUNCATED AT 250 WORDS)

The endogenous cofactors, thioctic acid and dihydrolipoic acid, are neuroprotective against NMDA and malonic acid lesions of striatum.

Thioctic acid (alpha-lipoic acid) and dihydrolipoic acid are endogenous, interconvertible cofactors of the mitochondrial pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase complexes. These compounds help to maintain glutathione and alpha-tocopherol in their reduced states, and they possess intrinsic free radical scavenging properties. We examined whether systemic treatment with thioctic acid or dihydrolipoic acid is protective against direct- and indirect-acting excitotoxins. Adult rats were treated for 10 days with intraperitoneal injections of vehicle, thioctic acid or dihydrolipoic acid, and on day 7 of treatment animals received unilateral stereotaxic injections of NMDA or malonic acid into the striatum. Histological assessment 3 days after the stereotaxic injections revealed a marked reduction in lesion volume in animals treated with thioctic acid or dihydrolipoic acid. We conclude that thioctic acid and dihydrolipoic acid are neuroprotective against direct and indirect excitotoxic insults.

Inhibition of succinate dehydrogenase by malonic acid produces an "excitotoxic" lesion in rat striatum.

Excitotoxicity and defects in neuronal energy metabolism have both been implicated in the pathogenesis of neurodegenerative disease. These two mechanisms may be linked through the NMDA receptor, activation of which is dependent on neuronal membrane potential. Because the ability to maintain membrane potential is dependent on neuronal energy metabolism, bioenergetic defects may affect NMDA receptor-mediated excitotoxicity. We now report that reversible inhibition of succinate dehydrogenase (SDH), an enzyme central to both the tricarboxylic acid cycle and the electron transport chain, produces an "excitotoxic" lesion in rat striatum that can be blocked by the NMDA antagonist MK-801. Male Sprague-Dawley rats received intrastriatal stereotaxic injections of the SDH inhibitor malonic acid (1 or 2 mumol) in combination with intraperitoneal injections of vehicle or MK-801 (5 mg/kg) 30 min before and 210 min after malonic acid. Animals were killed 72 h after surgery, and brains were processed for histology, cytochrome oxidase activity, and [3H]MK-801 and [3H]AMPA autoradiography. The higher dose of malonic acid (2 mumol) produced large lesions that were markedly attenuated by treatment with MK-801 (28.1 +/- 3.6 vs. 4.7 +/- 2.6 mm3; p < 0.001). [3H]MK-801 and [3H]AMPA binding were reduced in the lesions by 60 and 63%, respectively. One micromole of malonic acid produced smaller lesions that were almost completely blocked by MK-801 treatment (9.6 +/- 1.3 vs. 0.06 +/- 0.04 mm3; p < 0.0001). The toxic effects of malonic acid were due specifically to inhibition of SDH inasmuch as coinjection of a threefold excess of succinate with the malonic acid blocked the striatal lesions (p < 0.002).(ABSTRACT TRUNCATED AT 250 WORDS)

The cross-sectional legacy: an introduction to longitudinal studies of the climacteric.

Hitherto the bulk of psychosocial and sociological research on the menopause has been based on cross-sectional studies, in which the research strategy has been to compare women of different ages and/or different menopausal status by reference to various parameters. This paper summarizes the main findings of these studies, which relate to a variety of countries. A consistent finding has been that much of the variance in the symptoms and complaints reported by women during the climacteric can in fact be accounted for by a number of different adverse sociodemographic and psychosocial factors. It is postulated that the mechanism whereby these factors exercise their effect can best be conceptualized in terms of a vulnerability model. Although cross-sectional studies, have obvious limitations, their findings have now been complemented by those from a number of ongoing longitudinal studies in which the same cohort of women is being followed through this transitional period of their lives.