雌激素对喹啉酸致大鼠纹状体神经元损害的影响

目的 探讨雌激素对喹啉酸（QA）所致的大鼠纹状体神经元损伤的影响。方法 将成年雌性大鼠切除双侧卵巢后随机分为卵巢切除组（非替代组）和卵巢切除＋雌二醇组（替代组）;向纹状体注射QA,采用尼克酰胺腺嘌呤二核苷酸磷酸（NADPH）黄递酶组化法检测纹状体NADPH阳性神经元数,记录阿朴吗啡诱发大鼠旋转次数,分别用黄嘌呤氧化酶法和硫代巴比妥法测定纹状体超氧化物歧化酶（SOD）活性及丙二醛（MDA）含量。结果 与非替代组比较,替代组大鼠损伤侧纹状体NADPH阳性神经元明显增多（P〈0．05）,SOD活性明显增高（P〈0．05）,MDA含量明显降低（P〈0．01）,阿朴吗啡诱发的大鼠旋转次数明显减少（P〈0．01）。结论 雌激素可能通过减轻氧化应激反应对QA引起的纹状体神经元损害起保护作用。     

Effects of intracerebral injections of quinolinic acid on serotonergic neurons in the rat brain

The effects of intrastriatal and intrahippocampal injections of the excitotoxic amino acid, quinolinic acid (QUIN), were examined in the rat using immunohistochemical and neurochemical techniques. Serotonin and 5-hydroxyindoleacetic acid measurements at 90 min, 6 h, 4 and 11 days following QUIN administration revealed highly elevated levels of the metabolite in the injected nuclei, with peak increases occurring after 4 days. Serotonin levels remained largely unchanged over the same time period. Direct visualization of hippocampal serotonergic fibers by immunohistochemistry demonstrated morphological changes (varicosities, swellings) in otherwise undamaged serotonin-positive afferents 4 days following a local QUIN injection. Hippocampal serotonin turnover was assessed at 4 days after an intrahippocampal QUIN-application: following inhibition of aromatic amino acid decarboxylase, the accumulation of 5-hydroxytryptophan was twice as rapid in QUIN-lesioned hippocampi as in controls. Dose-response relationships, examination of brain regions distant from the two injection sites and the temporal sequence of the changes described here suggest a close association between QUIN-induced neuronal degeneration and alterations in the serotonergic system.     

Antagonism of seizures induced by the administration of the endogenous convulsant quinolinic acid into rat brain ventricles

Summary The effect of pretreatment with drugs on generalized clonic-tonic seizures induced by intracerebroventricular (i.c.v.) administration of the endogenous convulsant quinolinic acid (QUIN, 50g) was studies in rats. Of the inhibitory amino acids tested, only l-glycine (50 and 100g, i.c.v.) diminished the number of animals with seizures while taurine and GABA were ineffective. Of the kynurenine metabolites, only kynurenic acid (50g, i.c.v.) prevented seizures and lethality. Picolinic acid, nicotinic acid and nicotinamide were ineffective. The standard anticonvulsants phenobarbital, diphenylhydantoin and primidone were effective antagonists of QUIN-induced seizures at doses which did not influence pentylenetetrazol seizures. However, the only drug which completely prevented QUIN-induced seizures was diazepam (10 mg/kg). It also prevented pentylenetetrazol seizures in rats in a four times lower dose. The GABA derivatives sodium hydroxybutyrate and phenibut (beta-phenyl-GABA), which are effective QUIN-antagonists in mice, were found to be ineffective in rats. Species differences between rats and mice in the efficacy of antagonists QUIN are discussed.Reported at the 162nd Meeting of Leningrad Pharmacological Society (March 9, 1982).     

苯乙酸对大鼠原代皮质神经元的毒性作用

探讨苯乙酸 (phenylacetic acid,PAA)对大鼠原代培养的皮质神经元的毒性作用及其作用机制。实验结果显示 ,PAA加入无血清 Neurobasal培养基可剂量依赖地损伤原代培养的皮质神经元 ,且干预培养的时间越长 ,神经元存活率越低。而同浓度 (1 .1× 1 0 -3mol/L)的 PAA对皮质神经元的损伤明显高于海马。原代培养的皮质神经元补充一氧化氮合成酶抑制剂 L- NAME、NMDA受体拮抗剂 MR- 80 1及 L型钙通道阻滞剂尼莫地平可显著地抵抗 PAA的毒性作用。以上实验结果提示 ,PAA可选择性地损伤皮质神经元 ,其毒性作用可能与 NO过量产生、兴奋性氨基酸的堆积及钙超载有关     

Indoleamine 2,3 dioxygenase and quinolinic acid immunoreactivity in Alzheimer's disease hippocampus

The present immunohistochemical study provides evidence that the kynurenine pathway is up-regulated in Alzheimer's disease (AD) brain, leading to increases in the excitotoxin quinolinic acid (QUIN). We show that the regulatory enzyme of the pathway leading to QUIN synthesis, indoleamine 2,3 dioxygenase (IDO) is abundant in AD compared with controls. In AD hippocampus, both IDO- and QUIN-immunoreactivity (-IR) was detected in cortical microglia, astrocytes and neurones, with microglial and astrocytic expression of IDO and QUIN highest in the perimeter of senile plaques. QUIN-IR was present in granular deposits within the neuronal soma of AD cortex and was also seen uniformly labelling neurofibrillary tangles. Our data imply that QUIN may be involved in the complex and multifactorial cascade leading to neuro-degeneration in AD. These results may open a new therapeutic door for AD patients.     

Effect of PK11195 on attenuating the enhancement of glucose utilization induced by quinolinic acid infusion in the rat brain

PK11195, a selective PBR ligand, has been reported to exert a protective effect against the neuronal damage induced by the intrastriatal infusion of quinolinic acid, an excitatory amino acid. The neuroprotective effect of PK11195 observed at 48 h after the infusion was mediated by the inhibition of microglial activation. The aim of this study is to search the mechanism for the effect of PK11195 other than the inhibition of activation of microglia. In this study, the effect of PK11195 on glucose metabolism as well as neuroprotection in the early phase (2 h) after the injection of quinolinic acid was examined. Intrastriatal injection of quinolinic acid (60 nmol/microL) alone caused a significant enhancement of [(14)C]DG utilization in the infused striatum (about 160% vs. the contralateral side). This enhancement of glucose utilization might be due to an increase in phosphorylation rate of [(14)C]DG rather than delivery process from the plasma into the brain, since the initial uptake of [(14)C]DG (1 min) was not changed by quinolinic acid. Coinjection of PK11195 (10 nmol/microL) completely blocked the enhancement of [(14)C]DG uptake induced by quinolinic acid. The attenuating effect of PK11195 on glucose metabolic disturbance induced by quinolinic acid seemed to be related to voltage-dependent anion channels (VDAC), which are component of the PBR complex and associated with the regulation of hexokinase activity. PK11195 also showed neuroprotective effect at 2 h after the infusion of quinolinic acid, despite no significant activation of microglia was observed at this time-point. Thus, the neuroprotection of PK11195 might be related to normalization of the metabolic disturbance by the excitatory amino acid.     

Depletion of neutrophils reduces neuronal degeneration and inflammatory responses induced by quinolinic acid in vivo

The use of anti-neutrophil serum (anti-PMN) to induce neutropenia has been assessed for neuroprotection, modulation of microgliosis and astrogliosis, effects on oxidative stress, and intactness of blood-brain barrier (BBB) following injection of the excitotoxin quinolinic acid (QUIN) into rat striatum. At 1 day following QUIN injection, considerable striatal neurodegeneration was measured (Fluoro-Jade B marker). At this time, marked microgliosis (OX-42 marker) and astrogliosis (GFAP marker) were evident in QUIN-injected striatum. Treatment of QUIN-injected animals with anti-PMN protected neurons (48% reduction of striatal neuron loss) and inhibited microgliosis (61% reduction) and astrogliosis (43% reduction) compared with QUIN injection alone. Anti-PMN treatment was effective in decreasing levels of superoxide anions (by 42%) compared with QUIN alone; in addition, expressions of the neutrophil enzyme myeloperoxidase and the neutrophil oxidant 3-chlorotyrosine were markedly reduced (by 79 and 72%, respectively) with neutrophil depletion. QUIN-induced leakiness in BBB was indicated by elevated striatal levels of the blood protein fibrinogen, a result confirmed using Evans blue dye; anti-PMN was effective in reducing BBB permeability. Measurements from QUIN-injected animals directly confirmed anti-PMN efficacy in diminishing numbers of circulating neutrophils. Longer term neuroprotection and reduced microgliosis were also observed at 7 days post-injection of anti-PMN; at this time, anti-PMN-treated rats also demonstrated an improved apomorphine-induced rotational performance. We conclude that anti-PMN treatment could serve as a novel strategy to prevent leakiness to BBB, reduce gliosis, and protect striatal neurons in excitotoxin-injected brain.     

Neurotoxic effects of endogenous materials: quinolinic acid, L-pyroglutamic acid, and thyroid releasing hormone (TRH)

The previously reported, dose-related, and selective neurotoxic action of 100 to 200 nmol quinolinic acid on intrastriatal injection was confirmed. A slight neurotoxicity was obtained with 250 nmol thyroid releasing hormone but not with a similar dose of L-pyroglutamic acid.     

Kynurenic acid inhibits synaptic and acidic amino acid-induced responses in the rat hippocampus and spinal cord

Kynurenic acid, a tryptophan metabolite, inhibits excitatory synaptic transmission in the rat hippocampal slice and the isolated immature rat spinal cord, but does not affect membrane potential or input resistance of hippocampal CA1 pyramidal cells. Kynurenic acid also antagonizes responses induced in the dentate gyrus by excitatory amino acids, particularly N-methyl-DL-aspartate and the endogenous excitant quinolinic acid. These results indicate that kynurenic acid antagonizes synaptic transmission probably by blocking postsynaptic transmitter receptors at putative amino acid mediated synapses.     

Behavioral and anatomical effects of quinolinic acid in the striatum of the hemiparkinsonian rat

Parkinson's disease (PD), a hypokinetic disorder, and Huntington's disease (HD), a hyperkinetic disorder, share the fact that in the motor pathways the dysfunction starts in the striatum. In PD the projection neurons are overactive due to decreased inhibitory regulation by lost dopamine afferents, while in HD the output from the striatum is insufficient due to loss of projection neurons. This study aimed to determine whether the introduction of a mild HD condition in the PD striatum can counter the hypokinetic condition. The experiment was carried out in the 6-OHDA rat model for PD in which amphetamine, 5 mg/kg, evokes an asymmetric rotation response toward the side of the 6-OHDA lesion (ipsilateral rotation). The response to amphetamine in this study was fractionated into multiple components and measured automatically. After baseline measurements, the subjects were divided into four groups. Group I was unilaterally sham-lesioned in medial, central, and lateral striatum. Group II was injected quinolinic acid (QA) 20 nM in medial, central, and lateral striatum. Group III was injected QA 60 nM in central striatum. Group IV was injected QA 120 nM in central striatum. The effects of QA were measured weekly. The sham lesions in Group I had no effects. In Group II, ipsilateral rotation was reduced and replaced by oral stereotypy, a competitive behavior. In Group III, ipsilateral rotation decreased, but to a lesser degree than in Group II. In Group IV, QA had no effects. Histological findings show a unilateral loss of tyrosine immunoreactive (TH) neurons in substantia nigra and of fibers in striatum in all subjects. In addition, in Group II the striatum was atrophied. These findings suggest that the shift in Group II from ipsilateral rotation to oral stereotypy after QA was due to reduced striatal output caused by a loss of projection neurons, a loss insufficient to induce HD symptoms, but sufficient to counter the PD condition.     

Intracerebral quinolinic acid injection in the rat: Effects on dopaminergic neurons

Intrastriatal infusion of the endogenous excitotoxin quinolinic acid (QUIN) leads to the degeneration of neuronal cell bodies around the injection site. Dopaminergic afferents not only survive the toxic insult but react by increasing their activity in the acute and suba-cute phases following the injection of QUIN. Measurements of the tissue concentrations of acidic dopamine metabolites, and determinations ofl-DOPA accumulation after DOPA-decar☐ylase inhibition, indicate an increased dopamine turnover within 90 min after the administration of 50 μg QUIN. At the later timepoints examined (6 h, 4 and 11 days after QUIN), dopaminergic parameters are increased in the injected striatum only while no changes can be detected in the homolateral substantia nigra. Local norepinephrine levels are elevated 4 and 11 days after an intrastriatal QUIN injection but remain unchanged at distant sites or earlier postinjection periods. The acute increase in nigrostriatal activity may be mediated by an excessively stimulated, yet functional striatonigral feedback loop whereas subsequent changes represent local reactions of dopaminergic nerve terminals secondary to neuronal degeneration in the striatum. In accordance with this interpretation, no monoaminergic changes can be observed in the hypothalamus 4 days following the local injection of 50 μg QUIN, a dose which does not cause neuronal nercosis in this brain area. These data are concordant with, and are discussed in the context of, a possible involvement of QUIN in the pathogenesis of Huntington's disease.     

Physiology and pharmacology of olfactory bulb neurons in dissociated cell culture

Cells from olfactory bulbs of embryonic rats were grown in dissociated cell culture for up to 5 weeks. Both neurons and non-neuronal cells grew in these cultures, with a variety of neuronal populations appearing. A population of 20–25% of the neurons were GABAergic by the criterion of [³H]GABA uptake. Electrophysiologic measurements were made of the baseline activity of the cultured neurons. Cells showed a mean resting potential of60.1 ± 1.2mV and a mean input resistance of87.6 ± 9.5MΩ. All cells were sensitive to microperfusion of GABA with half-maximal effect occurring at about 20 μM. Glutamate was universally excitatory but with variations in degree. Carnosine (β-Ala-l-His), tested over the concentration range of 10 nM to 100 μM, had no effect on input resistance, resting potential, action potential shape, on-going synaptic activity or the responsiveness to either GABA or glutamate. These results are further evidence against a role for carnosine as the excitatory transmitter of the primary olfactory afferents.     

Glucocorticoid enhances the neurotoxic actions of quinolinic acid in the striatum in a cell-specific manner

This study demonstrates that corticosterone can exacerbate the damaging effects of infused quinolinic acid (QA) on the dorsal striatum. Adult adrenalectomised male rats were pretreated subcutaneously with graded doses of corticosterone (0, 0.5, 2, 5, 20 and 40 mg/kg/day) for 2 days and then received a unilateral infusion of QA (45 nmol) (under Isoflurane/N2O anaesthesia) into the dorsal striatum. A control infusion (vehicle) was made into the striatum on the other side. Corticosterone treatment was continued and they were killed 7 days later. Plasma corticosterone was measured by radioimmunoassay, and thymus weights were used as an integrated measure of glucocorticoid activity. Lesion volumes were measured on neuronal nuclei stained sections, dopamine and cyclic AMP-regulated phosphoprotein 32 (DARRP-32) was used to assess medium spiny neurone survival, NADPH-diaphorase histochemistry to assess medium aspiny neurones and, finally, choline acetyltransferase to assess large aspiny neurones. Adrenalectomised rats showed smaller lesions than control (sham-operated) rats, suggesting significant protection. Increasing doses of corticosterone resulted in larger lesions up to an apparent ceiling effect at higher doses; there was no evidence of a U-shaped dose-response. There was a differential effect of both QA and corticosterone on the cell populations of the striatum. Medium spiny neurones were most vulnerable to the effects of QA and to the exacerbating actions of corticosterone. Medium aspiny neurones were equally vulnerable to QA but corticosterone had no additional effect. Large aspiny neurones were relatively less sensitive to QA and there was no additional action of administered corticosterone. These results show that corticosterone has a selective neuroendangering action within the striatum, but there is no evidence for a protective action of glucocorticoids at lower doses.     

Neuropharmacological actions of kynurenic and quinolinic acids on horizontal cells of the isolated fish retina

We have studied the effects of naturally occurring metabolites of tryptophan, kynurenic and quinolinic acids, on the electrophysiological responses of retinal horizontal cells in the fish (Rutilus rutilus, the roach). Quinolinic acid usually hyperpolarizes the cells and reduces their light evoked responses (S-potentials) but on occasion, it causes a slight depolarization of the membrane potential. These actions are similar to those found with N-methyl-d-aspartate (NMDA) and our results are consistent with the proposal that it acts at NMDA binding sites. Kynurenic acid (1mM) invariably hyperpolarizes horizontal cells to their potassium Nernst equilibrium potential and, more significantly, blocks the depolarizing actions exerted on them by excitatory amino acids, such as kainic and quisqualic acids. We show that this action persists in the presence of the synaptic blocker, cobalt chloride, and thus is not mediated by chemical synaptic activity. Kynurenic acid does not reverse depolarization of horizontal cells by dopamine or γ-aminobutyric acid, thus its inhibitory effects are selective to the actions of excitatory amino acids. Neither xanthurenic acid, a close structural analogue of kynurenic acid, nor quinolinic acid are effective in blocking depolarizations by excitatory aino acids.     

Neuroprotective effect of interleukin-6 and IL6/IL6R chimera in the quinolinic acid rat model of Huntington's syndrome

Ciliary neurotrophic factor prevents behavioural deficits and striatal degeneration in rat and primate models of Huntington's disease. Interleukin-6, another member of the cytokine family, and the chimeric molecule (IL6/IL6R) in which interleukin-6 and its soluble receptor are fused, have been shown to exert trophic action on various neuronal populations in the central nervous system. Therefore, we investigated the neuroprotective effect of these two molecules in the quinolinic acid model of Huntington's disease. LacZ-, interleukin-6- and IL6/IL6R-expressing lentiviral vectors were stereotaxically injected into the striatum of Wistar rats. Three weeks later the animals were lesioned through the intrastriatal injection of 180 nmol of quinolinic acid. The extent of the striatal damage was significantly diminished in the rats that had been treated with interleukin-6 or IL6/IL6R. The neuroprotective effect was, however, more pronounced with the IL6/IL6R chimera than with interleukin-6 as indicated by the volume of the lesions (38.6 +/- 10% in the IL6/IL6R group, 63.3 +/- 3.6% in the IL-6 group and 84.3 +/-2.9% in the control group). Quantitative analysis of striatal interneurons further demonstrated that the IL6/IL6R chimera is more neuroprotective than IL-6 on ChAT- and NADPH-d-immunoreactive neurons. These results suggest that the IL6/IL6R chimera is a potential treatment for Huntington's disease.     

The excitotoxin quinolinic acid is present and unevenly distributed in the rat brain

The presence of quinolinic acid (2,3-pyridinedicarboxylic acid, QA) in the rat brain has been demonstrated using a mass-spectrometric method. Distribution studies indicate that this molecule is more concentrated in the cortex (2.1 nmol/g wet weight) than in other brain areas. Tryptophan, a possible QA precursor, administered in large doses, increases the cortical content of QA. The contrary occurs when rats are pretreated with p-chlorophenylalanine, a drug capable of decreasing brain tryptophan concentration. The neurotoxin 5,7-dihydroxytryptamine is inactive. Our findings support the idea that QA merits special attention as a potential transmitter and as an endogenous excitotoxin in brain.     

An iontophoretic investigation of the actions of convulsant kynurenines and their interaction with the endogenous excitant quinolinic acid

Kynurenine and related compounds are endogenous metabolites of tryptophan which have previously been found to be convulsant in animals. We now report that kynurenine, kynurenic acid, nicotinic acid do not excite neurones in the cerebral cortex, but that quinolinic acid is an effective excitant. Kynurenine had no effect on responses to quinolinic acid, glutamate or N-metyl-D-aspartate (NMDA) and did not block depressant responses to GABA, glycine or 5-hydroxytryptamine. Suprisingly kynurenic acid proved a powerful antagonist of quinolinic acid, NMDA and quisqualic acid, but showed no preferential antagonism to one agonist. It did, however, show a preference for reducing quinolinic acid responses compared to acetylcholine responses.     

Human oligodendrocytes in dissociated cell culture

Summary Human oligodendrocytes have been successfully maintained in cell cultures for 14 weeks using a modification of a method used previously for animal brain cell cultures. Dissociated cell cultures from spinal cords of human foetuses of 10 to 20 weeks gestional age were investigated for up to 98 days. Oligodendrocytes were identified by monoclonal human antiserum specific for myelin-associated glycoprotein, by polyclonal rabbit antiserum against myelin basic protein, and by the mouse monoclonal antibody 16G1. Astrocytes were identified by polyclonal antibodies against glial fibrillary acidic protein. Immunocytochemical cell identification was corroborated by electron microscopy, by which glial cells were investigated both in situ and in culture. Immunocytochemical staining of myelin-associated glycoprotein showed specifically labelled oligodendrocytes on electron microscopy. The present study indicates that human oligodendrocytes, a putative target in demyelinating disease, can be studied in dissociated cell culture of human foetal spinal cord for several weeks in vitro under stable conditions.Supported by the Swiss Multiple Sclerosis Society     

Effects of caffeic acid, rofecoxib, and their combination against quinolinic acid-induced behavioral alterations and disruption in glutathione redox status

Objective

The neuroprotective roles of cyclooxygenase (COX) and lipooxygenase (LOX) inhibitors have been well documented. Quinolinic acid (QA) is a well-known excitotoxic agent that could induce behavioral, morphological and biochemical alterations similar with symptoms of Huntington’s disease (HD), by stimulating NMDA receptors. However, the exact roles of COX and LOX inhibitors in HD have not yet been explained. The present study aims to elucidate the effects of caffeic acid (a specific inhibitor for LOX), rofecoxib (a specific inhibitor for COX-2), and their combination in ameliorating QAinduced neurotoxicity in rats.

Methods

QA was injected into the right striatum of rats to induce neurotoxicity. Caffeic acid and rofecoxib were then orally administered separately. In the combination study, caffeic acid and rofecoxib were administered together. After that, a series of behavioral assessments were conducted to determine the effects of caffeic acid and rofecoxib, respectively, and the co-effect of caffeic acid and rofecoxib, against QA-induced neurotoxicity.

Results

Intrastriatal QA administration (300 nmol) not only induced a significant reduction in body weight and motor incoordination, but also altered the redox status (decreased glutathione and increased oxidized glutathione level) in striatum, as compared to the sham group. Moreover, chronic treatment with caffeic acid (5 mg/kg and 10 mg/kg, respectively, p.o.) or rofecoxib (10 mg/kg, p.o.) could significantly attenuate QA-induced behavioral alterations and restore the redox status in striatum. However, at the dose of 2.5 mg/kg, caffeic acid did not show any significant effects on these parameters in QA-treated rats. Furthermore, the combination of rofecoxib (10 mg/kg) and caffeic acid (5 mg/kg) could significantly protect against QA neurotoxicity.

Conclusion

The in vivo study indicates that excitotoxic injury to the brain might affect oxidant/antioxidant equilibrium by eliciting changes in glutathione. Moreover, the LOX and the COX pathways may be both involved in quinolinic-induced neurotoxicity, which provides a promising target for HD treatment.