Modelagem celular in vitro da Doença de Huntington com ácido 3-nitropropiônico e ácido quinolínico & Avaliação das funções neuroprotetoras da adenosina / In vitro cell modeling of Huntington's Disease with 3-nitropropionic acid and quinolinic acid and evaluation of the neuroprotective functions of adenosine

A Doença de Huntington (Huntington's disease - HD) trata-se de uma patologia neurodegenerativa hereditária caracteriza por meio da expressão das proteínas huntingtinas mutantes (mHtt), das mortes dos neurônios espinhais médios (medium spiny neurons MSNs) GABAérgicos D2-positivos do striatum e da hipercinesia. Uma hipótese se refere à função das mHtts de potencializarem os efeitos excitotóxicos das estimulações dos receptores de NMDA (NMDAR) por meio da inibição da succinato desidrogenase, resultando em desequilibrio das [Ca2+]i, estresse oxidativo e apoptose. A adenosina agonista dos receptores purinérgicos P1 tem sido descrita por conta das suas funções neuroprotetoras e neuromodulatórias. Assim, estabelecemos dois modelos in vitro da HD fundamentados nas neurodiferenciações das linhagens murinas de célula-tronco embrionárias E14-TG2a e progenitoras neurais do hipocampo HT-22; seguidas pelos tratamentos com ácido quinolínico (QA) agonista seletivo dos NMDARs , na ausência e na presença do ácido 3-nitropropiônico (3-NP) inibidor irreversível da succinato desidrogenase. Estes modelos foram utilizados nas avaliações das funções neuroprotetoras da adenosina. Os neurônios pós-mitóticos das culturas de E14-TG2a diferenciadas foram caracterizados conforme os MSNs GABAérgicos do striatum; enquanto os neurônios HT-22 diferenciados foram caracterizados de modo inespecífico. Metodologia: imunofluorescência (microscopia e citometria); PCR em tempo real; análise das variações dos potenciais das membranas plasmáticas e das variações transientes das [Ca2+]i por microfluorimetria; e quantificações das reduções do AlamarBlue® (% de sobrevida celular) e das atividades extracelulares de LDH (U/L) (necrose) por espectrometria. Avaliamos a capacidade do 3-NP de potencializar os efeitos excitotóxicos do QA comparando dois grupos de neurônios HT-22 diferenciados: QA 8mM (EC50) (controle); e 3-NP 5mM/QA 8mM. Avaliarmos o potencial neuroprotetor da adenosina comparando quatro grupos de neurônios HT-22 diferenciados: QA 8mM; adenosina 250µM/QA 8mM; 3-NP 5mM/QA 8mM; 3-NP 5mM/adenosina 250µM/QA 8mM. Os neurônios pós-mitóticos derivados das E14TG2a foram classificados como MSNsGABAérgicos do striatum integrantes de uma cultura neuronal heterogênea semelhante às conexões nigroestriatais, corticoestriatais, striatonigral e striatopallidal. Os neurônios HT-22 diferenciados perfaziam uma cultura neuronal heterogênea, não totalmente madura, composta por neurônios glutamatérgicos, dopaminérgicos, colinérgicos e GABAérgicos. Os neurônios HT-22 diferenciados 3-NP 5mM apresentaram menores % de sobrevida celular após os tratamentos com QA 8mM por 24h (p<0.05); e maiores amplitudes das variações das [Ca2+]i dependentes do QA 8mM (p<0.05) (cinética 6 minutos). Por outro lado, os neurônios HT-22 diferenciados pré- tratados com 3-NP 5mM apresentaram menores atividades extracelulares de LDH após o tratamento com QA 8mM por 24h menor proporção de necrose. Os pré-tratamentos com adenosina 250µM indicaram uma tendência dos efeitos neuroprotetores (p>0.05) maiores % de sobrevida celular; menores atividades extracelulares de LDH; e menores amplitudes das variações transientes das [Ca2+]i. Em conjunto, nossos resultados indicam que a inibição da succinato desidrogenase potencializa os efeitos excitotóxicos dos NMDARs por meio da alteração das [Ca2+]i e, provavelmente, dos mecanismos de morte celular; enquanto a adenosina apenas tendeu à neuroproteção

Huntington's disease (HD) is a hereditary neurodegenerative pathology characterized by mutant huntingtin proteins (mHtt) expression, striatum D2-positive GABAergic medium spiny neurons (MSNs) cell death and hyperkinetic motor symptoms development. One hypothesis refers to the principle that mHtt potentiates the excitotoxic effects of NMDA receptor (NMDAR) stimulation by the inhibition of mitochondrial succinate dehydrogenase, resulting in [Ca2+]i imbalance, oxidative stress and apoptosis. Adenosine P1 purinergic receptor agonist is related to neuroprotective and neuromodulatory functions. Thus, we established two in vitro HD models based on the neurodifferentiation of murine embryonic stem cell lines E14-TG2a and hippocampal neuroprogenitor cell line HT-22 followed by treatment with quinolinic acid (QA) selective agonist of NMDARs , in the absence and in the presence of 3-nitropropionic acid (3-NP) irreversible inhibitor of succinate dehydrogenase. These models were used to assess the neuroprotective functions of adenosine. Post-mitotic neurons from differentiated E14-TG2a cultures were characterized according to striatum's GABAergic MSNs; while the differentiated HT-22 neurons were characterized in a non-specific way. Methodology included immunofluorescence (microscopy and cytometry); real-time PCR; analysis of variations in the plasma membrane potentials and of transient variations in the [Ca2+]i by microfluorimetry; and quantification of AlamarBlue® reductions (% cell survival) and of extracellular LDH activity (U/L) (necrosis) by spectrometry. We evaluated the ability of 3-NP to potentiate the excitotoxic effects of QA by comparing two groups of differentiated HT-22 neurons: 8mM QA (control); and 5mM 3-NP/8mM QA. We evaluated the neuroprotective potential of adenosine comparing four groups of differentiated HT-22 neurons: QA 8mM; 250µM adenosine/8mM QA; 5mM 3-NP/8mM QA; 5mM 3-NP/250µM adenosine/8mM QA. Postmitotic neurons derived from E14TG2a were classified as striatums GABAergic MSNs that are part of a heterogeneous neuronal culture similar to nigrostriatal, corticostriatal, striatonigral, and striatopallidal connections. Differentiated HT-22 neurons consisted of a heterogeneous neuronal culture and not fully mature glutamatergic,dopaminergic, cholinergic and GABAergic neurons. Differentiated HT-22 neurons following 5mM 3-NP treatment showed lower % cell survival after treatments with 8mM QA for 24h (p<0.05); and higher amplitudes of the variations of [Ca2+]i induced by 8mM QA (p<0.05) (kinetics 6 minutes). On the other hand, differentiated HT-22 neurons 5mM 3-NP showed lower extracellular LDH activities after treatment with 8mM QA for 24h indicating a lower proportion of necrotic cells. Pretreatments with 250µM adenosine indicated a trend towards neuroprotective effects, such as higher percentages of cell survival; lower extracellular LDH activities; and lower amplitudes of transient variations of [Ca2+]i. Taken together, our results indicate that succinate dehydrogenase inhibition potentiated the excitotoxic effects of NMDARs by altering [Ca2+]i and, probably, cell death mechanisms, while adenosine only to neuroprotection

The Protective Effect of Egb 761 Against 3-Nitropropionic Acid-Induced Hearing Loss: The Role of Sirtuin 1

OBJECTIVES: Local administration of 3-nitropropionic acid (3-NP) to the inner ear induces sensorineural hearing loss. Several studies have shown the otoprotective effects of ginkgo biloba extract EGb 761. Moreover, EGb 761 has been reported to activate Sirtuin 1 (SIRT1). The present study was designed to investigate whether EGb 761 prevents 3-NP-induced sensorineural hearing loss and determine its effects on the expression of SIRT1. METHODS: Sprague Dawley rats were divided into four experimental groups: control group receiving vehicle of 3-NP, EGb group receiving EGb 761, 3-NP group receiving 3-NP, and EGb+3-NP group receiving EGb 761 and 3-NP. EGb 761 was given orally for 5 days. The 3-NP solution was injected into the tympanum 3 days after the start of EGb 761 administration. The auditory brainstem response was recorded before and after the injection. At 4 weeks after the administration of 3-NP or vehicle of 3-NP, cochleae were harvested, and hematoxylin and eosin staining and immunohistochemistry for SIRT1 antibody were performed. RESULTS: EGb+3-NP group showed significantly lower threshold shifts than 3-NP group. There was a significant preservation of type II fibrocytes and spiral ganglion cells in EGb+3-NP group than in 3-NP group. In EGb+3-NP group, there was a significantly greater number of SIRT1 immunopositive type II fibrocytes and spiral ganglion cells than in 3-NP group. Calculating the percentage of SIRT1 immunoreactive type II fibrocytes and spiral ganglion cells in viable type II fibrocytes and spiral ganglion cells, respectively, EGb+3-NP group showed significantly higher SIRT1 immunoreactive cells than 3-NP group. CONCLUSION: These results suggest that EGb 761 may prevent hearing loss induced by 3-NP in an acute ototoxic animal model, which appears to be related with SIRT1 expression.

Neuroprotective effect of caffeic acid phenethyl ester in 3-nitropropionic acid-induced striatal neurotoxicity

Caffeic acid phenethyl ester (CAPE), derived from honeybee hives, is a bioactive compound with strong antioxidant activity. This study was designed to test the neuroprotective effect of CAPE in 3-nitropropionic acid (3NP)-induced striatal neurotoxicity, a chemical model of Huntington's disease (HD). Initially, to test CAPE's antioxidant activity, a 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) antioxidant assay was employed, and CAPE showed a strong direct radical-scavenging eff ect. In addition, CAPE provided protection from 3NP-induced neuronal cell death in cultured striatal neurons. Based on these observations, the in vivo therapeutic potential of CAPE in 3NP-induced HD was tested. For this purpose, male C57BL/6 mice were repeatedly given 3NP to induce HD-like pathogenesis, and 30 mg/kg of CAPE or vehicle (5% dimethyl sulfoxide and 95% peanut oil) was administered daily. CAPE did not cause changes in body weight, but it reduced mortality by 29%. In addition, compared to the vehicle-treated group, robustly reduced striatal damage was observed in the CAPE-treated animals, and the 3NP-induced behavioral defi cits on the rotarod test were signifi cantly rescued after the CAPE treatment. Furthermore, immunohistochemical data showed that immunoreactivity to glial fibrillary acidic protein (GFAP) and CD45, markers for astrocyte and microglia activation, respectively, were strikingly reduced. Combined, these data unequivocally indicate that CAPE has a strong antioxidant eff ect and can be used as a potential therapeutic agent against HD.

The microstructural effects of aqueous extract of Garcinia kola (Linn) on the hippocampus and cerebellum of malnourished mice

<p><b>OBJECTIVE</b>To assess the neuroprotective effects of aqueous extract of Garcinia kola on neurotoxin administered malnourished mice adopting histological procedure.</p><p><b>METHODS</b>The study was carried out using thirty-two adult malnourished mice which were randomly assigned into four groups (n=8): A, B, C and D. Group A served as control, while the other groups served as the experimental groups. Animals in group A were fed malnourished diet ad libitum and given water liberally. Animals in group B were administered with 3-Nitropropionic acid (3-NP) (neurotoxin) only at 20 mg/kg body weight, group C were given only Garcinia kola extracts, and group D were pre-treated with Garcinia kola extracts at 200 mg/kg for seven days prior to administration of neurotoxin at 20 mg/kg body weight. After three days of neurotoxins administration in the relevant groups, the brains were excised and fixed in formal calcium for histological processing.</p><p><b>RESULTS</b>The study showed that hippocampal and cerebellar neurons of animals in group B exhibited some cellular degeneration and blood vessel blockage, which were not seen in groups A, C and D. Cresyl violet staining was least intense in group B than in groups A, C and D. Despite the fact that animals in group D has equal administration of 3-Nitropropionic acid concentration, there were no traces of neural degeneration as it was evidenced in group B.</p><p><b>CONCLUSIONS</b>It is concluded that Garcinia kola has protective effects on the neurons of the hippocampus and cerebellum of malnourished mice.</p>

Preconditioning with 3-nitropropionic acid reduces myocardial apoptosis induced by ischemia-reperfusion injury / 中华急诊医学杂志

Objective To investigate the effects of preconditioning with 3-nitropropionie acid on myocardial apoptosis induced by ischemia-reperfusion injury.Method Twenty-four rabbits were randomly divided into control group(group C,n=8),precondition group(group 3-NPA,n=8)and 5-HD group(group 5-HD,n=8).The group 5-HD was treated intravenously with 5 mg·kg-1 5-HD(ATP-sensitive potassium channels blocker),group C and group 3-NPA received normal saline instead of 5-HD.Ten minutes later,5-HD group and 3-NPA group were injected with 3-NPA(3 mg·kg-1)and the group C was injected with normal saline.Twenty-four hours later,the left anterior descending coronary artery was ligated for 30 min and then unclamped for 120 min to estabhsh ischemi-a-reperfitsion injury model.After reperfusion,the infarct sizes of ventricular myocardium,apoptotic myocardial cells and the expressions of Bcl-2 and Bax protein were measured.Results Infarct sizes and apoptotic myocardial cells in group 3-NPA were less than those in the others(P＜0.01).The expressions of Bcl-2 in group 3-NPA.in-creased as compared with group C(P＜0.05)and group 5-HD(P＜0.05),whereas the expressions of Bax in group 3-NPA decreased as compared with group C(P＜0.05)and group 5-HD(P＜0.05).Conclusions Preconditioning with 3-nitmpropionie acid reduces myocardial apoptosis induced by isehemia-reporfusion injury which is attributed to the opening of mitochondrial KATP channels.

Behavior assessment and magnetic resonance imaging examination on premature rats with periventricular leukomalacia induced by intracerebral injection of 3-nitropropionic acid / 临床儿科杂志

Objective To establish the model for periventricular leukomalacia(PVL) by intracerebral injection of 3-nitropropionic acid (3-NPA) and explore realistic model for concerned studies and investigate the diagnostic method with magnetic resonance imaging (MRI) examination. MethodsSprague-Dawley rat pups of both sexes at the age of postnatal day 5 (P5) were randomized and divided into two groups: NPA group and PBS group , and they were injected the same volume of 3-NPA and PBS with a tip located at the corpus callosum above the left ventricle by stereotaxis instrument, respectively. One day (P6), two days (P7), three days (P8) and nine days (P14) after injection, the injections were transcardially perfused and brains were collected. Then sections of brains were undertaken HE staining; growth and the time of opening eyes of the rats in the two different groups were observed and compared. Neurobehaviorai activity and memory tests were performed on postnatal day 29 (P29) and day 30 (P30). MRI examination was performed on postnatal day 30 (P30). ResultsMore weight increase and slower opening eye time were found in the NPA group compared with PBS group (P < 0.05). In the NPA group, sub-cortical and periventricular white matter rarefaction were observed by HE staining on P6, P7 and P8, significant lateral ventricle enlargement was found on P14, while the same changes were not found in the PBS group, and no histological changes in gray matter were noted in both groups. The outcomes of neurobehavioral tests of NPA group were much more abnormal compared with the PBS group (P < 0.05). MRI examination disclosed the signal changes of brain tissue is worse in the NPA group than that of the PBS group in muscle strength of limbs, autocinesis, capability and white matter. ConclusionsThe model for PVL induced by intracerebral injection of 3-NPA is characterized by damage to the periventricular white matter. The model can stimulate the pathologic change factually in vivo. The neurobehavioral movement is consistent with.clinical symptom. It can be used as a model to investigate some related disease. MRI examination is a feasible diagnostic method to show anatomic changes of white matter injury of the brain.

Cerebral Ischemic Tolerance Induced by 3-nitropropionic Acid Is Associated with Increased Expression of Erythropoietin in Rats / 华中科技大学学报（医学）（英德文版）

To examine the changes in erythropoietin (Epo) protein and its mRNA expression in rat brain subjected to focal ischemia and possible mechanism of the preconditioning of mitochondrial toxin 3-nitropropionic acid (3-NPA), rats were administrated either vehicle or 3-NPA at a dose of 20 mg/kg,intraperitoneally (ip), 3 days prior to a 2-h middle cerebral artery occlusion followed by 24- h reperfusion. Infarct volumes were measured by using 2, 3, 5 triphenylte trazolinm chloride (TTC)staining, and Epo protein and its mRNA levels were assessed by immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR), respectively. Our results showed that after reperfusion, Epo was found to be expressed extensively in the rat brain. It was most apparent in the basal nuclei and hippocampus, and was, to some extent, present in cortex. Preconditioning with 3-NPA caused a reduction in infarct volume. The expression of both Epo protein and mRNA increased significantly in the different brain areas in the 3-NPA pretreated group as compared with the non-pretreated ischemia model group. These results suggested that preconditioning with low dose 3-NPA could induce ischemic tolerance and neuro-protective effects by increasing the Epo expression in the ischemic and ischemia-related areas.

Chemical Preconditioning by 3-nitropropionic Acid Reduces Ischemia-reperfusion Injury in Rat Heart / 华中科技大学学报（医学）（英德文版）

This study was designed to investigate the cardioprotective effects of preconditioning with 3-nitropropionic acid, an inhibitor of mitochondrial succinate dehydrogenase. 16 isolated rat hearts were randomly divided into two groups, a treatment group and a control group. The rats of the treatment group were treated intraperitoneally with 3-nitropropionic acid (3-NPA, 4 mg/kg)and the rats of the control group were treated with saline. 24 h after the treatment, the isolated hearts were mounted on a Langendorff apparatus. After 30 min, the hearts were subjected to 30-min ischemia and 60-min reperfusion. The HR, LVDP and ±dp/dtmax were measured at pre-ischemia and 30 min, 60 min after the reperfusion. Coronary effluent was collected 15 min after the reperfusion for the determination of CK and LDH. At the end of the 60-min reperfusion the heart was removed for the determination of myocardial SOD and MDA. Our results showed that in the 3-NPA group LVDP and ±dp/dtmax recovered significantly better, myocardial MDA, CK and LDH were significantly lower and the myocardial SOD was significantly higher than in the control group.It is concluded that chemical preconditioning by 3-nitropropionate has cardioprotective effects against ischemia-reperfusion injury.

Expression of Apurinic/apyrimidinic Endonuclease and Neuronal Apoptosis in the Striatum after Treatment of 3-Nitropropionic Acid in Mice

BACKGROUND: 3-Nitroporpionic acid (3-NP) is an irreVersible inhibitor of succinate dehydrogenase in mitochondria and can induce apoptosis-like cell death in the striatum. It has been reported that oxidative stress plays a role in the 3-NP induced neuronal damage. 3-NP induced striatal damage is implicated in the pathogenesis of several neurological diseases, such as chronic neurodegenerative diseases and stroke. The DNA repair enzyme, apurinic/apyrimidinic endonuclease (APE), is a multifunctional protein in the DNA base excision repair (BER) pathway. To clarify the relationship between APE and neuronal cell death associated with the apoptosis in the striatum was induced by 3-NP in vivo. METHODS: After intra-striatal injection of 3-NP, expression of the APE protein and mRNA were evaluated by Western blot, immunohistochemistry, RT-PCR and DNA fragmentation patterns. Oxidative DNA damage was investigated by detection of oxidized DNA, AP site and superoxide. RESULTS: Expression levels of APE was rapidly reduced as early as 1hr after injection of 3-NP. DNA fragmentation was observed 24 hours after 3-NP treatment but not 4 hours. APE gene expression was increased to 1hr after 3-NP treatment. The number of AP sites were reduced and the reduction of APE proteins were blocked by a superoxide scavenger, MnTBAP-treatment. CONCLUSIONS: These results suggest that the reduction of APE is the preceding event of DNA fragmentation that causes apoptosis and a decrease of APE may be induced by ROS after 3-NP treatment.

Involvement of Oxidative Stress and Poly (ADP-ribose) Polymerase Activation in 3-Nitropropionic Acid-induced Cytotoxicity in Human Neuroblastoma Cells

3-Nitropropionic acid (3-NP) inhibits electron transport in mitochondria, leading to a metabolic failure. In order to elucidate the mechanism underlying this toxicity, we examined a few biochemical changes possibly involved in the process, such as metabolic inhibition, generation of reactive oxygen species (ROS), DNA strand breakage, and activation of Poly (ADP-ribose) polymerase (PARP). Exposure of SK-N-BE (2) C neuroblastoma cells to 3-NP for 48 h caused actual cell death, while inhibition of mitochondrial function was readily observed when exposed for 24 h to low concentrations (0.2~2 mM) of 3-NP. The earliest biochemical change detected with low concentration of 3-NP was an accumulation of ROS (4 h after 3-NP exposure) followed by degradation of DNA. PARP activation by damaged DNA was also detectable, but at a later time. The accumulation of ROS and DNA strand breakage were suppressed by the addition of glutathione or N-acetyl-L-cysteine (NAC), which also partially restored mitochondrial function and cell viability. In addition, inhibition of PARP also reduced the 3-NP-induced DNA strand breakage and cytotoxicity. These results suggest that oxidative stress and activation of PARP are the major factors in 3-NP-induced cytotoxicity, and that the inhibition of these factors may be useful in protecting neuroblastoma cells from 3-NP-induced toxicity.

Effects of Shen-wu Capsule on motor function and content of dopamine in striatum in Huntington model rats / 中国康复理论与实践

@#ObjectiveTo investigate the role of mitochondrial dysfunction in neurodegenerative diseases and the pharmacological effects of Shen-wu Capsule.MethodsSD rats were divided into 6 groups with 10 rats each:model group was developed by peritoneal injection of 3-nitropropionic acid(3-NPA) 20mg/kg, once every another day. Haloferidoli was used as the positive drug (given to positive control group from 10μg/100g/day to 100μg/100g/day, during modeling). Low dose of Shen-wu Capsule is 0.45g/kg; middle dose of Shen-wu Capsule is 0.9g/kg; high dose of Shen-wu Capsule is 1.8g/kg (once a day for 25days). The rats in normal control group were peritoneally injected with saline and were fed with water.The open field test was used to test the space recognition ability and excitability of CNS of rats. The climbing test was used to test strength of muscles.The content of dopamine(DA) and it's metabolite dihydroxyphenylacetic acid (DOPAC),5-HT in stratium of all groups were detected by high performance liquid chromatograph (HPLC-ECD).ResultsThe 3-NPA model rats showed decrease of activity, rigidity and stagger just like clinical patients with Huntington's diseases. In open field test all indexes of 3-NPA model rats are decreased (P<0.05), while Shen-wu Capsule low dose and high dose can improve all the indexes. There is no difference in strength of muscles among all groups. The content of DA,DOPAC and 5-hydroxytryptamine (5-HT) were distinctly decreased in 3-NPA model rats(P<0.01); rats in three groups of Shen-wu Capsule and positive drug control groups showed increased content of these neurotransmitter(P<0.01). Conclusions Rats injected with 3-NPA can mimic the abnormal behaviors and changes of neurotransmitter in stratium of Huntington's patients, Shen-wu Capsule can ameliorate the behavior of model animals and improve the content of DA,DOPAC and 5-HT in stratium.

Relation between activation of hippocampal astrocytes and ischemic tolerance induced by 3-nitropropionic acid in gerbils / 中国临床药理学与治疗学

AIM: To investigate the relationship between astroglial activation state and ischemic tolerance induced by low dose of 3 nitropropionic acid (3 NPA) in gerbil hippocampus. METHODS: Transient forebrain ischemic model was induced by bilateral common carotid arteries occlution. HE staining and immunohistochemistry were used to identify neuronal and astrocyte response. RESULTS: Preconditioning with 3 NPA produced protective effects of CA 1neurons. The number of glial fibrillary acidic protein positive astrocyte in hippocampal CA 1 region increased slightly in control group, but increased significantly in preconditioning of the brain with 3 nitropropionic acid. CONCLUSION: The state of astroglial activation is related to neuronal survival in ischemic tolerance induced by low dose of 3 nitropropionic acid.

The Mechanism of Striatal Damage in Mice after Intraperitoneal Injection of 3-nitropropionic Acid

BACKGROUND: A newly-found mitochondrial toxin, 3-nitropropionic acid (3-NP), has been proved to induce apoptosis in the striatum. Although striatal lesions produced by 3-NP could develop through an excitotoxic mechanism, the exact relationship between apoptosis induction and excitotoxicity after 3-NP treatment is still not clear. We investigated the role of excitotoxicity and oxidative stress on apoptosis induction within the striatum following intra-peritoneal injection of 3-NP. METHODS: 3-NP was injected for 5 days intra-peritoneally in three month-old mice. One day after the last injection, animals were decapitated. To confirm the presence of apoptosis, we performed in-situ detection of DNA fragmentation by using TUNEL technique and agarose gel elctrophoresis after DNA extraction from striatum. To examine the effect of frontal cortex removal on 3-NP-indeced apoptosis, we removed left frontal cortex by aspiration. For excitotoxicity, NMDA-receptor antagonist-MK 801, non-NMDA antagonist-NBQX, and saline were injected intraperitoneally before 3-NP treatment To detect superoxide, we administered hydroethidium (HEt: 200 ul; 1mg/ml) into the jugular vein 2 days after 3-NP, and the density of oxidized HEt in samples were examined under flouscent microscope. We performed caspase staining to test immunoreactivity of caspase 3 in samples. RESULTS: The TUNEL positive cells were not observed in the striatum ipsilateral to the frontal cortex-removed side, but found in the contralateral striatum. Superoxide radicals measured by using HEt and caspase immunoreactivity were also significantly weaker in the striatum ipsilateral to the frontal cortex-removed side than the contralateral striatum. TUNEL staining revealed less apoptotic changes in the striatum of MK801-treated group than NBQX-or saline-treated groups. DNA laddering on agarose gel electrophoresis was observed in the striatum of NBQX- or saline-treated mice, but not found in MK 801-treated group. CONCLUSION: We demonstrated that removal of the corticostriatal glutamate pathway reduced superoxide production as well as apoptosis induced by 3-NP and NMDA receptor antogonist, but not non-NMDA antagonist, prevented 3-NP-induced apoptosis in the striatum. These results suggest that NMDA-mediated glutamatergic excitotokicity plays an important role in 3-NP related striatal damage.

Effects of 3-nitropropionic acid preconditioning on the Bcl-2 and Bax mRNA expressions following focal cerebral ischemia in rats / 临床神经病学杂志

Objective To investigate the involvement of Bcl-2 and Bax mRNA expressions in mitochondrial toxin 3-nitropropionic acid(3-NPA)induced ischemic tolerance to focal cerebral ischemia in rats.Methods Rats were administrated 3-NPA intraperitoneally at dose of 20 mg/kg 3 days prior to a 2 h middle cerebral artery occlusion followed by 1 h,6 h,12 h,24 h and 48 h of reperfusion.The Bcl-2 and Bax mRNA expressions were measured by reverse transcriptase polymerase chain reaction and compared to the sham operation group and ischemic reperfusion group.Results Compared to the sham operation group,the ischemic reperfusion and 3-NPA pretreated groups exhibited an increase in Bcl-2 and Bax mRNA after reperfusion every time point of focal cerebral ischemia(all(P