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NADPH-oxidase (NOX) is a multi-subunit enzyme complex. The upregulation of NOX causes massive production of superoxide (O2¯), which avidly reacts with nitric oxide (NO) and increases cellular reactive oxygen/nitrogen species (ROS/RNS). Increased ROS/RNS plays pivotal role in the sporadic Alzheimer's disease (sAD) development and brain damage following impaired insulin signaling. Hence, this study aimed to examine early-time course of changes in NOX and NOS expression, and apoptotic proteins in the rats hippocampi following insulin signaling impairment [induced by STZ injection; intraperitoneal (IP) or in cerebral ventricles (ICV)]. Early effects (1, 3, or 6 weeks) on the NOX activity, translocation of NOX subunits from cytosol to the membrane, NO-synthases [neuronal-, inducible- and endothelial-NOS; nNOS, iNOS and eNOS], The Rac-1 protein expression, levels of NO and O2¯, cytochrome c release, caspase-3 and 9 activations (cleavage) were studied. STZ injection (in both models) increased NOX activity, O2¯ production, and enhanced cytosolic subunits translocation into membrane. The iNOS but not nNOS and eNOS expression and NO levels were increased in STZ treated rats. Finally, STZ injection increased cytochrome c release, caspase-3 and 9 activations in a manner that was significantly associated with levels of O2¯ and NO in the hippocampus. ICV-STZ administration resulted in significant profound changes over the IP route. In conclusion, impairment in insulin function induces early changes in ROS/RNS contents through NOX and iNOS upregulation and neuronal apoptosis in the hippocampus. Our results could mechanistically explain the role of impaired insulin function in the development of sAD.
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Doença de Alzheimer , Apoptose , Hipocampo , Insulina , NADPH Oxidases , Óxido Nítrico Sintase Tipo II , Animais , Masculino , Ratos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Apoptose/fisiologia , Caspase 3/metabolismo , Citocromos c/metabolismo , Modelos Animais de Doenças , Hipocampo/metabolismo , Insulina/metabolismo , NADPH Oxidases/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Estreptozocina , Regulação para Cima/fisiologiaRESUMO
The core symptoms of attention deficit hyperactivity disorder (ADHD) are due to the hypofunction of the brain's adrenergic (NE) and dopamine (DA) systems. Drugs that enhance DA and NE neurotransmission in the brain by blocking their transporters or receptors are the current therapeutic strategies. Of late, the emerging results point out the serotonergic (5-HT) system, which indirectly modulates the DA activity in reducing the core symptoms of ADHD. On this basis, second-generation antipsychotics, which utilize 5-HT receptors, were prescribed to children with ADHD. However, it is not clear how serotonergic receptors modulate the DA activity to minimize the symptoms of ADHD. The present study investigates the efficacy of serotonergic and alpha-2 adrenergic receptor manipulation in tackling the core symptoms of ADHD and how it affects the DA neuroreceptors in the brain regions involved in ADHD. Fifteen-day-old male spontaneously hypertensive rats (SHRs) received 5-HT1A agonist (ipsapirone) or 5-HT2A antagonist (MDL 100907) (i.p.) or alpha-2 agonist (GFC) from postnatal days 15 to 42 along with age-matched Wistar Kyoto rats (WKY) (n = 8 in each group). ADHD-like behaviors were assessed using a battery of behavioral tests during postnatal days 44 to 65. After the behavioral tests, rat brains were processed to estimate the density of 5-HT1A, 5-HT2A, DA-D1, and DA-D2 neuroreceptors in the prefrontal cortex, the striatum, and the substantia nigra. All three neuroreceptor manipulations were able to minimize the core symptoms of ADHD in SHRs. The positive effect was mainly associated with the upregulation of 5-HT2A receptors in all three areas investigated, while 5-HT1A was in the prefrontal cortex and the substantia nigra. Further, the DA-D1 receptor expression was downregulated by all three neuroreceptor manipulations except for alpha-2 adrenergic receptor agonists in the striatum and 5-HT2A antagonists in the substantia nigra. The DA-D2 expression was upregulated in the striatum while downregulated in the prefrontal cortex and the substantia nigra. In this animal model study, the 5-HT1A agonist or 5-HT2A antagonist monotherapies were able to curtail the ADHD symptoms by differential expression of DA receptors in different regions of the brain.
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Transtorno do Deficit de Atenção com Hiperatividade , Humanos , Ratos , Animais , Criança , Masculino , Ratos Endogâmicos SHR , Adrenérgicos/uso terapêutico , Antagonistas do Receptor 5-HT2 de Serotonina/uso terapêutico , Agonistas do Receptor 5-HT1 de Serotonina/uso terapêutico , Dopamina/metabolismo , Ratos Endogâmicos WKYRESUMO
Oxidative stress with a depletion of glutathione is a key factor in the initiation and progression of Alzheimer's disease (AD). N-Acetylcysteine (NAC), a glutathione precursor, provides neuroprotective effects in AD animal models. Its amide form, N-Acetylcysteine amide (NACA), has an extended bioavailability compared to NAC. This study evaluates the neuroprotective effects of NACA against Aß1-42 peptide-induced AD-like pathology in rats. Male Wistar rats (2.5 months old) were divided into five groups: Normal Control (NC), Sham (SH), Aß, Aß + NACA and NACA + Aß + NACA (n = 8 in all groups). AD-like pathology was induced by the intracerebroventricular infusion of Aß1-42 peptide into the lateral ventricle. NACA (75 mg/kg) was administered either as a restorative (i.e., injection of NACA for 7 consecutive days after inducing AD-like pathology (Aß + N group)), or as prophylactic (for 7 days before and 7 days after inducing the pathology (N + Aß + N group)). Learning and memory, neurogenesis, expression of AD pathology markers, antioxidant parameters, neuroprotection, astrogliosis and microgliosis were studied in the hippocampus and the prefrontal cortex. All data were analyzed with a one-way ANOVA test followed by Bonferroni's multiple comparison test. NACA treatment reversed the cognitive deficits and reduced oxidative stress in the hippocampus and prefrontal cortex. Western blot analysis for Tau, Synaptophysin and Aß, as well as a histopathological evaluation through immunostaining for neurogenesis, the expression of neurofibrillary tangles, ß-amyloid peptide, synaptophysin, neuronal morphology and gliosis, showed a neuroprotective effect of NACA. In conclusion, this study demonstrates the neuroprotective effects of NACA against ß-amyloid induced AD-like pathology.
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Doença de Alzheimer , Fármacos Neuroprotetores , Masculino , Ratos , Animais , Acetilcisteína/farmacologia , Ratos Wistar , Doença de Alzheimer/induzido quimicamente , Doença de Alzheimer/tratamento farmacológico , Sinaptofisina , Fármacos Neuroprotetores/farmacologia , Peptídeos beta-Amiloides , Gliose/induzido quimicamente , Gliose/tratamento farmacológico , GlutationaRESUMO
Oxidative stress, caused by impaired insulin signaling, plays a pivotal role in the pathogenesis of sporadic Alzheimer's disease (sAD). We investigated the oxidative stress parameters in the synaptosomes prepared from the hippocampus tissue in order to identify their potential role in sAD development in intraperitoneal (IP) and intracerebroventricular (ICV) streptozotocin (STZ) injections models of insulin signaling impairment. Rats were harvested 1, 3, or 6 weeks post treatment. Spatial learning and memory, several antioxidants and oxidative stress markers were analyzed. Results showed a significant deficit in learning and memory in rats injected with STZ through IP and ICV routes. Glutathione, glutathione/oxidized glutathione, glutathione S-transferase, glutathione peroxidase, glutathione reductase, catalase, superoxide dismutase(SOD)-total, Zn/Cu(SOD), Mn/Fe(SOD) are significantly decreased in IP-STZ and ICV-STZ groups at 1, 3, and 6 weeks after STZ injection. Oxidized glutathione, thiobarbituric acid reactive species, glucose 6-Phosphate dehydrogenase, protein carbonyls, 4-Hydroxynonenal, and 3-Nitrotyrosine are significantly increased in IP-STZ and ICV-STZ groups at 1,3, and 6 weeks after STZ injection. Changes in oxidative stress parameters in ICV-STZ groups are greater than IP-STZ groups. STZ treatment induced cognitive impairments by 3-W and 6-W, and it was significantly correlated with the extent of oxidative damage. In conclusion, STZ administration through ICV route is deleterious in causing early synaptosomal oxidative damage that exacerbated with time and correlated with cognitive impairments. Our data implicate the involvement of oxidative stress as an early feature of sAD and provide insights into the behavioral and biochemical changes over the course of disease development.
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Doença de Alzheimer , Sinaptossomos , Animais , Ratos , Sinaptossomos/metabolismo , Doença de Alzheimer/metabolismo , Insulina/metabolismo , Dissulfeto de Glutationa/efeitos adversos , Dissulfeto de Glutationa/metabolismo , Ratos Wistar , Modelos Animais de Doenças , Estresse Oxidativo , Hipocampo/metabolismo , Estreptozocina/toxicidade , Superóxido Dismutase/metabolismo , Glutationa/metabolismo , Cognição , Aprendizagem em LabirintoRESUMO
BACKGROUND: In Ayurveda; an Indian system of traditional medicine, Ocimum sanctum is said to have remedial effect on hriddaurbalya (problems affecting the mind), aakshepayukta vikara (nervous disorders) and shiroroga (diseases of head). Hence, in Ayurvedic practice, it is profoundly used as an antistress medicine. Stress is known to affect neurons of functionally significant brain regions like substantia nigra. However, experimental evidence showing its effect on morphology of substantia nigral neurons is lacking. In addition, whether the O. sanctum treatment attenuates stress induced substantia nigral neuronal structural changes is not known. OBJECTIVES: To know the effect of stress on morphology of substantia nigral neurons and the effect of O. sanctum fresh leaf extract (OSE) on substantia nigral neurons of stressed rats. MATERIAL AND METHODS: Present study included three experiments. Experiment I: To study the effect of 3 and 6 weeks of foot shock stress in rats; Experiment II- To study the effect of 3 weeks of OSE treatment on 3 week-stress undergoing rats and on 3 week-stressed rats; Experiment III- To study the effect of 6 weeks of OSE treatment in 6 week-stress undergoing rats and in 6 week-stressed rats. RESULTS: In experiment I, stress had significant deleterious effect on dendritic arborization of substantia nigral neurons. Experiments II and III showed prevention and attenuation of the stress induced dendritic atrophy of substantia nigral neurons in both 2 ml and 4 ml OSE treatment groups. Protective effect of OSE was more pronounced in rats which are treated for a longer duration. CONCLUSIONS: Foot shock stress induces neuronal damage in the substantia nigra of rats. Treatment with fresh leaf extract of O. sanctum could prevent and attenuate the foot shock stress induced behavioral deficit and substantia nigral neuronal damage.
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We have recently reported the discovery of a series of oxazolidinone hydroxamic acid derivatives that are potent inhibitors of 5-lipoxygenase (5-LO) [arachidonate 5-lipoxygenase; EC 1.13.11.34]. We now report that one of the most active members of this series, compound PH-251, [(R)-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl) methyl)-N-hydroxyoctanamide], also possesses a unique and strong ability to concurrently inhibit mast cell degranulation. PH-251 inhibited the biosynthesis of leukotriene C4 (LTC4), as well as degranulation of IgE/allergen-activated bone marrow-derived mouse mast cells (BMMC) in vitro. In contrast, zileuton (the prototype 5-LO inhibitor) inhibited leukotriene generation, but not degranulation. Consistent with its dual activity, compound PH-251 also significantly inhibited both the early and the late anaphylactic contractions of guinea pig lung parenchymal strip, whereas zileuton inhibited only the late (leukotriene-dependent) contractions. Comparative structure-activity analysis of PH-251 and its structural analogues showed that the anti-degranulation effect appeared to be dependent on the length of the straight-chain hydrocarbon substitution on the hydroxamic acid moiety. In the in vivo studies, PH-251 (3-30 mg/kg s.c.) strongly inhibited various components of zymosan-induced peritonitis - a typical non-allergic LT-dependent animal model of inflammation. In the mouse allergic asthma model, the compound significantly inhibited allergen-induced bronchial eosinophilic inflammation and airway hyper-responsiveness to inhaled methacholine. These results show that PH-251 is a unique dual inhibitor of 5-LO and mast cell degranulation, with in vivo activity in animal models of disease and may therefore offer potential advantages over single-target drugs in the treatment of asthma and other allergic and inflammatory diseases.
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Antialérgicos , Antiasmáticos , Asma , Oxazolidinonas , Animais , Antialérgicos/uso terapêutico , Antiasmáticos/farmacologia , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Araquidonato 5-Lipoxigenase , Asma/tratamento farmacológico , Degranulação Celular , Cobaias , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Mastócitos , Camundongos , Camundongos Endogâmicos BALB C , Oxazolidinonas/farmacologia , Oxindóis/farmacologiaRESUMO
A negative association between blood Pb level (BPbL) and vitamin D metabolites in occupationally exposed populations has been reported, but data from the general population are scarce. Furthermore, the association between BPbL and vitamin D binding protein (DBP) and free 25-hydroxyvitamin D (25(OH)D) has not been reported. We investigated the association of BPbL with DBP, total and free 25(OH)D in healthy adolescents (n 1347; age range 11-16 years) cross-sectionally selected from all Governorates of Kuwait, utilising multi-stage cluster random sampling. Pb in whole blood was analysed by inductively coupled plasma MS, and DBP with ELISA. Plasma 25(OH)D was analysed by LC-MS/MS, and free 25(OH)D was calculated utilising the levels and binding affinities of DBP and albumin for 25(OH)D. DBP was positively associated with BPbL (ß = 0·81; 95 % CI 0·14, 0·22; P < 0·001). A negative association between BPbL and total 25(OH)D was non-significant (P = 0·24) when BPbL was used as a continuous variable but was significant when used as quartiles (P = 0·02). The negative association between BPbL and free 25(OH)D was significant whether BPbL was used as continuous, as quartiles or as cut-off point of <5 µg/dl (0·24 µmol/l). In multinomial logistic regression, the odds of vitamin D insufficiency and deficiency were more than two-fold higher in the upper quartiles of BPbL compared with the lowest quartile. The negative correlation of BPbL with free 25(OH)D was more robust than its correlation with total 25(OH)D. Future studies must consider the levels of DBP when assessing the association between Pb and vitamin D metabolites.
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Deficiência de Vitamina D , Proteína de Ligação a Vitamina D , Adolescente , Calcifediol , Criança , Cromatografia Líquida , Proteínas de Ligação a DNA , Humanos , Chumbo , Espectrometria de Massas em Tandem , Fatores de Transcrição , Vitamina D/análogos & derivados , Deficiência de Vitamina D/epidemiologiaRESUMO
Introduction: Diabetes Mellitus (DM) is the most common metabolic disease worldwide and is associated with many systemic complications. Muscle atrophy is one of the significant complications in DM patients, making routine tasks laborious as atrophy continues. It is known that heat stress stimulates heat shock proteins and other proteins that maintain muscle mass; however, it is not thoroughly studied in diabetic conditions. This study addressed whether heat therapy can attenuate muscle atrophy in STZ-induced diabetic rats and explored its mechanism of action on specific muscle proteins. Methods: Male Sprague Dawley rats were randomly divided into short-term (3 weeks) and long-term (6 weeks) experiments. In each experiment rats were divided into control, heat therapy, diabetic and diabetic + heat therapy groups. Rats in heat therapy groups were exposed to heat therapy for 30 min daily for three or six weeks in a temperature-controlled (42°C) chamber. Results: The attenuation of neuromuscular functions assessed by Rotarod, Kondziella's inverted screen, and extensor postural thrust tests showed that diabetic rats exposed to heat therapy performed significantly better than diabetic controls. Muscle cross sectional area data established that heat therapy reduced muscle atrophy by 34.3% within 3 weeks and 44.1% within 6 weeks in the diabetic groups. Further, heat therapy significantly decreased muscle atrophy markers (CD68, KLF, and MAFbx) and significantly elevated muscle hypertrophy markers (AKT, mTOR, and HSP70). Conclusions: This study shows the relevance and clinical significance of utilizing heat therapy as a viable treatment to attenuate muscle atrophy in diabetic patients.
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BACKGROUND: Cough hypersensitivity is a major characteristic feature associated with several types of cough, including chronic cough, but its underlying mechanisms remain to be fully understood. Inflammatory mediators, such as prostaglandin E2 (PGE2), have been implicated in both peripheral induction and sensitization of the cough reflex. In this study, using a conscious guinea pig model of cough, we investigated whether PGE2 can sensitize the cough reflex via central actions and, if so, via which mechanisms. METHODS: All drugs were administered by intracerebroventricular (i.c.v.) route and whole-body plethysmograph set-up was used for both induction, using aerosolized citric acid (0.2 M), and recording of cough. Immunohistochemistry was performed to confirm the expression of NaV 1.8 channels in the nucleus tractus solitarius (nTS). RESULTS: We show that both PGE2 and the non-selective EP1/EP3 agonist, sulprostone, dose-dependently enhanced the citric acid-induced cough (P ≤ 0.001, P ≤ 0.01, respectively). Pretreatment with the EP1 antagonist, ONO-8130, did not affect the sulprostone-induced cough sensitization, whilst the EP3 antagonist, L-798,106, dose-dependently inhibited this effect (P ≤ 0.05). Furthermore, treatment with either the EP2 agonist, butaprost or the EP4 agonist, L-902,688, had no effect on cough sensitization. Additionally, pretreatment with either the TRPV1 antagonist, JNJ-17203212 or the TRPA1 antagonist, HC-030031, alone or in combination, nor with the NaV 1.1, 1.2, 1.3, 1.4, 1.6 and 1.7 channel blocker, tetrodotoxin, had any effect on the cough. In contrast, pretreatment with the NaV 1.8 antagonist, A-803467, dose-dependently inhibited this effect (P ≤ 0.05). Furthermore, NaV 1.8 channels were shown to be expressed in the nTS. CONCLUSION: Collectively, our findings show that PGE2 sensitizes the cough reflex centrally via EP3 receptor-dependent activation of NaV 1.8 but independently of TRPV1,TRPA1 and TTX-sensitive sodium channel activation. These results indicate that PGE2 plays an important role in central sensitization of the cough reflex and suggest that central EP3 receptors and/or NaVv 1.8 channels may represent novel antitussive molecular targets.
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Tosse/fisiopatologia , Dinoprostona/farmacologia , Receptores de Prostaglandina E Subtipo EP3/metabolismo , Animais , Tosse/metabolismo , Modelos Animais de Doenças , Feminino , Cobaias , Masculino , Ocitócicos/farmacologiaRESUMO
BACKGROUND: Elevated blood lead level (EBLL) is a public health problem in both developing and industrialized countries. Being a petrochemical-based economy, lead (Pb) levels are expected to be high in Kuwait, but systematic data on population exposure are lacking. This study aimed at determining the prevalence of EBLL in adolescents in Kuwait. METHODS: Adolescents (N = 1385; age range 11-16 years) were cross-sectionally selected from public middle schools from all Governorates of Kuwait, utilizing multistage cluster random sampling. Pb in whole blood was analyzed by inductively coupled plasma mass spectrometry. Distribution of blood Pb levels (BLL) among Governorates and sexes were compared by non-parametric tests and the prevalence of EBLL (defined as BLL above the CDC reference level of ≥5 µg/dL) was estimated by χ2 test. Binary logistic regression was used for assessing the association between EBLL and Governorate. RESULTS: Median (IQR) BLL was 5.1(3.6-7.1) µg/dL [4.9 (3.8-6.5) µg/dL in males and 5.4 (3.3-7.6) µg/dL in females; p = 0.001]. In the overall sample, 51% had BLL ≥5 µg/dL; 13% had ≥10 µg/dL and 3% > 20 µg/dL. Prevalence of EBLL was 47% in males and 56% in females (p < 0.001). EBLLs were clustered in Al-Asima, Al-Ahmadi (in both sexes); Al-Jahra (in males) and Mubarak Al-Kabeer (in females) Governorates. CONCLUSIONS: EBLL is a significant public health problem in adolescents in Kuwait. Urgent public health intervention is required in areas with EBLL, and the sources of exposure need to be identified for prevention.
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Intoxicação por Chumbo , Chumbo , Adolescente , Criança , Estudos Transversais , Exposição Ambiental/efeitos adversos , Feminino , Humanos , Kuweit/epidemiologia , Intoxicação por Chumbo/epidemiologia , Masculino , Prevalência , Fatores de RiscoRESUMO
Thymoquinone (TQ) and Ferulic Acid (FA) are natural ingredients from Nigella sativa and Ferula asafetida, respectively. Individually both TQ and FA have shown anticancer properties in a variety of cancer cell lines. We investigated the combination effect of lower doses of TQ and FA on proliferation, apoptosis, and cell cycle of a breast cancer cell line MDA-MB 231. Cells were treated with various concentrations of TQ, FA and various combinations of TQ + FA for 48 hrs. Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, whereas cell cycle and apoptosis were measured with flow-cytometry after propidium iodide staining and Annexin-V/FITC staining, respectively. TQ (50 and 100 µM) and FA (450 µM), and all the doses of TQ and FA in combination significantly decreased cell proliferation. 25 µM TQ and 250 µM FA individually did not affect cell proliferation but in combination significantly reduced cell proliferation. TQ at 50 µM caused significant apoptosis, whereas, FA did not affect apoptosis. These in vitro data suggest that in combination, relatively lower doses of TQ and FA are effective in decreasing cancer cell proliferation, and thus have anticancer therapeutic potential. Further research is needed to corroborate these findings in an animal model of breast cancer.
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Adenocarcinoma , Neoplasias da Mama , Animais , Apoptose , Benzoquinonas/farmacologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células , Ácidos Cumáricos , Feminino , Humanos , Células MCF-7RESUMO
Among the many factors responsible for the cognitive decline in Alzheimer's disease, beta amyloid protein and plaque formation is crucial. This amyloid pathology is associated with activation of glial cells and oxidative stress but whether oxidative stress activates beta amyloid protein in the neurons is not clear. Further the expression of microglia is also known to vary during pathogenesis of beta amyloid plaques. The aim of the present study is to evaluate the antioxidant effect of NAC on amyloid pathology and cognition and also to investigate the link between amyloid pathology and glial cells activation. Intracerebroventricular colchicine in rats known mimics human AD in many aspects including memory loss, oxidative stress, and hyper phosphorylation of tau protein. The animal groups consisted of age matched control, sham operated, AD, and NAC treated in AD models of rats. Cognitive function was evaluated in active avoidance test; beta amyloid protein, beta amyloid plaques, astrocytes, and microglia cells were quantified using immunohistochemistry in hippocampal and prefrontal cortices. Colchicine has resulted in significant cognitive loss, increased intraneuronal beta amyloid protein expression, increased reactive astrocytes, and activated microglia in all the regions of the hippocampus and prefrontal cortices. The antioxidant NAC has reversed the cognitive deficits and inhibited microglia activation but failed to inhibit BAP expression and astrocytosis. Intraneuronal BAP accumulation is deleterious and known to adversely affect cognition, but in this study in spite of intraneuronal BAP accumulation, the cognition is restored. It can be postulated that NAC might have reversed the effect of intraneuronal beta amyloid protein by acting on some downstream compensatory mechanisms which needs to be explored.
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Quinolinic acid (QA) is an excitotoxic metabolite of the kynurenine pathway of tryptophan metabolism produced in response to inflammation and oxidative stress. Lead (Pb) causes oxidative stress and thus may produce neurotoxicity by increasing QA production. We investigated the in vitro cytotoxic effects of Pb and QA and the protective effects of the NMDA receptor antagonist memantine. Primary cultures of embryonic hippocampal cells from Wistar rats were treated with different concentrations of Pb, QA, and Pb + QA with and without memantine. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Apoptosis was analyzed by flow cytometry after Annexin-V/propidium iodide staining. The numbers of immunostained neurons (with ß3-Tubulin; Tuj1) and astrocytes (with glial fibrillary acidic protein) were counted. Pb at 20 µg/dL (0.97 µM) and QA at 500 nM concentrations showed significant cytotoxic effects, as evidenced by decreased cell viability, increased apoptosis, and a decrease in the number of both astrocytes and neurons. The combination of Pb and QA showed significant synergistic apoptotic effects at lower doses. Memantine (500 nM) was largely protective against the cytotoxic effects of both Pb and QA, suggesting that Pb's and QA's cytotoxicity involves NMDA receptor activation. Whereas the neuroprotection by memantine from QA-induced toxicity has been previously reported, this is the first study reporting the protection by memantine against Pb-induced cytotoxicity in cultured hippocampal cells. Protection by memantine against these neurotoxicants in vivo needs to be investigated.
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Astrócitos/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Chumbo/toxicidade , Memantina/farmacologia , Neurônios/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Ácido Quinolínico/toxicidade , Animais , Apoptose/efeitos dos fármacos , Astrócitos/patologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Hipocampo/patologia , Masculino , Neurônios/patologia , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/agonistas , Relação Estrutura-AtividadeRESUMO
Alzheimer's disease (AD) is characterized by the accumulation of neurofibrillary tangles (NFT), deposition of beta amyloid plaques, and consequent neuronal loss in the brain tissue. Oxidative stress to the neurons is often attributed to AD, but its link to NFT and ß-amyloid protein (BAP) still remains unclear. In an animal model of AD, we boosted the oxidative defense by N-Acetyl cysteine (NAC), a precursor of glutathione, a powerful antioxidant and free radical scavenger, to understand the link between oxidative stress and NFT. In mimicking AD, intracerebroventricular (ICV) colchicine, a microtubule disrupting agent also known to cause oxidative stress was administered to the rats. The animal groups consisted of an age-matched control, sham operated, AD, and NAC treated in AD models of rats. Cognitive function was evaluated in a passive avoidance test; neuronal degeneration was quantified using Nissl staining. NFT in the form of abnormal tau expression in different regions of the brain were evaluated through immunohistochemistry using rabbit anti-tau antibody. ICV has resulted in significant cognitive and neuronal loss in medial prefrontal cortex (MFC) and all the regions of the hippocampus. It has also resulted in increased accumulation of intraneuronal tau in the hippocampus and MFC. NAC treatment in AD model rats has reversed the cognitive loss and neuronal degeneration. The intraneuronal tau expression also minimized with NAC treatment in AD model rats. Thus, our findings suggest that an antioxidant supplement during the progression of AD is likely to prevent neuronal degeneration by minimizing the neurofibrillary degeneration in the form of tau accumulation.
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BACKGROUND: Lead (Pb), a heavy metal, and quinolinic acid (QA), a metabolite of the kynurenine pathway of tryptophan metabolism, are known neurotoxicants. Both Pb and QA impair spatial learning and memory. Pb activates astrocytes and microglia, which in turn induce the synthesis of QA. We hypothesized increased QA production in response to Pb exposure as a novel mechanism of Pb-neurotoxicity. METHODS: Two experimental paradigms were used. In experiment one, Wistar rat pups were exposed to Pb via their dams' drinking water from postnatal day 1 to 21. Control group was given regular water. In the second protocol, QA (9 mM) or normal saline (as Vehicle Control) was infused into right lateral ventricle of 21-day old rats for 7 days using osmotic pumps. Learning and memory were assessed by Morris water maze test on postnatal day 30 or 45 in both Pb- and QA-exposed rats. QA levels in the Pb exposed rats were measured in blood by ELISA and in the brain by immunohistochemistry on postnatal days 45 and 60. Expression of various molecules involved in learning and memory was analyzed by Western blot. Means of control and experimental groups were compared with two-way repeated measure ANOVA (learning) and t test (all other variables). RESULTS: Pb exposure increased QA level in the blood (by ~ 58%) and increased (p < 0.05) the number of QA-immunoreactive cells in the cortex, and CA1, CA3 and dentate gyrus regions of the hippocampus, compared to control rats. In separate experiments, QA infusion impaired learning and short-term memory similar to Pb. PSD-95, PP1, and PP2A were decreased (p < 0.05) in the QA-infused rats, whereas tau phosphorylation was increased, compared to vehicle infused rats. CONCLUSION: Putting together the results of the two experimental paradigms, we propose that increased QA production in response to Pb exposure is a novel mechanism of Pb-induced neurotoxicity.
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Intoxicação do Sistema Nervoso por Chumbo/complicações , Transtornos da Memória/induzido quimicamente , Ácido Quinolínico/toxicidade , Aprendizagem Espacial/efeitos dos fármacos , Análise de Variância , Animais , Animais Recém-Nascidos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Proteína de Ligação a CREB/metabolismo , Modelos Animais de Doenças , Reação de Fuga/efeitos dos fármacos , Injeções Intraventriculares , Intoxicação do Sistema Nervoso por Chumbo/patologia , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/metabolismo , Ácido Quinolínico/sangue , Ratos , Ratos Wistar , Tempo de Reação/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas tau/metabolismoRESUMO
Dibutyryl cyclic adenosine monophosphate (dBcAMP), a cell-permeable synthetic analog of cAMP, has been shown to induce astrogliosis in culture. However, the exact mechanism underlying how dBcAMP exerts its function in situ is not clear. The objective of this study was to examine the effects of dBcAMP on astrogliosis and survival of neurons in stab wound and kainic acid models of brain injury. Stab wound was done in cerebral cortex of BALB/c male mice. Kainic acid lesion was induced in hippocampus by injecting 1µl kainic acid into the lateral ventricle. Animals in both models of injury were divided into L+dBcAMP and L+PBS groups and treated with dBcAMP or PBS for 3, 5, and 7 days respectively. The brain sections were stained for Cresyl violet and Fluro jade-B to assess the degenerating neurons. Immunostaining for GFAP and Iba-1 was done for assessing the astrogliosis and microglial response respectively. Expression of GFAP and BDNF levels in the tissue were estimated by Western blotting and ELISA respectively. The results showed a gradual increase in the number of both astrocytes and microglia in both injuries with a significant increase in dBcAMP-treated groups. The number of degenerating neurons significantly decreased in dBcAMP treated groups. In addition, it was found that dBcAMP stimulated the expression of GFAP and BDNF in both stab wound and kainic acid injuries. Treatment with BDNF receptor inhibitor AZ-23, showed an increase in the degenerating neurons suggesting the role of BDNF in neuroprotection. This study indicates that dBcAMP protects neurons from degeneration by enhancing the production of BDNF and may be considered for use as therapeutic agent for treatment of brain injuries.
RESUMO
Lead (Pb) is a neurotoxic heavy metal, but the mechanism of its neurotoxicity is not clearly understood. Expression of metallothioneins (MTs) is induced in response to heavy metal exposure as a protective mechanism against heavy metal toxicity. There are several isoforms of MTs (MT-1 to 4), of which MT-3 is the neuron specific isoform, which also has neurite growth inhibitory effects. Whereas, the induction of MT-1 and 2 in response to Pb has been reported, the effect of Pb on the expression of MT-3 in the brain has not been documented. This study aimed at investigating the effect of Pb exposure on the expression of MT-3 in the cerebrum and hippocampus. Wistar rat pups were exposed to Pb via their dams' drinking water (0.2% lead acetate in deionized water) from postnatal day (PND) 0 to 21 and directly via drinking water until PND30. Expression of MT-3 was measured by Western blot and quantitative RT-PCR. MT-3 localization was done by immunohistochemistry. Divalent metal ions were analysed by atomic absorption spectrophotometry. Levels of Pb in blood and cerebrum were significantly increased, while that of copper (Cu), zinc (Zn) and manganese (Mn) were significantly decreased in the Pb-exposed rats at both PND21 and PND30. MT-3 protein was significantly increased in the cerebrum (by 2.5-fold) and in hippocampus (1.4 to 3.2-fold) in both PND21 and PND30 Pb-exposed rats over controls. MT-3 gene expression also increased in the cerebrum (by 42%), and in the hippocampus (by 65% and 43% in the PND21 and PND30 rats, respectively), in the Pb-exposed rats over controls, but the increase was statistically significant (pâ¯<â¯0.05) only in the PND30 rats. Pb exposure significantly increased (pâ¯<â¯0.05) percentage of MT-3 immunoreactive cells in Cornu Ammonis and dentate gyrus regions in the PND21 rats, and in the Cornu Ammonis 1, dentate gyrus and cortex regions in the PND30 rats. Our data thus provide convincing evidence that exposure to low levels of Pb during preweaning period increases the expression of MT-3 in the brain of rats.
Assuntos
Cérebro/metabolismo , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Compostos Organometálicos/toxicidade , Administração Oral , Fatores Etários , Animais , Cobre/sangue , Cobre/metabolismo , Feminino , Chumbo/sangue , Chumbo/metabolismo , Masculino , Manganês/sangue , Manganês/metabolismo , Exposição Materna , Metalotioneína 3 , Compostos Organometálicos/administração & dosagem , Ratos , Zinco/sangue , Zinco/metabolismoRESUMO
Monoamine oxidase-B (MAOB), a flavin adenine dinucleotide (FAD), is an enzyme which catalyzes the oxidation of amines. MAOB is proposed to play a major role in the pathogenesis of neurodegeneration through the production of reactive oxygen species (ROS) and neurotoxins. The present study was designed to outline the effects of the MAOB inhibitor (MAOB-I) on neuroprotection of spinal neurons, regeneration of sciatic nerve fibers, and recovery of sensory-motor functions in the sciatic nerve crush injury model. Male Wistar rats (4-months-old) were assigned to i) Naïve (N), ii) Sham (S), iii) Sciatic nerve crush and treated with saline (CRUSH + SALINE) and iv) Sciatic nerve crush and treated with MAOB inhibitor (CRUSH + MAOB-I) groups (n = 10/group). In groups iii and iv, the crush injury was produced by crushing the sciatic nerve followed by treatment with saline or MAOB-I (Selegiline® 2.5 mg/kg) intraperitoneally for 10 days. Behavioral tests were conducted from week 1 to week 6. At the end of the study, sciatic nerve and lumbar spinal cord were examined by immunohistochemistry, light and electron microscopy. MAOB-I treatment showed significant improvement in sensory and motor functions compared to saline treatment (p < 0.05-0.001) in injured nerves. The morphological study showed a significantly increased number of nerve fibers in sciatic nerve distal to the site of injury (p < 0.05), with better myelination pattern in CRUSH + MAOB-I treated group compared to CRUSH + SALINE group. Spinal cord ventral horns showed a significant increase in the number of NeuN-immunoreactive neurons in the MAOB-I treated group compared to Saline treated group (p < 0.01). MAOB-I has a significant potential for protecting the degenerating spinal cord neurons and enhancing the regeneration of injured sciatic nerve fibers following crush injury.
Assuntos
Inibidores da Monoaminoxidase/uso terapêutico , Degeneração Neural/prevenção & controle , Regeneração Nervosa/efeitos dos fármacos , Recuperação de Função Fisiológica/efeitos dos fármacos , Neuropatia Ciática/complicações , Medula Espinal/patologia , Animais , Células do Corno Anterior/efeitos dos fármacos , Células do Corno Anterior/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Movimento/efeitos dos fármacos , Proteína Básica da Mielina/metabolismo , Fibras Nervosas Mielinizadas/efeitos dos fármacos , Fibras Nervosas Mielinizadas/patologia , Fibras Nervosas Mielinizadas/ultraestrutura , Limiar da Dor/efeitos dos fármacos , Fosfopiruvato Hidratase/metabolismo , Ratos , Ratos Wistar , Neuropatia Ciática/tratamento farmacológico , Neuropatia Ciática/patologia , Selegilina/farmacologia , Selegilina/uso terapêutico , Suporte de Carga/fisiologiaRESUMO
Lead (Pb), a known environmental toxicant, adversely affects almost all organ systems. In this study, we investigated the effects of maternal lead exposure on fetal rat cerebellum. Female Sprague-Dawley rats were given lead nitrate in drinking water (0, 0.5, and 1%) for two weeks before conception, and during pregnancy. Fetuses were collected by caesarian section on gestational day 21 and observed for developmental abnormalities. The fetal cerebellar sections from control and 1% lead group were stained with cresyl violet. Immunohistochemical expressions of p53, Bax, Bcl-2, and caspase 3 were quantified by AnalySIS image analyzer (Life Science, Germany). Lead exposure induced developmental abnormalities of eyes, ear, limbs, neck and ventral abdominal wall; however, these abnormalities were commonly seen in the 1% lead-treated group. In addition, lead also caused fetal mortality and reduced body growth in both dose groups and reduced brain weight in the 1% lead-treated group. The fetal cerebella from the 1% lead-treated group showed unorganized cerebellar cortical layers, and degenerative changes in granule and Purkinje cells such as the formation of clumps of Nissl granules. An increase in Bax and caspase 3, and a decrease in Bcl-2 (p < 0.05), but not in p53, showed apoptosis of the neurons. In conclusion, gestational lead exposure in rats induces fetal toxicity and developmental abnormalities. The lead exposure also impairs development of cerebellar layers, induces structural changes, and apoptosis in the fetal cerebellar cortex. These results suggest that lead exposure during gestation is extremely toxic to developing cerebellum in rats.
Assuntos
Apoptose/efeitos dos fármacos , Córtex Cerebelar/efeitos dos fármacos , Anormalidades Congênitas/etiologia , Poluentes Ambientais/toxicidade , Chumbo/toxicidade , Exposição Materna/efeitos adversos , Nitratos/toxicidade , Organogênese/efeitos dos fármacos , Animais , Córtex Cerebelar/embriologia , Córtex Cerebelar/metabolismo , Córtex Cerebelar/patologia , Anormalidades Congênitas/metabolismo , Anormalidades Congênitas/patologia , Feminino , Imuno-Histoquímica , Gravidez , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Ratos Sprague-Dawley , Ratos Wistar , Proteína Supressora de Tumor p53/biossíntese , Proteína X Associada a bcl-2/biossínteseRESUMO
Our previous studies have established that (-)-epigallocatechin-3-gallate (EGCG) has both neuroprotective and -regenerative capacity after sciatic nerve injury. Moreover, this improvement was evident on the behavioral level. The aim of this study was to investigate the central effects of ECGC on spinal cord motor neurons after sciatic nerve injury. Our study showed that administering 50 mg/kg intraperitoneally i.p. of EGCG to sciatic nerve-injured rats improved their performance on different motor functions and mechanical hyperesthesia neurobehavioral tests. Histological analysis of spinal cords of EGCG-treated sciatic nerve-injured (CRUSH+ECGC) animals showed an increase in the number of neurons in the anterior horn, when compared to the naïve, sham, and saline-treated sciatic nerve-injured (CRUSH) control groups. Additionally, immunohistochemical study of spinal cord sections revealed that EGCG reduced the expression of glial fibrillary acidic protein and increased the expression of growth-associated protein 43, a marker of regenerating axons. Finally, EGCG reduced the ratio of B-cell lymphoma 2 (Bcl-2)-associated X protein/Bcl-2 and increased the expression of survivin gene. This study may shed some light on the future clinical use of EGCG and its constituents in the treatment of peripheral nerve injury.