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1.
J Toxicol Sci ; 45(8): 503-513, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32741900

RESUMO

Creatine kinase (CK) and lactate dehydrogenase (LDH) serve as biomarkers for skeletal muscle injury in preclinical toxicity studies, but have a limitation regarding tissue specificity. Circulating miR-206 was recently reported to be a useful biomarker for skeletal muscle disorders in humans. Here, we sought to determine whether serum miR-206 can be used as a biomarker in preclinical toxicity studies to detect drug-induced skeletal muscle injury with higher sensitivity and specificity than the biomarkers CK, LDH, skeletal troponin I (sTnI), and myosin light chain 3 (Myl3). We established rat models of skeletal muscle injury through treatment with the muscle toxicant 2,3,5,6-tetramethyl-p-phenylenediamine (TMPD) as well as four in-house compounds. We found that serum miR-206 levels significantly increased after treatment with TMPD, and tended to be higher in rats treated with in-house compounds than in control rats. ROC analysis revealed that the specificity of serum miR-206 for detection of skeletal muscle injury was higher compared with those of other markers. Further, serum miR-206 levels were unchanged in rats with isoproterenol-induced cardiotoxicity. These findings demonstrate that serum miR-206 may serve as a highly specific biomarker for preclinical analysis of rats with drug-induced skeletal muscle injuries.


Assuntos
MicroRNAs/sangue , Músculo Esquelético , Doenças Musculares/induzido quimicamente , Doenças Musculares/diagnóstico , Picolinas/toxicidade , Animais , Biomarcadores/sangue , Modelos Animais de Doenças , Masculino , Especificidade de Órgãos , Curva ROC , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Sensibilidade e Especificidade
2.
PLoS One ; 15(7): e0235338, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32609782

RESUMO

The goal of this study was to use liquid chromatography mass spectrometry to assess metabolic changes of two different diets in three distinct rat strains. Sprague-Dawley, Fischer 344, and Brown-Norway male rats were maintained on a high-fat, or regular diet for 24 weeks. Liver tissue was collected at 4, 12, and 24 weeks to assess global small molecule metabolite changes using high resolution accurate mass spectrometry coupled to ultra-high-performance liquid chromatography. The results of the global metabolomics analysis revealed significant changes based on both age and diet within all three strains. Principal component analysis revealed that the influence of diet caused a greater variation in significantly changing metabolites than that of age for the Brown Norway and Fisher 344 strains, whereas diet had the greatest influence in the Sprague Dawley strain only at the 4-week time point. As expected, metabolites involved in lipid metabolism were changed in the animals maintained on a high fat diet compared to the regular diet. There were also significant changes observed in the concentration of Tri carboxylic acid cycle intermediates that were extracted from the liver of all three strains based on diet. The results of this study showed that a high fat diet caused significant liver and metabolic changes compared to a regular diet in multiple rat strains. The inbred Fisher 344 and Brown Norway rats were more metabolically sensitive to the diet changes than outbred Sprague Dawley strain. The study also showed that age, as was the case for Sprague Dawley, is an important variable to consider when assessing metabolic changes.


Assuntos
Fatores Etários , Dieta Hiperlipídica/efeitos adversos , Fígado/metabolismo , Metaboloma , Metabolômica/métodos , Animais , Cromatografia Líquida de Alta Pressão , Ciclo do Ácido Cítrico , Metabolismo dos Lipídeos , Masculino , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem/métodos
3.
PLoS One ; 15(6): e0229053, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32569277

RESUMO

PURPOSE: To identify key dosimetric parameters that have close associations with tumor treatment response and body weight change in SFRT treatments with a large range of spatial-fractionation scale at dose rates of several Gy/min. METHODS: Six study arms using uniform tumor radiation, half-tumor radiation, 2mm beam array radiation, 0.3mm minibeam radiation, and an untreated arm were used. All treatments were delivered on a 320kV x-ray irradiator. Forty-two female Fischer 344 rats with fibrosarcoma tumor allografts were used. Dosimetric parameters studied are peak dose and width, valley dose and width, peak-to-valley-dose-ratio (PVDR), volumetric average dose, percentage volume directly irradiated, and tumor- and normal-tissue EUD. Animal survival, tumor volume change, and body weight change (indicative of treatment toxicity) are tested for association with the dosimetric parameters using linear regression and Cox Proportional Hazards models. RESULTS: The dosimetric parameters most closely associated with tumor response are tumor EUD (R2 = 0.7923, F-stat = 15.26*; z-test = -4.07***), valley (minimum) dose (R2 = 0.7636, F-stat = 12.92*; z-test = -4.338***), and percentage tumor directly irradiated (R2 = 0.7153, F-stat = 10.05*; z-test = -3.837***) per the linear regression and Cox Proportional Hazards models, respectively. Tumor response is linearly proportional to valley (minimum) doses and tumor EUD. Average dose (R2 = 0.2745, F-stat = 1.514 (no sig.); z-test = -2.811**) and peak dose (R2 = 0.04472, F-stat = 0.6874 (not sig.); z-test = -0.786 (not sig.)) show the weakest associations to tumor response. Only the uniform radiation arm did not gain body weight post-radiation, indicative of treatment toxicity; however, body weight change in general shows weak association with all dosimetric parameters except for valley (minimum) dose (R2 = 0.3814, F-stat = 13.56**), valley width (R2 = 0.2853, F-stat = 8.783**), and peak width (R2 = 0.2759, F-stat = 8.382**). CONCLUSIONS: For a single-fraction SFRT at conventional dose rates, valley, not peak, dose is closely associated with tumor treatment response and thus should be used for treatment prescription. Tumor EUD, valley (minimum) dose, and percentage tumor directly irradiated are the top three dosimetric parameters that exhibited close associations with tumor response.


Assuntos
Fracionamento da Dose de Radiação , Fibrossarcoma/radioterapia , Animais , Peso Corporal/efeitos da radiação , Modelos Animais de Doenças , Feminino , Fibrossarcoma/patologia , Radiometria , Ratos , Ratos Endogâmicos F344 , Resultado do Tratamento , Carga Tumoral/efeitos da radiação
4.
J Toxicol Sci ; 45(6): 339-347, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32493876

RESUMO

The metabolomic profiles of rat primary hepatocytes following treatment with rotenone, FCCP, or (+)-usnic acid were determined using liquid chromatography-mass spectrometry/mass spectrometry and gas chromatography-mass spectrometry. Significant and similar changes in the levels of 283 biochemical metabolites were associated with the three treatments compared with solvent control samples. Overall, the three treatments generated similar global biochemical profiles, with some minor differences associated with rotenone treatment. All three treatments resulted in a shift in energy metabolism as demonstrated by decreased glycogen stores and glycolysis. A reduced antioxidant response was detected in cells following all treatments. In addition, bile acid biosynthesis decreased as a potential consequence of increased oxidative stress by all three treatments. Conversely, rotenone treatment induced a number of changes after 1 hr, which were not detected in FCCP- or (+)-usnic acid-treated samples; these changes were not sustained over time and included increased NAD+ salvage and lysine degradation. In conclusion, these biochemical profiles could provide new insights into the mechanism(s) of mitochondrial toxicity.


Assuntos
Benzofuranos/efeitos adversos , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/efeitos adversos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Rotenona/efeitos adversos , Animais , Ácidos e Sais Biliares/metabolismo , Células Cultivadas , Cromatografia Líquida , Metabolismo Energético/efeitos dos fármacos , Cromatografia Gasosa-Espectrometria de Massas , Glicogênio/metabolismo , Glicólise/efeitos dos fármacos , Metabolômica , Mitocôndrias Hepáticas/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ratos Endogâmicos F344
5.
PLoS One ; 15(5): e0233617, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469935

RESUMO

OBJECTIVES: The aim of the study was to investigate therapeutic efficacy of single- or two-fraction radiotherapy in conjunction with IDO1-inhibition in a syngeneic rat glioblastoma model. IDO is known to cause immunosuppression through breakdown of tryptophan in the tumor microenvironment. METHODS: Gene expression analyses of IDO in glioblastoma were performed with data from publicly available datasets. Fractionation studies were done on animals to evaluate tumor size, immune cell infiltration of tumors and serum profile on day 18 after tumor inoculation. Survival analyses were done with animals carrying intracranial glioblastomas comparing two-fraction radiotherapy+IDO1-inhibition to controls. IDO inhibition was achieved by administration of 1-methyl tryptophan (1-MT), and radiotherapy (RT) was delivered in doses of 8Gy. RESULTS: The expression of IDO1 was increased on gene level in glioblastoma stem cells. Tumor size was significantly reduced in animals treated with 1-MT+RTx 2 (both long and short intervals, i.e. 7 and 4 days between the treatments) as compared to control animals, animals treated with only 1-MT or animals treated with 1-MT+RTx1. Serum levels of IL-1A were significantly altered in all treated animals as compared to control animals. Survival was significantly increased in the animals treated with 1-MT+RTx2 (7-day interval) compared to control animals. CONCLUSIONS: Addition of two-fraction RT to IDO1 inhibition with 1-MT significantly reduced tumor size in animals with glioblastoma. Survival was significantly increased in animals treated with two-fractioned RT+1-MT as compared to untreated controls increased significantly. ADVANCES IN KNOWLEDGE: The currently used combination of only two fractions of radiotherapy and immune therapy is a promising area of research, increasing efficacy compared to single fraction irradiation, while potentially lowering radiation side effects compared to radiation in current clinical practice.


Assuntos
Neoplasias Encefálicas/radioterapia , Inibidores Enzimáticos/uso terapêutico , Glioblastoma/radioterapia , Triptofano/análogos & derivados , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Ratos , Ratos Endogâmicos F344 , Triptofano/uso terapêutico , Microambiente Tumoral/efeitos dos fármacos
6.
PLoS One ; 15(5): e0233736, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32470002

RESUMO

INTRODUCTION: The liver is well known for its enormous regenerative capacity. If the hepatocytes are compromised the reserve stem cells can regrow the lost tissue by means of oval cells differentiating into hepatocytes. We were curious whether this standby system was able to compensate for ontogenic liver growth arrested by 2-acetylaminofluorene (AAF) treatment or if it can be influenced by cholic acid, known to promote liver growth in several reactions. METHODS: (i) Four weeks-old (60-70g) male F344 rats were kept on standard chow and treated with solvent only, (ii) others were kept on 0,2% cholic acid (CA) enriched diet, (iii) treated with AAF, or (iiii) given a combination of CA diet and AAF treatment (AAF/CA). The proliferative response of epithelial cells was characterized by pulse bromodeoxyuridine labelling. The relative gene expression levels of senescence-related factors and bile acid receptors were determined by quantitative real-time polymerase chain reaction analysis. RESULTS: AAF administration efficiently inhibited the physiological proliferation of hepatocytes in young, male F344 rats after weaning. The activation of stem cells was indicated by the increased proliferation of periportal biliary/oval cells (B/OC). If the rats were fed additionally by cholic acid enriched diet, typical oval cell reaction emerged, subsequently the oval cells differentiated into hepatocytes restituting liver growth. This reaction was mediated by increased production of HGF, IL-6 and SCF by the damaged liver. Moreover, upregulation of FXR expression on B/OC made them competent for bile acids. Our results indicate that endogenous, autocrine mechanisms involved in liver ontogeny are also able to activate the backup regenerative machinery of stem cells.


Assuntos
Sistema Biliar/crescimento & desenvolvimento , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Hepatócitos/metabolismo , Regeneração Hepática , Fígado/crescimento & desenvolvimento , 2-Acetilaminofluoreno/toxicidade , Animais , Sistema Biliar/patologia , Diferenciação Celular , Proliferação de Células , Ácido Cólico/farmacologia , Hepatócitos/patologia , Fígado/patologia , Masculino , Ratos , Ratos Endogâmicos F344 , Células-Tronco/citologia , Células-Tronco/metabolismo
7.
J Vasc Res ; 57(4): 178-184, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32434183

RESUMO

BACKGROUND: Lysophosphatidic acid (LPA) is a small phospholipid-signaling molecule, which can alter responses to stress in the central nervous system. OBJECTIVE: We hypothesized that exogenous LPA would increase the size of infarct and reduce microregional O2 supply/consumption balance after cerebral ischemia-reperfusion. METHODS: This was tested in isoflurane-anesthetized rats with middle cerebral artery blockade for 1 h and reperfusion for 2 h with or without LPA (1 mg/kg, at 30, 60, and 90 min after reperfusion). Regional cerebral blood flow was determined using a C14-iodoantipyrine autoradiographic technique. Regional small-vessel (20-60 µm in diameter) arterial and venous oxygen saturations were determined microspectrophotometrically. RESULTS: There were no significant hemodynamic or arterial blood gas differences between groups. The control ischemic-reperfused cortex had a similar O2 consumption to the contralateral cortex. However, microregional O2 supply/consumption balance was significantly reduced in the ischemic-reperfused cortex with many areas of low O2 saturation (43 of 80 veins with O2 saturation below 50%). LPA did not significantly alter cerebral blood flow, but it did significantly increase O2 extraction and consumption of the ischemic-reperfused region. It also significantly increased the number of small veins with low O2 saturations in the reperfused region (76 of 80 veins with O2 saturation below 50%). This was associated with a significantly increased cortical infarct size after LPA administration (11.4 ± 0.5% control vs. 16.4 ± 0.6% LPA). CONCLUSION: This suggests that LPA reduces cell survival and that it is associated with an increase in the number of small microregions with reduced local oxygen balance after cerebral ischemia-reperfusion.


Assuntos
Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/efeitos dos fármacos , Circulação Cerebrovascular/efeitos dos fármacos , Infarto da Artéria Cerebral Média/patologia , Lisofosfolipídeos/toxicidade , Microcirculação/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Oxigênio/sangue , Traumatismo por Reperfusão/patologia , Animais , Morte Celular/efeitos dos fármacos , Córtex Cerebral/patologia , Veias Cerebrais/efeitos dos fármacos , Veias Cerebrais/patologia , Veias Cerebrais/fisiopatologia , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/sangue , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Ratos Endogâmicos F344 , Traumatismo por Reperfusão/sangue , Traumatismo por Reperfusão/fisiopatologia
8.
Cell Prolif ; 53(5): e12808, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32347601

RESUMO

OBJECTIVES: Although the hepatomitogenic activity of triiodothyronine (T3) is well established, the wide range of harmful effects exerted by this hormone precludes its use in liver regenerative therapy. Selective agonists of the beta isoform of thyroid hormone receptor (TRß) do not exhibit T3-induced cardiotoxicity and show a good safety profile in patients with NASH. The aim of this study was to investigate whether two novel TRß agonists, the prodrug TG68 and the active compound IS25 could stimulate hepatocyte proliferation without T3/TRα-dependent side effects. METHODS: Rats were treated with three different doses (12.5, 25 and 50 µg/100 g body weight) for one week. Hepatocyte proliferation, liver injury and serum biochemical parameters were measured by immunohistochemistry, qRT-PCR and Western blot. RESULTS: Both drugs increased hepatocyte proliferation as assessed by bromodeoxyuridine incorporation (from 14% to 28% vs 5% of controls) and mitotic activity. Enhanced proliferation occurred in the absence of significant signs of liver injury as shown by lack of increased serum transaminase levels or of apoptosis. No cardiac or renal hypertrophy typically associated with treatment with T3 was observed. Importantly, no proliferation of pancreatic acinar cells, such as that seen after administration of T3 or the TRß agonist GC1 was detected following either TG68 or IS25, demonstrating the hepato-specificity of these novel TRß agonists. CONCLUSIONS: The present study shows that TG68 and IS25 induce massive hepatocyte proliferation without overt toxicity. Hence, these agents may have a significant clinical application for regenerative therapies in liver transplantation or other surgical settings.


Assuntos
Regeneração Hepática/efeitos dos fármacos , Fígado/efeitos dos fármacos , Pró-Fármacos/farmacologia , Receptores beta dos Hormônios Tireóideos/agonistas , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fígado/metabolismo , Masculino , Ratos , Ratos Endogâmicos F344 , Tri-Iodotironina/metabolismo
9.
J Neurosci ; 40(18): 3675-3691, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32238479

RESUMO

The transcription factor Nurr1 has been identified to be ectopically induced in the striatum of rodents expressing l-DOPA-induced dyskinesia (LID). In the present study, we sought to characterize Nurr1 as a causative factor in LID expression. We used rAAV2/5 to overexpress Nurr1 or GFP in the parkinsonian striatum of LID-resistant Lewis or LID-prone Fischer-344 (F344) male rats. In a second cohort, rats received the Nurr1 agonist amodiaquine (AQ) together with l-DOPA or ropinirole. All rats received a chronic DA agonist and were evaluated for LID severity. Finally, we performed single-unit recordings and dendritic spine analyses on striatal medium spiny neurons (MSNs) in drug-naïve rAAV-injected male parkinsonian rats. rAAV-GFP injected LID-resistant hemi-parkinsonian Lewis rats displayed mild LID and no induction of striatal Nurr1 despite receiving a high dose of l-DOPA. However, Lewis rats overexpressing Nurr1 developed severe LID. Nurr1 agonism with AQ exacerbated LID in F344 rats. We additionally determined that in l-DOPA-naïve rats striatal rAAV-Nurr1 overexpression (1) increased cortically-evoked firing in a subpopulation of identified striatonigral MSNs, and (2) altered spine density and thin-spine morphology on striatal MSNs; both phenomena mimicking changes seen in dyskinetic rats. Finally, we provide postmortem evidence of Nurr1 expression in striatal neurons of l-DOPA-treated PD patients. Our data demonstrate that ectopic induction of striatal Nurr1 is capable of inducing LID behavior and associated neuropathology, even in resistant subjects. These data support a direct role of Nurr1 in aberrant neuronal plasticity and LID induction, providing a potential novel target for therapeutic development.SIGNIFICANCE STATEMENT The transcription factor Nurr1 is ectopically induced in striatal neurons of rats exhibiting levodopa-induced dyskinesia [LID; a side-effect to dopamine replacement strategies in Parkinson's disease (PD)]. Here we asked whether Nurr1 is causing LID. Indeed, rAAV-mediated expression of Nurr1 in striatal neurons was sufficient to overcome LID-resistance, and Nurr1 agonism exacerbated LID severity in dyskinetic rats. Moreover, we found that expression of Nurr1 in l-DOPA naïve hemi-parkinsonian rats resulted in the formation of morphologic and electrophysiological signatures of maladaptive neuronal plasticity; a phenomenon associated with LID. Finally, we determined that ectopic Nurr1 expression can be found in the putamen of l-DOPA-treated PD patients. These data suggest that striatal Nurr1 is an important mediator of the formation of LID.


Assuntos
Corpo Estriado/metabolismo , Discinesia Induzida por Medicamentos/metabolismo , Levodopa/toxicidade , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/biossíntese , Transtornos Parkinsonianos/metabolismo , Idoso , Animais , Corpo Estriado/efeitos dos fármacos , Discinesia Induzida por Medicamentos/patologia , Feminino , Humanos , Masculino , Oxidopamina/toxicidade , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/patologia , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Ratos Sprague-Dawley
10.
Artigo em Inglês | MEDLINE | ID: mdl-32233792

RESUMO

We previously demonstrated that the combined exposure of human pulmonary microvascular endothelial cells (HPMECs) to morphine and viral protein(s) results in the oxidative stress-mediated induction of autophagy, leading to shift in the cells from early apoptotic to apoptosis-resistant proliferative status associated with the angioproliferative remodeling observed in pulmonary arterial hypertension (PAH). In this study, we tried to delineate the major source of HIV-1 protein Tat and morphine induced oxidative burst in HPMECs and its consequences on vascular remodeling and PAH in an in vivo model. We observed switch from the initial increased expression of NADPH oxidase (NOX) 2 in response to acute treatment of morphine and HIV-Tat to later increased expression of NOX4 on chronic treatment in the endoplasmic reticulum of HPMECs without any alterations in the mitochondria. Furthermore, NOX-dependent induction of autophagy was observed to play a pivotal role in regulating the endothelial cell survival. Our in vivo findings showed significant increase in pulmonary vascular remodeling, right ventricular systolic pressure, and Fulton index in HIV-transgenic rats on chronic administration of morphine. This was associated with increased oxidative stress in lung tissues and rat pulmonary microvascular endothelial cells. Additionally, endothelial cells from morphine-treated HIV-transgenic rats demonstrated increased expression of NOX2 and NOX4 proteins, inhibition of which ameliorated their increased survival upon serum starvation. In conclusion, this study describes NADPH oxidases as one of the main players in the oxidative stress-mediated endothelial dysfunction on the dual hit of HIV-viral protein(s) and opioids.


Assuntos
Analgésicos Opioides/farmacologia , Células Endoteliais/efeitos dos fármacos , Morfina/farmacologia , NADPH Oxidase 2/genética , NADPH Oxidase 4/genética , Hipertensão Arterial Pulmonar/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Proliferação de Células/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Regulação da Expressão Gênica , HIV-1/genética , HIV-1/metabolismo , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/metabolismo , Estresse Oxidativo , Hipertensão Arterial Pulmonar/induzido quimicamente , Hipertensão Arterial Pulmonar/metabolismo , Ratos , Ratos Endogâmicos F344 , Ratos Transgênicos , Espécies Reativas de Oxigênio/metabolismo , Remodelação Vascular/efeitos dos fármacos , Remodelação Vascular/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologia
11.
Life Sci ; 250: 117549, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32179073

RESUMO

AIM: To evaluate physical fitness and cardiovascular effects in rats with renovascular hypertension, two kidneys, one clip (2K1C) submitted to voluntary exercise (ExV). MAIN METHODS: 24 h after surgery (SHAM and 2K1C) rats were submitted to ExV for one week (adaptation). ExV adherent rats were separated into exercise (2K1C-EX and SHAM-EX) or sedentary (2K1C-SED and SHAM-SED) groups. After 4 weeks, exhaustion test, plasma lactate, cardiovascular parameters were evaluated and gastrocnemius muscle was removed for evaluation of gene expression of muscle metabolism markers (PGC1α; AMPK, SIRT-1, UCP-3; MCP-1; LDH) and of the redox process. KEY FINDINGS: ExV decreased blood lactate concentration and increased SOD and CAT activity and a SIRT-1 and UCP-3 gene expression in the gastrocnemius muscle of 2K1C-ExV rats compared to 2K1C-SED rats. Gene expressions of PGC1α, UCP-3, MCT-1, AMPK were higher in 2K1C-ExV rats compared to SHAM-SED rats. Blood pressure in 2K1C-ExV was lower compared to 2K1C-SED and higher in SHAM-SED rats. Reflex bradycardia in 2K1C-EX rats increased compared to 2K1C-SED and was similar to SHAM-SED. The variation in mean blood pressure induced by ganglion blocker hexamethonium and Ang II AT1 receptor antagonist, losartan in the 2K1C-ExV rats was smaller compared to the 2K1C-SED rats and it was similar to the SHAM-SED rats. SIGNIFICANCE: O ExV induced adaptive responses in 2K1C-ExV rats by decreasing sympathetic and Ang II activities and stimulating intracellular signaling that favors redox balance and reduced blood lactate concentration. These adaptive responses, then, contribute to reduced arterial pressure, improved baroreflex sensitivity and physical fitness of 2K1C rats.


Assuntos
Hipertensão Renovascular/metabolismo , Músculo Esquelético/metabolismo , Condicionamento Físico Animal , Transdução de Sinais , Animais , Barorreflexo/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Bradicardia , Modelos Animais de Doenças , Frequência Cardíaca/efeitos dos fármacos , Rim/efeitos dos fármacos , Losartan/farmacologia , Masculino , Oxirredução , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Ratos , Ratos Endogâmicos F344 , Sirtuína 1/metabolismo , Sistema Nervoso Simpático/efeitos dos fármacos , Proteína Desacopladora 3/metabolismo
12.
J Vis Exp ; (156)2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32176203

RESUMO

Recent advances in neuroprosthetics have enabled those living with extremity loss to reproduce many functions native to the absent extremity, and this is often accomplished through integration with the peripheral nervous system. Unfortunately, methods currently employed are often associated with significant tissue damage which prevents prolonged use. Additionally, these devices often lack any meaningful degree of sensory feedback as their complex construction dampens any vibrations or other sensations a user may have previously depended on when using more simple prosthetics. The composite regenerative peripheral nerve interface (C-RPNI) was developed as a stable, biologic construct with the ability to amplify efferent motor nerve signals while providing simultaneous afferent sensory feedback. The C-RPNI consists of a segment of free dermal and muscle graft secured around a target mixed sensorimotor nerve, with preferential motor nerve reinnervation of the muscle graft and sensory nerve reinnervation of the dermal graft. In rats, this construct has demonstrated the generation of compound muscle action potentials (CMAPs), amplifying the target nerve's signal from the micro- to milli-volt level, with signal to noise ratios averaging approximately 30-50. Stimulation of the dermal component of the construct generates compound sensory nerve action potentials (CSNAPs) at the proximal nerve. As such, this construct has promising future utility towards the realization of the ideal, intuitive prosthetic.


Assuntos
Regeneração Nervosa/fisiologia , Nervos Periféricos/fisiologia , Potenciais de Ação , Animais , Feminino , Masculino , Ratos , Ratos Endogâmicos F344
13.
Artigo em Inglês | MEDLINE | ID: mdl-32159371

RESUMO

Over 2,000 mutations have been reported in the cystic fibrosis transmembrane conductance regulator (cftr) gene, many of which cause disease but are rare and have no effective treatment. Thus, there is an unmet need for new, mutation-agnostic therapies for cystic fibrosis (CF). Phosphodiesterase (PDE) inhibitors are one such class of therapeutics that have been shown to elevate intracellular cAMP levels and stimulate CFTR-dependent anion secretion in human airway epithelia; however, the number of people with CF that could be helped by PDE inhibitors remains to be determined. Here we used Fisher rat thyroid (FRT) cells stably transduced with rare human CFTR mutants and studied their responsiveness to the dual phosphodiesterase 3/4 inhibitor RPL554 (Verona Pharma). Through its inhibitory effect on PDE4D, we find that RPL554 can elevate intracellular cAMP leading to a potentiation of forskolin-stimulated current mediated by R334W, T338I, G551D, and S549R mutants of CFTR when used alone or in combination with CFTR modulators. We also were able to reproduce these effects of RPL554 on G551D-CFTR when it was expressed in primary human bronchial epithelial cells, indicating that RPL554 would have stimulatory effects on rare CFTR mutants in human airways and validating FRT cells as a model for PDE inhibitor studies. Furthermore, we provide biochemical evidence that VX-809 causes surprisingly robust correction of several class III and IV CFTR mutants. Together, our findings further support the therapeutic potential of RPL554 for patients with CF with class III/IV mutations and emphasize the potential of PDEs as potential drug targets that could benefit patients with CF.


Assuntos
AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Isoquinolinas/farmacologia , Inibidores da Fosfodiesterase 3/farmacologia , Inibidores da Fosfodiesterase 4/farmacologia , Pirimidinonas/farmacologia , Células Epiteliais da Tireoide/efeitos dos fármacos , Aminopiridinas/farmacologia , Animais , Benzodioxóis/farmacologia , Brônquios/citologia , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Linhagem Celular , Colforsina/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/agonistas , Regulador de Condutância Transmembrana em Fibrose Cística/classificação , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Mutação , Cultura Primária de Células , Ratos , Ratos Endogâmicos F344 , Células Epiteliais da Tireoide/citologia , Células Epiteliais da Tireoide/metabolismo , Transgenes
14.
Transplant Proc ; 52(3): 992-1000, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32122662

RESUMO

BACKGROUND: Macrophages may be important in chronic rejection after organ transplantation. This study aimed to investigate the possibility of depleting macrophages for a certain amount of time to alleviate chronic rejection in a heart transplant model of Fischer to Lewis rats. METHODS: Clodronate liposome was injected abdominally to deplete macrophages for 2 time frames. The expression levels of ectodysplasin 1, arginase 1 (Arg1), chitinase-like lectin (Ym1), interferon gamma, tumor necrosis factor α (TNF-α), smooth muscle α-actin (α-SMA), monocyte chemoattractant protein 1 (MCP-1), and interleukin 10 (IL-10) were detected. RESULTS: 1. The expression levels of α-SMA, interferon gamma, TNF-α, and MCP-1 and the transformation of peripheral T cells were lower after macrophage depletion for 2 or 4 weeks. 2. The expression levels of α-SMA, TNF-α, and MCP-1 and the transformation of peripheral T cells were even lower after 4 weeks compared with 2 weeks, except for interferon gamma. 3. A higher level of expression of Arg1 and Ym1 after macrophage depletion for 2 weeks was observed. 4. A higher level of expression of IL-10 after macrophage depletion for 2 weeks, but not 4 weeks, was also observed. CONCLUSIONS: Macrophage clearance after heart transplantation alleviated chronic rejection probably via M2 polarization of regenerated macrophages, reduced T-lymphocyte proliferation, and changed the expression levels of interferon gamma, TNF-α, MCP-1, and IL-10.


Assuntos
Rejeição de Enxerto/imunologia , Transplante de Coração/métodos , Macrófagos , Aloenxertos/imunologia , Aloenxertos/metabolismo , Animais , Rejeição de Enxerto/prevenção & controle , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Transplante Homólogo
15.
Toxicol Lett ; 324: 12-19, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32035981

RESUMO

APC mutation is the first event triggering colon carcinogenesis (CRC). The contribution of APC to colon mucosa DNA damage is not well characterized yet. Similarly, the role of genotoxin-producer gut microorganisms is unclear. DNA strand breaks and oxidative damage were measured in Pirc rats, mutated in Apc, with the COMET assay at age 1 (T1) and 11 months (T11), i.e. in absence and presence of colon adenomas. In Pirc colon mucosa a 2-fold increase in the mean level of DNA oxidative damage was found at T11 compared to T1. Moreover, the analysis of DNA damage distribution showed that the proportion of Pirc mucosa cells in the highest DNA damage class was increased compared to wt rats at T1 and T11 months (p < 0.05 and <0.001, respectively). The analysis of colon mucosa-associated microbiota composition showed that this result was not attributable to the presence of genotoxin-producer bacteria B. fragilis nor E. coli. However, Pirc colon mucosa was enriched in Clostridium cluster XI, harmful bacteria in the large intestine, while the wt colon mucosa was enriched in Clostridium cluster IV. This work provides an original way to investigate the interplay between Apc and gut microbiota in affecting DNA stability during CRC.


Assuntos
Colo/metabolismo , Neoplasias do Colo/etiologia , Dano ao DNA , Genes APC , Mucosa Intestinal/metabolismo , Mutação , Animais , Neoplasias do Colo/genética , Modelos Animais de Doenças , Microbioma Gastrointestinal , Ratos , Ratos Endogâmicos F344
16.
Mutat Res ; 849: 503130, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32087857

RESUMO

Human risk assessment of the toxic potency of chemicals typically includes genotoxicity assays for predicting carcinogenicity. Gene mutation frequency and chromosomal aberration are two major genotoxicity endpoints in standardized in vitro and in vivo assays. The weight-of-evidence approach in risk assessment is more focused on in vivo assay results; however, animal welfare considerations are aimed at the reduction, replacement, and refinement (3R's) of animal experiments, including a reduction in the number of experimental animals. Proposals to reduce experimental animals in genotoxicity testing include the incorporation of genotoxicity endpoint(s) into other toxicological studies and the combination of two or more assays detecting different genotoxicity endpoints in the same animals. In this study, we used 1,2-dimethylhydrazine as a model chemical of colon carcinogen to assess gene mutation frequency and chromosomal aberration in vivo simultaneously. Specifically, a gene mutation frequency assay was combined with a multiple-organ micronucleus test (peripheral blood, bone marrow, liver, and colon) in F344 gpt delta transgenic rats. Both gpt mutant frequency and micronucleated cell frequency significantly increased in colon and liver but not in bone marrow. Interestingly, we found that the colon carcinogen induced both gene mutations and micronuclei in the targeted colon tissue. Thus, we demonstrated that the mechanism of a carcinogen could be derived from an animal experiment using a lower number of experimental animals as currently recommended. Moreover, a significant increase in mutant frequency in colon and liver was already observed on the first day after treatment completion, as well as on the third day, which is the guideline-recommended period. Thus, this endpoint is compatible with other genotoxicity assays. We confirmed that performing the micronucleus assay in combination with a gene mutation assay in F344 gpt delta transgenic rats is useful to evaluate different genotoxic endpoints simultaneously in the same animals, which reduces the number of experimental animals.


Assuntos
1,2-Dimetilidrazina/toxicidade , Carcinógenos/toxicidade , Aberrações Cromossômicas/efeitos dos fármacos , Neoplasias do Colo/diagnóstico , Determinação de Ponto Final , Testes de Mutagenicidade , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Taxa de Mutação , Especificidade de Órgãos , Ratos , Ratos Endogâmicos F344 , Ratos Transgênicos
17.
Int J Nanomedicine ; 15: 749-760, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32099363

RESUMO

Background: n-Butylidenephthalide (BP) has anti-tumor effects on glioblastoma. However, the limitation of BP for clinical application is its unstable structure. A polycationic liposomal polyethylenimine (PEI) and polyethylene glycol (PEG) complex (LPPC) has been developed to encapsulate BP for drug structure protection. The purpose of this study was to investigate the anti-cancer effects of the BP/LPPC complex on glioblastoma in vitro and in vivo. Methods: DBTRG-05MG tumor bearing xenograft mice were treated with BP and BP/LPPC and then their tumor sizes, survival, drug biodistribution were measured. RG2 tumor bearing F344 rats also treated with BP and BP/LPPC and then their tumor sizes by magnetic resonance imaging for evaluation blood-brain barrier (BBB) across and drug therapeutic effects. After treated with BP/LPPC in vitro, cell uptake, cell cycle and apoptotic regulators were analyzed for evaluation the therapeutic mechanism. Results: In athymic mice, BP/LPPC could efficiently suppress tumor growth and prolong survival. In F334 rats, BP/LPPC crossed the BBB and led to tumor shrinkage. BP/LPPC promoted cell cycle arrest at the G0/G1 phase and triggered the extrinsic and intrinsic cell apoptosis pathways resulting cell death. BP/LPPC also efficiently suppressed VEGF, VEGFR1, VEGFR2, MMP2 and MMP9 expression. Conclusion: BP/LPPC was rapidly and efficiently transported to the tumor area across the BBB and induced cell apoptosis, anti-angiogenetic and anti-metastatic effects in vitro and in vivo.


Assuntos
Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Anidridos Ftálicos/uso terapêutico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Barreira Hematoencefálica/patologia , Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Lipossomos/química , Camundongos , Camundongos Nus , Anidridos Ftálicos/farmacologia , Polietilenoglicóis/química , Polietilenoimina/química , Ratos Endogâmicos F344 , Tela Subcutânea/efeitos dos fármacos , Tela Subcutânea/patologia , Distribuição Tecidual/efeitos dos fármacos
18.
Int J Nanomedicine ; 15: 943-952, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32103948

RESUMO

Background: Malignant pleural effusion (MPE) is the accumulation of fluid in the pleural cavity as a result of malignancies affecting the lung, pleura and mediastinal lymph nodes. Curcumin, a compound found in turmeric, has anti-cancer properties that could not only treat MPE accumulation but also reduce cancer burden. To our knowledge, direct administration of curcumin into the pleural cavity has never been reported, neither in animals nor in humans. Purpose: To explore the compartmental distribution, targeted pharmacokinetics and the safety profile of liposomal curcumin following intrapleural and intravenous administration. Methods: Liposomal curcumin (16 mg/kg) was administered into Fischer 344 rats by either intrapleural injection or intravenous infusion. The concentration of curcumin in plasma and tissues (lung, liver and diaphragm) were measured using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Blood and tissues were examined for pathological changes. Results: No pleural or lung pathologies were observed following intrapleural liposomal curcumin administration. Total curcumin concentration peaked 1.5 hrs after the administration of intrapleural liposomal curcumin and red blood cell morphology appeared normal. A red blood cells abnormality (echinocytosis) was observed immediately and at 1.5 hrs after intravenous infusion of liposomal curcumin. Conclusion: These results indicate that liposomal curcumin is safe when administered directly into the pleural cavity and may represent a viable alternative to intravenous infusion in patients with pleural-based tumors.


Assuntos
Curcumina/administração & dosagem , Curcumina/farmacocinética , Cavidade Pleural/efeitos dos fármacos , Administração Intravenosa , Animais , Cromatografia Líquida , Feminino , Lipossomos/administração & dosagem , Lipossomos/farmacocinética , Pulmão/efeitos dos fármacos , Masculino , Neoplasias Pleurais/patologia , Ratos Endogâmicos F344 , Espectrometria de Massas em Tandem , Distribuição Tecidual
19.
Cardiovasc Diabetol ; 19(1): 13, 2020 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-32035482

RESUMO

BACKGROUND AND AIMS: Sodium-glucose linked cotransporter-2 (SGLT2) inhibitors reduce the likelihood of hospitalization for heart failure and cardiovascular death in both diabetic and non-diabetic individuals with reduced ejection fraction heart failure. Because SGLT2 inhibitors lead to volume contraction with reductions in both preload and afterload, these load-dependent factors are thought to be major contributors to the cardioprotective effects of the drug class. Beyond these effects, we hypothesized that SGLT2 inhibitors may also improve intrinsic cardiac function, independent of loading conditions. METHODS: Pressure-volume (P-V) relationship analysis was used to elucidate changes in intrinsic cardiac function, independent of alterations in loading conditions in animals with experimental myocardial infarction, a well-established model of HFrEF. Ten-week old, non-diabetic Fischer F344 rats underwent ligation of the left anterior descending (LAD) coronary artery to induce myocardial infarction (MI) of the left ventricle (LV). Following confirmation of infarct size with echocardiography 1-week post MI, animals were randomized to receive vehicle, or the SGLT2 inhibitor, empagliflozin. Cardiac function was assessed by conductance catheterization just prior to termination 6 weeks later. RESULTS: The circumferential extent of MI in animals that were subsequently randomized to vehicle or empagliflozin groups was similar. Empagliflozin did not affect fractional shortening (FS) as assessed by echocardiography. In contrast, load-insensitive measures of cardiac function were substantially improved with empagliflozin. Load-independent measures of cardiac contractility, preload recruitable stroke work (PRSW) and end-systolic pressure volume relationship (ESPVR) were higher in rats that had received empagliflozin. Consistent with enhanced cardiac performance in the heart failure setting, systolic blood pressure (SBP) was higher in rats that had received empagliflozin despite its diuretic effects. A trend to improved diastolic function, as evidenced by reduction in left ventricular end-diastolic pressure (LVEDP) was also seen with empagliflozin. MI animals treated with vehicle demonstrated myocyte hypertrophy, interstitial fibrosis and evidence for changes in key calcium handling proteins (all p < 0.05) that were not affected by empagliflozin therapy. CONCLUSION: Empagliflozin therapy improves cardiac function independent of loading conditions. These findings suggest that its salutary effects are, at least in part, due to actions beyond a direct effect of reduced preload and afterload.


Assuntos
Compostos Benzidrílicos/farmacologia , Glucosídeos/farmacologia , Insuficiência Cardíaca/tratamento farmacológico , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Volume Sistólico/efeitos dos fármacos , Função Ventricular Esquerda/efeitos dos fármacos , Pressão Ventricular/efeitos dos fármacos , Animais , Sinalização do Cálcio/efeitos dos fármacos , Modelos Animais de Doenças , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Infarto do Miocárdio/complicações , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Miocárdio/metabolismo , Miocárdio/patologia , Ratos Endogâmicos F344
20.
Life Sci ; 243: 117226, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31904366

RESUMO

Hypertension is a risk factor for premature death and roughly 50% of hypertensive patients are salt-sensitive. The incidence of salt-sensitive hypertension increases with age. However, the mechanisms of salt-sensitive hypertension are not well understood. We had demonstrated decreased renal sodium­hydrogen exchanger regulatory factor 1 (NHERF1) expression in old salt-resistant F344 rats. Based on those studies we hypothesized that NHERF1 expression is required for the development of some forms of salt-sensitive hypertension. To address this hypothesis, we measured blood pressure in NHERF1 expressing salt-sensitive 4-mo and 24-mo-old male and female Fischer Brown Norway (FBN) rats male and female 18-mo-old NHERF1 knock-out (NHERF1-/-) mice and wild-type (WT) littermates on C57BL/6J background after feeding high salt (8% NaCl) diet for 7 days. Our data demonstrate that 8% salt diet increased blood pressure in both male and female 24-mo-old FBN rats but not in 4-mo-old FBN rats and in 18-mo-old male and female WT mice but not in NHERF1-/- mice. Renal dopamine 1 receptor (D1R) expression was decreased in 24-mo-old rats, compared with 4-mo-old FBN rats. However, sodium chloride cotransporter (NCC) expression increased in 24-mo-old FBN rats. In FBN rats, age had no effect on NaK ATPase α1 and NKCC2 expression. By contrast, high salt diet increased the renal expressions of NKCC2, and NCC in 24-mo-old FBN rats. High salt diet also increased NKCC2 and NCC expression in WT mice but not NHERF1-/- mice. Our data suggest that renal NHERF1 expression confers salt sensitivity with aging, associated with increased expression of sodium transporters.


Assuntos
Envelhecimento/metabolismo , Hipertensão/metabolismo , Fosfoproteínas/fisiologia , Cloreto de Sódio na Dieta/administração & dosagem , Trocadores de Sódio-Hidrogênio/fisiologia , Animais , Pressão Sanguínea , Modelos Animais de Doenças , Feminino , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/genética , Ratos , Ratos Endogâmicos F344 , Trocadores de Sódio-Hidrogênio/genética
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