Preactivation of neutrophils and systemic oxidative stress in dogs with hyperleptinemia.

High occurrence of obesity currently constitutes the main nutritional disease of the canine species. There is evidence that leptin increases during obesity in dogs. Hyperleptinemia is associated with increased neutrophil oxidative metabolism in obese humans and contributes to oxidative stress. However, in obese dogs, the probable relationship between this condition and the activation of the oxidative metabolism of neutrophils has yet to be established. Thus, we investigated the hypothesis that neutrophil activation and systemic oxidative stress occur in dogs with hyperleptinemia. A control group of 24 healthy dogs with a body condition score (BCS) of 4-5, an overweight group of 25 dogs with a BCS of 6-7, and 27 obese dogs with a BCS of 8-9, were composed. Two subgroups were formed composed of dogs with and without hyperleptinemia, grouped according to the 95% confidence interval obtained for plasma leptin values of the control group. Changes in obesity markers (body condition score, adiponectin and plasma leptin) and plasma oxidative stress (lipid peroxidation, total antioxidant and oxidant capacities and oxidative stress index) were measured in all the dogs selected. Neutrophil oxidative metabolism was evaluated in flow cytometry by superoxide production with the probe hydroethidine and by hydrogen peroxide production with the probe 2',7'-dichlorofluorescein diacetate, with or without phorbol myristate acetate (PMA) stimulation. Apoptosis and neutrophil viability were quantified in a capillary flow cytometer using Annexin VPE, with or without camptothecin apoptosis inducing effect. Obese dogs presented higher systemic oxidative stress, hyperleptinemia and preactivated neutrophils with accelerated apoptosis. Dogs with hyperleptinemia and obese dogs presented higher neutrophil superoxide production under PMA stimulation and the presence of systemic oxidative stress compared with control. To our knowledge, this is probably the first evidence that preactivation of the oxidative metabolism of circulating neutrophils occurs in dogs with hyperleptinemia, a condition that can induce systemic oxidative stress in the canine species.

Hemólise interfere na mensuração dos biomarcadores plasmáticos de estresse oxidativo em cães / Hemolysis interferes in the measurement of plasma biomakers of oxidative stress in dogs

Considerando que, entre todas as fontes de erro analítico, a hemólise é a mais importante na rotina laboratorial, o presente estudo teve como objetivo investigar o efeito da hemólise in vitro sobre os principais biomarcadores plasmáticos de estresse oxidativo mensurados (BPEO) de cães. Para tal, amostras de sangue total de 19 cães clinicamente saudáveis foram hemolisadas em diferentes graus por ação mecânica. Amostras controle contendo baixa concentração de hemoglobina (Hb) no plasma foram comparadas com quatro graus de hemólise (<0,36; 0,36-0,60; 0,61-1,0; 1,1-4g/L Hb). Imediatamente após a hemólise, foram mensuradas as concentrações plasmáticas de ácido úrico (AU), albumina, bilirrubina, gamaglutamiltransferase (GGT), capacidade antioxidante total (TAC) e concentração de oxidante total (TOC). Os erros relativos causados pelos diferentes graus de hemólises foram calculados e confrontados com o erro total aceitável (ETA) e com o limite de erro permitido (LEP) empregados nos programas de controle de qualidade de exames laboratoriais. Foi observado que mesmo pequeno grau de hemólise gera algum erro analítico não aceitável (ETA e/ou LEP) nos BPEO mensurados, exceto na bilirrubina. Foi possível concluir que a hemólise é um fator limitante para avaliação do estresse oxidativo sistêmico mensurado no plasma, podendo causar erros que potencialmente comprometem o diagnóstico clínico.(AU)

Among all the various sources of analytical error, hemolysis is the most important in the laboratory routine. The present study aimed to investigate the effect of hemolysis "in vitro" on the main plasma biomarkers of oxidative stress (BPEO) dogs. For this purpose, whole blood samples from 19 healthy dogs were hemolyzed in different degrees by mechanical action. Control samples containing low concentration of hemoglobin (Hb) levels in plasma were compared with four degrees of hemolysis (<0.36, from 0.36 to 0.60, 0.61 to 1.0, 1.1 to 4g/L Hb). Immediately after causing hemolysis, plasma concentrations of uric acid (UA), albumin, bilirubin, gamma glutamyl transferase (GGT), total antioxidant capacity (TAC), and total oxidant concentration (TOC) were measured. The relative errors caused by several levels of hemolysis were calculated and compared with the total acceptable error (TAE) and allowed error limit (LEP) by employees in quality control programs for laboratory tests. Even small levels of hemolysis generate unacceptable analytical error (TAE and / or LEP) in BPEO measured, except for bilirubin. Hemolysis is a limiting factor for the assessment of systemic oxidative stress measured in plasma and may cause errors that potentially compromise clinical diagnosis.(AU)

Resveratrol atenua o estresse oxidativo e a lesão muscular de ratos sedentários submetidos ao exercício físico / Resveratrol attenuates oxidative stress and muscle damage in sedentary rats subjected to physical exercise

O sedentarismo é um problema de saúde pública e um dos maiores males da sociedade moderna. Já está bem estabelecido que esforço físico em excesso ou em indivíduos não condicionados acarreta estresse oxidativo e lesões musculares. No presente estudo, foi testada a hipótese de que um único esforço físico é capaz de causar estresse oxidativo e lesão muscular em indivíduos sedentários. Aditivamente foi avaliado efeito antioxidante do polifenol resveratrol (RV) quanto a sua capacidade de atenuar o estresse oxidativo e a lesão muscular causados pelo esforço físico. Para tal, 40 ratos (Rattus norvegicus albinus, Wistar), machos, adultos e sedentários foram aleatoriamente submetidos ou não a 90 minutos de natação, com e sem tratamento com RV (100mg/kg/PV/14dias): N-RV- (n=10) grupo mantido em repouso e não tratado com RV; N-RV+ (n=10) grupo mantido em repouso e tratado com RV; N+RV- (n=10) grupo submetido ao esforço físico de natação e não tratado com RV e N+RV+ (n=10) grupo submetido ao esforço físico de natação e tratado com RV. Em ratos sedentários, o esforço físico da natação promoveu estresse oxidativo (aumento da peroxidação lipídica e diminuição da capacidade antioxidante total do plasma) e aumento significativo da atividade plasmática de creatina quinase (CK) e lactato desidrogenase (LDH). O tratamento com RV diminuiu a peroxidação lipídica e a concentração dos marcadores de lesão muscular (CK e LDH) de ratos sedentários submetidos à natação. Essa é uma das primeiras evidências de que um único esforço físico pode causar estresse oxidativo em indivíduos sedentários e que o RV pode ser uma alternativa para atenuar a lesão muscular causada por esse estresse.(AU)

Physical inactivity is a public health problem when a sedentary population practices physical activity sporadically. Exercise in unconditioned individuals causes oxidative stress and muscle damage. This study tested the hypothesis that a single physical exertion can cause muscle damage and oxidative stress in sedentary individuals, and resveratrol can attenuate it. For this, 40 sedentary adult male rats were equally and randomized into four groups subjected to 90min swimming or rest and administered aqueous resveratrol (100mg/kg/day) or saline for 14 days: N-RV-, rats maintained at rest and administered saline; N-RV+, rats maintained at rest and treated with resveratrol; N+RV-, rats subjected to physical exercise and administered saline; and N+RV+, rats subjected to physical exercise and treated with resveratrol. In sedentary rats, the physical exertion of swimming promoted oxidative stress, i.e. increased lipid peroxidation and decreased plasma total antioxidant capacity, and significant increases in CK and LDH plasma activities. Resveratrol diminished lipid peroxidation and the concentrations of muscle damage markers (CK and LDH) in sedentary rats subjected to swimming. The results provide evidence that a single sudden physical exertion can cause oxidative stress in sedentary rats. Resveratrol showed good results as a treatment for minimizing muscle damage caused by this stress.(AU)

Estresse oxidativo em cães com doença periodontal: comparação dos biomarcadores plasmáticos e salivares / Oxidative stress in dogs with periodontal disease: comparison of plasmatic and salivary biomarkers

A doença periodontal (DP) é a enfermidade inflamatória mais comum da cavidade oral dos cães. A quantificação de biomarcadores do plasma e da saliva tem sido utilizada para avaliar o estresse oxidativo sistêmico (EOS) e local (EOL) da DP humana. Na DP canina, os mecanismos do estresse oxidativo não estão bem caracterizados e estabelecidos. O objetivo do presente estudo foi investigar a hipótese de que o EOS ocorre na DP canina e de que a saliva pode ser utilizada para avaliar o EOL. Analisou-se, também, a hipótese de que a ativação do metabolismo oxidativo dos neutrófilos contribui para EOS na DP dos cães. Para tal, foram selecionados 20 cães adultos portadores de DP, agrupados de acordo com o grau de lesão: gengivite (n=6), periodontites leve (n=8) e avançada (n=6). O grupo controle foi composto pelos mesmos 20 cães, 30 dias após o tratamento periodontal. Para avaliar o metabolismo oxidativo dos neutrófilos circulantes foi quantificada a produção de superóxido pelo teste de redução do tetrazólio nitroazul (NBT). As concentrações de oxidante total (TOC) e de espécies reativas ao ácido tiobartbitúrico (TBARS) no plasma foram quantificadas para avaliar o EOS. Para a avaliação do estresse oxidativo local, foi quantificado o TOC salivar e a concentração dos principais antioxidantes da saliva (albumina, ácido úrico e bilirrubina total). O EOS na DP foi confirmado pelo aumento da produção de superóxido dos neutrófilos circulantes, TOC e TBARS plasmático. Foi possível quantificar todos os biomarcadores na saliva de cães, porém nenhum foi capaz de expressar o EOL da DP canina. Esta é uma das primeiras evidências de que o EOS ocorre em cães com DP e que a ativação do metabolismo oxidativo dos neutrófilos pode contribuir para desequilíbrio entre antioxidantes e oxidantes. Este estudo ressalta a importância da higiene bucal dos cães para a prevenção da DP e de lesões degenerativas crônicas de diversos tecidos causadas pelo EOS.(AU)

Periodontal disease (PD) is the most common inflammatory disease of the oral cavity of dogs. Quantitation of plasma and salivary biomarkers have been used to assess the systemic oxidative stress (SOS) and local (LOS) of human PD. In canine PD, oxidative stress mechanisms are not well characterized and established. Our objective was to investigate the hypothesis that SOS occurs in dog PD and saliva can be used to evaluate the LOS. We also investigated the hypothesis that the activation of neutrophil oxidative metabolism contributes to SOS in dog SD. For this purpose, 20 adult dogs were selected PD patients, grouped according to the degree of injury: gingivitis (n=6), light periodontitis (n=8) and advanced periodontitis (n=6). The control group was composed of the same 20 dogs, 30 days after periodontal treatment. To assess oxidative metabolism of circulating neutrophils superoxide production was measured by test nitroblue tetrazolium reduction (NBT). The total oxidant concentrations (TOC) and reactive species to tiobartbitúrico acid (TBARS) in plasma were quantified to evaluate SOS. For the evaluation of local oxidative stress were quantified salivary TOC and concentration of the main antioxidant in saliva (albumin, uric acid, and total bilirubin). EOS in dogs with PD was confirmed by increased superoxide production of circulating neutrophils, TOC, and plasma TBARS. It was possible to quantify all the biomarkers in the saliva of dogs, but none was able to express the LOS canine PD. This is the first evidence that SOS occurs in dogs with PD and that activation of the oxidative metabolism of neutrophils may contribute to an imbalance between oxidants and antioxidants. This study highlights the importance of oral hygiene of dogs to prevent PD and chronic degenerative lesions of various tissues caused by SOS.(AU)

Induction of haem oxygenase-1 increases infection of dog macrophages by L. infantum.

We aimed to induce and inhibit HO-1, ascertaining its effect on infection rate, parasite load and the levels of superoxide, reactive oxygen species (ROS), nitric oxide (NO), TNF-alpha and IL-10 in cultured macrophages from healthy dogs infected by Leishmania infantum. Macrophages obtained from 15 healthy dogs were cultured alone or infected with L. infantum, with or without association of HO-1 inducer and inhibitor. The infection rate and the parasite load were determined by the number of infected macrophages and number of promastigotes per macrophage, respectively. HO-1 levels and gene expression, as well as IL-10 and TNF-alpha levels were also measured in these cultures. Superoxide, ROS and NO levels in macrophages were measured through flow cytometry. Induction of HO-1 increased the infection rate and parasite load, while its inhibition decreased the infection rate and IL-10 production. There was a positive correlation between HO-1 and infection rate or parasite load. Increased infection rate was associated with decreased superoxide, ROS and NO levels. Induction of HO-1 metabolism in dogs infected by L. infantum is possibly one of the mechanisms responsible for increasing the infection of macrophages, mainly through reduction in the oxidative and nitrosative metabolisms of these cells.

The uremic toxin methylguanidine increases the oxidative metabolism and accelerates the apoptosis of canine neutrophils.

We investigated the hypothesis that the increased concentration of plasma methylguanidine (MG) increases oxidative metabolism and accelerates apoptosis of neutrophils from dogs with chronic kidney disease (CKD). To achieve this, the levels of MG were quantified in healthy (n=16) and uremic dogs with CKD stage 4 of according to the guidelines of the International Renal Interest Society (IRIS, 2015) (n=16) using high performance liquid chromatography (HPLC). To evaluate the isolated effect of MG on neutrophil oxidative metabolism and apoptosis, neutrophils isolated from 12 healthy dogs were incubated with the highest concentration of plasma MG (0.005g/L) observed in dogs with CKD. Neutrophil oxidative metabolism was assessed by flow cytometry, using the probes hydroethidine for superoxide production and 2',7'-dichlorofluorescein diacetate for hydrogen peroxide production, with or without phorbol myristate acetate (PMA) stimulus. Neutrophil apoptosis and viability were also evaluated in flow cytometer using the Annexin V-PE system, with or without the apoptosis-inducing effect of camptothecin. Uremic dogs presented higher concentrations of MG (p<0.0001), increased oxidative stress and primed neutrophils with higher apoptosis rate. The neutrophil abnormalities observed in vivo were also reproduced in vitro, using cells isolated from healthy dogs and incubated with MG. We obtained strong evidence that in dogs with CKD, increased MG levels contributed to oxidative stress and potentially compromised the non-specific immune response by altering the oxidative metabolism and viability of canine neutrophils.

Correlations between peripheral parasite load and common clinical and laboratory alterations in dogs with visceral leishmaniasis.

Intensity of peripheral parasite infection has an important role in the transmission of Leishmania spp. from one host to another. As parasite load quantification is still an expensive procedure to be used routinely in epidemiological surveillance, the use of surrogate predictors may be an important asset in the identification of dogs with high transmitting ability. The present study examined whether common clinical and laboratory alterations can serve as predictors of peripheral parasitism in dogs naturally infected with Leishmania spp. Thirty-seven dogs were examined in order to establish correlations between parasite load (PL) in multiple peripheral tissues and common clinical and laboratory findings in canine visceral leishmaniasis (CVL). Quantitative polymerase chain reaction was employed to determine PL in conjunctival swabs, ear skin, peripheral blood and buffy coat. Additionally, a series of hematological, biochemical and oxidative stress markers were quantified. Correlations between net peripheral infection and severity of clinical alterations and variation in laboratory parameters were assessed through a new analytical approach, namely Compressed Parasite Load Data (CPLD), which uses dimension reduction techniques from multivariate statistics to summarize PL across tissues into a single variable. The analysis revealed that elevation in PL is positively correlated with severity of clinical sings commonly observed in CVL, such as skin lesions, ophthalmic alterations, onycogriphosis, popliteal lymphadenomegaly and low body mass. Furthermore, increase in PL was found to be followed by intensification of non-regenerative anemia, neutrophilia, eosinopenia, hepatic injury and oxidative imbalance. These results suggest that routinely used clinical and laboratory exams can be predictive of intensity of peripheral parasite infection, which has an important implication in the identification of dogs with high transmitting ability.

Leishmaniasis causes oxidative stress and alteration of oxidative metabolism and viability of neutrophils in dogs.

The aim of the present study was to test the hypothesis that oxidative stress and alteration of oxidative metabolism and apoptosis of neutrophils in dogs vary with the stage of leishmaniasis and to determine the contribution of uremia to such alterations. Dogs with leishmaniasis were classified into two stages: moderate (Leish II, n=20) or very severe (i.e. with concurrent uremia; Leish IV, n=20) according to the LeishVet Consensus. The two leishmaniasis groups were compared with uremic dogs without leishmaniasis (Uremic, n=10) and to healthy dogs (Control, n=30). To determine oxidative stress, total antioxidant/oxidant capacity, lipid peroxidation, total glutathione and the plasma antioxidants albumin, uric acid and bilirubin were quantified. Superoxide production was determined using the hydroethidine probe and viability and apoptosis were measured using annexin V-PE by capillary flow cytometry. Oxidative stress was present in both uremia and leishmaniasis with reduced total antioxidant capacity and was associated with increased induced production of superoxide and apoptosis. The greatest amount of oxidants was observed in animals with moderate disease only. Neutrophils from uremic dogs with and without leishmaniasis had decreased viability and an increased apoptosis rate in addition to increased lipid peroxidation. In conclusion, oxidative stress occurs in both stages of leishmaniasis with differences in intensity and levels of plasma markers; however, uremia does contribute to the decreased spontaneous viability of neutrophils in dogs in the final stage of the disease.

Neutrophil dysfunction varies with the stage of canine visceral leishmaniosis.

Canine visceral leishmaniosis (CVL) causes a dependent-stage alteration in neutrophil oxidative metabolism. When production of reactive oxygen species (ROS) exceeds the antioxidant capacity of neutrophils, apoptosis is triggered, impairing the viability and function of these cells, which can predispose dogs to infection. However, the uremic condition observed in late-stage CVL can also alter the viability and function of human neutrophils. To more clearly understand this relationship, the apoptosis rate and oxidative metabolism of neutrophils from control dogs (n=20) were compared to dogs in moderate (n=15) and very severe (n=15) stage CVL, classified according to LeishVet Consensus. To assess neutrophil oxidative metabolism, superoxide production was measured using the nitroblue tetrazolium reduction test (NBT) in isolated neutrophils. The apoptosis rate of neutrophils was estimated using the morphological method. Moderate-stage dogs presented increased superoxide production, while dogs with very severe stage CVL presented decreased superoxide production and an increase neutrophil apoptosis rate. Leishmaniosis causes differential neutrophil dysfunction according to disease stage. In moderate stage CVL, increased superoxide production is observed with no change in neutrophil viability. However, in very severe stage CVL, decreased superoxide production and increased apoptosis occur associated with uremia.