Adapted technique for demonstrating argyrophilic nucleolar organizer regions in the cytologic samples of canine mast cell tumors.

INTRODUCTION: Argyrophilic nucleolar organizer regions (AgNORs) are nonhistone argyrophilic nucleolar proteins associated with ribosomal genes found in the nucleolar organizer region that reflect cell proliferation and have an affinity for silver. AgNOR staining may be useful to evaluate prognosis in several neoplasms because higher AgNOR counts are related to higher grade tumors, metastases, and shorter survival times. OBJECTIVE: We aimed to report on a quick and practical technique to identify AgNORs adapted for use in routine cytology. MATERIALS AND METHODS: The cytopathologic diagnosis of mast cell tumor (MCT) in samples collected by fine-needle aspiration (FNA) was determined. Next, slides were impregnated with a solution containing silver nitrate; the main modification of our technique included incubation of these slides at a controlled temperature of 25 °C. Some slides were previously stained with Diff-Quik and others were only fixed with methanol. The slides were analyzed under a microscope, and the number of blackened intranuclear points (AgNORs) was counted. RESULTS: Slides prestained with Diff-Quik were easily counted compared with slides only fixed in methanol. Technical issues encountered with the methanol-fixed slides included insufficient cellularity, background precipitation, and an absence of silver impregnation. CONCLUSIONS: The technique reported in this study showed satisfactory results for AgNOR counting in cytologic smears from MCT, such as good impregnation and the elimination of background interferents. Further evaluation of this method comparing AgNOR counts with histologic examinations, tumor grades, other prognostic markers, and survival times are needed to fully evaluate the benefit of this technique.

Amikacin prevents platelet aggregation in feline venous blood samples.

INTRODUCTION: Physiologically, feline platelets are more reactive and prone to aggregation, which interferes with platelet counts using automated counters and manual methods. The use of aminoglycoside amikacin in association with EDTA has proven to be efficient in preventing platelet aggregates in cases of pseudo thrombocytopenia (PTP) in people. OBJECTIVES: This study evaluated the efficacy of amikacin in preventing platelet aggregation in EDTA-containing feline blood samples and investigated the possible effects on hematologic measurands. MATERIALS AND METHODS: Blood samples (1.0 mL) collected from 100 healthy cats were stored in two EDTA tubes: 0.5 mL in a microtube containing 10 µL of 250 mg/mL amikacin (EDTA-AMK group) and 0.5 mL in a microtube containing only K2 EDTA 10% (EDTA group). A CBC was executed with an automated impedance blood analyzer, and a microscopic examination of the blood smears was performed. RESULTS: Platelet clumps were observed in 56% of samples from the EDTA group and 5% of samples from the EDTA-AMK group. Platelet counts (PLT), plateletcrit (PCT), and WBC counts were significantly higher (P < .001) in the EDTA-AMK group compared withi the EDTA group. CONCLUSIONS: Amikacin prevents platelet aggregation in feline venous blood samples and does not cause clinically relevant changes in other hematologic measurands. To our knowledge, this is the first report showing the use of amikacin in preventing platelet aggregation in feline blood samples. Based on this study, amikacin could be added to EDTA collection tubes for complete blood counts in cats.

Effects of Heated Pneumoperitoneum on Inflammation, Oxidative Stress, and Peritoneal Histology in Female Dogs That Underwent Video-Assisted Ovariohysterectomy.

Laparoscopic procedures require the creation of pneumoperitoneum. CO2, which must be cold and dry, is the standard gas used in such surgeries. The type of gas used, its temperature, and moisture may change the peritoneal surface and cause systemic and local oxidative stress. Our objective is to evaluate the influence of pneumoperitoneum heating on the occurrence of histological lesions in the peritoneum, inflammation, plasma oxidative stress, and on the mesothelial surface in patients undergoing video-assisted ovariohysterectomy. Twenty canine females were included and distributed evenly into two groups: heated CO2 (HG) and unheated CO2 (UHG). The biomarkers of inflammation and oxidative stress were evaluated before insufflation (T0), at 30 min (T1), and at 60 min (T2) of exposure to CO2. Biopsies of the peritoneal tissue for histological evaluation were performed at T0 and T2. Regarding plasma parameters, acetylcholinesterase (AChE) showed a greater activity in the HG at T1 (p=0.0268) and T2 (p=0.0423); in turn, butyrylcholinesterase (BChE) showed a greater activity at T2 in the HG (p=0.0175) compared with T0. Catalase activity (CAT) was different between HG times; it was higher at T1 (p=0.0253). There was a decrease in the levels of substances reactive to thiobarbituric acid (TBARS) (p=0.0117) and in glutathione (GSH) (p=0.0114) between T0 and T2 in the UHG. Regarding tissue oxidative stress, the CAT in the HG showed a greater activity at T2 than T1 (p=0.0150). By comparing the groups at each time, there was a difference only at T2 (p=0.0288), being greater in the HG. Regarding the activity of superoxide dismutase (SOD) in the HG, there was a difference between T2 in relation to T0 and T1 (p=0.0181); finally, there was an increase only at T1 (p=0.0287) in the UHG when comparing groups at the same time. There were no differences in the histological parameters evaluated. Our study demonstrates that the heating of CO2 generates a greater inflammatory response and forms reactive oxygen species (ROS) at the plasma and peritoneal levels.

Lipotoxicity-associated inflammation is prevented by guarana (Paullinia cupana) in a model of hyperlipidemia.

Hyperlipidemia causes lipotoxicity which prompts an inflammatory response linked to the development of cardiovascular diseases. Natural compounds have been receiving special attention for its potential to treat diseases, inexpensiveness, and safety. Guarana (Paullinia cupana) has demonstrated notable anti-inflammatory and antioxidant effects, which may prevent chronic diseases caused by changes in lipid profile. Thus, this study aims to evaluate the effect of guarana powder (Paullinia cupana) in the purine metabolism and inflammatory profile in lymphocytes and serum of rats with Poloxamer-407-induced hyperlipidemia. Pretreatment with guarana 12.5, 25, and 50 mg/kg/day or caffeine (0.2 mg/kg/day) by gavage was applied to adult male Wistar rats for a period of 30 days. As a comparative standard, we used simvastatin (0.04 mg/kg) post-induction. Hyperlipidemia was acutely induced with intraperitoneally injection of Poloxamer-407 (500 mg/kg). Guarana powder and caffeine increased the activity of the E-NTPDase (ecto-apyrase), and all pretreatments decreased the E-ADA (ecto-adenosine deaminase) activity, reducing the inflammatory process caused by lipotoxicity. In hyperlipidemic rats, ATP levels were increased while adenosine levels were decreased, guarana and caffeine reverted these changes. Guarana powder, caffeine, and simvastatin also prevented the increase in INF-γ and potentiated the increase in IL-4 levels, promoting an anti-inflammatory profile. Guarana promoted a more robust effect than caffeine. Our results show that guarana powder and caffeine have an anti-inflammatory as seen by the shift from a proinflammatory to an anti-inflammatory profile. The effects of guarana were more pronounced, suggesting that guarana powder may be used as a complementary therapy to improve the lipotoxicity-associated inflammation.

Impacts of dose and length of exposure to boldenone and stanazolol on enzymatic antioxidant systems, myeloperoxidase and NAGase activities, and glycogen and lactate levels in rat liver.

We investigated the adverse effects of the anabolic androgenic steroids (AAS) boldenone (BOL) and stanazolol (ST) on the enzymatic antioxidant systems of the rat liver. Male Wistar rats were divided in three protocols (P): PI, 5 mg/kg BOL or ST once a week for 4 weeks; PII, 2.5 mg/kg BOL or ST once a week for 8 weeks; PIII, 1.25 mg/kg BOL or ST once a week for 12 weeks. AAS were administered intramuscularly (0.2 ml, olive oil vehicle) once a week in all protocols. Activities of the enzymes glutathione peroxidase (GPx), glutathione S-transferase (GST), and glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), were investigated. We assessed the content of hydrogen peroxide (H2O2), glycogen and lactate; and enzyme markers of neutrophils (myeloperoxidase, MPO) and macrophages (NAGase). PI and PII altered the SOD and CAT activities and increased the H2O2 content. PI led to increases in the MPO and NAGase activities. In contrast, changes in GPx, GST and, GR were observed under PII and, to a greater extend, under PIII. Following PIII, GPx, GR, and GST exhibited reduced activities. All protocols altered the glycogen and lactate content. The use of high doses of AAS for a short duration first alters SOD/CAT activity. In contrast, at lower doses of AAS for long periods is associated with changes in the glutathione system. Protocols with high doses of AAS for a short duration exert the most deleterious effects on redox status, markers of cellular infiltration, and the metabolic functioning of hepatic tissues.

Astrocaryum aculeatum fruit improves inflammation and redox balance in phytohemagglutinin-stimulated macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruit of Astrocaryum aculeatum G.Mey. (tucumã) is highly consumed by riverside communities in the Amazonian region. These communities have recently been shown to have increased longevity and reduced prevalence of age-related morbidity. Tucumã, which is locally used in their diet and traditional medicine may contribute to these features. AIM OF THE STUDY: To investigate the anti-inflammatory and antioxidant properties of A. aculeatum extract against phytohemagglutinin-induced inflammation in cell cultures. MATERIALS AND METHODS: Cell viability and cytotoxicity assays, gene expression of interleukins IL-1ß, IL-6, IL-10, levels of reactive oxygen species (ROS), nitric oxide (NO) and thiols were employed, as well as the activities of antioxidant enzymes in RAW 264.7 cells stimulated with phytohemagglutinin to mimic inflammation. RESULTS: The extract of A. aculeatum fruit inhibited macrophage proliferation (P < 0.05), arrested the cell cycle in G0/G1 phase (P < 0.001), increased antioxidant defenses (P < 0.01), reduced oxidative stress (P < 0.01), and modulated genes related to the inflammatory response (P < 0.001). CONCLUSION: Our results demonstrate that A. aculeatum fruit has anti-inflammatory and antioxidant capacities. These beneficial effects of tucumã on cells are also likely to be seen in vivo, thereby suggesting that its extract is a suitable therapeutic adjuvant in the prevention or treatment of inflammatory diseases.

Trypanosoma evansi impacts on embryonic neural progenitor cell functions.

Trypanosoma evansi appears to have a significant tropism for brain tissue in its chronic and acute phases. The most common symptoms of this brain infection are motor incoordination, meningoencephalitis, demyelination, and anemia. There have only been few studies of the effects of T. evansi infection on neuronal differentiation and brain plasticity. Here, we investigated the impact of the congenital T. evansi infection on brain development in mice. We collected telencephalon-derived neural progenitor cells (NPCs) from T. evansi uninfected and infected mice, and cultivated them into neurospheres. We found that T. evansi significantly decreased the number of cells during development of neurospheres. Analysis of neurosphere differentiation revealed that T. evansi infection significantly increased neural migration. We also observed that T. evansi promoted expression of glial fibrillary acidic protein (GFAP) in infected cells. These data suggest that congenital T. evansi infection may affect embryonic brain development.

Effect of free and nano-encapsulated curcumin on treatment and energetic metabolism of gerbils infected by Listeria monocytogenes.

Bacterial infections require special care since the indiscriminate use of antibiotics to treat them has been linked to the emergence of resistant strains. In this sense, phytoterapeutic alternatives such as curcumin and its nanocapsules have emerged as a promising supplement in optimizing availability of bioactives and reducing the development of antimicrobial resistance. Thus, the aim of this study was to verify the effects of pure and nanoencapsulated curcumin in the treatment of experimental listeriosis in gerbils regarding many aspects including antibacterial effect, antioxidant mechanisms involved and the energetic metabolism. Four groups were used containing 6 animals each: T0 (control), T1 (infected), T2 (infected and treated with free curcumin - dose of 30 mg/kg/day) and T3 (infected and treated with nanocapsules containing curcumin - a dose of 3 mg/kg/day). Treated animals received curcumin for 6 consecutive days starting 24 h after Listeria monocytogenes infection. All animals were euthanized on the 12th day after L. monocytogenes infection. Quantitative polymerase chain reaction (qPCR) identified L. monocytogenes DNA in the spleens of all animals of the T1 group, as well as T2 (2 out of 6) and T3 (5 out of 6). The weight of the spleens confirmed the infection, since it was larger in the T1 group, differing statistically from T0, and similarly to T2 and T3. Hepatic histopathological examination showed mild infiltration of neutrophils and macrophages, except for the T3 group (only 1/6). In the liver, the pyruvate kinase activity was higher in T1 and T2 compared to T0 and T3. The adenylate kinase activity did not differ between groups. The Na+/K+ATPase activity was lower in T1 group compared to T0 and T3. Lipoperoxidation was lower in the T3 group compared to groups T0, T1 and T2. The antioxidant capacity against peroxyl radicals was higher in T1, T2 and T3 groups compared to T0. In conclusion, free curcumin showed potent antibacterial effects; however, the nanoencapsulated form was able to minimize the effects caused by L. monocytogenes regarding tissue injury, changes on enzymes of the energetic metabolism, in addition to an antioxidant effect against lipoperoxidation.

Effects of resveratrol on the differentiation fate of neural progenitor cells of mouse embryos infected with Trypanosoma cruzi.

Chagas disease (CD) affecting about 7 million people is caused by the flagellate protozoan Trypanosoma cruzi. The central nervous system (CNS) is an important site for T. cruzi persistence in the host during the chronic phase of infection, because the protozoan may pass the blood-brain barrier and may cause motor and cognitive neuronal damage. Thinking about avoiding or minimizing these negative effects, it is hypothesized that resveratrol (RSV), a component with several medicinal properties has beneficial effects on the CNS. The objective of this study was to investigate, whether T. cruzi infection interferes with neurogenesis and gliogenesis of embryos of infected mice females, and whether RSV would be able to avoid or minimize these changes caused by CD. RSV is a polyphenol found in grapes and widely studied for its neuroprotective and antioxidant properties. In addition, we investigated the role caused by the parasite during congenital infection and CNS development. Embryos and their brains were PCR-positive for T. cruzi. For this study, NPCs obtained from telencephalon of infected and uninfected embryos and were cultured in presence of resveratrol for forming neurospheres. The results demonstrated that the congenital transmission of T. cruzi influences CNS formation and neural fate, decreasing the number of neuroespheres and causing an elongation in the phases of the cell cycle. In addition, the parasite promoted an increase in neugliogenesis. Resveratrol was neuroprotective and prevented negative effects of the infection. Thus, we suggest the use of resveratrol as a therapeutic target for the treatment of neuroinflammation or as neuroprotective agent during Chagas disease, as it improves gliogenesis and restores neural migration.

Rutin and curcumin reduce inflammation, triglyceride levels and ADA activity in serum and immune cells in a model of hyperlipidemia.

Hyperlipidemia is associated with endothelial dysfunction and inflammatory disorders. Adenosine and adenosine deaminase (ADA) modulate immune responses and lipid metabolism. Curcumin and rutin are polyphenols with antioxidant, anti-inflammatory, and hypolipidemic effects. We evaluated the action of rutin and curcumin in the lipid levels and inflammation, as well as their effect on ADA activity in serum, lymphocytes, platelets, and neutrophils of hyperlipidemic rats. Adult male Wistar rats pretreated with curcumin and/or rutin for 30 days were submitted to Poloxamer-407- induced hyperlipidemia. Biochemical, hematological, and oxidative stress parameters, as well as serum and extracellular ADA activity, were performed 36h post-induction. Hyperlipidemia was confirmed by the increase in total cholesterol (TC) and triglycerides (TG). Hematological alterations, elevated reactive oxygen species (ROS) levels, and increased myeloperoxidase (MPO) and ADA activities were observed in hyperlipidemic rats. Curcumin and the curcumin/rutin association decreased TG and increased high-density lipids (HDL) levels. The pretreatments prevented changes in the hematological parameters, decreased the activities of MPO in plasma and ADA in serum and cells. Cholesterol and ROS levels were not altered by the pretreatments. Our results show that pretreatments with rutin and/or curcumin prevent the hyperlipidemia-induced inflammation. Pretreatments with curcumin and/or rutin are potential complementary therapies in the prevention of hypertriglyceridemia and inflammation.

Anti-erythrocyte IgG in hamsters with acute experimental infection by Leptospira interrogans serovar Canicola.

The aim of this study was to evaluate the behavior of erythrocyte and platelet, as immunological markers, as well as evaluate the involvement of these factors in hemolytic and hemorrhagic reactions in hamsters experimentally infected by Leptospira interrogans Serovar Canicola. Our experimental design was composed by two randomized groups: Infected Group (IG) (n = 12) and control group (CG) (n = 6). Ninety-six hours after the inoculation, the presence of immunoglobulins (IgG and IgM) and complement C3 levels, related to erythrocytes and platelets, was assessed. Platelet's microparticles marked by CD61, reticulocytes and reticulated platelets were also quantified. Additionally, fibrinogen, prothrombin time, partially activated thromboplastin time and sera levels of IgG and IgM were assessed. Our results showed that levels of platelet decreased in IG (P < 0.001); as well as, there was presence of IgG and C3 associated with erythrocyte surface in the infected animals (P < 0.01, P < 0.05, respectively). Levels of prothrombin time and Activated Partial Thromboplastin Time were increased, while fibrinogen level was decreased (P < 0.01) in IG. CD61 microparticles were higher (P < 0.05) in IG due to platelet activation. Thus, it was established a positive correlation (P < 0.01) between platelets count and fibrinogen (Figure 3, R = 0.84, P < 0.001). Therefore, the platelet consumption component was preponderant in relation to autoimmune causes. Finally, regarding the erythrocytes, the autoimmune component played an important role, did not causing hemolytic reaction in this acute experimental time.

Neuroprotective effects of pretreatment with quercetin as assessed by acetylcholinesterase assay and behavioral testing in poloxamer-407 induced hyperlipidemic rats.

Hyperlipidemia is a group of disorders characterized by excessive lipids in the bloodstream. It is associated with the incidence of cardiovascular diseases and recognized as the most important factor underlying the occurrence of atherosclerosis. This study was conducted to investigate whether pretreatment with quercetin can protect against possible memory impairment and deterioration of the cholinergic system in hyperlipidemic rats. Animals were divided into ten groups (n=7): saline/control, saline/quercetin 5mg/kg, saline/quercetin 25mg/kg, saline/quercetin 50mg/kg, saline/simvastatin (0.04mg/kg), hyperlipidemia, hyperlipidemia/quercetin 5mg/kg, hyperlipidemia/quercetin 25mg/kg, hyperlipidemia/quercetin 50mg/kg and hyperlipidemia/simvastatin. The animals were pretreated with quercetin by oral gavage for a period of 30days and hyperlipidemia was subsequently induced by intraperitoneal administration of a single dose of 500mg/kg of poloxamer-407. Simvastatin was administered after the induction of hyperlipidemia. The results demonstrated that hyperlipidemic rats had memory impairment compared with the saline control group (P<0.001). However, pretreatment with quercetin and simvastatin treatment attenuated the damage caused by hyperlipidemia compared with the hyperlipidemic group (P<0.05). Acetylcholinesterase (AChE) activity in the cerebral hippocampus was significantly (P<0.001) reduced in the hyperlipidemic group compared with the control saline group. Pretreatment with quercetin and simvastatin treatment in the hyperlipidemic groups significantly (P<0.05) increased AChE activity compared with the hyperlipidemic group. Our results thus suggest that quercetin may prevent memory impairment, alter lipid metabolism, and modulate AChE activity in an experimental model of hyperlipidemia.

Impacts of dose and time of boldenone and stanazolol exposure in inflammatory markers, oxidative and nitrosative stress and histopathological changes in the rat testes.

The present study was conducted to analyze the adverse effects of the anabolic steroids boldenone (BOL) and stanazolol (ST) in the reproductive function of male rats. These molecules were administered using three different protocols. In Protocol I, BOL and ST were administered in a higher dose than what is recommended but for a short period. In Protocol II, a moderate dose of these compounds was applied for an intermediate period, whereas in Protocol III a reduced dose was administered but for an extended period. Notably, Protocol I and III resulted in increased levels of reactive oxygen specimens (ROS [I, p < 0.01] [III, p < 0.001)]) and nitrite plus nitrate (NOx [I, p < 0.01] [II, p < 0.01] [III,p < 0.05]), respectively, whereas non-protein thiols (NPSH) levels were decreased only after Protocol III (p < 0.01). Myeloperoxidase activity was significantly increased after treatment with BOL in protocol II (p < 0.01) and III (p < 0.05) than with ST in protocol III (p < 0.05). Boldenone and ST also caused a significant up-regulation in the levels of serum testosterone when protocols I (p < 0.01) and II (p < 0.05) were performed. There were also visible histopathological alterations in the testes induced by treatment with BOL, namely degenerative changes primarily characterized by a decrease in the germinal epithelium. Together, these results suggest that the administration of BOL or ST exerts a significantly harmful effect in the testes of male rats. Moreover, all the treatment protocols used in this study induced deleterious effects on the testes, as indicated by the different biochemical parameters investigated. However, only the protocols of longer exposure time (II and III) induced morphological changes compatible with infertility.

Curcumin attenuates memory deficits and the impairment of cholinergic and purinergic signaling in rats chronically exposed to cadmium.

This study investigated the protective effect of curcumin on memory loss and on the alteration of acetylcholinesterase and ectonucleotidases activities in rats exposed chronically to cadmium (Cd). Rats received Cd (1 mg/kg) and curcumin (30, 60, or 90 mg/kg) by oral gavage 5 days a week for 3 months. The animals were divided into eight groups: vehicle (saline/oil), saline/curcumin 30 mg/kg, saline/curcumin 60 mg/kg, saline/curcumin 90 mg/kg, Cd/oil, Cd/curcumin 30 mg/kg, Cd/curcumin 60 mg/kg, and Cd/curcumin 90 mg/kg. Curcumin prevented the decrease in the step-down latency induced by Cd. In cerebral cortex synaptosomes, Cd-exposed rats showed an increase in acetylcholinesterase and NTPDase (ATP and ADP as substrates) activities and a decrease in the 5'-nucleotidase activity. Curcumin was not able to prevent the effect of Cd on acetylcholinesterase activity, but it prevented the effects caused by Cd on NTPDase (ATP and ADP as substrate) and 5'-nucleotidase activities. Increased acetylcholinesterase activity was observed in different brain structures, whole blood and lymphocytes of the Cd-treated group. In addition, Cd increased lipid peroxidation in different brain structures. Higher doses of curcumin were more effective in preventing these effects. These findings show that curcumin prevented the Cd-mediated memory impairment, demonstrating that this compound has a neuroprotective role and is capable of modulating acetylcholinesterase, NTPDase, and 5'-nucleotidase activities. Finally, it highlights the possibility of using curcumin as an adjuvant against toxicological conditions involving Cd exposure. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 70-83, 2017.

Bradykinin-induced inhibition of proliferation rate during neurosphere differentiation: consequence or cause of neuronal enrichment?

Neural stem cells proliferate and differentiate into neurons and glial cells, being responsible for embryonic and postnatal development of the central nervous system (CNS) as well as for regeneration in the adult brain. These cells also play a key role in maintaining the physiological integrity of the CNS in face of injury or disease. The previous study has demonstrated that bradykinin (BK) treatment simultaneously induces neuronal enrichment (indicating that BK contributes to neurogenesis) and reduced proliferation rates during in vitro differentiation of rat embryonic telencephalon neural precursor cells (NPCs). Here, we provide a mechanism for the unresolved question whether (i) the low rate of proliferation is owed to enhanced neurogenesis or, conversely, (ii) the alteration of the population ratio could result from low proliferation of NPCs and glial cells. In agreement with the previous study, BK promoted neuron-specific ß3-tubulin and MAP2 expression in differentiating embryonic mouse neurospheres, whereas glial protein expression and global proliferation rates decreased. Furthermore, BK augmented the global frequency of cells in G0 -phase of cell cycle after differentiation. Heterogeneous cell populations were observed at this stage, including neurons that always remaining a quiescent state (G0 -phase). It is noteworthy that BK did not interfere with proliferation of any particular cell type, evidenced by coimmunostaining for nestin, ß3-tubulin, glial fibrillary acidic protein (GFAP), and 5-ethynyl-2'-deoxyuridine (EdU). Thus, we conclude that neuronal enrichment is owing only to the fostering of neurogenesis, and that the low proliferation rate on the seventh day of differentiation is a consequence and not the cause of BK-induced neuronal enrichment.