Reversal by methylene blue of tetanic fade induced in cats by nitric oxide

Previous data from our laboratory have indicated that nitric oxide (NO) acting at the presynaptic level increases the amplitude of muscular contraction (AMC) of the phrenic-diaphragm preparations isolated from indirectly stimulated rats, but, by acting at the postsynaptic level, it reduces the AMC when the preparations are directly stimulated. In the present study we investigated the effects induced by NO when tetanic frequencies of stimulation were applied to in vivo preparations (sciatic nerve-anterior tibial muscle of the cat). Intra-arterial injection of NO (0.75-1.5 mg/kg) induced a dose-dependent increase in the Wedensky inhibition produced by high frequencies of stimulation applied to the motor nerve. Intra-arterial administration of 7.2 µg/kg methylene blue did not produce any change in AMC at low frequencies of nerve stimulation (0.2 Hz), but antagonized the NO-induced Wedensky inhibition. The experimental data suggest that NO-induced Wedensky inhibition may be mediated by the guanylate cyclase-cGMP pathway

Blockade of the action of nitric oxide in human septic shock increases systemic vascular resistance and has detrimental effects on pulmonary function after a short infusion of methylene blue

To investigate the role of nitric oxide in human sepsis, ten patients with severe septic shock requiring vasoactive drug therapy and mechanical ventilation were enrolled in a prospective, open, non-randomized clinical trial to study the acute effects of methylene blue, an inhibitor of guanylate cyclase. Hemodynamic and metabolic variables were measured before and 20, 40, 60, and 120 min after the start of a 1-h intravenous infusion of 4 mg/kg of methylene blue. Methylene blue administration caused a progressive increase in mean arterial pressure (60 [55-70] to 70 [65-100] mmHg, median [25-75th percentiles]; P<0.05), systemic vascular resistance index (649 [479-1084] to 1066 [585-1356] dyne s-1 cm-5 m-2; P<0.05) and the left ventricular stroke work index (35 [27-47] to 38 [32-56] g m-1 m-2; P<0.05) from baseline to 60 min. The pulmonary vascular resistance index increased from 150 [83-207] to 186 [121-367] dyne s-1 cm-5 m-2 after 20 min (P<0.05). Mixed venous saturation decreased from 65 [56-76] to 63 [55-69]% (P<0.05) after 60 min. The PaO2/FiO2 ratio decreased from 168 [131-215] to 132 [109-156] mmHg (P<0.05) after 40 min. Arterial lactate concentration decreased from 5.1 ± 2.9 to 4.5 ± 2.1 mmol/l, mean ± SD (P<0.05) after 60 min. Heart rate, cardiac filling pressures, cardiac output, oxygen delivery and consumption did not change. Methylene blue administration was safe and no adverse effect was observed. In severe human septic shock, a short infusion of methylene blue increases systemic vascular resistance and may improve myocardial function. Although there was a reduction in blood lactate concentration, this was not explained by an improvement in tissue oxygenation, since overall oxygen availability did not change. However, there was a significant increase in pulmonary vascular tone and a deterioration in gas exchange. Further studies are needed to demonstrate if nitric oxide blockade with methylene blue can be safe for patients with septic shock and, particularly, if it has an effect on pulmonary function.

Anxiolytic effect of methylene blue microinjected into the dorsal periaqueductal gray matter

The dorsal periaqueductal gray (DPAG) has been implicated in the behavioral and autonomic expression of defensive reactions. Several results suggest that, along with GABA, glutamate and serotonin, nitric oxide (NO) may play a role in defense reactions mediated by this region. To further investigate this possibility we microinjected methylene blue (MB; 10, 30 or 100 nmol/0.5 µl) into the DPAG of rats submitted to the elevated plus-maze test, an animal model of anxiety. MB has been used as an inhibitor of soluble guanylate cyclase (sGC) to demonstrate cGMP-mediated processes, and there is evidence that NO may exert its biological effects by binding to the heme part of guanylate cyclase, causing an increase in cGMP levels. The results showed that MB (30 nmol) significantly increased the percent of time spent in the open arms (saline = 11.57 ± 1.54, MB = 18.5 ± 2.45, P<0.05) and tended to do the same with the percentage of open arm entries (saline = 25.8 ± 1.97, MB = 33.77 ± 3.07, P<0.10), but did not change the number of enclosed arm entries. The dose-response curve, however, had an inverted U shape. These results indicate that MB, within a limited dose range, has anxiolytic properties when microinjected into the DPAG.

Kallikrein-like amidase activity in renal ischemia and reperfusion

We assessed a kallikrein-like amidase activity probably related to the kallikrein-kinin system, as well as the participation of leukocyte infiltration in renal ischemia and reperfusion. Male C57BL/KSJmdb mice were subjected to 20 or 60 min of ischemia and to different periods of reperfusion. A control group consisted of sham-operated mice, under similar conditions, except for ischemia induction. Kallikrein-like amidase activity, Evans blue extravasation and myeloperoxidase activity were measured in kidney homogenates, previously perfused with 0.9% NaCl. Plasma creatinine concentration increased only in the 60-min ischemic group. After 20 min of ischemia and 1 or 24 h of reperfusion, no change in kallikrein-like amidase activity or Evans blue extravasation was observed. In the mice subjected to 20 min of ischemia, edema was evident at 1 h of reperfusion, but kidney water content returned to basal levels after 24 h of reperfusion. In the 60-min ischemic group, kallikrein-like amidase activity and Evans blue extravasation showed a similar significant increase along reperfusion time. Kallikrein-like amidase activity increased from 4 nmol PNA mg protein-1 min-1 in the basal condition to 15 nmol PNA mg protein-1 min-1 at 10 h of reperfusion. For dye extravasation the concentration measured was near 200 µg of Evans blue/g dry tissue in the basal condition and 1750 µg of Evans blue/g dry tissue at 10 h of reperfusion. No variation could be detected in the control group. A significant increase from 5 to 40 units of DAbs 655 nm g wet tissue-1 min-1 in the activity of the enzyme myeloperoxidase was observed in the 60-min ischemic group, when it was evaluated after 24 h of reperfusion. Histological analysis of the kidneys showed migration of polymorphonuclear leukocytes from the vascular bed to the interstitial tissue in the 60-min ischemic group after 24 h of reperfusion. We conclude that the duration of ischemia is critical for the development of damage during reperfusion and that the increase in renal cortex kallikrein-like amidase activity probably released from both the kidney and leukocytes may be responsible, at least in part, for the observed effects, probably through direct induction of increased vascular permeability.

Environmental blue light prevents stress in the fish Nile tilapia

The present study aimed to test the effects of blue, green or white light on the stress response of the Nile tilapia, Oreochromis niloticus (L.). Each color was tested on two groups of isolated adult Nile tilapia (8 replicates each): one being subjected to confinement stress, and the other not (control). A different environmental color was imposed on each compartment by covering the light source with cellophane of the respective color (green or blue; no cellophane was used for white light). The intensity of green, white and blue lights was 250, 590 and 250 lux, respectively. Basal plasma cortisol levels were determined for each fish prior to the experimental procedures. The fish were confined by being displaced toward one side of the aquarium using an opaque partition for 1 h both in the morning and the afternoon of the two consecutive days of the test. At the end of this 48-h period, plasma cortisol levels were measured again. Basal cortisol levels (ng/ml) were similar for each group (ANOVA, F(2;42) = 0.77, P = 0.47). Thus, plasma cortisol levels were analyzed in terms of variation from their respective basal level. After confinement, plasma cortisol levels were not increased in fish submitted to a blue light environment. Thus, blue light prevents the confinement-induced cortisol response, an effect not necessarily related to light intensity.

Evaluation by blue native polyacrylamide electrophoresis colorimetric staining of the effects of physical exercise on the activities of mitochondrial complexes in rat muscle

Blue native polyacrylamide electrophoresis (BN-PAGE) is a technique developed for the analysis of membrane complexes. Combined with histochemical staining, it permits the analysis and quantification of the activities of mitochondrial oxidative phosphorylation enzymes using whole muscle homogenates, without the need to isolate muscle mitochondria. Mitochondrial complex activities were measured by emerging gels in a solution containing all specific substrates for NADH dehydrogenase and cytochrome c oxidase enzymes (complexes I and IV, respectively) and the colored bands obtained were measured by optique densitometry. The objective of the present study was the application of BN-PAGE colorimetric staining for enzymatic characterization of mitochondrial complexes I and IV in rat muscles with different morphological and biochemical properties. We also investigated these activities at different times after acute exercise of rat soleus muscle. Although having fewer mitochondria than oxidative muscles, white gastrocnemius muscle presented a significantly higher activity (26.7 ± 9.5) in terms of complex I/V ratio compared to the red gastrocnemius (3.8 ± 0.65, P < 0.05) and soleus (9.8 ± 0.9, P < 0.001) muscles. Furthermore, the complex IV/V ratio of white gastrocnemius muscle was always significantly higher when compared to the other muscles. Ninety-five minutes of exhaustive physical exercise induced a decrease in complex I/V and complex IV/V ratios after all resting times (0, 3 and 6 h) compared to control (P < 0.05), probably reflecting the oxidative damage due to increasing free radical production in mitochondria. These results demonstrate the possible and useful application of BN-PAGE-histochemical staining to physical exercise studies.

A model of hemorrhagic cystitis induced with acrolein in mice

Acrolein is a urinary metabolite of cyclophosphamide and ifosfamide, which has been reported to be the causative agent of hemorrhagic cystitis induced by these compounds. A direct cytotoxic effect of acrolein, however, has not yet been demonstrated. In the present study, the effects of intravesical injection of acrolein and mesna, the classical acrolein chemical inhibitor, were evaluated. Male Swiss mice weighing 25 to 35 g (N = 6 per group) received saline or acrolein (25, 75, 225 µg) intravesically 3, 6, 12, and 24 h before sacrifice for evaluation of bladder wet weight, macroscopic and histopathological changes by Gray's criteria, and 3 and 24 h for assessment of increase in vascular permeability. In other animals, mesna was administered intravesically (2 mg) or systemically (80 mg/kg) 1 h before acrolein. Intravesical administration of acrolein induced a dose- and time-dependent increase in vascular permeability and bladder wet weight (within 3 h: 2.2- and 21-fold increases in bladder wet weight and Evans blue dye exuded, respectively, at doses of 75 µg/bladder), as confirmed by Gray's criteria. Pretreatment with mesna (2-mercaptoethanesulfonic acid), which interacts with acrolein resulting in an inactive compound, inhibited all changes induced by acrolein. Our results are the first demonstration that intravesical administration of acrolein induces hemorrhagic cystitis. This model of acrolein-induced hemorrhagic cystitis in mice may be an important tool for the evaluation of the mechanism by which acrolein induces bladder lesion, as well as for investigation of new uroprotective drugs.

Hind limb ischemic preconditioning induces an anti-inflammatory response by remote organs in rats

Ischemic preconditioning (IPC), a strategy used to attenuate ischemia-reperfusion injury, consists of brief ischemic periods, each followed by reperfusion, prior to a sustained ischemic insult. The purpose of the present study was to evaluate the local and systemic anti-inflammatory effects of hind limb IPC in male Wistar rat (200-250 g) models of acute inflammation. IPC was induced with right hind limb ischemia for 10 min by placing an elastic rubber band tourniquet on the proximal part of the limb followed by 30 min of reperfusion. Groups (N = 6-8) were submitted to right or left paw edema (PE) with carrageenan (100 µg) or Dextran (200 µg), hemorrhagic cystitis with ifosfamide (200 mg/kg, ip) or gastric injury (GI) with indomethacin (20 mg/kg, vo). Controls received similar treatments, without IPC (Sham-IPC). PE is reported as variation of paw volume (mL), vesical edema (VE) as vesical wet weight (mg), vascular permeability (VP) with Evans blue extravasation (µg), GI with the gastric lesion index (GLI; total length of all erosions, mm), and neutrophil migration (NM) from myeloperoxidase activity. The statistical significance (P < 0.05) was determined by ANOVA, followed by the Tukey test. Carrageenan or Dextran-induced PE and VP in either paw were reduced by IPC (42-58.7%). IPC inhibited VE (38.8%) and VP (54%) in ifosfamide-induced hemorrhagic cystitis. GI and NM induced by indomethacin were inhibited by IPC (GLI: 90.3%; NM: 64%). This study shows for the first time that IPC produces local and systemic anti-inflammatory effects in models of acute inflammation other than ischemia-reperfusion injury.

Trypan blue exclusion assay by flow cytometry

Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.

Recuperação da solução de soda cáustica usada no tratamento do couro bovino na produção de gelatina

No tratamento de couro bovino para a produção de gelatina utiliza-se uma solução de soda cáustica com função de dissolver substâncias orgânicas indesejáveis, como proteínas e gorduras. Para evitar seu descarte como efluente, procurou-se viabilizar um processo de purificação da soda cáustica, evitando seu desperdício e ainda tornando-o adequado para reutilização no processo. A microfiltração, a ultrafiltração e a nanofiltração são técnicas potenciais para esta separação, dependendo do tipo e tamanho dos sólidos existentes. Experimentos de ultrafiltração foram realizados na unidade de micro/ultrafiltração Koch Membrane System Model Protosep modified IV, nas pressões transmembrana de 2,5; 3,5 e 4,5 kgf/cm² e temperaturas de 25 e 50 °C. Utilizaram-se membranas cerâmicas (material TiO2/alfa-Al2O3) tubulares com diâmetro médio de corte de 0,01, 0,05 e 0,10 µm. O trabalho foi dividido em duas etapas: na primeira selecionou-se a melhor pressão para cada membrana, e na segunda adotou-se a pressão de 3,5 kgf/cm², usou-se uma alimentação centrifugada e outra peneirada para então definir a membrana. As melhores condições operacionais foram determinadas em termos de fluxo de permeado e qualidade de produto. Com os resultados obtidos, observaram-se as melhores condições operacionais: pressão de 3,5 kgf/cm², temperatura de 25 °C e membrana com diâmetro médio de poros de 0,01 µm.

Effect of different calcium sources on the antioxidant stability of tortilla chips from extruded and nixtamalized blue corn (Zea mays L.) flours

This research aimed to develop tortilla chips (TC) high in antioxidants from extruded and nixtamalized blue corn flours prepared with calcium hydroxide Ca(OH)2 and calcium lactate C6H10O6Ca. Tortilla chips were made with extruded flours [0.1% Ca(OH)2; 0.9% C6H10O6Ca; without calcium] and nixtamalized flours [1% Ca(OH)2; 2.95% C6H10O6Ca] using the frying process. Total anthocyanin, total phenolics content, antioxidant activity, color, texture, and oil content were determined. The color of tortilla chips from extruded flours (TCEF) showed high values of the parameters a* and b* indicating a reduction in the blue color. These color parameters were significantly different from those observed in tortilla chips from nixtamalized flours (TCNF), which tended to be more blue. The TCEF retained 15% anthocyanins, 34% phenolics, and 54% antioxidant activity. Pearson's correlation analysis indicated that anthocyanins and phenolics correlated significantly with antioxidant activity and color. TCEF with both calcium sources showed higher fracturability compared with that of TCNF. Oil absorption showed an opposite effect, with lower oil content in TCEF. Nixtamalization and extrusion with C6H10O6Ca resulted in flours and TC high in anthocyanins and antioxidant activity, representing an alternative production process for corn snack high in antioxidants.

Quality changes during frozen storage of blue shrimp (Litopenaeus stylirostris) with antioxidant, α-tocopherol, under different conditions

Fresh blue shrimp (Litopenaeus stylirostris) muscle was stored with antioxidants under different conditions: ANTIOX 2%, packed in bilayer film of polyamide-low density polyethylene film (PA-LDPE) with 2% α-tocopherol; ANTIOX 4%, packed in PA-LDPE film with 4% α-tocopherol; and ANTIOX-GLAZED, samples stored glazed with 2% α-tocopherol. Shrimps packed in PA-LDPE without α-tocopherol were used as CONTROL. All samples were stored at –20 °C for 120 days. As compared to the CONTROL, the shrimp stored with the antioxidant showed lower lipid oxidation (0.10-0.14 vs 1.58 mgMA/kg of muscle), lost less firmness and astaxanthin content. ANTIOX 2% and ANTIOX-GLAZED showed the lowest concentrations of formaldehyde (0.081-0.083 μM/g). There were no significant differences in color and sensory properties, but differences in the integrity of the muscle fibers were observed. The treatments with α-tocopherol maintained the shrimp muscle quality during frozen storage. However, no significant differences were found between these treatments.