The influence of methylene blue infusion on cytokine levels during severe sepsis

The aim of our study was to assess the effect of methylene blue infusion on plasma levels of cytokines in severe sepsis. In a prospective, randomized, double-blind, placebo-controlled study, patients received either methylene blue 0.5 mg.kg-1.h-1 (MB group, n = 15) or similar volume of isotonic saline (control group, n = 15) i.v. for 6 hours. Plasma concentrations of tumour necrosis factor-alpha, interleukin-1, interleukin-2 receptor, interleukin-6, interleukin-8 were measured by sensitive immunoassays at basal (15 min before start of the study), immediately after, and at 24 and 48 hours after methylene blue infusion. We evaluated haemodynamic parameters (mean arterial pressure, heart rate), blood gases, methaemoglobin levels, and biochemical parameters at the same time. Methylene blue administration had no significant effect on plasma cytokine levels, blood gases and biochemical parameters. When compared to placebo infusion in controls, methylene blue administration resulted in significantly higher mean arterial pressure (85 +/- 14 mmHg vs 74.1 +/- 10.3 mmHg; P < 0.01), and methaemoglobin levels (1.06 +/- 0.22% vs 0.9 +/- 0.05%; P < 0.05). Furthermore, comparison with baseline levels revealed significantly increased both mean arterial pressure (85 +/- 14 mmHg and 74.1 +/- 10.2 mmHg; P < 0.05) and methaemoglobin levels (1.06 +/- 0.22% and 0.88 +/- 0.06%; P < 0.05) in MB group. There was no difference in mortality rates between the groups. We found that methylene blue infusion did not change cytokine levels or outcome in severe sepsis. The administration of methylene blue, however, resulted in a transient increase in arterial pressure. Because of the limited size of the present study, and the short period of observation, our findings need to be confirmed by larger clinical trials of methylene blue infused in a dose-titrated manner.     

Potential hazard of methylene blue

The administration of methylene blue to assist the identification of insulin secreting pancreatic adenomata, during surgery in a patient with normal haemoglobin and red cells, was associated with an increase in the methaemoglobin concentration from 0.6% to 7.1%. In patients with unstable haemoglobins or abnormalities of the hexose monophosphate pathway the administration of large amounts of methylene blue is potentially dangerous.     

THE CHEMISTRY OF THE IN VIVO REACTION BETWEEN HAEMOGLOBIN AND VARIOUS OXIDES OF NITROGEN

Massive conversion of oxyhaemoglobin to methaemoglobin has beenfound in a series of anaesthetized dogs exposed to nitric oxideor nitrogen dioxide in concentrations of 2 per cent, 100 percent conversion occurring in one case. Less marked changes occurredwith inspired concentrations in the range 0.1–0.5 percent. Reduction in oxygen capacity contributed to the deathof some dogs in the series. Almost complete reconversion tooxyhaemoglobin was accomplished with methylene blue. There wasno evidence for the formation of other compounds such as nitricoxide-haemoglobin, nitric oxide-methaemoglobin and nitrite-methaemoglobin.     

Methaemoglobinaemia due to accidental sodium nitrite poisoning. Report of 10 cases

Nitrates and nitrites are widely used in the food and chemical industry. Poisoning with these agents may be potentially life-threatening as a result of the production of methaemoglobin. A group of 10 patients suffering from moderate to severe methaemoglobinaemia after accidental intoxication with a sodium nitrite salt is described. One patient died but the other 9 recovered rapidly. The low mortality rate was attributed to prompt diagnosis and institution of appropriate therapy with methylene blue and ascorbic acid.     

Nitrite-induced methaemoglobinaemia - aetiology, diagnosis and treatment

A 41-year-old woman presented with the clinical features of methaemoglobinaemia after drinking Chinese herbal medicine. A life threatening methaemoglobin level of 68% was measured. Both clinical and laboratory diagnostic difficulties were encountered. The pitfalls of pulse oximeter, blood gas analysis and co-oximeter interpretation during diagnosis and after methylene blue administration are discussed.     

Reduction in formation of peritoneal adhesions by methylene blue in rats: a dose response study.

OBJECTIVE: To find out if the ability of methylene blue given intraperitoneally to reduce surgically-induced adhesions in rats is concentration dependent. DESIGN: Random, controlled prospective study. SETTING: Teaching hospital, Israel. ANIMALS: Seventy female Wistar rats. INTERVENTIONS: Intraperitoneal adhesions were generated in rats by scraping the anterior wall of the uterine horn. They were then given either saline or serially diluted methylene blue intraperitoneally. Additional rats received identical treatments, but without the serosal damage. OUTCOME MEASURES: Graded formation of adhesions, two weeks later. RESULTS: In the rats treated with methylene blue 1%, adhesion formation was similar to that in sham operated rats and was significantly lower (p < 0.001) than in all other groups. CONCLUSION: Methylene blue 1%, and to lesser degree 0.5%, had the best anti-adhesion potential. Lower methylene blue concentrations were only partially effective, and much high concentrations may cause adverse effects.     

Methylene blue in the evaluation of gastrointestinal tract integrity: potential limitations

INTRODUCTION: The integrity of the gastrointestinal tract can be evaluated by oral or rectal application of methylene blue. In the presence of anastomotic leaks or fistulas, methylene blue can be recovered in adjacent drains. However, parts of the dye can biochemically be reduced by intestinal bacteria to its colorless form leucomethylene blue, limiting the prediction of the test. MATERIALS AND METHODS: Diluted methylene blue was added to different concentrations of bacterial suspensions of Escherichia coli and Enterococcus faecalis. The time for discoloration of the suspension was measured. RESULTS: Reduction of methylene blue to leucomethylene blue was observed in both types of bacterial suspension. A 10(8) bacterial concentration discolorated the dye within 1 h in the E. faecalis suspension, respectively 2.5 h in the E. coli suspension. Longer bacterial interaction with methylene blue reduced the bacterial concentration required to achieve complete discoloration. DISCUSSION: Methylene blue can reliably be used as routine diagnostic test for the assessment of upper gastrointestinal integrity, where bacterial load is low. In the lower gastrointestinal tract, where bacterial load is generally higher, the dye can indicate leaks, only if extended intestinal passage after oral ingestion is avoided. In all other cases, the examiner has to be aware of false-negative results by bacterial discoloration of methylene blue.     

Patent blue versus methylene blue and indigo carmine as a better dye for chromodiscography: in vitro staining efficacy and cytotoxicity study using bovine coccygeal intervertebral discs

BACKGROUND CONTEXTChromodiscography is an integral part of full-endoscopic discectomy (FED), comprising ordinary discography with radiopacity produced by contrast medium and intradiscal stain for visualizing annular defects in the endoscopic field. Nevertheless, concerns remain about the cytotoxicity of the stains used. The study of their staining efficacy is also lacking.PURPOSETo evaluate the feasibility of methylene blue, patent blue, and indigo carmine for intradiscal injection, investigate the effectiveness of each dye, and define critical concentration with adequate staining efficacy and tolerable cytotoxicity for use in chromodiscography during FED.STUDY DESIGNAn experimental in vitro study.METHODSDye stock solutions were prepared from powder. The stock was diluted with culture medium or balanced saline and used for cytotoxicity or intervertebral disc staining assays, respectively. Bovine tails were obtained from the local slaughterhouse and functional spine units of intervertebral discs were acquired by transverse incision at the disc level. Each disc was punctured over the posterolateral aspect using a surgical knife to simulate an annular defect. The intradiscal injection was performed with each dye at different concentrations using a 22G needle from the contralateral aspect of the punctured site. Staining efficacy was quantified using ImageJ software. Primary cells of bovine tails were cultivated in each dye at different concentrations. Cytotoxicity was assessed 24 hours after stain exposure using the CCK-8 toxicity assay.RESULTSStaining efficacy and cytotoxicity were proportional to the concentration of tested dyes. Lower limits of concentration producing significant staining efficacy of indigo carmine, methylene blue, and patent blue were 0.25 mg/mL, 0.25 mg/mL, and 0.05 mg/mL, respectively. Compared with controls, concentrations showing significant toxicity for indigo carmine, methylene blue, and patient blue were 1 mg/mL, 0.5 mg/mL, and 2.5 mg/mL, respectively.CONCLUSIONSPatent blue can serve as a more suitable tissue stain than either indigo carmine or methylene blue due to the widest range of tradeoff concentration within 0.05 to 2.5 mg/mL.CLINICAL SIGNIFICANCEPatent blue with the characteristic of good staining efficacy and lower cytotoxicity may be a promising option for chromodiscography during FED.     

A study into the incidence of methaemoglobinaemia after ''three-in-one'' block with prilocaine

A 34-year-old female patient developed methaemoglobinaemia following a femoral nerve block using prilocaine. The concentrations of methaemoglobin in the blood of the next eight patients receiving this block were assayed, and found to be significantly increased above baseline concentrations, but very significantly less than the concentration in the patient with symptomatic methaemoglobinaemia. Pulse oximetry was of value in making the diagnosis.     

Bactericide effect of methylene blue associated with low-level laser therapy in <Emphasis Type="Italic">Escherichia coli</Emphasis> bacteria isolated from pressure ulcers

The present study analyzed the bactericidal effect of methylene blue associated with low-level lasers on Escherichia coli isolated from a pressure ulcer. Microbiological material from a pressure ulcer was isolated using an aseptic swab, and antimicrobial activity was verified using the diffusion disc method. Methylene blue was used at concentrations of 0.001 and 0.005%, and low-level lasers of 670, 830, and 904 nm, with the energy densities of 4, 8, 10, and 14 J/cm², were tested on three plates each and combined with methylene blue of each concentration. In addition, three control plates were used, with each concentration and energy density separated without any interventions. The results were analyzed using the paired sample t test to determine the bactericidal effect of the methylene blue and using the ANOVA test to compare the effects of the energy densities and wavelengths among the low-level laser treatment protocols. The results showed bacterial reduction at wavelengths of 830 and 904 nm and more proliferation in wavelengths of 670 nm. In wavelength of 830 nm, a bacterial reduction was observed in the conditions with 0.001% methylene blue in all energy density utilized, with 0.005% methylene blue in energy density of 10 J/cm², and without methylene blue in energy density at 10 J/cm². And in a wavelength of 904 nm, all condition showed bacterial reduction with or without methylene blue. We concluded that the low-level lasers of 904 and 830 nm have bactericidal effects and at better energy densities (10 and 14 J/cm²).     

The interaction of lipopolysaccharides with phenothiazine dyes

BACKGROUND AND OBJECTIVES: The difference in the photobactericidal efficacy of methylene blue and toluidine blue against gram-negative bacteria may result from their primary reaction with lipopolysaccharides (LPS) of the outer bacterial membrane. The aim of the present study was to compare the reactivity of these dyes with LPS extracted from different gram-negative bacteria. STUDY DESIGN/MATERIALS AND METHODS: The interactions of methylene blue and toluidine blue with LPS from Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae (K. pneumoniae), and Serratia marcescens (S. marcescens) were studied spectrophotometrically in 0.45% saline. The dyes were used at the concentration of 10 microM. The concentrations of LPS ranged from 5-100 microg/ml. RESULTS: Methylene blue and toluidine blue enter into a metachromatic reaction with the LPS resulting the in generation of dimers of methylene blue and higher aggregates of toluidine blue. The more significant hypochromic and hypsochromic effects in the reaction of the latter with LPS indicate a greater metachromatic efficacy of toluidine blue than methylene blue. The equilibrium constants of the metachromatic complex between toluidine blue and different LPS were calculated. The spectrophotometric titration of LPS with the dyes was used to estimate the equivalent weight of LPS. CONCLUSIONS: Toluidine blue interacts with LPS more significantly than methylene blue in vitro. This may be one of the main factors determining its greater photobactericidal efficacy against gram-negative bacteria.     

超声引导美蓝双重标记与经皮缝线牵引联合切除触诊阴性乳腺肿物

目的 探讨应用超声引导美蓝双重标记与经皮缝线牵引联合微创切除触诊阴性乳腺肿物的价值.方法 对46例直径0.5～1.0 cm的触诊阴性乳腺肿物,术前1 h超声引导下应用美蓝对肿物及肿物四周行双重标记,术中经皮缝线牵引外提肿物,行手术准确切除肿物.术后1个月超声复查有无残留或误切.结果 46例58个肿物均准确切除,无残留和误切.切除部分手术时间为10～20 min.结论 超声引导美蓝双重标记与经皮缝线牵引联合微创切除触诊阴性乳腺肿物,定位准确,手术时间短,创伤减轻,操作简单,便于普及和推广.     

Methylene blue as an inhibitor of stone formation

Summary Kinetics of growth and dissolution of calcium oxalate monohydrate were examined in the presence of small concentrations of methylene blue. The data presented show moderate retardation in growth and dissolution rates. It was also found that methylene blue decreased the decalcification rate of calcium oxalate renal calculi. The implications of these findings in the treatment of urolithiasis are discussed.This work was supported by the Medical Research Council of Canada.     

The effect of methylene blue on histological damage after spermatic cord torsion in a rat model.

OBJECTIVE: To investigate, in a rat model, whether methylene blue (a free-radical scavenger) is effective in alleviating the histological testicular damage caused by reperfusion after spermatic cord torsion. MATERIALS AND METHODS: Male Wistar rats underwent a clockwise 720 degrees left spermatic cord torsion lasting 3 h; they were then randomly assigned to treatment and control groups. In the treated group, 2 mg/kg methylene blue was injected intraperitoneally 20 min before the cord was untwisted, and an additional 1 mg/kg injected into the penile vein 1--2 min beforehand. After 14 days, the left testicle was removed and assessed histologically. The control group was not treated. RESULTS: Thirty-three rats in the treatment group and 35 untreated controls were suitable for evaluation. Treatment with methylene blue was not significantly predictive of histological damage; the colour of the testis before detorsion was not correlated with histological damage. CONCLUSION: Anti-oxidant treatment with methylene blue had no palliative effect on the histological changes after torsion and detorsion of the spermatic cord in this rat model.     

直肠充气试验＋美蓝试验在直肠癌TME术中的应用研究

目的探讨直肠充气试验十美蓝试验在直肠癌全直肠系膜切除（TME）术中检测吻合口漏的作用。方法2010年3月至2013年3月期间笔者所在医院科室收治直肠癌患者132例,将其随机分为直肠充气试验组（充气组）65例和直肠充气试验＋美蓝试验组（充气＋美蓝组）67例,2组均遵循TME原则行Dixon术。充气组在行TME术及肠吻合完毕后,用肠钳在吻合口上端2cm处钳夹肠管,于盆腔内倒人蒸馏水500mL;经肛门置入24#Foley尿管,向尿管球囊注水使球囊膨胀封闭肛门,使吻合肠段处于相对封闭状态;再经尿管注入气体50mL,观察水面有无气泡出现,若有气泡则表示存在吻合口漏,并予以修补,至无气泡出现。充气＋美蓝组患者则在充气组的基础上加行美蓝试验,吸出盆腔内的蒸馏水和直肠内气体后,经尿管注入美蓝1支（20mg）和生理盐水50mL,观察吻合口有无美蓝漏出。结果充气组术中发现吻合口漏3例（4．62％）,均给予了修补;术后发生吻合口漏9例（13．85％）,5例经充分引流、禁食及对症治疗后治愈,3例经肛门缝合修补后治愈,1例经横结肠造口、局部引流后治愈。充气＋美蓝组经充气试验发现吻合口漏2例,后经美蓝试验发现13例存在吻合口漏,共发现吻合口漏15例（22．39％）,均给予修补;术后无一例患者发生吻合口漏。与充气组比较,充气＋美蓝组术中吻合口漏的检出率较高（P〈0．05）,术后吻合口漏的发生率较低（P〈0．05）。结论充气试验可发现较大的吻合口漏,而美蓝试验可发现小的吻合口漏和隐性漏,两种试验联合应用可发现吻合口缺陷与漏;直视下修补可有效防止TME术后吻合口漏的发生。     

Die methämoglobinämie bei verbrennungen

Zusammenfassung 5 Fälle von Met-Hb-ämie nach Verbrennungen über 15% der gesamten Körperoberfläche werden beschrieben. Es wird auf ein direkte toxische, jedoch reversible Ursache auf Grund der Verbrennungskrankheit hingewiesen und schließlich eine prophylaktische Therapie mit hohen Dosen von Vitamin C täglich empfohlen.

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Methaemoglobinaemia in burns

Summary The diagnosis of methaemoglobinaemia after burns is often very difficult because of its lack of clinical symptoms. In a series of 64 patients (including 31 children) with burns of over 15% of the whole body surface, 5 children with a methaemoglobinaemia have been diagnosed and treated. Only in one patient, a four years old girl with 20% of burned body surface, were all clinical symptoms observed, while the methaemoglobin in the blood rose to 57%. The therapy in this case consisted of 1 mg/kg body weight/day of methylene blue and 20 g of Vitamin C intravenously. The grave and dangerous symptoms dissapeared after 5 hours. For the next 20 days the therapy was continued by high dosage of Vitamin C intravenously till the methaemoglobin in the blood became normal.The other four cases were treated with high dosage of Vitamin C intravenously (4 to 8 g/d) until the Met-Hb concentration in the blood fell to normal level. A hereditary defect of the erythrocytes, which is considered to be the most frequent cause of this disease, has been excluded through laboratory investigations of the various enzymes. Amino-benzol-sulfonamide has been used only in two cases, but the content of sulfonamide in the urine was not increased.In conclusion, we consider methaemoglobinaemia following burns (especially in children) to be a not very rare complication, and is a reversible toxical consequence of burns caused by the oxydation of haemoglobin.Therefore we recommend for all cases of burns (over 10 to 15%) a prophylactic therapy of 4 to 5 g/d Vitamin C intravenously.

     

左布比卡因和罗哌卡因与亚甲蓝的配比液对肛周术后镇痛效果的比较研究

为比较左布比卡因和罗哌卡因与亚甲蓝的配比液在肛周手术后的镇痛效果,探讨肛周手术后最佳的镇痛方式,将196例肛门手术病人随机分为A组和B组各98例,按照3：2的等效比率,A组使用0．75％罗哌卡因5ml加1％亚甲蓝2ml的配比液,B组使用0．5％左布比卡因5ml加1％亚甲蓝2ml的配比液,组成长效镇痛剂进行肛周皮下局部注射。在术后1～7d采用疼痛视觉评分系统（VAS）对病人疼痛程度进行评分。结果显示,术后1～3dA组与B组VAS评分无显著性差异（P〉0．05）,术后4～7dB组VAS评分低于A组（P〈0．05）。结果表明,用左布比卡因与亚甲蓝的配比液能够更好地进行肛周术后的镇痛。     

Oxalate toxicity in cultured mouse inner medullary collecting duct cells

PURPOSE: Oxalate, a metabolic end product and a major constituent of the majority of renal stones, has been shown to be toxic to renal epithelial cells of cortical origin. However, to our knowledge it is unknown whether inner medullary collecting duct (IMCD) cells, which are physiologically exposed to higher concentrations of oxalate, also behave in a similar manner. MATERIALS AND METHODS: A line of IMCD cells was exposed to oxalate (0.2 to 10 mM) for various time points. Trypan blue, and hematoxylin and eosin stains were used to assess cell morphology and membrane integrity. The production of reactive oxidative species was determined using the nitro blue tetrazolium reaction and crystal violet staining was used to measure cell density. RESULTS: Exposure of IMCD cells to oxalate produced time and concentration dependent changes in the light microscopic appearance of the cells. Long-term exposure to oxalate resulted in alterations in cell viability with net cell loss following exposure to concentrations of 2 mM and greater. Free radical production was time and concentration dependent. Crystal formation occurred in less than 1 hour and cells in proximity to crystals lost membrane integrity. Compared to IMCD cells LLC-PK1 and HK2 cells showed significant toxicity starting at lower oxalate concentrations (0.4 mM and above). CONCLUSIONS: To our knowledge the results provide the first direct demonstration of toxic effects of oxalate in IMCD cells, a line of renal epithelial cells of the inner medullary collecting duct, and suggest that cells lining the collecting duct are relatively resistant to oxalate toxicity.     

The effect of biological dyes and contrast media on the vas deferens in Long Evans rats

The conventional diagnostic procedure of vasography utilizes a contrast medium to evaluate the patency of the vas deferens. With the development of microsurgical reconstruction for obstructive azoospermia in the past two decades, intraoperative vasography with saline or biological dye injection has replaced the use of radiographic contrast media. However, there are few reports on the effect of biological dyes on the healthy vas deferens. Therefore, we used experimental vasography to evaluate histological changes and functional patency of the vas deferens after infusion with a contrast medium and biological dye.
Four groups of 10 Long Evans male rats were injected by vasopuncture with 1% methylene blue, 1% gentian violet and 38% Urografin or saline into the vas deferens. The animals were killed 30 days later, and the vasa deferentia were excised and examined for histological changes and for functional patency. Vasopuncture with saline injection induced minimal change both at the puncture site and in the distal vas deferens. In both the Urografin- and methylene blue-injected groups, inflammation at the puncture site was found in 20–22% of cases, and 10–11% of cases revealed functional obstruction of the vasal lumen. In the gentian violet-injected group, severe histological and obliterated changes were found in all cases. Leakage of the dye and contrast medium or the sperm reaction may be responsible for the inflammation; otherwise, methylene blue and urografin did not seem to be harmful to the vas deferens. Although gentian violet is a blue dye, as is methylene blue, it has marked destructive effects on the vas deferens. It is concluded that some biological dyes used for vasal injection can cause occlusion of the vasal lumen, while inflammatory responses can occur from placing a needle transmurally.     

直肠上动脉亚甲蓝灌注法判断全直肠系膜切除术后标本系膜的完整性

目的探讨经直肠上动脉灌注亚甲蓝对低位直肠癌行全直肠系膜切除术（TME）后标本系膜完整性判断的临床意义。方法将按照TME原则进行根治性手术的20例低位直肠癌患者的切除标本．按随机数字表法分为亚甲蓝组和常规检测组．每组10例。常规检测组术后肉眼观察直肠系膜完整性情况．亚甲蓝组经直肠上动脉灌注肝素和亚甲蓝．观察有无亚甲蓝从系膜表面溢出．并进一步对亚甲蓝溢出处系膜及系膜淋巴结情况进行镜检。结果所有病例标本肉眼下观察直肠系膜均完整．亚甲蓝组经直肠上动脉灌注亚甲蓝后，直肠系膜染色明显．8例直肠系膜面无亚甲蓝溢出．提示直肠系膜完整．2例出现亚甲蓝溢出，提示系膜存在肉眼观察无法辨别的缺损，并在镜下得到证实。此外，亚甲蓝组淋巴结平均检出数目（17．3±2．4）枚，而常规检测组为（12．4±5．4）枚，两组差异有统计学意义（P=0．016）。结论结直肠外科医生应在TME术后常规对直肠系膜完整性进行细致评价，亚甲蓝灌注染色是判断系膜微小缺损的一种简便、有效的方法．并能提高淋巴结检出率。