Red blood cell transfusion-induced non-transferrin-bound iron promotes Pseudomonas aeruginosa biofilms in human sera and mortality in catheterized mice.

Transfusion of storage-damaged red blood cells (RBCs) increases non-transferrin-bound iron (NTBI) levels in humans. This can potentially enhance virulence of microorganisms. In this study, Pseudomonas aeruginosa replication and biofilm production in vitro correlated with NTBI levels of transfused subjects (R2 = 0·80; P < 0·0001). Transfusion of stored RBCs into catheterized mice enhanced P. aeruginosa virulence and mortality in vivo, while pre-administration of apotransferrin reduced NTBI levels improving survival (69% vs 27% mortality; P < 0·05). These results suggest that longer RBC storage, by modulating the bioavailability of iron, may increase the risk of P. aeruginosa biofilm-related infections in transfused patients.

Testing Alternative Surface Disinfection Agents for Zebrafish (Danio rerio) Embryos.

Pathogen transmission into zebrafish colonies is controlled through vigilant biosecurity practices. One such practice is embryo surface disinfection, which often uses sodium hypochlorite. However, if sodium hypochlorite is used at an inappropriate pH, concentration, or exposure time, zebrafish embryos can experience significant mortality and morbidity. Reagent-grade sodium hypochlorite is often used for embryo surface disinfection because commercial-grade sodium hypochlorite has additional ingredients that may have deleterious effects on the embryo. In addition, chlorine dioxide and the combination of sodium chloride and potassium peroxymonosulfate (SCPP) are effective equipment disinfectants; however, the effects of these chemical agents on zebrafish embryos during surface disinfection are unknown. In this study, we exposed strain 5D zebrafish embryos (ages, 6 and 24 h postfertilization) to 4 chlorine-containing agents (reagent-grade sodium hypochlorite [bleach], commercial-grade sodium hypochlorite [bleach], SCPP, and chlorine dioxide) at either 50- or 100- ppm for 5 or 10 min. All groups were evaluated at 5 d postfertilization for survival, hatching rate, and morphologic defect rate. The experimental group with the highest survival and hatching rates and the lowest morphologic defect rate was the 24-h postfertilization embryos exposed to 50 ppm SCPP for 5 min. The survival, hatching rate, and defect rate did not differ significantly among age-matched controls; however, the hatching rate after exposure to 50 ppm SCPP was significantly higher than that of embryos exposed to 50 ppm reagent-grade sodium hypochlorite for 5 min (100% compared with 23% respectively). SCPP solution may provide an alternative surface disinfectant for zebrafish embryos because it maximizes survival and hatching rates and minimizes morphologic defect rates. However, in vivo efficacy against common zebrafish pathogens requires further testing. Use of chlorine dioxide at 50 ppm or greater is not recommended for zebrafish embryo surface disinfection due to significant mortality among 6 and 24 h postfertilization embryos.

Effect of 3 Euthanasia Methods on Serum Yield and Serum Cortisol Concentration in Zebrafish (Danio rerio).

Zebrafish are an important model in neuroscience and developmental biology and are also an emerging model in hematology and immunology. Little information is available for zebrafish regarding the physiologic impact of different euthanasia methods and whether a chosen method of euthanasia can impact serum yield. These parameters could impact the choice of euthanasia method for a study. To that end, the current study compared 3 methods of adult zebrafish euthanasia and their effects on 3 distinct criteria; time to loss of opercular movement, volume of serum obtained, and serum cortisol concentration. Blood was collected using a postmortem tail amputation and centrifugation blood collection technique. Time to loss of opercular movement differed significantly among euthanasia methods, with animals undergoing rapid chilling displaying the shortest time (mean Rapid Chilling: 40 s; Benzocaine: 86 s; MS222: 96 s). All methods of euthanasia resulted in a comparable average serum yield (Rapid Chilling = 7.5 µL; Benzocaine = 8.5 µL; MS222 = 7.5 µL per fish). None of the euthanasia methods tested resulted in average cortisol concentrations above the reported physiologic range. Although no significant differences were observed in serum yield or serum cortisol concentration, rapid chilling remains the preferred method for painless, humane euthanasia.

Sustained-Release Buprenorphine Improves Postsurgical Clinical Condition but Does Not Alter Survival or Cytokine Levels in a Murine Model of Polymicrobial Sepsis.

Cecal ligation and perforation (CLP) is a common technique for studying sepsis in mice. Because of the invasiveness of the procedure and its effects on clinical condition, many animal care and use committees require the use of analgesics with CLP. However, some analgesics have immunomodulatory effects and thus can hinder the overall research outcomes of a project. Here we sought to determine the effects of buprenorphine hydrochloride (Bup HCl) compared with sustained-release buprenorphine (Bup SR) on clinical condition, plasma concentrations of monocyte chemoattractant protein (MCP) 1 and IL6, and overall mortality in a murine CLP model of sepsis. Male C57/BL6 mice underwent CLP surgery and received Bup HCl or Bup SR as a component of an IACUCapproved analgesic dosing regimen. Mice were observed twice daily for clinical condition scoring by the same blinded investigator for the duration of the study. MCP1 and IL6 levels and mortality did not differ significantly between the 2 groups. Scoring of clinical condition revealed a significant decrease in behaviors associated with perceived pain at 12 and 24 h postoperatively in mice in the Bup SR group compared with the Bup HCl group. Because of the lack of significant effect on MCP1 and IL6 levels and mortality and the superior analgesic effects of Bup SR, we recommend the use of Bup SR for analgesia during the murine CLP model of sepsis.

Determination of RBC Survival in C57BL/6 and C57BL/6-Tg(UBC-GFP) Mice.

Although several methods for determining erythrocyte lifespan are used in research studies that involve mice, all involve the alteration of RBC to allow for its tracking over time, which may affect overall RBC survival. The aims of this study were to determine 1) whether sex affects RBC survival; 2) whether RBC survival differs between the biotin method and an alternative method that uses GFP; and 3) whether repeat exposure of mice to biotin results in an antibiotin antibody response or decreased RBC survival. The results suggest no difference in the RBC half-life between male and female C57BL/6 mice (22.9 ± 1.2 and 22.4 ± 0.9 d, respectively). In addition, RBC half-life did not differ between the biotin- and GFP-based methods (20.5 ± 2.1 d and 22.7 ± 2.1 d, respectively). Finally, retransfusion of mice 90 d after an initial transfusion with biotin-labeled RBC did not induce detectable antibiotin antibodies nor alter the half-life of transfused biotin-labeled RBC (initial transfusion, 22.0 ± 1.2 d; subsequent transfusion, 23.4 ± 1.4 d, respectively).

Efficacy of enrofloxacin in a mouse model of sepsis.

We examined the efficacy of enrofloxacin administered by 2 different routes in a mouse model of sepsis. Male CD1 mice were infected with a bioluminescent strain of enteropathogenic Escherichia coli and treated with enrofloxacin either by injection or in drinking water. Peak serum levels were evaluated by using HPLC. Mice were monitored for signs of clinical disease, and infections were monitored by using bioluminescence imaging. Serum levels of enrofloxacin and the active metabolite ciprofloxacin were greater in the group treated by injection than in controls or the groups treated by administration in drinking water. Survival of the group treated with enrofloxacin injection was greater than that of controls and groups treated with enrofloxacin in the drinking water. Bioluminescence in the group treated with enrofloxacin injection was less than that in the groups treated with oral administration at 12 h and in the groups treated orally and the control group at 16 h. According to these findings, we recommend the use of injectable enrofloxacin at 5 mg/kg SC for mice with systemic infections.

Transfusion of stored blood impairs host defenses against Gram-negative pathogens in mice.

BACKGROUND: Although human red blood cell (RBC) units may be refrigerator stored for up to 42 days, transfusion of older RBCs acutely delivers a large bolus of iron to mononuclear phagocytes. Similarly, iron dextran circulates in plasma for hours to days and is progressively cleared by mononuclear phagocytes, which return iron to plasma. Finally, malaria infection continuously delivers iron to macrophages by intra- and extravascular hemolysis. Studies suggest that iron administration increases infectious risk. STUDY DESIGN AND METHODS: To assess the effects of increased iron availability on susceptibility to infection, we infected mice with model Gram-negative intracellular or extracellular pathogens (Salmonella typhimurium or Escherichia coli, respectively), accompanied by RBC transfusion, iron dextran administration, or malarial coinfection. RESULTS: In our mouse models, transfusion of older RBCs exacerbates infection with both Gram-negative pathogens. Although iron dextran exacerbates E. coli infection to a similar extent as transfusion of corresponding amounts of iron, higher iron doses are required to produce comparable effects with S. typhimurium. Coinfection of mice with Plasmodium yoelii and S. typhimurium produces overwhelming Salmonella sepsis. Finally, treating mice with antibiotics abrogates the enhancing effect on E. coli infection of both older RBC transfusion and iron dextran administration. CONCLUSIONS: Transfusion of older RBCs exacerbates Gram-negative infection to a similar extent as malaria coinfection or iron dextran administration. Appropriate antibiotic therapy abrogates the effect of older RBC transfusions on infection with E. coli. Iron delivery to macrophages may be an underappreciated mechanism mediating, at least some, adverse effects of RBC transfusions.

Using reduced personal protective equipment in an endemically infected mouse colony.

Personal protective equipment (PPE) frequently is used to reduce the risk of spreading adventitial diseases in rodent colonies. The PPE worn often reflects the historic practices of the research institution rather than published performance data. Standard PPE for a rodent facility typically consists of a disposable hair bonnet, gown, face mask, shoe covers, and gloves, which are donned on facility entry and removed on exiting. This study evaluated the effect of a reduced PPE protocol on disease spread within an endemically infected mouse colony. In the reduced protocol, only the parts of the wearer that came in direct contact with the mice or their environment were covered with PPE. To test the reduced PPE protocol, proven naïve mice were housed in a facility endemically infected with murine norovirus and mouse hepatitis virus for 12 wk. During that time, routine husbandry operations were conducted by using either the standard or reduced PPE protocols. All study mice remained free of virus antibody when reduced PPE was implemented. These results indicate that reduced PPE is adequate for disease containment when correct techniques for handling microisolation caging are used. Reducing the amount of PPE used in an animal facility affords considerable cost savings yet limits the risk of disease spread.

Effect of dietary iron on fetal growth in pregnant mice.

Iron deficiency is the most common nutritional disorder. Children and pregnant women are at highest risk for developing iron deficiency because of their increased iron requirements. Iron-deficiency anemia during pregnancy is associated with adverse effects on fetal development, including low birth weight, growth retardation, hypertension, intrauterine fetal death, neurologic impairment, and premature birth. We hypothesized that pregnant mice fed an iron-deficient diet would have a similar outcome regarding fetal growth to that of humans. To this end, we randomly assigned female C57BL/6 mice to consume 1 of 4 diets (high-iron-low-bioavailability, high-iron-high-bioavailability, iron-replete, and iron-deficient) for 4 wk before breeding, followed by euthanasia on day 17 to 18 of gestation. Compared with all other groups, dams fed the high-iron-high-bioavailability diet had significantly higher liver iron. Hct and Hgb levels in dams fed the iron-deficient diet were decreased by at least 2.5 g/dL as compared with those of all other groups. In addition, the percentage of viable pups among dams fed the iron-deficient diet was lower than that of all other groups. Finally, compared with all other groups, fetuses from dams fed the iron-deficient diet had lower fetal brain iron levels, shorter crown-rump lengths, and lower weights. In summary, mice fed an iron-deficient diet had similar hematologic values and fetal outcomes as those of iron-deficient humans, making this a useful model for studying iron-deficiency anemia during pregnancy.

Increased Cell-Cell Coupling Increases Infarct Size and Does not Decrease Incidence of Ventricular Tachycardia in Mice.

Increasing connexin43 (Cx43) gap junctional conductance as a means to improve cardiac conduction has been proposed as a novel antiarrhythmic modality. Yet, transmission of molecules via gap junctions may be associated with increased infarct size. To determine whether maintaining open gap junction channels impacts on infarct size and induction of ventricular tachycardia (VT) following coronary occlusion, we expressed the pH- and voltage-independent connexin isoform connexin32 (Cx32) in ventricle and confirmed Cx32 expression. Wild-type (WT) mice injected with adenovirus-Cx32 (Cx32inj) were examined following coronary occlusion to determine infarct size and inducibility of VT. There was an increased infarct size in Cx32inj hearts as compared to WT (WT 22.9 ± 4%; Cx32inj 44.3 ± 5%; p < 0.05). Programmed electrical stimulation showed no difference in VT inducibility in WT and Cx32inj mice (VT was reproducibly inducible in 55% of shams and 50% of Cx32inj mice (p > 0.05). Following coronary occlusion, improving cell-cell communication increased infarct size, and conferred no antiarrhythmic benefit.

Expression of skeletal muscle sodium channel (Nav1.4) or connexin32 prevents reperfusion arrhythmias in murine heart.

AIMS: acute myocardial ischaemia induces a decrease in resting membrane potential [which leads to reduction of action potential (AP) V(max)] and intracellular acidification (which closes gap junctions). Both contribute to conduction slowing. We hypothesized that ventricular expression of the skeletal muscle Na(+) channel, Nav1.4 (which activates fully at low membrane potentials), or connexin32 (Cx32, which is less pH-sensitive than connexin43) would support conduction and be antiarrhythmic. We tested this hypothesis in a murine model of ischaemia and reperfusion arrhythmias. METHODS AND RESULTS: empty adenovirus (Sham) or adenoviral constructs expressing either SkM1 (gene encoding Nav1.4) or Cx32 genes were injected into the left ventricular wall. Four days later, ventricular tachycardia (VT) occurred during reperfusion following a 5 min coronary occlusion. In Nav1.4- and Cx32-expressing mice, VT incidence and duration were lower than in Sham (P < 0.05). In vitro multisite microelectrode mapping was performed in the superfused right ventricular wall. To simulate ischaemic conditions, [K(+)] in solution was increased to 10 mmol/L and/or pH was decreased to 6.0. Western blots revealed Cx32 and Nav1.4 expression in both ventricles. Nav1.4 APs showed higher V(max) and conduction velocity (CV) than Shams at normal and elevated [K(+)]. Exposure of tissue to acid solution reduced intracellular pH to 6.4. There was no difference in CV between Sham and Cx32 groups in control solution. Acid solution slowed CV in Sham (P < 0.05) but not in Cx32. CONCLUSION: Nav1.4 or Cx32 expression preserved normal conduction in murine hearts and decreased the incidence of reperfusion VT.

Cardiac expression of skeletal muscle sodium channels increases longitudinal conduction velocity in the canine 1-week myocardial infarction.

BACKGROUND: Skeletal muscle sodium channel (Nav1.4) expression in border zone myocardium increases action potential upstroke velocity in depolarized isolated tissue. Because resting membrane potential in the 1-week canine infarct is reduced, we hypothesized that conduction velocity (CV) is greater in Nav1.4 dogs compared with in control dogs. OBJECTIVE: The purpose of this study was to measure CV in the infarct border zone border in dogs with and without Nav1.4 expression. METHODS: Adenovirus was injected in the infarct border zone in 34 dogs. The adenovirus incorporated the Nav1.4- and a green fluorescent protein (GFP) gene (Nav1.4 group, n = 16) or only GFP (n = 18). After 1 week, upstroke velocity and CV were measured by sequential microelectrode recordings at 4 and 7 mM [K(+)] in superfused epicardial slabs. High-density in vivo epicardial activation mapping was performed in a subgroup (8 Nav1.4, 6 GFP) at three to four locations in the border zone. Microscopy and antibody staining confirmed GFP or Nav1.4 expression. RESULTS: Infarct sizes were similar between groups (30.6% +/- 3% of left ventricle mass, mean +/- standard error of the mean). Longitudinal CV was greater in Nav1.4 than in GFP sites (58.5 +/- 1.8 vs. 53.3 +/- 1.2 cm/s, 20 and 15 sites, respectively; P <.05). Transverse CV was not different between the groups. In tissue slabs, dV/dt(max) was higher and CV was greater in Nav1.4 than in control at 7 mM [K(+)] (P <.05). Immunohistochemical Nav1.4 staining was seen at the longitudinal ends of the myocytes. CONCLUSION: Nav1.4 channels in myocardium surviving 1 week infarction increases longitudinal but not transverse CV, consistent with the increased dV/dt(max) and with the cellular localization of Nav1.4.