Nephroblastoma in a Sprague Dawley rat unrelated to titanium dioxide nanoparticle exposure in utero.

Nephroblastoma is an embryonal tumour that has rarely been reported in laboratory rats. In this case report, a large nephroblastoma with peritoneal seeding was found during necropsy in an 11-month-old, female, Sprague Dawley rat. The rat had a history of indirect exposure to nano-TiO2 (titanium dioxide nanoparticles) during maternal gestation. A firm mass in the upper right abdominal quadrant was palpated. Four weeks later, the animal quickly declined. Nephroblastoma was confirmed by histopathology. Only one rat developed nephroblastoma among the ten littermates. Nephroblastomas in Sprague Dawley rats are typically spontaneous tumours with non-malignant mesenchymal elements. The capability to induce a nephroblastoma with nano-TiO2 is less likely in this case.

Adverse Effects Profile of Dicycloplatin (DCP) Offers Chemotherapeutic Advantage Over Cisplatin and Carboplatin.

BACKGROUND/AIM: Platinum-based chemotherapy often fails due to its severe adverse effects. The aim of this study was to examine the adverse effects profile and efficacy of dicycloplatin and compare them to those of cisplatin and carboplatin. MATERIALS AND METHODS: Cystoscopy surveillance of the first American cancer patient treated with dicycloplatin was performed quarterly. In vitro and in vivo studies were conducted using immunoblotting and flow cytometry to assess immune status of spleen and bone marrow of mice treated with dicycloplatin, cisplatin and carboplatin. RESULTS: The American patient did not suffer clinically significant myelosuppression; dicycloplatin has sustained remission in this patient to date. Experimental studies showed that dicycloplatin is less toxic to bone marrow and spleen of mice than cisplatin and carboplatin. CONCLUSION: Dicycloplatin is a promising drug in cancer chemotherapy with less aggressive side-effects than those typically associated with cisplatin and carboplatin. This is an important therapeutic advantage in cancer chemotherapy. Clinical investigation of dicycloplatin as an alternative to cisplatin or carboplatin is warranted.

Ammonia Levels and Urine-Spot Characteristics as Cage-Change Indicators for High-Density Individually Ventilated Mouse Cages.

Mouse cage and bedding changes are potentially stressful to mice and are also labor- and resource-intensive. These changes are often performed on a calendar-based schedule to maintain a clean microenvironment and limit the concentrations of ammonia to which mice and workers are exposed. The current study sought to establish a performance-based approach to mouse cage-changing that uses urine spot characteristics as visual indicators of intracage ammonia levels. Colorimetric ammonia indicators were used to measure ammonia levels in individually-ventilated cages (IVC) housing male or female mice (n =5 per cage) of various strains at 1 to 16 d after cage change. Urine spot characteristics were correlated with ammonia levels to create a visual indicator of the cage-change criterion of 25 ppm ammonia. Results demonstrated a consistent increase in ammonia levels with days since cage change, with cages reaching the cage-change criterion at approximately 10 d for IVC containing male mice and 16 d for those with female mice. Ammonia levels were higher for male than female mice but were not correlated with mouse age. However, urine spot diameter, color, and edge characteristics were strongly correlated with ammonia levels. Husbandry practices based on using urine spot characteristics as indicators of ammonia levels led to fewer weekly cage changes and concomitant savings in labor and resources. Therefore, urine spot characteristics can be used as visual indicators of intracage ammonia levels for use of a performance (urine spot)-based approach to cage-changing frequency that maintains animal health and wellbeing.

Correlation of Versican Expression, Accumulation, and Degradation during Embryonic Development by Quantitative Immunohistochemistry.

Versican, a chondroitin sulfate proteoglycan, is important in embryonic development, and disruption of the versican gene is embryonically lethal in the mouse. Although several studies show that versican is increased in various organs during development, a focused quantitative study on versican expression and distribution during lung and central nervous system development in the mouse has not previously been performed. We tracked changes in versican (Vcan) gene expression and in the accumulation and degradation of versican. Vcan expression and quantitative immunohistochemistry performed from embryonic day (E) 11.5 to E15.5 showed peak Vcan expression at E13.5 in the lungs and brain. Quantitative mRNA analysis and versican immunohistochemistry showed differences in the expression of the versican isoforms in the embryonic lung and head. The expression of Vcan mRNA and accumulation of versican in tissues was complementary. Immunohistochemistry demonstrated co-localization of versican accumulation and degradation, suggesting distinct roles of versican deposition and degradation in embryogenesis. Very little versican mRNA or protein was found in the lungs of 12- to 16-week-old mice but versican accumulation was significantly increased in mice with Pseudomonas aeruginosa lung infection. These data suggest that versican plays an important role in fundamental, overlapping cellular processes in lung development and infection.

Iterative development of visual control systems in a research vivarium.

The goal of this study was to test the hypothesis that reintroduction of Continuous Performance Improvement (CPI) methodology, a lean approach to management at Seattle Children's (Hospital, Research Institute, Foundation), would facilitate engagement of vivarium employees in the development and sustainment of a daily management system and a work-in-process board. Such engagement was implemented through reintroduction of aspects of the Toyota Production System. Iterations of a Work-In-Process Board were generated using Shewhart's Plan-Do-Check-Act process improvement cycle. Specific attention was given to the importance of detecting and preventing errors through assessment of the following 5 levels of quality: Level 1, customer inspects; Level 2, company inspects; Level 3, work unit inspects; Level 4, self-inspection; Level 5, mistake proofing. A functioning iteration of a Mouse Cage Work-In-Process Board was eventually established using electronic data entry, an improvement that increased the quality level from 1 to 3 while reducing wasteful steps, handoffs and queues. A visual workplace was realized via a daily management system that included a Work-In-Process Board, a problem solving board and two Heijunka boards. One Heijunka board tracked cage changing as a function of a biological kanban, which was validated via ammonia levels. A 17% reduction in cage changing frequency provided vivarium staff with additional time to support Institute researchers in their mutual goal of advancing cures for pediatric diseases. Cage washing metrics demonstrated an improvement in the flow continuum in which a traditional batch and queue push system was replaced with a supermarket-type pull system. Staff engagement during the improvement process was challenging and is discussed. The collective data indicate that the hypothesis was found to be true. The reintroduction of CPI into daily work in the vivarium is consistent with the 4P Model of the Toyota Way and selected Principles that guide implementation of the Toyota Production System.

The a3 problem solving report: a 10-step scientific method to execute performance improvements in an academic research vivarium.

The purpose of this study was to illustrate the application of A3 Problem Solving Reports of the Toyota Production System to our research vivarium through the methodology of Continuous Performance Improvement, a lean approach to healthcare management at Seattle Children's (Hospital, Research Institute, Foundation). The Report format is described within the perspective of a 10-step scientific method designed to realize measurable improvements of Issues identified by the Report's Author, Sponsor and Coach. The 10-step method (Issue, Background, Current Condition, Goal, Root Cause, Target Condition, Countermeasures, Implementation Plan, Test, and Follow-up) was shown to align with Shewhart's Plan-Do-Check-Act process improvement cycle in a manner that allowed for quantitative analysis of the Countermeasure's outcomes and of Testing results. During fiscal year 2012, 9 A3 Problem Solving Reports were completed in the vivarium under the teaching and coaching system implemented by the Research Institute. Two of the 9 reports are described herein. Report #1 addressed the issue of the vivarium's veterinarian not being able to provide input into sick animal cases during the work day, while report #7 tackled the lack of a standard in keeping track of weekend/holiday animal health inspections. In each Report, a measurable Goal that established the basis for improvement recognition was present. A Five Whys analysis identified the Root Cause for Report #1 as historical work patterns that existed before the veterinarian was hired on and that modern electronic communication tools had not been implemented. The same analysis identified the Root Cause for Report #7 as the vivarium had never standardized the process for weekend/holiday checks. Successful outcomes for both Reports were obtained and validated by robust audit plans. The collective data indicate that vivarium staff acquired a disciplined way of reporting on, as well as solving, problems in a manner consistent with high level A3 Thinking.

Extrauterine listeriosis in the gravid mouse influences embryonic growth and development.

Gravid mice and other rodents inoculated with Listeria monocytogenes typically fail to clear an intrauterine infection and either succumb or expel their intrauterine contents. We took advantage of this property to investigate the effects of an extrauterine infection on parameters of pregnancy success. Pregnant mice were selected for our study if they showed no clinical signs of listeriosis following oral inoculation at 7.5 gestational days (gd), and had no detectable intrauterine colony forming units (cfu) at near term (18.5 gd). The range of oral doses employed was 106-108 cfu per mouse for two listerial serotype strains (4nonb and 1/2a). At all doses, inoculation resulted in a decrease in average near-term (18.5 gd) fetal weight per litter compared to sham inoculated controls. Additionally, embryonic death (indicated by intrauterine resorptions) was exhibited by some inoculated mice but was absent in all sham inoculated animals. In parallel experiments designed to detect possible loss of placental function, gravid uteruses were examined histopathologically and microbiologically 96 h after oral inoculation. Placental lesions were associated with high (> 106), but not low (< 10²) or absent intrauterine cfu. In vitro, mouse embryonic trophoblasts were indistinguishable from mouse enterocytes in terms of their sensitivity to listerial exposure. A model consistent with our observations is one in which products (host or bacterial) generated during an acute infection enter embryos transplacentally and influences embryonic survival and slows normal growth in utero.

Clean technique for prolonged nonsurvival cardiothoracic surgery in swine (Sus scrofa).

Laboratory animal regulations provide little guidance regarding duration of nonsurvival surgery requiring aseptic technique. We hypothesized that swine would experience no sepsis during nonsurvival cardiothoracic surgery accomplished by using clean technique and lasting 8 h or less. Incision sites of 5 male farm pigs (Sus scrofa) were shaved and then cleaned with alcohol and povidone-iodine. The surgeon wore sterile gloves, clean scrubs, and hair bonnet; assistants wore clean scrubs and nonsterile gloves; most instruments were autoclaved. A median sternotomy incision was used for thoracic cavity exposure, and the skull was exposed to allow induction of brain death. Heart rate, body temperature, and blood samples were obtained before surgery (0 h; baseline) and at 2, 4, 5 or 6, and 7 or 8 h thereafter. Statistical analysis by t-tests showed that heart rate was unchanged and body temperature increased after the 0-h (baseline) time point. Aerobic blood cultures were negative except for 2 samples that were positive for coagulase-negative Staphylococcus spp. at 4 h. RBC, Hgb, and Hct levels were decreased at 2 and 4 h, but WBC and platelets were unchanged. Other alterations included decreased glucose (at 7 or 8 h), increased BUN (at 5 or 6 h and 7 or 8 h) and creatinine (at 5 or 6 h), decreased Na(+) and Ca and increased K(+) (most time points), decreased total protein and albumin (most time points), and decreased globulin (at 7 or 8 h). Liver enzymes and bilirubin typically were unchanged, and cholesterol consistently was decreased. Together our results indicate a lack of sepsis for 8 h or less in pigs undergoing cardiothoracic surgery by using clean technique. These findings provide new and specific data regarding the use of aseptic technique during prolonged nonsurvival surgeries.

Transforming growth factor-ß signaling in myogenic cells regulates vascular morphogenesis, differentiation, and matrix synthesis.

OBJECTIVE: Transforming growth factor-ß (TGF-ß) signaling is required for normal vascular development. We aimed to discover the role of TGF-ß signaling in embryonic smooth muscle cells (SMCs). METHODS AND RESULTS: We bred mice with smooth muscle (SM) 22α-Cre and Tgfbr2(flox) alleles to generate embryos in which the type II TGF-ß receptor (TGFBR2; required for TGF-ß signaling) was deleted in SMCs. Embryos were harvested between embryonic day (E) 9.5 and E18.5 and examined grossly, microscopically, and by histochemical and RNA analyses. SM22α-Cre(+/0) Tgfbr2(flox/flox) (knockout [KO]) embryos died before E15.5 with defects that included cardiac outflow tract abnormalities, persistence of the right dorsal aorta, and dilation of the distal aorta. Histological analyses suggested normal expression of SMC differentiation markers in KO aortas; however, RNA analyses showed that SMC differentiation markers were increased in KO cardiac outflow vessels but decreased in the descending aorta. KO aortas had only rare mature elastin deposits and contained abnormal aggregates of extracellular matrix proteins. Expression of several matrix proteins was significantly decreased in KO descending aortas but not in cardiac outflow vessels. CONCLUSIONS: TGF-ß signaling in SMCs controls differentiation, matrix synthesis, and vascular morphogenesis. Effects of TGF-ß on SMC gene expression appear to differ depending on the location of SMCs in the aorta.

Dose- and route-dependent teratogenicity, toxicity, and pharmacokinetic profiles of the hedgehog signaling antagonist cyclopamine in the mouse.

The Hedgehog (Hh) signaling pathway is an essential regulator of embryonic development and appears to play important roles in postnatal repair and cancer progression and metastasis. The teratogenic Veratrum alkaloid cyclopamine is a potent Hh antagonist and is used experimentally both in vitro and in vivo to investigate the role of Hh signaling in diverse biological processes. Here, we set out to establish an administration regimen for cyclopamine-induced teratogenicity in the mouse. The dysmorphogenic concentration of cyclopamine was determined in vitro via mouse whole-embryo culture assays to be 2.0 microM. We administered cyclopamine to female C57BL/6J mice at varied doses by oral gavage, ip injection, or osmotic pump infusion and assessed toxicity and pharmacokinetic (PK) models. Bolus administration was limited by toxicity and rapid clearance. In vivo cyclopamine infusion at 160 mg/kg/day yielded a dam serum steady-state concentration of approximately 2 microM with a corresponding amniotic fluid concentration of approximately 1.5 microM. Gross facial defects were induced in 30% of cyclopamine-exposed litters, with affected embryos exhibiting cleft lip and palate. This is the first report describing the PKs and teratogenic potential of cyclopamine in the mouse and demonstrates that transient Hh signaling inhibition induces facial clefting anomalies in the mouse that mimic common human birth defects.

Objective measures of health and well-being in laboratory rhesus monkeys (Macaca mulatta).

BACKGROUND: Non-human primates are an invaluable part of biomedical research. Strict regulations insure animals have a maximum likelihood of well-being and optimum health during the course of experimental procedures. Objective assessment of well-being is a critical component of these assurances. METHODS: Here we describe an objective and quantitative system we used to identify two well-being concerns in laboratory rhesus monkeys (Macaca mulatta). We provide a series of indicators for use by laboratory personnel to promote laboratory primate well-being. The indicators measure (1) potentially life threatening clinical concerns, (2) developing clinical issues, (3) atypical behaviors, and (4) laboratory performance. We include specific criteria to facilitate veterinary intervention. RESULTS: The assessment, applied to two case studies reported here, enabled swift veterinary intervention returning the animals to a healthy state. CONCLUSIONS: The measures described here provide a battery of observable and objective measures across multiple dimensions that can further ensure both excellent science and veterinary care.