Assessment of Pain Associated with the Injection of Sodium Pentobarbital in Laboratory Mice (Mus musculus).

The AVMA Guidelines for the Euthanasia of Animals considers injection of barbiturates to be an acceptable method of euthanasia in rodents but states there is a potential for pain when administered intraperitoneally. This study examined the potential for pain in mice by assessing visceral pain after intraperitoneal administration and acute pain by using a paw-lick test. Male and female mice (n = 160) intraperitoneally received a euthanizing dose of sodium pentobarbital at a concentration of 5, 50, or 390 mg/mL and were observed for writhing, peritoneum-directed behaviors (PDB), loss of righting reflex, and time to death. Writhing was not observed in any animal. There was no significant difference in the number of mice exhibiting PDB or in the rate of PDB for responders receiving either saline or the 390-mg/mL solution. There was a significant treatment effect on time, with greater concentration and dose resulting in more rapid loss of righting reflex and death. In the second set of experiments, the same solutions were injected subcutaneously into the plantar hindpaw of male and female mice (n = 84). The number of responders, latency until the first lick, and the number of licks per responder were recorded. The number of responders was increased in the 50-mg/mL group; however, there was no difference in latency or the number of licks per responder. These results show that intraperitoneal injection of sodium pentobarbital for euthanasia in mice did not result in increased behavioral signs of pain, and animals lose consciousness more rapidly than the onset of pain seen in the pawlick test. Therefore, although sodium pentobarbital is capable of inducing inflammation, euthanasia through intraperitoneal administration is rapid and does not result in overt signs of pain when compared with injection of saline.

Case study: polycystic livers in a transgenic mouse line.

Three mice (2 male, 1 female; age, 5 to 16 mo) from a mouse line transgenic for keratin 14 (K14)-driven LacZ expression and on an outbred Crl:CD1(ICR) background, were identified as having distended abdomens and livers that were diffusely enlarged by numerous cysts (diameter, 0.1 to 2.0 cm). Histopathology revealed hepatic cysts lined by biliary type epithelium and mild chronic inflammation, and confirmed the absence of parasites. Among 21 related mice, 5 additional affected mice were identified via laparotomy. Breeding of these 5 mice (after 5 mo of age) did not result in any offspring; the K14 mice with polycystic livers failed to reproduce. Affected male mice had degenerative testicular lesions, and their sperm was immotile. Nonpolycystic K14 control male mice bred well, had no testicular lesions, and had appropriate sperm motility. Genetic analysis did not identify an association of this phenotype with the transgene or insertion site.

Predictive observation-based endpoint criteria for mice receiving total body irradiation.

Total body irradiation of mice is a commonly used research technique; however, humane endpoints have not been clearly identified. This situation has led to the inconsistent use of various endpoints, including death. To address this issue, we refined a cageside observation-based scoring system specifically for mice receiving total body irradiated. Male and female C57BL/6 mice (age, 8 wk) received 1 of 3 doses of radiation from 1 of 2 different radiation sources and were observed for progression of clinical signs. All mice were scored individually by using cageside observations of their body posture (score, 0 to 3), eye appearance (0 to 3), and activity level (0 to 3). Retrospective analysis of the observation score data indicated that death could be predicted accurately with total scores of 7 or greater, and observation scores were consistent between observers. This scoring system can be used to increase the consistent use of endpoint criteria in total body murine irradiation studies and ultimately to improve animal welfare.

Endpoint refinement for total body irradiation of C57BL/6 mice.

Acute radiation syndrome is a life-threatening condition that has the potential to affect large populations of humans. Although several animal models of this syndrome are available, the total-body-irradiated mouse has emerged as an important tool to evaluate the efficacy of prospective prophylaxis, mitigation, and treatment compounds. Despite the widespread use of this model, humane endpoints have not been clearly identified. To address this issue, we developed a cageside observation-based scoring system specifically for total-body-irradiated mice to assess the progression of clinical signs associated with acute radiation syndrome. Male C57BL/6 mice (n=175; age, 8 to 9 wk) received an anticipated LD50 dose of radiation and were observed for progression of clinical signs of acute radiation syndrome for 30 d. All mice were scored individually through cageside observation of their body posture (score, 0 to 3), eye appearance (0 to 3), and activity level (0 to 3). Retrospective analysis of the score data indicated that death could be predicted accurately by using increasing cumulative scores (0 to 9). Total scores of 6, 7, 8, and 9 were associated with mortality rates of 78.6%, 86.4%, 93.3%, and 100%, respectively. Furthermore, scores of 6, 7, and 8 predicted death within 3, 1.5, and 0.5 d, respectively. The use of this scoring system provides investigators and IACUCs with predictive humane, surrogate endpoints for total-body-irradiated mice. This system allows preemptive euthanasia of mice before they become moribund, thereby minimizing pain and distress associated with acute radiation syndrome and improving animal welfare.

Leaching of heavy metals from water bottle components into the drinking water of rodents.

Providing high-quality, uncontaminated drinking water is an essential component of rodent husbandry. Acidification of drinking water is a common technique to control microbial growth but is not a benign treatment. In addition to its potential biologic effects, acidified water might interact with the water-delivery system, leading to the leaching of heavy metals into the drinking water. The goal of the current study was to evaluate the effects of water acidification and autoclaving on water-bottle assemblies. The individual components of the system (stainless-steel sipper tubes, rubber stoppers, neoprene stoppers, and polysulfone water bottles) were acid-digested and analyzed for cadmium, chromium, copper, iron, lead, magnesium, manganese, selenium, and zinc to quantify the metal composition of each material. In addition the amounts of these metals that leached into tap and acidified water with and without autoclaving were quantified after 1 wk of contact time. On a weight basis, sipper tubes contained the largest quantities of all metals except magnesium and zinc, which were greatest in the neoprene stoppers. Except for cadmium and selenium, all metals had leached into the water after 1 wk, especially under the acidified condition. The quantities of copper, lead, and zinc that leached into the drinking water were the most noteworthy, because the resulting concentrations had the potential to confound animal experiments. On the basis of these findings, we suggest that water-quality monitoring programs include heavy metal analysis at the level of water delivery to animals.

A novel peptide nanomedicine against acute lung injury: GLP-1 in phospholipid micelles.

PURPOSE: Treatment of acute lung injury (ALI) observed in Gram-negative sepsis represents an unmet medical need due to a high mortality rate and lack of effective treatment. Accordingly, we developed and characterized a novel nanomedicine against ALI. We showed that when human glucagon-like peptide 1(7-36) (GLP-1) self-associated with PEGylated phospholipid micelles (SSM), the resulting GLP1-SSM (hydrodynamic size, ~15 nm) exerted effective anti-inflammatory protection against lipopolysaccharide (LPS)-induced ALI in mice. METHODS: GLP1-SSM was prepared by incubating GLP-1 with SSM dispersion in saline and characterized using fluorescence spectroscopy and circular dichroism. Bioactivity was tested by in vitro cAMP induction, while in vivo anti-inflammatory effects were determined by lung neutrophil cell count, myeloperoxidase activity and pro-inflammatory cytokine levels in LPS-induced ALI mice. RESULTS: Amphipathic GLP-1 interacted spontaneously with SSM as indicated by increased α-helicity and fluorescence emission. This association elicited increased bioactivity as determined by in vitro cAMP production. Correspondingly, subcutaneous GLP1-SSM (5-30 nmol/mouse) manifested dose-dependent decrease in lung neutrophil influx, myeloperoxidase activity and interleukin-6 in ALI mice. By contrast, GLP-1 in saline showed no significant anti-inflammatory effects against LPS-induced lung hyper-inflammatory responses. CONCLUSIONS: GLP1-SSM is a promising novel anti-inflammatory nanomedicine against ALI and should be further developed for its transition to clinics.

Effects of weekly blood collection in C57BL/6 mice.

We assessed hematologic recovery, body weight, and behavior after serial blood collection in 10- to 14-wk-old C57BL/6 mice. Male and female mice (5 to 11 mice for pilot groups, 23 to 35 mice for full study groups) had either 15%, 20%, or 25% of their estimated total blood volume (TBV) collected once weekly for 6 wk. Except for those of the 25% TBV male pilot group, the weights of all mice recovered or increased from one collection to the next. The behavior of all mice, with the exception of the 25% TBV male pilot group, appeared normal throughout the study. Erythrogram value changes from baseline were analyzed at each weekly blood collection. Recovery was defined as the return of mean hemoglobin values to within 2 SD of mean baseline values. According to this definition, mice in the 15% TBV male group and 15%, 20%, and 25% TBV female groups recovered hematologically. To support the statistical definition of recovery, we compared our data with human anemia categories to assess the clinical relevance of the mouse hemoglobin values. On the basis of these data, we conclude that as much as 25% TBV can be collected once weekly from female mice for 6 wk and as much as 15% TBV can be collected once weekly from male mice for 6 wk without producing weight loss, behavioral changes, or clinically significant anemia.

The use of cross-foster rederivation to eliminate murine norovirus, Helicobacter spp., and murine hepatitis virus from a mouse colony.

Over 10 mo, 287 mouse litters were cross-fostered by using 1 of 2 paradigms to eliminate murine norovirus (MNV), Helicobacter spp., murine hepatitis virus (MHV), and Syphacia obvelata. Paradigm 1 involved cross-fostering litters at younger than 48 h with no attention to the changing of bedding material. Paradigm 2 involved cross-fostering litters at younger than 24 h from cages in which the bedding material was changed within 24 h before cross-fostering. After cross-foster rederivation, mice were tested for the presence of Helicobacter spp. by means of fecal PCR at 4, 8, and 12 wk. Surrogates also were tested for MNV by use of multiplex fluorometric assay serology at 4 wk and fecal PCR at 12 wk. Surrogate mice were tested for MHV by means of MFIA at 4 wk and for pinworms by perianal tape test and fecal flotation at 4 and 12 wk. Compared with those from paradigm 1, litters from paradigm 2 were less likely to be positive for MHV and Helicobacter spp. The use of cross-foster rederivation alone was unsuccessful for the elimination of Syphacia obvelata. For cross-foster rederivation, we recommend that litters be younger than 24 h and from cages in which the bedding material was changed within 24 h before cross-fostering. The presence of MNV, Helicobacter spp., and MHV can be predicted reliably at 12, 8, and 4 wk, respectively.

Concentration and emission of airborne contaminants in a laboratory animal facility housing rabbits.

Characterization of animal housing conditions can determine the frequency of bedding and cage changes, which are not standardized from facility to facility. Rabbits produce noticeable odors, and their excreta can scald and stain cages. Our facility wanted to document measurable airborne contaminants in a laboratory rabbit room in which excreta pans were changed weekly and cages changed biweekly. Contaminants included particulate, endotoxin, ammonia, carbon dioxide, and a rabbit salivary protein as a marker for rabbit allergen. Concentrations were measured daily over a 2-wk period in a laboratory animal facility to determine whether they increased over time and on days considered to be the dirtiest. Except for ammonia, concentrations of all airborne contaminants did not differ between clean and dirty days. Concentrations were lower than occupational health exposure guidelines for all contaminants studied, including ammonia. After measurement of concentration, a model was applied to calculate mean emission factors in this rabbit room. Examples of emission factor utilization to determine airborne contaminant concentration in rabbit rooms under various environmental conditions and housing densities are provided.

Importance of signaling via the IFN-alpha/beta receptor on host cells for the realization of the therapeutic benefits of cyclophosphamide for mice bearing a large MOPC-315 tumor.

Here we show that low-dose cyclophosphamide (CY), that depends for its therapeutic effectiveness on the immunopotentiating activity of the drug for T cell-mediated tumor-eradicating immunity, is curative for approximately 80% of wild-type (WT) mice bearing a large s.c. MOPC-315 tumor, but only for approximately 10% of IFN-alpha/betaR-/- mice bearing a large s.c. MOPC-315 tumor. Histopathological examination of the s.c. tumors of such mice on day 4 after the chemotherapy revealed that the low dose of CY led to accumulation of T lymphocytes in both the WT and the IFN-alpha/betaR-/- mice. However, in the CY treated tumor bearing WT mice the T lymphocytes were present throughout the tumor mass and in direct contact with tumor cells, but in the CY treated tumor bearing IFN-alpha/betaR-/- mice most of the T lymphocytes remained in blood vessels. In addition to being important for CY-induced transendothelial migration of T lymphocytes into the tumor mass, we show here that signaling via the IFN-alpha/betaR is also important for CY-induced control of metastatic tumor progression in the spleen and liver of the tumor bearing mice. Finally, CY cured tumor bearing WT mice were resistant to a subsequent challenge with MOPC-315 tumor cells, but the few CY cured tumor bearing IFN-alpha/betaR-/- mice were not. Thus, signaling via the IFN-alpha/betaR on host cells in MOPC-315 tumor bearers is important for CY-induced: (a) transendothelial migration of T lymphocytes into the tumor mass and the eradication of the primary tumor, (b) control of metastatic tumor progression, and (c) resistance to a subsequent tumor challenge.

An evaluation of preparation methods and storage conditions of tribromoethanol.

This reports the in vitro portion of a study designed to establish guidelines for the preparation, storage, and use of tribromoethanol (TBE). We evaluated: 1) the purity of TBE powder from three suppliers; 2) nine methods of preparation of a 25-mg/ml (working) solution for formation of particulates and breakdown products; 3) formation of particulates and breakdown products and pH change in 1-g/ml (stock) solutions and working solutions stored under four conditions (25 degrees C and 5 degrees C in light and in dark); and 4) stock and working solutions of TBE that caused lethal effects in mice. These objectives were met by using nuclear magnetic resonance spectroscopy, gas chromatography-mass spectroscopy, particle-size and turbidity analyses, and pH strips. TBE powder from three suppliers varied in purity. No significant differences in breakdown product formation, particle size, or turbidity were noted between the nine preparation methods evaluated. Stock solutions and the working solution stored at 5 degrees C in the dark maintained a pH of 6.5 to 7.0, whereas the pH dropped for all other working solutions. A low level of dibromoacetaldehyde (DBA), a potential breakdown product reported to cause toxic effects, was detectable in all newly prepared solutions. Regardless of the storage condition or pH, DBA concentration did not increase measurably in any of the solutions after 8 weeks. The stock and working solutions that demonstrated lethal effects in mice had a pH of 6.5 and did not differ notably from newly prepared, non-lethal solutions, when evaluated for DBA. A decrease in pH could not be correlated to an increase in DBA or potential lethality, as suggested in the literature. The toxicity associated with the lethal TBE in this study appears to be a result of a chemical reaction or breakdown product that has not yet been reported.

Efficacy and safety of stored and newly prepared tribromoethanol in ICR mice.

This study, performed in conjunction with an in vitro evaluation of tribromoethanol (TBE), consisted of three trials with three objectives. The first objective was to compare anesthetic efficacy and short-term pathologic findings of TBE, ketamine-xylazine (K-X), and sodium pentobarbital (NaP). The second objective was to evaluate how changes of TBE that occur during the perceived most favorable and least favorable storage conditions (8 weeks at 5 degrees C in the dark [5D] and 25 degrees C with exposure to light [25L], respectively) affect anesthetic efficacy and short-term pathology when compared to newly prepared TBE. The third objective was to perform a 6-week clinical assessment of animals that received newly prepared TBE. All animals that received TBE (400 mg/kg) and 14 of 15 that received K-X (K, 120 mg/kg; X, 16 mg/kg) were anesthetized, as defined by loss of pedal reflex. In comparison, only 8 of 15 animals administered NaP (60 mg/kg) were anesthetized. Anesthetic duration for animals that received K-X was 31.7 min, which was significantly (P = 0.0085) longer than animals that received TBE (18.5 min). Recovery times for TBE and K-X were not significantly different (26.5 and 27.5 min, respectively). Pathologic lesions associated with TBE administration were significantly (P = 0.001) greater than those associated with K-X. NaP was not associated with any pathologic lesions. The pH of newly prepared and 5D TBE was 6.5 to 7.0, whereas that for 25L TBE was 3.0. Anesthetic induction, duration, recovery times, and pathologic lesions were not significantly different, regardless of the pH or storage condition of the solution. It was noted, however, that the average anesthetic duration for animals administered newly prepared TBE in the second trial was longer (37.7 min) than the first trial that used newly prepared TBE. For the third trial (long-term clinical assessment), the average anesthetic duration for TBE was 46.5 min, significantly (P < 0.025) longer when compared to the first trial that used newly prepared TBE. During the third trial, 10 animals were found dead or moribund. All animals that were found moribund were necropsied and found to exhibit a marked ileus. Because of the variability in anesthetic effectiveness, pathology, and morbidity and mortality associated with the use of TBE, we do not recommend the use of this anesthetic agent in ICR mice.

Effects of Freund's complete adjuvant on the physiology, histology, and activity of New Zealand White Rabbits.

The purpose of this study was to determine whether Freund's complete adjuvant causes adverse effects on the physiology, histology, and activity of rabbits used for polyclonal antibody production. Rabbits in the experimental groups were immunized intradermally and subcutaneously with keyhole limpet hemacyanin with or without Freund's adjuvant. Booster immunizations were administered 28 days after the initial immunizations. No immunizations were administered to rabbits in the control group. Body weight, food consumption, activity, rectal temperature, white blood cell count, corticosterone concentration, and induration around immunization sites were measured. Histologic changes in the lung, kidney, liver, lymph node, and skin were evaluated after euthanasia. There were significant differences in white blood cell count, induration around immunization sites, and lipid droplet deposition in pulmonary microgranulomas in some rabbits that received Freund's adjuvant. These differences did not affect well-being of the rabbits. Freund's complete adjuvant caused no adverse effects on physiologic parameters and activity levels in rabbits; thus, its use in polyclonal antibody production should not be discouraged.

Environmental Conditions in a Chamber with Dry Ice.

Dry ice is used in research laboratories as a source of CO2 for euthanasia of rodents. The present study was performed to evaluate environmental conditions created in a standard (9-liter) bell jar. By using a portable gas analyzer and measuring at 7.5 cm above the platform, we evaluated the chamber filling rate at room temperature with a known quantity of dry ice, maintenance of 70% CO2, and concentration of CO2 maintained when the top of the chamber was removed and replaced. Ambient temperature in the chamber and temperature of the platform were also measured. Results indicated that 500 g dry ice was required to maintain the filling rate and CO2 concentration at levels recommended by the American Veterinary Medical Association Panel on Euthanasia; that after removal of an animal, the lid should be replaced for 1 min before placing another animal in the chamber; and that although ambient temperature in the chamber never fell below 178C during an 80-min period, the platform temperature decreased to 08C. If a chamber must be used as described above for more than 1 h, consideration should be given to alternating between two chambers and allowing a chamber to warm to room temperature before reuse.