Helicobacter hepaticus is well established as an unwanted variable in laboratory rodent colonies. Historically, cesarean section and embryo transfer have been used to derive Helicobacter-free mouse colonies. Neonatal transfer of newborn mice onto Helicobacter-free foster dams was recently reported as an alternative method of deriving Helicobacter-free mice, but until now, the age by which pups must be fostered to remain Helicobacter-free was unknown. The purpose of the study reported here was to determine the age by which mouse pups must be fostered to remain free of H. hepaticus. Beginning on the day of birth, 20 C57BL/6 mice were fostered from H. hepaticus-positive parents onto Helicobacter-free BALB/c dams each day for 14 days for a total of 280 pups fostered. Fecal specimens collected at weaning, and fecal, liver, and cecal specimens collected at euthanasia were analyzed by use of polymerase chain reaction (PCR) analysis. No pup fostered within 24 h of birth became infected with H. hepaticus; however, many of those fostered after 24 h became infected. These results were supported by those of a large field trial, in which 201 litters representing 71 strains of mice were fostered within 24 h of birth. Follow-up fecal PCR analysis was performed on 52 mice or their progeny that were randomly sampled from the 201 fostered litters. All mice tested remained free of H. hepaticus approximately 100 days after fostering. The results indicate that mouse pups must be fostered within 24 h of birth to remain free of H. hepaticus. In addition, cecal and fecal PCR analyses detected more infections, than did liver PCR analysis, thus indicating that those specimens are preferred for detection of H. hepaticus infection.

Genetically altered mice are important research tools for the study of human development and disease. Occasionally, whether or not related to the genetic mutation, mice may become infertile with age and, thus, risk loss of the mutant line. Under conditions in which assisted reproduction techniques (ARTs), such as in vitro fertilization, are unsuccessful, a new strategy, intracytoplasmic sperm injection (ICSI), may be applicable. This technique has been perfected for use in the mouse and is now considered a reliable, effective, and efficient ART. In the study reported here, we “rescued” (i.e., produced offspring, using ICSI from a “last-of-line” mutant male mouse) four lines that otherwise had become infertile and unresponsive to conventional ART's. A total of 26 live pups were produced from eight pregnant recipient foster mothers. Five mutant male mice were derived (one each from three lines, and two from one line), and all survived to adulthood. We found that live born mice could be successfully derived by use of ICSI that subsequently could breed by natural mating to reestablish the mutant line. Because of its effectiveness and reliability under these conditions, ICSI should be considered a powerful addition to the armamentarium of ART's applicable in the genetically-altered mouse, especially when only one male may still be available.

Adequate pain control is necessary for optimal postsurgical recovery and humane treatment of laboratory and companion animals. Opioid drugs are currently the most potent analgesic agents available in human and veterinary medicine. Long-acting formulations of opioid drugs confer several important advantages over standard pharmaceutical preparations, especially for use in animals. A long-acting formulation of oxymorphone hydrochloride was produced by encapsulation into liposomes. Liposome-encapsulated (LE) oxymorphone was tested in a rat model of visceral postoperative pain. Rats were given one subcutaneous injection of LE oxymorphone (1.2 or 1.6 mg/kg of body weight) or standard oxymorphone (0.3 mg/kg) at the time of intestinal transection or resection. A single administration of LE oxymorphone hydrochloride was as effective for relief of postoperative pain in rats (P = 0.18), as were multiple (q4 h or q8 h) injections of 0.3 mg/kg of the standard pharmaceutical preparation. The rats given LE oxymorphone prior to intestinal resection also had significantly higher body weight at three and seven days after surgery than did rats that were given standard oxymorphone. In conclusion, LE oxymorphone was effective in treating visceral pain associated with intestinal surgery in rats. On the basis of body weight gain, rats treated with LE oxymorphone had improved recovery outcome, compared with rats treated with repeated injections of standard oxymorphone.

An extended-release formulation of oxymorphone was produced by encapsulation into liposomes, using a novel technique. Liposome-encapsulated morphine was produced, using a standard technique These preparations were tested in an animal model of neuropathic pain. Male Sprague-Dawley rats (approx. 300 g) were allotted to control (non-loaded liposomes) and treatment (liposome-encapsulated oxymorphone or morphine) groups. Drugs were administered subcutaneously to all rats immediately prior to sciatic nerve ligation. Thermal withdrawal latencies were measured at baseline and daily for seven days after sciatic nerve ligation. A second experiment involved subcutaneous administration of non-loaded liposomes, morphine, or oxymorphone to rats that did not undergo sciatic nerve ligation. Thermal withdrawal latencies in sciatic nerve-ligated rats given non-loaded liposomes decreased significantly by day four, with maximal decrease at day seven after surgery, indicating development of full hyperalgesia. In contrast, ligated rats given liposome-encapsulated morphine or liposome-encapsulated oxymorphone had no decrease in thermal withdrawal latency by day four, indicating that these long-acting preparations prevented development of hyperalgesia after a single injection. This treatment effect persisted to day seven. Non-ligated rats treated with vehicle or liposome-encapsulated morphine had no change in thermal withdrawal latencies. Non-ligated rats treated with liposome-encapsulated oxymorphone had a small, but significant increase in thermal withdrawal latency from day four through day seven. One subcutaneous injection of liposome-encapsulated oxymorphone or morphine was effective in preventing hyperalgesia in this pain model for up to seven days. These results suggest that liposome-encapsulation of oxymorphone offers a novel, convenient, and effective means to provide long-term analgesia.

Purpose: The objective of the study reported here was to investigate whether massage-like stroking of the thorax and cranial portion of the abdomen might relax unanesthetized rats sufficiently to permit in vivo echocardiography. Methods: Nine-month-old spontaneously hypertensive rats (SHR) were first conditioned to being held by hand for 10 to 15 min twice a day for seven to 10 days. During each session, the animal was placed in supine position, and the thorax and cranial abdominal area were gently stroked (approx. 5 cm/s, 12 to 14 times/min). After the conditioning period, echocardiography was initiated. We obtained serial transthoracic two-dimensional (2-D) and M-mode echocardiograms from nine-month-old SHR that were treated with isoproterenol (60 mg/kg of body weight, s.c., x 1, followed by 30 mg/kg/d x3), and from old (20 to 24 months old) SHR, studied when labored breathing, suggestive of heart failure, was evident (SHR-F). Measurements included end-diastolic volume (EDV) and end-systolic volume (ESV). Results: In the isoproterenol-treated SHR, mean ± SD echocardiographically derived EDV (2-D, 0.29 ± 0.05; M-mode, 0.28 ± 0.01 ml) was not significantly different from volume at necropsy (0.33 ± 0.04 ml). Measurements of EDV and ESV by use of M-mode and 2-D echocardiography were significantly correlated (EDV R² = 0.48, P = 0.05; ESV R² = 0.39, P = 0.02). Echocardiography revealed pleuropericardial effusions (4/6), atrial thrombi (5/6), and left and right ventricular enlargement (6/6). The EDV and ESV were increased fivefold (P < 0.01) and threefold (P < 0.05), respectively, versus values for SHR not in heart failure (SHR-NF). Left ventricular ejection fraction of hearts from SHR-F was markedly decreased, compared with that in SHR-NF (44 ± 7 versus 74 ± 2%, respectively; P < 0.05). The presence or absence of left atrial thrombi and fluid in the thoracic cavity was confirmed at necropsy in SHR-F and SHR-NF. Conclusion: Thoracic massage permits use of echocardiography in unanesthetized rats, thereby providing a simple, non-invasive technique for assessment of cardiac structure and function in rats without the potentially adverse effects of anesthesia.

Influenza is a respiratory tract disease of viral origin that can cause major epidemics in humans. The influenza virus infects and damages epithelial cells of the respiratory tract and causes pneumonia. Lung lesions of mice infected with influenza virus resembles those seen in humans with influenza, and can result in severe and even fatal pneumonia. In contrast, experimental infection of rats with the virus induces a milder form of the disease, with no mortality. The purpose of the study reported here was to determine the time course of influenza infection and lung injury in Brown Norway (BN), Fischer-344 (F344), and Sprague-Dawley (SD) rats to ascertain whether genetic background impacts susceptibility to infection and host responses. Rats of each strain were inoculated intranasally with 10,000 plaque-forming units of rat-adapted influenza virus (RAIV), and lungs were assessed at postinoculation hour (PIH) 2, 24, 48, 72, and 144 for viral titer, inflammatory cells, pro-inflammatory cytokines, and biochemical indicators of lung edema (protein) and injury (lactate dehydrogenase [LD] activity). Virus titer peaked at PIH 24, and was 100-fold higher in the F344 and SD, compared with the BN strain. Alveolar macrophages, LD activity, and total protein concentration were higher in the BN rats, whereas neutrophil numbers and interleukin 6 and tumor necrosis factor-alpha activities were greatest in the bronchoalveolar lavage fluid of F344 and SD rats. The results indicate that F344 and SD rats respond in similar manner to viral infection, whereas viral replication was more limited in BN rats and was associated with a different profile of pulmonary cells.

Wood is often used as a contact bedding material for laboratory animals. It has been established that wood, particularly softwood, has the potential to induce hepatic drug-metabolizing enzymes. However, to the authors' knowledge, changes in enzyme activity after removal of animals from bedding have not been characterized. The purpose of the study reported here was to determine how hepatic ethoxyresorufin-O-deethylation and p-nitrophenol hydroxylation activities alter over time after removal of rats from pine bedding. Male rats, three to four months old, raised in cages containing pine bedding, were transferred to wire-bottomed cages. At various times thereafter (up to 84 days), groups of rats were euthanized and the liver was processed to obtain microsomes. The microsomal protein and total cytochrome P450 (CYP) content and enzyme activities were determined. Significant differences in total microsomal protein or total CYP values were not observed over the 84 days, but a decrease in ethoxyresorufin-O-deethylation and p-nitrophenol hydroxylation activities was detected. For p-nitrophenol hydroxylation, the decrease was exponential, with a half-life of approximately nine days, whereas for ethoxyresorufin-O-deethylation, a rapid decrease in activity in the first week was followed by a reduced rate of decrease thereafter. Enzyme activities did not stabilize for at least six weeks. Researchers using laboratory animals should, therefore, be aware that it takes several weeks for enzyme activities to stabilize once animals are removed from the bedding.

Over a period of ten months, five mice submitted to our service (the Pathology Section of the Veterinary Resources Program, Office of Research Services at the National Institutes of Health, Bethesda, Md.) were diagnosed with disseminated trichosporonosis. These mice had pyogranulomatous inflammation in multiple organs, including lung, liver, lymph nodes, salivary gland, and skin. Fungal elements in many of the lesions were identified, using special histochemical stains, and Trichosporon beigelii was obtained by use of culture of specimens at affected sites. This saprophytic fungus has caused disseminated disease in immunosuppressed humans. However, despite widespread use of immunosuppressed rodents in research, to the authors' knowledge, this organism had not previously been reported to cause spontaneous disseminated disease in laboratory mice. All affected mice had a genetically engineered defect in p47^phox, a critical component of the nicotinamide dinucleotide phosphate (NADPH) oxidase, the enzyme responsible for generating the phagocyte oxidative burst. These animals are used as a murine model of human chronic granulomatous disease. We discuss the lesions, differential diagnosis, identification of the organism, and the role of NADPH oxidase in protecting against disseminated trichosporonosis.

An adult male cynomolgous macaque (Macaca fascicularis) died suddenly after anesthesia for a positron emission tomography scan. Bacteriologic culture of the mucopurulent secretions recovered from the endotracheal tube yielded heavy growth of Pseudomonas putida, a known endotoxin producer. Histologically, the lungs had severe, diffuse perivascular edema and neutrophils marginating to the endothelium. The sudden death and the pathologic findings were consistent with peracute endotoxic shock. Numerous environmental swab specimens of the surgical suite and equipment were submitted for bacteriologic culture, as were swab specimens of endotracheal secretions from a control animal; however, Pseudomonas putida was not isolated from any specimen. The animal in this report may have carried Pseudomonas putida as a commensal in the oropharynx, and the stress of anesthesia may have resulted in increased sensitivity to the endotoxin.