Both the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act and Regulations require animals in research to receive adequate analgesia unless an exception can be scientifically justified and IACUC approved. Extended- release buprenorphine (BUP-XR) is a pharmaceutical-grade formulation that is FDA-indexed for use in mice and rats. However, this new formulation has not been evaluated in adult Mongolian gerbils (Meriones unguiculatus). Our goal was to determine whether the extrapolated dose (1 mg/kg SC) would achieve plasma buprenorphine concentrations above the murine therapeutic threshold (> 1.0 ng/mL) in male and female gerbils. We hypothesized that BUP-XR administered at 1 mg/kg would achieve the murine therapeutic threshold in both male and female gerbils until at least 48 h after injection. Gerbils received one injection of BUP-XR (1 mg/kg SC) and underwent 4 serial blood collections (0.5, 1, 2, and 4, or 0.5, 24, 48, and 72 h after injection). The average plasma buprenorphine concentrations were above 1 ng/mL within 30 min of administration for both males and females. Plasma buprenorphine concentrations remained above 1.0 ng/mL for 48 h after administration. In males, plasma buprenorphine concentrations were significantly higher at 1 h after injection as compared with females; no other significant differences were observed between sexes. Mild to moderate injection-site granulomas were observed in five of nine gerbils, presumably due to the lipid matrix of the BUP-XR formulation. Our findings demonstrate that a single BUP-XR dose (1 mg/kg SC) achieves plasma buprenorphine levels that remain above the murine therapeutic threshold of 1.0 ng/mL for up to 48 h in both sexes.

Pathogen monitoring and colony health management are critical components of any rodent research program. From an operational perspective, rodent facilities are protected from unwanted infectious agents by facility-specific bioexclusion criteria, sanitation of the physical environment, and personal protective equipment. Another important preventative measure is the use of room health levels to provide traffic patterns for animal care and research staff as they move between rooms of differing health status. For mice, our institution uses a tiered room level system with 6 defined categories, ranging from level 1 (strictest entry criteria) to 6 (least stringent entry criteria). Level 6 is defined as rooms with mice that have tested positive for mouse parvovirus (MPV) or mouse rotavirus (MRV) or both on sentinel serology at any point in time in the past and no decontamination. Because many of our mouse rooms had historically been positive for MPV and/or MRV and because of the high financial and logistic challenges of using repeated test-and-cull for elimination, we had tolerated the potential presence of MPV and MRV and had developed management practices that would promote 'burnout' (that is, elimination of infectious agents due to absence of susceptible hosts) of these pathogens. Analysis of sentinel data showed that we had 28 rooms in 4 facilities for which excluded pathogens had not been identified in 3 y or more. We therefore developed a hybrid testing strategy involving both PCR analysis and serology and implemented it in sentinels and in select colony mice to determine whether the rooms had undergone successful burnout and were free of MPV and MRV. All test results obtained during the assessment were negative for both viruses, and the rooms were subsequently upgraded to level 5 (free from excluded pathogens and allowing two-way movement in and out of housing room). All upgraded rooms have remained negative on subsequent quarterly routine sentinel serology for over 3 y. Our testing strategy for confirming pathogen burnout may be a useful and cost-efficient model for other academic rodent research programs that face a similar situation.

Benign and malignant pulmonary tumors have been reported in both Old World and New World monkeys but are uncommon. Hemangiomas are also rarely reported in nonhuman primates. Here we present a case of two primary neoplasms (a papillary adenocarcinoma of bronchioloalveolar origin and multiple cavernous subcutaneous hemangiomas) arising in an aged squirrel monkey (Saimiri sciureus).

Insulin resistance occurs during various stages of the estrus cycle in dogs. To quantify the effects of proestrus-estrus (PE) and determine whether PE affects liver insulin sensitivity, 11 female mongrel dogs were implanted with sampling and intraportal infusion catheters. Five of the dogs (PE group) entered proestrus after surgery; those remaining in anestrus were controls. The dogs were fasted overnight, [3-(3)H]glucose and somatostatin were infused through peripheral veins, and glucagon was infused intraportally. Insulin was infused intraportally, with the rate adjusted to maintain arterial plasma glucose at basal levels (PE, 294±25 μU/kg/min; control, 223±21 μU/kg/min). Subsequently the insulin infusion rate was increased by 0.2 mU/kg/min for 120 min (P1) and then to 1.5 mU/kg/min for the last 120 min (P2); glucose was infused peripherally as needed to maintain euglycemia. Insulin concentrations did not differ between groups at any time; they increased 3 μU/mL over baseline during P1 and to 3 times baseline during P2. The glucose infusion rate in PE dogs during P2 was 63% of that in control dogs. Net hepatic glucose output and the endogenous glucose production rate declined 40% to 50% from baseline in both groups during P1; during P2, both groups exhibited a low rate of net hepatic glucose uptake with full suppression of endogenous glucose production. The glucose disappearance rate during P1 and P2 was 35% greater in control than PE dogs. Therefore, PE in canines is associated with loss of nonhepatic (primarily muscle) but not hepatic insulin sensitivity.

Both respiratory syncytial virus (RSV) and influenza A virus induce nucleotide/P2Y purinergic receptor-mediated impairment of alveolar fluid clearance (AFC), which contributes to formation of lung edema. Although genetically dissimilar, both viruses generate double-stranded RNA replication intermediates, which act as Toll-like receptor (TLR)-3 ligands. We hypothesized that double-stranded RNA/TLR-3 signaling underlies nucleotide-mediated inhibition of amiloride-sensitive AFC in both infections. We found that addition of the synthetic double-stranded RNA analog poly-inosinic-cytidylic acid [poly(I:C)] (500 ng/ml) to the AFC instillate resulted in nucleotide/P2Y purinergic receptor-mediated inhibition of amiloride-sensitive AFC in BALB/c mice but had no effect on cystic fibrosis transmembrane regulator (CFTR)-mediated Cl(-) transport. Poly(I:C) also induced acute keratinocyte cytokine-mediated AFC insensitivity to stimulation by the β-adrenergic agonist terbutaline. Inhibitory effects of poly(I:C) on AFC were absent in TLR-3(-/-) mice and were not replicated by addition to the AFC instillate of ligands for other TLRs except TLR-2. Intranasal poly(I:C) administration (250 μg/mouse) similarly induced nucleotide-dependent AFC inhibition 2-3 days later, together with increased lung water content and neutrophilic inflammation. Intranasal treatment of BALB/c mice with poly(I:C) did not induce airway hyperresponsiveness at day 2 but did result in insensitivity to airway bronchodilation by β-adrenergic agonists. These findings suggest that viral double-stranded RNA replication intermediates induce nucleotide-mediated impairment of amiloride-sensitive AFC in both infections, together with β-adrenergic agonist insensitivity. Both of these effects also occur in RSV infection. However, double-stranded RNA replication intermediates do not appear to be sufficient to induce either adenosine-mediated, CFTR-dependent Cl(-) secretion in the lung or severe, lethal hypoxemia, both of which are features of influenza infection.

beta-Adrenergic agonists (beta-agonists) are commonly used to treat respiratory syncytial virus (RSV) bronchiolitis but are generally ineffective for unknown reasons. We have previously shown that RSV strain A2 inhibits bronchoalveolar epithelial responses to beta-agonists in a BALB/c mouse model by inducing heterologous keratinocyte cytokine (KC)/CXCR2-mediated desensitization of epithelial beta(2)-adrenergic receptors. The aim of the current study was to determine whether RSV also induces airway insensitivity to beta-agonists. Total lung resistance (R) was measured in anesthetized female BALB/c mice undergoing mechanical ventilation on a flexiVent computer-controlled piston ventilator. Data were analyzed using the single-compartment model. Infection with RSV A2 did not induce airway hyperresponsiveness to increasing doses of the nebulized cholinergic agonist methacholine (MCh) at any time point after RSV infection. Prenebulization with the beta-agonist terbutaline (100 muM) significantly attenuated bronchoconstrictive responses to 20 and 50 mg/ml MCh in uninfected mice and in mice infected with RSV 4-8 days postinfection (d.p.i.). However, in mice infected with replication-competent, but not UV-inactivated, RSV for 2 days, significant terbutaline insensitivity was found. Terbutaline insensitivity at 2 d.p.i. could be reversed by systemic preinfection treatment with neutralizing anti-CXCR2 antibodies, which reduced bronchoalveolar lavage (BAL) neutrophil counts but did not alter viral replication, BAL KC levels, or lung edema. Terbutaline insensitivity was also reversed by postinfection nebulization with neutralizing anti-KC or anti-CXCR2 antibodies and could be replicated in normal, uninfected mice by nebulization with recombinant KC. These data suggest that KC/CXCR2-mediated airway insensitivity to beta-agonists may underlie the modest utility of these drugs as bronchodilators in therapy for acute RSV bronchiolitis.

High tidal volume ventilation is detrimental to alveolar fluid clearance (AFC), but effects of ventilation pressure (P) on AFC are unknown. In anesthetized BALB/c mice ventilated at constant tidal volume (8 ml/kg), mean AFC rate was 12.8% at 6 cmH(2)O P, but increased to 37.3% at 18 cmH(2)O P. AFC rate declined at 22 cmH(2)O P, which also induced lung damage. Increased AFC at 18 cmH(2)O P did not result from elevated plasma catecholamines, hypercapnia, or hypocapnia, but was due to augmented Na(+) and Cl(-) absorption. PKA agonists and beta-agonists stimulated AFC at 10 cmH(2)O P by upregulating amiloride-sensitive Na(+) transport. However, at 18 cmH(2)O P, PKA agonists and beta-agonists reduced AFC. At 15 cmH(2)O P, the AFC rate was intermediate (mean 26.6%), and forskolin and beta-agonists had no effect. Comparable P dependency of AFC and beta-agonist responsiveness was found in C57BL/6 mice. The effect on AFC of increasing P to 18 cmH(2)O was blocked by adenosine deaminase or an A(2b)-adenosine receptor antagonist, and could be mimicked by adenosine in mice ventilated at 10 cmH(2)O P. Modulation of adenosine signaling also resulted in altered responsiveness to beta-agonists. These findings indicate that, in the normal mouse lung, basal AFC rates and responses to beta-agonists are impacted by ventilation pressure in an adenosine-dependent manner.

RATIONALE Pulmonary infections can impair alveolar fluid clearance (AFC), contributing to formation of lung edema. Effects of influenza A virus (IAV) on AFC are unknown. OBJECTIVES To determine effects of IAV infection on AFC, and to identify intercellular signaling mechanisms underlying influenza-mediated inhibition of AFC. METHODS BALB/c mice were infected intranasally with influenza A/WSN/33 (10,000 or 2,500 focus-forming units per mouse). AFC was measured in anesthetized, ventilated mice by instilling 5% bovine serum albumin into the dependent lung. MEASUREMENTS AND MAIN RESULTS Infection with high-dose IAV resulted in a steady decline in arterial oxygen saturation and increased lung water content. AFC was significantly inhibited starting 1 hour after infection, and remained suppressed through Day 6. AFC inhibition at early time points (1-4 h after infection) did not require viral replication, whereas AFC inhibition later in infection was replication-dependent. Low-dose IAV infection impaired AFC for 10 days, but induced only mild hypoxemia. High-dose IAV infection increased bronchoalveolar lavage fluid ATP and UTP levels. Impaired AFC at Day 2 resulted primarily from reduced amiloride-sensitive AFC, mediated by increased activation of the pyrimidine-P2Y purinergic receptor axis. However, an additional component of AFC impairment was due to activation of A(1) adenosine receptors and stimulation of increased cystic fibrosis transmembrane regulator-mediated anion secretion. Finally, IAV-mediated inhibition of AFC at Day 2 could be reversed by addition of beta-adrenergic agonists to the AFC instillate. CONCLUSIONS AFC inhibition may be an important feature of early IAV infection. Its blockade may reduce the severity of pulmonary edema and hypoxemia associated with influenza pneumonia.