Guidelines on severity assessment and classification of genetically altered mouse and rat lines.

Genetic alterations can unpredictably compromise the wellbeing of animals. Thus, more or less harmful phenotypes might appear in the animals used in research projects even when they are not subjected to experimental treatments. The severity classification of suffering has become an important issue since the implementation of Directive 2010/63/EU on the protection of animals used for scientific purposes. Accordingly, the breeding and maintenance of genetically altered (GA) animals which are likely to develop a harmful phenotype has to be authorized. However, a determination of the degree of severity is rather challenging due to the large variety of phenotypes. Here, the Working Group of Berlin Animal Welfare Officers (WG Berlin AWO) provides field-tested guidelines on severity assessment and classification of GA rodents. With a focus on basic welfare assessment and severity classification we provide a list of symptoms that have been classified as non-harmful, mild, moderate or severe burdens. Corresponding monitoring and refinement strategies as well as specific housing requirements have been compiled and are strongly recommended to improve hitherto applied breeding procedures and conditions. The document serves as a guide to determine the degree of severity for an observed phenotype. The aim is to support scientists, animal care takers, animal welfare bodies and competent authorities with this task, and thereby make an important contribution to a European harmonization of severity assessments for the continually increasing number of GA rodents.

Acute Lung Injury Causes Asynchronous Alveolar Ventilation That Can Be Corrected by Individual Sighs.

RATIONALE: Improved ventilation strategies have been the mainstay for reducing mortality in acute respiratory distress syndrome. Their unique clinical effectiveness is, however, unmatched by our understanding of the underlying mechanobiology, and their impact on alveolar dynamics and gas exchange remains largely speculative. OBJECTIVES: To assess changes in alveolar dynamics and associated effects on local gas exchange in experimental models of acute lung injury (ALI) and their responsiveness to sighs. METHODS: Alveolar dynamics and local gas exchange were studied in vivo by darkfield microscopy and multispectral oximetry in experimental murine models of ALI induced by hydrochloric acid, Tween instillation, or in antibody-mediated transfusion-related ALI. MEASUREMENTS AND MAIN RESULTS: Independent of injury mode, ALI resulted in asynchronous alveolar ventilation characteristic of alveolar pendelluft, which either spontaneously resolved or progressed to a complete cessation or even inversion of alveolar ventilation. The functional relevance of the latter phenomena was evident as impaired blood oxygenation in juxtaposed lung capillaries. Individual sighs (2 × 10 s at inspiratory plateau pressure of 30 cm H2O) largely restored normal alveolar dynamics and gas exchange in acid-induced ALI, yet not in Tween-induced surfactant depletion. CONCLUSIONS: We describe for the first time in detail the different forms and temporal sequence of impaired alveolar dynamics in the acutely injured lung and report the first direct visualization of alveolar pendelluft. Moreover, we identify individual sighs as an effective strategy to restore intact alveolar ventilation by a mechanism independent of alveolar collapse and reopening.

Mechanical ventilation causes airway distension with proinflammatory sequelae in mice.

The pathogenesis of ventilator-induced lung injury has predominantly been attributed to overdistension or mechanical opening and collapse of alveoli, whereas mechanical strain on the airways is rarely taken into consideration. Here, we hypothesized that mechanical ventilation may cause significant airway distension, which may contribute to the pathological features of ventilator-induced lung injury. C57BL/6J mice were anesthetized and mechanically ventilated at tidal volumes of 6, 10, or 15 ml/kg body wt. Mice were imaged by flat-panel volume computer tomography, and central airways were segmented and rendered in 3D for quantitative assessment of airway distension. Alveolar distension was imaged by intravital microscopy. Functional dead space was analyzed in vivo, and proinflammatory cytokine release was analyzed in isolated, ventilated tracheae. CT scans revealed a reversible, up to 2.5-fold increase in upper airway volume during mechanical ventilation compared with spontaneous breathing. Airway distension was most pronounced in main bronchi, which showed the largest volumes at tidal volumes of 10 ml/kg body wt. Conversely, airway distension in segmental bronchi and functional dead space increased almost linearly, and alveolar distension increased even disproportionately with higher tidal volumes. In isolated tracheae, mechanical ventilation stimulated the release of the early-response cytokines TNF-α and IL-1ß. Mechanical ventilation causes a rapid, pronounced, and reversible distension of upper airways in mice that is associated with an increase in functional dead space. Upper airway distension is most pronounced at moderate tidal volumes, whereas higher tidal volumes redistribute preferentially to the alveolar compartment. Airway distension triggers proinflammatory responses and may thus contribute relevantly to ventilator-induced pathologies.

Angiotensin-(1-7) protects from experimental acute lung injury.

OBJECTIVES: Recently, recombinant angiotensin-converting enzyme 2 was shown to protect mice from acute lung injury, an effect attributed to reduced bioavailability of angiotensin II. Since angiotensin-converting enzyme 2 metabolizes angiotensin II to angiotensin-(1-7), we hypothesized that this effect is alternatively mediated by angiotensin-(1-7) and activation of its receptor(s). DESIGN: To test this hypothesis, we investigated the effects of intravenously infused angiotensin-(1-7) in three experimental models of acute lung injury. SETTING: Animal research laboratory. SUBJECTS: Male Sprague-Dawley rats, Balb/c mice, and C57Bl6/J mice. INTERVENTIONS: Angiotensin-(1-7) was administered with ventilator- or acid aspiration-induced lung injury in mice or 30 minutes after oleic acid infusion in rats. In vitro, the effect of angiotensin-(1-7) on transendothelial electrical resistance of human pulmonary microvascular endothelial cells was analyzed. MEASUREMENTS AND MAIN RESULTS: Infusion of angiotensin-(1-7) starting 30 minutes after oleic acid administration protected rats from acute lung injury as evident by reduced lung edema, myeloperoxidase activity, histological lung injury score, and pulmonary vascular resistance while systemic arterial pressure was stabilized. Such effects were largely reproduced by the nonpeptidic angiotensin-(1-7) analog AVE0991. Infusion of angiotensin-(1-7) was equally protective in murine models of ventilator- or acid aspiration-induced lung injury. In the oleic acid model, the two distinct angiotensin-(1-7) receptor blockers A779 and D-Pro-angiotensin-(1-7) reversed the normalizing effects of angiotensin-(1-7) on systemic and pulmonary hemodynamics, but only D-Pro-angiotensin-(1-7) blocked the protection from lung edema and protein leak, whereas A779 restored the infiltration of neutrophils. Rats were also protected from acute lung injury by the AT1 antagonist irbesartan; however, this effect was again blocked by A779 and D-Pro-angiotensin-(1-7). In vitro, angiotensin-(1-7) protected pulmonary microvascular endothelial cells from thrombin-induced barrier failure, yet D-Pro-angiotensin-(1-7) or NO synthase inhibition blocked this effect. CONCLUSIONS: Angiotensin-(1-7) or its analogs attenuate the key features of acute lung injury and may present a promising therapeutic strategy for the treatment of this disease.

Hypoxic pulmonary vasoconstriction requires connexin 40-mediated endothelial signal conduction.

Hypoxic pulmonary vasoconstriction (HPV) is a physiological mechanism by which pulmonary arteries constrict in hypoxic lung areas in order to redirect blood flow to areas with greater oxygen supply. Both oxygen sensing and the contractile response are thought to be intrinsic to pulmonary arterial smooth muscle cells. Here we speculated that the ideal site for oxygen sensing might instead be at the alveolocapillary level, with subsequent retrograde propagation to upstream arterioles via connexin 40 (Cx40) endothelial gap junctions. HPV was largely attenuated by Cx40-specific and nonspecific gap junction uncouplers in the lungs of wild-type mice and in lungs from mice lacking Cx40 (Cx40-/-). In vivo, hypoxemia was more severe in Cx40-/- mice than in wild-type mice. Real-time fluorescence imaging revealed that hypoxia caused endothelial membrane depolarization in alveolar capillaries that propagated to upstream arterioles in wild-type, but not Cx40-/-, mice. Transformation of endothelial depolarization into vasoconstriction involved endothelial voltage-dependent α1G subtype Ca2+ channels, cytosolic phospholipase A2, and epoxyeicosatrienoic acids. Based on these data, we propose that HPV originates at the alveolocapillary level, from which the hypoxic signal is propagated as endothelial membrane depolarization to upstream arterioles in a Cx40-dependent manner.