CIRS-LAS - a novel approach to increase transparency in laboratory animal science for improving animal welfare by reducing laboratory animal distress.

The 3Rs principle is highly topical in animal-based research. These include, above all, new scientific methods for conducting experiments without an animal model, by using non-animal models (Replace), reducing the number of laboratory animals (Reduction) or taking measures to keep the stress on the laboratory animal as low as possible (Refinement). Despite numerous modern alternative approaches, the complete replacement of animal experiments is not yet possible. The exchange in the team about the daily work with laboratory animals, about open questions and problems, contributes to a reflection of one's own work and to a better understanding of the work of the others. CIRS-LAS (Critical Incident Reporting System in Laboratory Animal Science) represents a reporting system for incidents in laboratory animal science. It is urgently needed because the lack of transparency about incidents leads to the repetition of failed experiments. Negative experiences from animal-based experiments are often not mentioned in publications, and the fear of hostility is still very high. Therefore, a constructive approach to errors is not a matter of course. To overcome this barrier, CIRS-LAS was created as a web-based database. It addresses the areas of reduction and refinement of the 3Rs principle by providing a platform to collect and analyze incidents. CIRS-LAS is open to all individuals working with laboratory animals worldwide and currently exists with 303 registered members, 52 reports, and an average of 71 visitors per month. The development of CIRS-LAS shows, that an open and constructive error culture is difficult to establish. Nevertheless, the upload of a case report or the search in the database leads to an active reflection of critical occurrences. Thus, it is an important step towards more transparency in laboratory animal science. As expected, the collected events in the database concern different categories and animal species and are primarily reported by persons involved in an experiment. However, reliable conclusions about observed effects require further analysis and continuous collection of case reports. Looking at the development of CIRS-LAS, its high potential is shown in considering the 3Rs principle in daily scientific work.

Altered Cerebral Blood Flow and Potential Neuroprotective Effect of Human Relaxin-2 (Serelaxin) During Hypoxia or Severe Hypovolemia in a Sheep Model.

Specific neuroprotective strategies to minimize cerebral damage caused by severe hypoxia or hypovolemia are lacking. Based on previous studies showing that relaxin-2/serelaxin increases cortical cerebral blood flow, we postulated that serelaxin might provide a neuroprotective effect. Therefore, we tested serelaxin in two emergency models: hypoxia was induced via inhalation of 5% oxygen and 95% nitrogen for 12 min; thereafter, the animals were reoxygenated. Hypovolemia was induced and maintained for 20 min by removal of 50% of the total blood volume; thereafter, the animals were retransfused. In each damage model, the serelaxin group received an intravenous injection of 30 µg/kg of serelaxin in saline, while control animals received saline only. Blood gases, shock index values, heart frequency, blood pressure, and renal blood flow showed almost no significant differences between control and treatment groups in both settings. However, serelaxin significantly blunted the increase of lactate during hypovolemia. Serelaxin treatment resulted in significantly elevated cortical cerebral blood flow (CBF) in both damage models, compared with the respective control groups. Measurements of the neuroproteins S100B and neuron-specific enolase in cerebrospinal fluid revealed a neuroprotective effect of serelaxin treatment in both hypoxic and hypovolemic animals, whereas in control animals, neuroproteins increased during the experiment. Western blotting showed the expression of relaxin receptors and indicated region-specific differences in relaxin receptor-mediated signaling in cortical and subcortical brain arterioles, respectively. Our findings support the hypothesis that serelaxin is a potential neuroprotectant during hypoxia and hypovolemia. Due to its preferential improvement of cortical CBF, serelaxin might reduce cognitive impairments associated with these emergencies.

Pulmonary hemodynamic effects and pulmonary arterial compliance during hypovolemic shock and reinfusion with human relaxin-2 (serelaxin) treatment in a sheep model.

BACKGROUND: Previous studies on the recombinant form of human relaxin-2 (serelaxin) have shown a decrease of pulmonary hemodynamics after serelaxin injection. Currently, the effect of serelaxin treatment during hypovolemia in a large animal model remains mostly unknown. METHODS: 12 sheep were randomly assigned to a sham or serelaxin (30µg/kg serelaxin) group and underwent right heart catheterization. 50% of the estimated total blood volume were removed to induce hypovolemia, and subsequently retransfused 20 min later (reinfusion). Blood gases, heart rate, peripheral and pulmonary arterial oxygen saturation, systolic, diastolic and mean values of both pulmonary artery pressure (PAP) and pulmonary capillary wedge pressure (PCW) were measured. Cardiac output (CO), pulmonary vascular resistance (PVR), pulmonary arterial compliance (PAcompl) and systemic vascular resistance (SVR) were calculated. RESULTS: Hypovolemia and shock led to a similar decrease of PAP and PCW in both groups (p≤0.001). CO, SV and PAcompl decreased only in the control group (p≤0.05) and remained higher in the serelaxin-treated group. The results of this study suggest that serelaxin treatment did not negatively influence hemodynamic parameters during hypovolemic shock. CONCLUSION: The main conclusion of this study is that cardiopulmonary adaption mechanisms are not critically altered by serelaxin administration during severe hypovolemia and retransfusion.

Pulmonary arterial compliance and pulmonary hemodynamic effects of Serelaxin in a sheep model.

BACKGROUND: The influence of the recombinant form of human relaxin-2 (serelaxin) on pulmonary hemodynamics under physiologic conditions have not been the subject of studies in an animal model up until now. METHODS: We therefore utilised the large animal model sheep, convenient in its similar cardiovascular physiology, to investigate said influence. All animals underwent right heart catheterization, a safe and reliable invasive procedure for the assessment of pulmonary hemodynamics, and then received either 30µg/kg serelaxin (n = 11) or saline (n = 13). Systolic, diastolic and mean values of both pulmonary artery pressure (respectively, PAPs, PAPd, PAPm) and pulmonary capillary wedge pressure (respectively, PCWs, PCWd, PCWm) blood gases, heart rate (HR) and both peripheral and pulmonary arterial oxygen saturation were obtained. Cardiac output (CO), pulmonary vascular resistance (PVR), pulmonary arterial compliance (PAcompl) and systemic vascular resistance (SVR) were calculated. RESULTS: The key findings of the current study are that 20 min after serelaxin injection a rapid decrease of the PAPm, PCWPm, SVR and an decrease of the PAcompl was observed (P < 0.01). CONCLUSION: These findings suggest that serelaxin might be suitable to improve pulmonary hemodynamics in clinically relevant scenarios, like acute heart failure or pulmonary hypertension.

Evaluation of a vital staining protocol with 2,3,5-triphenyltetrazolium chloride for cancellous bone in a sheep model.

Decision making on the optimal surgical treatment of fractures often is hampered by the lack of a method for direct assessment of bone vitality. In various contexts, for example to determine the extents of cerebral insults or of myocardial infarctions in experimental studies, tetrazolium based staining procedures of vital cells are widely used. Here, we set out to test the applicability of tetrazolium based staining on bone samples. 8 brains and 26 femoral heads from sheep were used to prepare tissue slices, which were stained with 2,3,5-triphenyltetrazolium chloride (TTC) at various times (1 to 12h) after explantation. Staining of tissue slices was quantified by densitometric image analysis. Spectrophotometry was used for quantification in cultured cells. TTC-staining of tissue slices indicated detectability of vital cells in slices from both tissues up to 4h after explantation. Staining intensity at later time-points was indistinguishable from the staining of untreated samples or sodium azide treated (necrotic cells) controls. We provide experimental evidence that the choice of the optimal surgical approach for the treatment of fractures involving cancellous bone could be aided by a simple staining procedure for vital bone. However, the described procedure depends on the availability of bone specimens (slices). Therefore, search for an improved stain directly applicable to the bone surface is needed.

Increase of cortical cerebral blood flow and further cerebral microcirculatory effects of Serelaxin in a sheep model.

Serelaxin, recombinant human relaxin-2, modulates endothelial vasodilatory functionality and is under evaluation for treatment of acute heart failure. Little is known about acute effects on cerebral perfusion. We tested the hypothesis that Serelaxin might also have effects on the cerebral microcirculation in a sheep model, which resembles human brain structure quite well. We used laser Doppler flowmetry and sidestream dark-field (SDF) imaging techniques, which are reliable tools to continuously assess dynamic changes in cerebral perfusion. Laser Doppler flowmetry shows that bolus injection of 30 µg Serelaxin/kg body wt induces an increase (P = 0.006) to roughly 150% of cortical cerebral blood flow (CBF), whereas subcortical CBF remains unchanged (P = 0.688). The effects on area-dependent CBF were significantly different after the bolus injection (P = 0.042). Effects on cortical CBF were further confirmed by SDF imaging. The bolus injection of Serelaxin increased total vessel density to 127% (P = 0.00046), perfused vessel density to 145% (P = 0.024), and perfused capillary density to 153% (P = 0.024). Western blotting confirmed the expression of relaxin receptors RXFP1 and truncated RXFP2-variants in the respective brain regions, suggesting a possible contribution of RXFP1 on the effects of Serelaxin. In conclusion, the injection of a high dose of Serelaxin exerts quick effects on the cerebral microcirculation. Therefore, Serelaxin might be suitable to improve cortical microcirculation and exert neuroprotective effects in clinically relevant scenarios that involve cortical hypoperfusion. These findings need to be confirmed in relevant experimental settings involving cerebral cortical hypoperfusion and can possibly be translated into clinical practice.

Renal glucose release during hypoglycemia is partly controlled by sympathetic nerves - a study in pigs with unilateral surgically denervated kidneys.

Catecholamines are known to increase renal glucose release during hypoglycemia. The specific extent of the contribution of different sources of catecholamines, endocrine delivery via circulation or release from autonomous sympathetic renal nerves, though, is unknown. We tested the hypothesis that sympathetic renal innervation plays a major role in the regulation of renal gluconeogenesis. For this purpose, instrumented adolescent pigs had one kidney surgically denervated while the other kidney served as a control. A hypoglycemic clamp with arterial blood glucose below 2 mmol/L was maintained for 75 min. Arteriovenous blood glucose difference, inulin clearance, p-aminohippurate clearance, and sodium excretion were measured in intervals of 15 min separately for both kidneys. Blood glucose was lowered to 0.84 ± 0.33 mmol/L for 75 min. The side-dependent renal net glucose release (SGN) decreased significantly after the unilateral ablation of renal nerves. In the linear mixed model, renal denervation had a significant inhibitory effect on renal net glucose release (P = 0.036). The SGN of the ablated kidney decreased by 0.02 mmol/min and was equivalent to 43.3 ± 23.2% of the control (nonablated) kidney in the pigs. This allows the conclusion that renal glucose release is partly controlled by sympathetic nerves. This may be relevant in humans as well, and could explain the increased risk of severe hypoglycemia of patients with diabetes mellitus and autonomous neuropathy. The effects of denervation on renal glucose metabolism should be critically taken into account when considering renal denervation as a therapy in diabetic patients.

Laryngeal pacing in minipigs: in vivo test of a new minimal invasive transcricoidal electrode insertion method for functional electrical stimulation of the PCA.

Functional electrical stimulation (FES) of the posterior cricoarytenoid muscle (PCA) to restore respiratory function of the larynx may become an option for the treatment of bilateral recurrent laryngeal nerve paralysis (RLNP) in the near future. The feasibility of this has been shown in several animal trials and in a human pilot study. The common open surgical inferolateral approach for electrode insertion into the PCA for FES has a risk of damaging the recurrent laryngeal nerve (RLN) and may result in postoperative swelling and scaring of the larynx. Therefore, a minimal invasive electrode insertion technique is needed. A new miniaturized bipolar spiral tip electrode and a new electrical stimulatable insertion needle were tested in a short-term trial for an endoscopically guided and functionally controlled transcricoidal electrode insertion in eight Göttingen minipigs with bilateral normal RLN function. The feasibility of this technique was evaluated and the achieved positions of the electrodes in the PCA were analyzed using intraoperative stimulation threshold data and 3D-CT reconstructions. In seven cases it was possible to place two well-performing electrodes into the PCA. They were positioned one on either side. In one animal no functioning electrode position could be achieved because the PCA was missed. Thresholds of the electrode tips varied between 0.2 and 2.5 mA (mean 0.71 mA). In any case maximal glottal opening could be reached before adductors were co-activated. The majority of electrodes were placed into the central lower part of the PCA with no apparent correlation between threshold and electrode position. Surgical trauma might be further reduced by using endoscopy via a laryngeal mask avoiding the temporary tracheostomy used in this trial. If the implanted electrodes remain stable in long-term tests, we suggest that this method could soon be transferred into human application.

Effects of early- and late-gestational maternal stress and synthetic glucocorticoid on development of the fetal hypothalamus-pituitary-adrenal axis in sheep.

Prenatal maternal stress (PMS) programs dysregulation of the hypothalamus-pituitary-adrenal axis (HPAA) in postnatal life, though time periods vulnerable to PMS, are still unclear. We evaluated in pregnant sheep the effect of PMS during early gestation [30-100 days of gestation (dGA); term is 150 dGA] or late gestation (100-120 dGA) on development of fetal HPAA function. We compared the effects of endogenous cortisol with synthetic glucocorticoid (GC) exposure, as used clinically to enhance fetal lung maturation. Pregnant sheep were exposed to repeated isolation stress twice per week for 3 h in a separate box with no visual, tactile, or auditory contact with their flock-mates either during early (n = 7) or late (n = 7) gestation. Additional groups received two courses of betamethasone (BM; n = 7; 2 × 110 µg kg(- 1) body weight, 24 h apart) during late gestation (106/107 and 112/113 dGA, n = 7) or acted as controls (n = 7). Fetal cortisol responses to hypotensive challenge, a physiological fetal stressor, were measured at 112 and 129 dGA, i.e. before and during maturation of the HPAA. Hypotension was induced by fetal infusion of sodium nitroprusside, a potent vasodilator. At 112 dGA, neither PMS nor BM altered fetal cortisol responses. PMS, during early or late gestation, and BM treatment increased fetal cortisol responses at 129 dGA with the greatest increase achieved in stressed early pregnant sheep. Thus, development of the HPAA is vulnerable to inappropriate levels of GCs during long periods of fetal life, whereas early gestation is most vulnerable to PMS.