Clicker Training in Minipigs to Reduce Stress during Blood Collection-An Example of Applied Refinement.

Pigs (and minipigs) are often restrained with a maxillary sling for blood collection. They mainly produce strong vocalisations and show resistance to the procedure, which subjectively appears to be stressful for the animals. The present study investigated whether minipigs can be trained to tolerate aversive stimuli and whether training can reduce stress during blood collection. Blood was taken from 12 Ellegaard minipigs with fixation; thereafter, the animals were trained for 3 weeks using clicker training. Then, blood was taken again, but without fixation. Before and after each blood sample, saliva samples were taken. The cortisol concentration was determined using ELISAs. Serum cortisol was not significantly different before and after training (paired-sample t-test, t (9) = 2.052, p = 0.07). However, salivary cortisol was significantly lower after training (ANOVA (analysis of variance), p-value < 0.001, F-value 6.181). In addition, trained minipigs showed a significantly lower heart rate after blood sampling (paired-sample t-test, t (11) = 4.678, p = 0.001) as well as significantly lower heart rate variability (t (11) = 3.704, p = 0.003) compared to before training. The minipigs could be trained to tolerate aversive stimuli. This contributed to stress reduction when taking blood samples.

Clicker Training as an Applied Refinement Measure in Chickens.

When using chickens in animal studies, the handling of these animals for sample collection or general examinations is considered stressful due to their prey nature. For the study presented here, plasma and salivary corticosterone as well as New Area Test behavior and fecal output were used to evaluate whether it is possible to influence this stress perception using a three-week clicker training program. The results indicate that clicker training seems to be a suitable refinement measure in the sense of cognitive enrichment for the husbandry of this species. However, since it was also shown that three-week training was not sufficient to sustainably reduce the stress perception with regard to prolonged stressor exposure, and since it was also evident that manipulations such as routine blood sampling are perceived as less stressful than assumed, further studies with prolonged training intervals and situations with higher stressor potential are warranted. Also, further parameters for training assessment must be considered. For the general use of training as a supportive measure in animal experiments, its proportionality must be considered, particularly considering the expected stress and adequate training time.

Animal Welfare during Transport and Slaughter of Cattle: A Systematic Review of Studies in the European Legal Framework.

Literature related to European transport and slaughter processes were included in this systematic review. The publication period is limited to the past twelve years since the European Animal Welfare Transport Regulation was enacted in 2009. Three different databases were used. The final screening resulted in the inclusion of 19 articles in this review. When handling cattle during transport and slaughter, personnel have an important impact and may inflict stress on the animals. Other factors, such as the group composition and health status prior to transport, can have a strong negative effect on animal welfare. At the abattoir, constructional conditions and the resulting environmental influences can have a negative impact on welfare as well. These include increased noise levels due to the lack of noise dampening and changing light conditions. Stress in cattle can be assessed, e.g., by measuring stress hormones or heart rate. Effective stunning is an important welfare-relevant step in the slaughtering process. Some signs of unconsciousness, such as immediate body collapse or absence of the corneal reflex, can be easily assessed. Expertise and continuous training of all personnel involved are important measures in stress reduction.

Psychological stress and strain in laboratory animal professionals - a systematic review.

Laboratory animal professionals (LAP) are faced with various situations and tasks influencing their mental well-being. A systematic review has been conducted to investigate whether there are specific stressors for LAP and which moderators are relevant for the development of psychological strain. A comprehensive search following PRISMA Guidelines was carried out in June 2021. Results include 12 studies and have been summarized qualitatively in narrative synthesis and tabular presentation. Available literature indicates that LAP are facing stressors but does not allow for conclusions on specific stressful job duties other than euthanasia.Signs of strain are present in LAP. Specifically, participants in qualitative studies reported acute symptoms, while chronic manifestations were in focus in quantitative studies. Although a wide variety of moderating factors have been investigated, only social support has been rated as relevant by multiple qualitative and quantitative studies without contrasting results.According to current data, there is a risk for psychological strain in LAP. However, there is limited understanding of specific stressors and data on moderators is diverse. Further studies that focus on domain-specific knowledge and clearly distinguish stressors from moderators are necessary to set up institutional programmes addressing psychological strain in LAP.

Stress hormones or general well-being are not altered in immune-deficient mice lacking either T- and B- lymphocytes or Interferon gamma signaling if kept under specific pathogen free housing conditions.

It is controversially discussed whether immune-deficient mice experience severity in the absence of infection. Because a comprehensive analysis of the well-being of immune-deficient mice under specific pathogen free conditions is missing, we used a multi-parametric test analyzing, corticosterone, weight, nest building and facial expression over a period of 9 month to determine the well-being of two immune-deficient mouse lines (recombination activating gene 2- and interferon gamma receptor-deficient mice). We do not find evidence for severity when comparing immune-deficient mice to their heterozygous immune-competent littermates. Our data challenge the assumption that immune-deficiency per se regardless of housing conditions causes severity. Based on our study we propose to use objective non-invasive parameters determined by laboratory animal science for decisions concerning severity of immune-deficient mice.

Direct angiotensin type 2 receptor (AT2R) stimulation attenuates T-cell and microglia activation and prevents demyelination in experimental autoimmune encephalomyelitis in mice.

In the present study, we evaluated stimulation of the angiotensin type 2 receptor (AT2R) by the selective non-peptide agonist Compound 21 (C21) as a novel therapeutic concept for the treatment of multiple sclerosis using the model of experimental autoimmune encephalomyelitis (EAE) in mice. C57BL-6 mice were immunized with myelin-oligodendrocyte peptide and treated for 4 weeks with C21 (0.3 mg/kg/day i.p.). Potential effects on myelination, microglia and T-cell composition were estimated by immunostaining and FACS analyses of lumbar spinal cords. The in vivo study was complemented by experiments in aggregating brain cell cultures and microglia in vitro. In the EAE model, treatment with C21 ameliorated microglia activation and decreased the number of total T-cells and CD4+ T-cells in the spinal cord. Fluorescent myelin staining of spinal cords further revealed a significant reduction in EAE-induced demyelinated areas in lumbar spinal cord tissue after AT2R stimulation. C21-treated mice had a significantly better neurological score than vehicle-treated controls. In aggregating brain cell cultures challenged with lipopolysaccharide (LPS) plus interferon-γ (IFNγ), AT2R stimulation prevented demyelination, accelerated re-myelination and reduced the number of microglia. Cytokine synthesis and nitric oxide production by microglia in vitro were significantly reduced after C21 treatment. These results suggest that AT2R stimulation protects the myelin sheaths in autoimmune central nervous system inflammation by inhibiting the T-cell response and microglia activation. Our findings identify the AT2R as a potential new pharmacological target for demyelinating diseases such as multiple sclerosis.

Composition of intestinal microbiota in immune-deficient mice kept in three different housing conditions.

BACKGROUND: Abundance of commensals constituting the intestinal microbiota (IM) affects the immune system and predisposes to a variety of diseases, including intestinal infections, cancer, inflammatory and metabolic disorders. Housing conditions determine the IM and can hence influence the immune system. We analyzed how both variables affect the IM of four immune-compromized mouse lines kept under different housing conditions. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the IM composition in mice by quantitative 16S rRNA RT-PCR analysis of the main fecal bacterial groups (Enterobacteriaceae, enterococci, lactobacilli, bifidobacteria, Bacteroides/Prevotella (BP) spp., Clostridium leptum and coccoides groups). Mice were homozygous (HO) or heterozygous (HE) for a targeted inactivating mutation of either the IFN-γ Receptor (R), IFN-γ, Rag1 or IL-4 genes. Overall, differences in IM composition were subtle. However, in the SPF-barrier, total eubacterial loads were higher in Rag1 HE versus Rag1 HO mice as well as in IFN-γR HE versus IFN-γR HO and WT animals. Although absent in WT mice, bifidobacterial loads were higher in HO and HE IFN-γ and Rag1 as well as IL-4 HO mice. Furthermore, BP was slightly lower in HO and HE IFN-γR and IFN-γ mice as well as in IL-4 HO mice as compared to WT controls. Interestingly, IM compositions were comparable in WT mice when kept in individual ventilated cages (IVC) or open cages (OC). IFN-γ HO and HE mice, however, had higher enterobacteria and BP loads, but lacked bifidobacteria when kept in OC versus IVC, as was the case in HO and HE Rag1 mice. In addition, Rag1 HO mice harbored higher clostridial loads when housed in OC as compared to IVC. Unexpectedly, lactobacilli levels were higher in IFN-γR mice when kept in OC versus IVC. CONCLUSION/SIGNIFICANCE: Housing-dependent and immune-deficiency mediated changes in intestinal microbiota composition were rather subtle but may nevertheless impact immunopathology in experimental models.

Differences in serum cytokine levels between wild type mice and mice with a targeted mutation suggests necessity of using control littermates.

To enhance protection from pathogens, housing conditions have been improved constantly. We wanted to test whether various environmental conditions and caging systems affect serum cytokine levels of immunodeficient mice differently than they affect immunocompetent control animals. We compared serum cytokine levels of immunodeficient and immunocompetent mice kept in three different environments: a specific pathogen free (SPF) breeding barrier with open cages. An SPF experimental unit with individually ventilated cages. An experimental semi-barrier with open cages. Serum from Rag1(-/-), µMT(-/-), IFN-γR(-/-), IFN-γ(-/-), IL-4(-/-), the heterozygous controls and wild type C57BL/6 or BALB/c mice was analyzed for the presence of 10 cytokines (IL-1α, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, IFN-γ, TNF-α and GM-CSF). No major changes in cytokine levels were detected in mice exposed to different housing conditions. However, irrespective of immunodeficiency at 4 weeks of age a number of mice from the breeding colonies with a targeted mutation (TM), both -/- and +/- mice, showed a statistically significant elevation of some cytokines (primarily IL-1α, IL-5) when compared to wild type BALB/c and C57BL/6 mice. We conclude that under SPF conditions, immunodeficient mice can be kept either in open caging or IVC systems without affecting serum cytokine levels. The more important conclusion, however, stems from the observation that there is a significant difference in serum cytokine levels between wild type and mice carrying either one or two alleles of a targeted mutation (either -/- and +/- mice). This suggests an altered base-line inflammatory responsiveness in the TM-breeding colonies.

Prevention and intervention studies with telmisartan, ramipril and their combination in different rat stroke models.

OBJECTIVES: The effects of AT1 receptor blocker, telmisartan, and the ACE inhibitor, ramipril, were tested head-to head and in combination on stroke prevention in hypertensive rats and on potential neuroprotection in acute cerebral ischemia in normotensive rats. METHODS: Prevention study: Stroke-prone spontaneously hypertensive rats (SHR-SP) were subjected to high salt and randomly assigned to 4 groups: (1) untreated (NaCl, n = 24), (2) telmisartan (T; n = 27), (3) ramipril (R; n = 27) and (4) telmisartan + ramipril (T+R; n = 26). Drug doses were selected to keep blood pressure (BP) at 150 mmHg in all groups. Neurological signs and stroke incidence at 50% mortality of untreated SHR-SP were investigated. Intervention study: Normotensive Wistar rats were treated s.c. 5 days prior to middle cerebral artery occlusion (MCAO) for 90 min with reperfusion. Groups (n = 10 each): (1) sham, (2) vehicle (V; 0.9% NaCl), (3) T (0.5 mg/kg once daily), (4) R (0.01 mg/kg twice daily), (5) R (0.1 mg/kg twice daily) or (6) T (0.5 mg/kg once daily) plus R (0.01 mg/kg twice daily). Twenty-four and 48 h after MCAO, neurological outcome (NO) was determined. Forty-eight h after MCAO, infarct volume by MRI, neuronal survival, inflammation factors and neurotrophin receptor (TrkB) were analysed. RESULTS: Stroke incidence was reduced, survival was prolonged and neurological outcome was improved in all treated SHR-SP with no differences between treated groups. In the acute intervention study, T and T+R, but not R alone, improved NO, reduced infarct volume, inflammation (TNFα), and induced TrkB receptor and neuronal survival in comparison to V. CONCLUSIONS: T, R or T+R had similar beneficial effects on stroke incidence and NO in hypertensive rats, confirming BP reduction as determinant factor in stroke prevention. In contrast, T and T+R provided superior neuroprotection in comparison to R alone in normotensive rats with induced cerebral ischemia.

Novel therapy approach in primary stroke prevention: simultaneous inhibition of endothelin converting enzyme and neutral endopeptidase in spontaneously hypertensive, stroke-prone rats improves survival.

OBJECTIVES: Stroke, frequently a consequence of hypertension, is one of the leading causes of death and neurological disabilities worldwide. In the ischemic brain, levels of endothelin-1, one of the most potent vasoconstrictors, are raised. Anti-inflammatory and neuroprotective effects of endothelin antagonists after stroke have been described in literature. Based on these findings, we investigated the protective effect of the endothelin converting enzyme/neutral endopeptidase blocker, SLV 338, in salt-loaded, stroke-prone, spontaneously hypertensive rats. METHODS: Male, 8-week-old spontaneously hypertensive stroke-prone rats were put on a high salt diet and treated with either 30 mg/kg or 100 mg/kg SLV 338 or vehicle for 27 weeks. Blood pressure, neurological outcome, body weight, and mortality were investigated throughout treatment. In weeks 1 and 9, animals were housed in metabolic cages for collection of urinary and blood samples and assessment of salt water and food intake. In weeks 22 and 27, additional blood samples were taken. At the end of the study, all brains were analyzed using magnetic resonance imaging. RESULTS: SLV 338 was well tolerated in all animals. Neurological outcome and infarct size were similar in all groups. Albuminuria was considerably delayed and the incidence of stroke significantly lowered in treated animals. In spontaneously hypertensive stroke-prone rats, treatment with SLV 338 significantly (P = 0·01) improved survival in comparison to the vehicle treated group in a blood pressure-independent manner. DISCUSSION: Our data in spontaneously hypertensive stroke-prone rats demonstrate that combined endothelin converting enzyme/neutral endopeptidase inhibition could offer a new therapeutic approach for primary stroke prevention and improvement of mortality. The mechanism seems to be blood pressure-independent.

Effects of aliskiren on stroke in rats expressing human renin and angiotensinogen genes.

OBJECTIVE: Pre-treatment with angiotensin receptor blockers is known to improve neurological outcome after stroke. This study investigated for the first time, whether the renin inhibitor aliskiren has similar neuroprotective effects. METHODS: Since aliskiren specifically blocks human renin, double transgenic rats expressing human renin and angiotensinogen genes were used. To achieve a systolic blood pressure of 150 or 130 mmHg animals were treated with aliskiren (7.5 or 12.5 mg/kg*d) or candesartan (1.5 or 10 mg/kg*d) via osmotic minipump starting five days before middle cerebral artery occlusion with reperfusion. Infarct size was determined by magnetic resonance imaging. mRNA of inflammatory marker genes was studied in different brain regions. RESULTS: The mortality of 33.3% (7 of 21 animals) in the vehicle group was reduced to below 10% by treatment with candesartan or aliskiren (p<0.05). Aliskiren-treated animals had a better neurological outcome 7 days post-ischemia, compared to candesartan (Garcia scale: 9.9±0.7 vs. 7.3±0.7; p<0.05). The reduction of infarct size in the aliskiren group did not reach statistical significance compared to candesartan and vehicle (24 h post-ischemia: 314±81 vs. 377±70 and 403±70 mm(3) respectively). Only aliskiren was able to significantly reduce stroke-induced gene expression of CXC chemokine ligand 1, interleukin-6 and tumor necrosis factor-alpha in the ischemic core. CONCLUSIONS: Head-to-head comparison suggests that treatment with aliskiren before and during cerebral ischemia is at least as effective as candesartan in double transgenic rats. The improved neurological outcome in the aliskiren group was blood pressure independent. Whether this effect is due to primary anti-inflammatory mechanisms has to be investigated further.

No effects of human ghrelin on cardiac function despite profound effects on body composition in a rat model of heart failure.

BACKGROUND: Ghrelin, was observed to have treatment-potential for severe chronic heart failure (CHF) and cardiac cachexia based on anti-cachectic and cardio-protective effects. METHODS: We performed two studies to assess the effects of human ghrelin on food intake, body weight and body composition, as well as heart function in a rat model of CHF. Study-1 (50 or 500 nmole/kg/d ghrelin by pump infusion) was focused on food intake and body composition, study-2 (50 or 100 nmole/kg/d ghrelin by subcutaneous injection (3-times daily) was focused on heart function due to a lack of cardiac effects observed in study-1. In both studies, myocardial infarction was induced by LAD ligation. On day 28 after surgery, rats were randomized and treated with ghrelin or placebo for 4 weeks. Food intake (study-1), body composition (NMR) cardiac function (echocardiography and invasive hemodynamics (study-2 only) were assessed. RESULTS: In study-1, CHF rats treated with high dose ghrelin showed an increase in body weight (+25%, p<0.001), lean mass (+16%, p<0.01) and fat mass (+17%, p=0.001) vs placebo. In study-2, CHF rats treated with both low- and high dose ghrelin showed an increase in body weight (both +18%, p

Comparison between single and combined treatment with candesartan and pioglitazone following transient focal ischemia in rat brain.

Angiotensin AT1 receptor blockers (ARBs) and thiazolidinediones (TZDs) have become well established drugs for the treatment of major risk factors of stroke. Since several studies provided evidence that ARBs and TZDs also have additional anti-inflammatory effects, we hypothesized that a combined treatment with the ARB, candesartan, and the TZD, pioglitazone, ameliorates ischemia-induced brain injury and inflammation by synergistic anti-inflammatory actions. Normotensive Wistar rats were pre-treated for 5 days with vehicle (0.9% NaCl), 0.2 mg/kg/day candesartan (s.c.), and/or 2 and/or 20 mg/kg/day pioglitazone (p.o.), respectively and underwent 90 min of middle cerebral artery occlusion (MCAO) with successive reperfusion. Neurological deficits and infarct size were determined 24 h and 48 h after MCAO, respectively, followed by tissue sampling. Animals treated with candesartan, pioglitazone, and the combination of candesartan and pioglitazone had reduced neurological deficits 24 h and 48 h after MCAO, respectively (P<0.05-0.01). Infarct size was reduced by treatment of candesartan, pioglitazone, and their respective combination (each P<0.05) 48 h after stroke compared to vehicle. Treatment with candesartan, pioglitazone, and their combination resulted in significantly reduced mRNA expression of the inflammatory markers CXCL1 and TNFalpha in vivo (P<0.01). The combination of candesartan plus pioglitazone is equally effective compared to their single applications concerning neuroprotection and attenuation of inflammation after MCAO. Therefore, we conclude that a direct synergistic neuroprotective action of parallel ARB and TZD treatment is unlikely.