Defining piscine endpoints: Towards score sheets for assessment of clinical signs in fish research.

The seminar 'Severity and humane endpoints in fish research' organized by the University of Bergen, the Industrial and Aquatic Laboratory, together with Fondazione Guido Bernadini, took place on 4 October 2019 in Bergen, Norway. The seminar was followed by a workshop, 'Establishing score sheets and defining endpoints in fish experiments', held on 28 January 2020, also in Bergen. The purpose of the seminar was to raise awareness about fish ethics together with severity classification and humane endpoints in fish studies, using examples from farmed fish, mainly salmonids and lumpfish. The overall aim of the workshop was to better define humane endpoints in fish experiments, as well as to discuss suggestions for development and use of score sheets for assessing clinical signs related to endpoints. Endpoints for fish should not only be based on what we know about fish diseases and the lesions they induce but should also take into consideration knowledge about fish species and life stage, fish anatomy, physiology and the general state and behaviour of the fish. For this reason, to reinforce that endpoints should come from the animal's perspective and needs, we renamed humane endpoints for fish to piscine endpoints. This paper reports the main messages from the workshop sessions including advice on development and use of score sheets.

Airway inflammation and bronchial remodelling in toluene diisocyanate-exposed BALB/c mouse model.

UNLABELLED: Toluene diisocyanate (TDI), a highly reactive industrial chemical, is one of the leading causes of occupation-related asthma in industrialized countries. The pathogenesis of TDI-induced asthma, however, remains not fully understood, in part due to lack of appropriate animal models. Twenty five female BALB/c mice (age: 8 weeks) were randomly divided into 5 groups: Ovabumin (OVA); OVA peptide amino acid residues No. 323-339 (Pep); TDI; alum and physiological saline. Mice were intraperitoneally injected with 25 microg OVA or pep absorbed on 300 microg alum, 300 microg alum or saline on days 0, 7 and 14. For the TDI group, mice were sensitized subcutaneously with 20 microl neat TDI on day 0; 20 microl of TDI in olive oil (1:10) on days 7 and 14; on days 21-23. Then each group was challenged intranasally with 20 microl of 1% OVA, 1% Pep, 1% TDI, 10% alum and saline respectively. On day 28, mice were killed under pentothal anesthesia. The results demonstrated that neutrophil-dominant inflammation with a few eosinophil infiltration occurred in the peri-bronchial and peri-vascular regions of the lungs. This was accompanied by hyperplasia/hypertrophy of cells lining the airways and mucus production as shown by HE staining. Positive immunohistochemical MBP staining in parenchyma was also shown. Th2 cytokine IL-4 and IgE production were significant increased 5 days after last challenge while IFN-gamma level was below the detection limit. CONCLUSION: the clear elevation of IL-4 and IgE could allow to conclude a possible Th2-like dominated allergic response in TDI-exposed BALB/c mouse model.

Effect of the cytoskeletal fixation agent phalloidin on transcapillary albumin transport and interstitial fluid pressure following subdermal prostaglandin E1 administration in the rat.

AIM: Lowering of interstitial fluid pressure (Pif) facilitates fluid filtration across the capillary membrane and oedema formation in acute inflammation. The cellular mechanism behind this lowering of Pif involves beta1-integrins mediating contact between dermal cells and the extracellular matrix fibres, and also the cell cytoskeleton as disruption of actin filaments using cytochalasin-D induced a lowering of Pif and oedema formation. Fixation of actin with phalloidin attenuates oedema formation and abolishes lowering of Pif in anaphylaxis in the rat. The objective of this study was to determine whether phalloidin modifies lowering of Pif and albumin extravasation in rat skin also after prostaglandin E1 (PGE1). METHODS: Pif was measured using micropipettes connected to a servo-controlled counterpressure system. Microvascular permeability was estimated as the albumin extravasation (Ealb) using radiolabelled human serum albumin. RESULTS: Subdermal injection of PGE1 (0.85 mg mL-1) lowered Pif from -0.8 +/- 0.8 mmHg (SD) in control to -3.5 +/- 0.9 mmHg (P < 0.05) within 30 min. Pre-treatment with phalloidin (500 microg kg-1) before PGE1 resulted in Pif of -1.7 +/- 1.0 mmHg (P < 0.05 compared with PGE1). Ealb after subdermal saline was 0.07 +/- 0.04 mL g-1 DW and increased to 0.32 +/- 0.32 mL g-1 DW with PGE1 (P < 0.05) but was unaffected by pre-treatment with phalloidin given before PGE1 0.32 +/- 0.35 mL g-1 DW (P > 0.05 compared with PGE1 alone). CONCLUSION: These results are consistent with the concept that the cytoskeleton actin filaments participate in control of Pif.

Continuous measurements of plasma protein extravasation with microdialysis after various inflammatory challenges in rat and mouse skin.

This study describes the use of microdialysis technique for continuous measurement of plasma protein extravasation (PPE) in rat and mouse skin with drug application either intravenously or via the microdialysis fiber. Hollow plasmapheresis fibers (3-cm length, 0.4-mm diameter, cutoff 3,000 kDa) were placed subcutaneously on the back of anesthetized mice and rats. Intravenous injection of dextran (Macrodex, 60 mg/ml) increased PPE by 355% from baseline within 30 min in rats with ligated kidneys (n = 6; P < 0.05) but not in animals with intact kidneys. Phalloidin (500 microg/kg iv 40 min before dextran, n = 6; P < 0.05) did not change the response to dextran in either group. Animals receiving PGE1, compound 48/80 (mice), paclitaxel, docetaxel, and cremophor EL via the microdialysis fiber were also provided with a control fiber receiving vehicle. Both rats and mice had constant PPE in the control fiber, and there was no change in PPE in the NaCl-treated groups (rats, n = 4; mice, n = 6). Application via the fiber of PGE1 (20 microg/ml), compound 48/80 (mice; 4 mg/ml), and docetaxel (0.5 mg/ml) increased PPE compared with baseline within 60 min by 139% (n = 6; P < 0.05), 273% (n = 6; P < 0.05), and 325% (n = 5; P < 0.05), respectively. Phalloidin alone did not increase PPE (n = 5; P < 0.05). Pretreatment with phalloidin did not inhibit the increase after PGE1 or compound 48/80 but inhibited that after docetaxel (n = 6). Paclitaxel (0.6 mg/ml, n = 5) or vehicle (Cremophor) (n = 5) gave no increase in PPE. The results demonstrate that microdialysis can be used to continuously measure changes in PPE after inflammatory challenges in skin of rats and mice.