Our goal was the visualizing the vascular damage and acute inflammatory response to micro- and minibeam irradiation in vivo. Microbeam (MRT) and minibeam radiation therapies (MBRT) are tumor treatment approaches of potential clinical relevance, both consisting of parallel X-ray beams and allowing the delivery of thousands of Grays within tumors. We compared the effects of microbeams (25–100 μm wide) and minibeams (200–800 μm wide) on vasculature, inflammation and surrounding tissue changes during zebrafish caudal fin regeneration in vivo. Microbeam irradiation triggered an acute inflammatory response restricted to the regenerating tissue. Six hours post irradiation (6 hpi), it was infiltrated by neutrophils and fli1a+ thrombocytes adhered to the cell wall locally in the beam path. The mature tissue was not affected by microbeam irradiation. In contrast, minibeam irradiation efficiently damaged the immature tissue at 6 hpi and damaged both the mature and immature tissue at 48 hpi. We demonstrate that vascular damage, inflammatory processes and cellular toxicity depend on the beam width and the stage of tissue maturation. Minibeam irradiation did not differentiate between mature and immature tissue. In contrast, all irradiation-induced effects of the microbeams were restricted to the rapidly growing immature tissue, indicating that microbeam irradiation could be a promising tumor treatment tool.

BackgroundResearchers evaluating angiomodulating compounds as a part of scientific projects or pre-clinical studies are often confronted with limitations of applied animal models. The rough and insufficient early-stage compound assessment without reliable quantification of the vascular response counts, at least partially, to the low transition rate to clinics.ObjectiveTo establish an advanced, rapid and cost-effective angiogenesis assay for the precise and sensitive assessment of angiomodulating compounds using zebrafish caudal fin regeneration. It should provide information regarding the angiogenic mechanisms involved and should include qualitative and quantitative data of drug effects in a non-biased and time-efficient way.Approach & ResultsBasic vascular parameters (total regenerated area, vascular projection area, contour length, vessel area density) were extracted from in vivo fluorescence microscopy images using a stereological approach. Skeletonization of the vasculature by our custom-made software Skelios provided additional parameters including “graph energy” and “distance to farthest node”. The latter gave important insights into the complexity, connectivity and maturation status of the regenerating vascular network. The employment of a reference point (vascular parameters prior amputation) is unique for the model and crucial for a proper assessment. Additionally, the assay provides exceptional possibilities for correlative microscopy by combining in vivo-imaging and morphological investigation of the area of interest. The 3-way correlative microscopy links the dynamic changes in vivo with their structural substrate at the subcellular level.ConclusionsThe improved zebrafish fin regeneration model with advanced quantitative analysis and optional 3-way correlative morphology is a promising in vivo angiogenesis assay, well-suitable for basic research and preclinical investigations.

IntroductionHemodynamic parameters in zebrafish receive increasing attention because of their important role in cardiovascular processes such as atherosclerosis, hematopoiesis, sprouting and intussusceptive angiogenesis. To study underlying mechanisms, the precise modulation of parameters like blood flow velocity or shear stress is centrally important. Questions related to blood flow have been addressed in the past in either embryonic or ex vivo-zebrafish models but little information is available for adult animals. Here we describe a pharmacological approach to modulate cardiac and hemodynamic parameters in adult zebrafish in vivo.Materials and MethodsAdult zebrafish were paralyzed and orally perfused with salt water. The drugs isoprenaline and sodium nitroprusside were directly applied with the perfusate, thus closely resembling the preferred method for drug delivery in zebrafish, namely within the water. Drug effects on the heart and on blood flow in the submental vein were studied using electrocardiograms, in vivo-microscopy and mathematical flow simulations.ResultsUnder control conditions, heart rate, blood flow velocity and shear stress varied less than ± 5%. Maximal chronotropic effects of isoprenaline were achieved at a concentration of 50 μmol/L, where it increased the heart rate by 22.6 ± 1.3% (n = 4; p < 0.0001). Blood flow velocity and shear stress in the submental vein were not significantly increased. Sodium nitroprusside at 1 mmol/L did not alter the heart rate but increased blood flow velocity by 110.46 ± 19.64% (p = 0.01) and shear stress by 117.96 ± 23.65% (n = 9; p = 0.03).DiscussionIn this study, we demonstrate that cardiac and hemodynamic parameters in adult zebrafish can be efficiently modulated by isoprenaline and sodium nitroprusside. Together with the suitability of the zebrafish for in vivo-microscopy and genetic modifications, the methodology described permits studying biological processes that are dependent on hemodynamic alterations.

Nucleated circulating red blood cells (RBCs) of developing zebrafish, chick and mouse embryos can actively proliferate. While marrow- or organ-mediated erythropoiesis has been widely studied, transforming processes of circulating RBCs are under little scrutiny. We employed confocal, stereo- and electron microscopy to document the maturation of intravascular RBCs In zebrafish embryos (32-72 h post-fertilization), RBC splitting in the caudal vein plexus follows a four-step program: (i) nuclear division with continued cytoplasmic connection between somata; (ii) dumbbell-shaped RBCs tangle at transluminal vascular pillars; (iii) elongation; and (iv) disruption of soma-to-soma connection. Dividing RBCs of chick embryos, however, retain the nucleus in one of their somata. Here, RBC splitting acts to pinch off portions of cytoplasm, organelles and ribosomes. Dumbbell-shaped primitive RBCs re-appeared as circulation constituents in mouse embryos. The splitting of circulating RBCs thus represents a biologically relevant mechanism of RBC division and maturation during early vertebrate ontogeny.

Cutaneous melanoma represents a major cause of cancer death in Europe. Without adequate therapy, the 5-year survival rate is 15-20% in distant metastatic disease. Evaluating the status quo of treatment standards in advanced melanoma and rationale for therapy decisions in Switzerland between January 2016 and September 2018. In this retrospective, anonymized registry, data of male and female patients with unresectable advanced/ metastatic BRAF-positive cutaneous melanoma treated in first-, second-and third-line with registered substances were analyzed using descriptive statistics. Forty-one patients (56.1% male) were included providing a total of 70 treatment lines (first-line: n = 41; second-line: n = 18; and third-line: n = 11). Within the patients presenting with stage III or IV melanoma, immunotherapy with checkpoint inhibitors was more frequently administered as first-line treatment than targeted therapy (TT) (70.7% vs. 29.3%). Across all lines, patients received TT in 47.1% (predominantly combined BRAF-MEK-inhibition) and immunotherapy in 52.9% of the cases (anti-PD-1 monotherapy in 62.2% and anti-PD-1/anti-CTLA-4 combinations in 37.8%). Most commonly, the treatment type was switched from TT to immunotherapy or vice versa upon disease progression. The most frequent rationales for prescribing either TT or immunotherapy were physician's preference (40.0%) or remission pressure (28.6%), respectively. Disease progression led to treatment discontinuation more frequently than undesired events. Patients in Switzerland with unresectable advanced or metastatic BRAF-mutant melanoma predominantly receive guideline-recommended treatments. IO was used as predominant front-line therapy, with TT/immunotherapy switch being the predominant treatment principle. Sequencing studies are underway to identify the optimal treatment regimen for those patients.