The anabolic effect of exercise on muscles and bones is well documented. In teleost fish, exercise has been shown to accelerate skeletogenesis, to increase bone volume, and to change the shape of vertebral bodies. Still, increased swimming has also been reported to induce malformations of the teleost vertebral column, particularly lordosis. This study examines whether zebrafish (Danio rerio) develops lordosis as a result of continuous physical exercise. Zebrafish were subjected, for 1 week, to an increased swimming exercise of 5.0, 6.5 or 8.0 total body lengths (TL) per second. Control and exercise group zebrafish were examined for the presence of vertebral abnormalities, by in vivo examination, whole mount staining for bone and cartilage and histology and micro‐computed tomography (CT) scanning. Exercise zebrafish developed a significantly higher rate of lordosis in the haemal part of the vertebral column. At the end of the experiment, the frequency of lordosis in the control groups was 0.5 ± 1.3% and that in the exercise groups was 7.5 ± 10.6%, 47.5 ± 10.6% and 92.5 ± 6.0% of 5.0, 6.5 and 8.0 TL∙s−1, respectively. Histological analysis and CT scanning revealed abnormal vertebrae with dorsal folding of the vertebral body end plates. Possible mechanisms that trigger lordotic spine malformations are discussed. This is the first study to report a quick, reliable and welfare‐compatible method of inducing skeletal abnormalities in a vertebrate model during the post‐embryonic period.
Targeting in zebrafish fast growth, high survival rates and improved reproductive performance has led over the last years in variable feeding regimes between different facilities. Despite its significance on fish function and welfare, normal skeletal development has rarely been evaluated in establishing the best feeding practices for zebrafish. The aim of this study was to establish a protocol for normal skeletal development, growth and survival of zebrafish larvae through live feed‐to‐microdiet transition at an appropriate rate. Four feeding regimes including feeding exclusively on Artemia nauplii (A) or dry microdiet (D), and feeding on both Artemia and microdiet at two different transition rates (slow (B) or fast (C)) were applied from 5 to 24 dpf (days post‐fertilization). Results demonstrated a significant effect of feeding regimes on the incidence of skeletal abnormalities (gill cover, fins and vertebral column, p < .05) in zebrafish larvae. The A and B experimental groups presented the highest (88 ± 3 and 84 ± 17%, respectively), but the C and D the lowest (18 ± 14 and 11 ± 2%, respectively), rates of normal fish (fish without any abnormality). Similarly, growth rate was comparatively elevated in A and B groups. No significant differences were observed in fish survival between A, B and C groups. However, D group presented a significantly lower survival rate. To our knowledge, this is the first study to show that the live feed‐to‐microdiet transition rate influences larval growth, survival and abnormality rates in a non‐homogenous pattern.
Microplastics (MPs)’ ingestion has been demonstrated in several aquatic organisms. This process may facilitate the hydrophobic waterborne pollutants or chemical additives transfer to biota. In the present study the suitability of a battery of biomarkers on oxidative stress, physiology, tissue function and metabolic profile was investigated for the early detection of adverse effects of 21-day exposure to polystyrene microplastics (PS-MPs, sized 5–12 μm) in the liver and gills of zebrafish Danio rerio and perch, Perca fluviatilis, both of which are freshwater fish species. An optical volume map representation of the zebrafish gill by Raman spectroscopy depicted 5 μm diameter PS-MP dispersed in the gill tissue. Concentrations of PS-MPs close to the EC50 of each fish affected fish physiology in all tissues studied. Increased levels of biomarkers of oxidative damage in exposed fish in relation to controls were observed, as well as activation of apoptosis and autophagy processes. Malondialdehyde (MDA), protein carbonyls and DNA damage responses differed with regard to the sensitivity of each tissue of each fish. In the toxicity cascade gills seemed to be more liable to respond to PS-MPs than liver for the majority of the parameters measured. DNA damage was the most susceptible biomarker exhibiting greater response in the liver of both species. The interaction between MPs and cellular components provoked metabolic alterations in the tissues studied, affecting mainly amino acids, nitrogen and energy metabolism. Toxicity was species and tissue specific, with specific biomarkers responding differently in gills and in liver. The fish species that seemed to be more susceptible to MPs at the conditions studied, was P. fluviatilis compared to D. rerio. The current findings add to a holistic approach for the identification of small sized PS-MPs’ biological effects in fish, thus aiming to provide evidence regarding PS-MPs’ environmental impact on wild fish populations and food safety and adequacy.
Adrenal cortical carcinoma (ACC) is a rare cancer with poor prognosis that needs to be distinguished from adrenocortical adenomas (ACAs). Although, the recently developed transcriptome analysis seems to be a reliable tool for the differential diagnosis of adrenocortical neoplasms, it is not widely available in clinical practice. We aim to evaluate histological and immunohistochemical markers for the distinction of ACCs from ACAs along with assessing their prognostic role. Clinical data were retrospectively analyzed from 37 patients; 24 archived, formalin-fixed, and paraffin-embedded ACC samples underwent histochemical analysis of reticulin and immunohistochemical analysis of p27, p53, Ki-67 markers and were compared with 13 ACA samples. Weiss and Helsinki scores were also considered. Kaplan−Meier and univariate Cox regression methods were implemented to identify prognostic effects. Altered reticulin pattern, Ki-67% labelling index and overexpression of p53 protein were found to be useful histopathological markers for distinguishing ACAs from ACCs. Among the studied markers, only pathological p53 nuclear protein expression was found to reach statistically significant association with poor survival and development of metastases, although in a small series of patients. In conclusion, altered reticulin pattern and p53/Ki-67 expression are useful markers for distinguishing ACCs from ACAs. Immunohistopathology alone cannot discriminate ACCs with different prognosis and it should be combined with morphological criteria and transcriptome analysis.
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