Steven Van Cruchten

Incubation at 32.5 °C and above causes malformations in the zebrafish embryo

Zebrafish embryos are increasingly used for developmental toxicity screening of candidate drugs and are occasionally co-incubated with a metabolic activation system at 32°C for 1, 2 or 4h, depending on their developmental stage. As this temperature is higher than the optimal temperature for zebrafish embryonic development (26-28.5°C), we investigated whether continuous incubation of zebrafish embryos from 2.5 until 96h post fertilization (hpf) at high temperatures (30.5-36.5°C) causes malformations. At 32.5°C tail malformations were observed as early as 24hpf, and these became even more prominent at 34.5 and 36.5°C. Cardiovascular and head malformations, edema and blood accumulations throughout the body were present at 36.5°C. Finally, temperatures higher than 28.5°C accelerated embryonic development except for 36.5°C, at which a lower hatching rate and hatching enzyme activity were observed. In conclusion, incubation of zebrafish embryos at 32.5°C and above from 2.5 until 96hpf causes malformations as early as 24hpf.

Ontogeny of CYP3A and P‐Glycoprotein in the Liver and the Small Intestine of the Göttingen Minipig: An Immunohistochemical Evaluation

Despite the increasing use of the minipig as a non‐rodent species in general and juvenile toxicity studies, knowledge on their biotransformation processes and their ontogeny is scarce. Such data are prerequisite for the correct interpretation of non‐clinical studies in this species. Therefore, the aim of our investigation was to immunohistochemically document the presence of the drug transporter P‐glycoprotein (Pgp) and the metabolizing cytochrome P450 (CYP) 3A subfamily in the livers (n = 115) and the small intestines (n = 74) of foetal, neonatal, juvenile and adult Göttingen minipigs. Pgp was expressed in the liver in all age groups, whereas its presence in the jejunum was detected from 86 days of gestation onwards. Low expression of CYP3A was detected in the jejunums and livers from foetal and neonatal piglets. During postnatal development, the immunoreactivity for CYP3A increased in both organs. A centrilobular pattern, with a more intense staining for CYP3A of the hepatocytes surrounding the central vein, was noticed in the postnatal livers. In conclusion, the presented data suggest that the intestinal and hepatic ontogeny of P‐glycoprotein and CYP3A in minipigs corresponds to that in man, in which a similar spatio‐temporal expression has been reported.

Pre- and Postnatal Development of the Eye: A Species Comparison

In this review paper, literature data on pre‐ and postnatal eye development are compared between humans and nonclinical species that are commonly used for human safety assessment, namely, mouse, rat, rabbit, dog, minipig, and nonhuman primates. Some new data on rat and minipig ocular development are also included. This compiled information can be helpful for species selection in juvenile toxicity studies or assist in the interpretation of (non)clinical data during pediatric drug development. Despite some differences in developmental windows and anatomical peculiarities, such as the lack of a fovea centralis in nonprimate species or the presence of a nictitating membrane in some nonclinical species, the functioning and development of the eye is strikingly similar between humans and other mammals. As such, all commonly used nonclinical species appear to be relatively good models for human eye development, although some practical constraints such as size may be a limiting factor. Birth Defects Research 109:1540–1567, 2017.

M cell specific markers in man and domestic animals : valuable tools in vaccine development

M cells play a pivotal role in the induction of immune responses within the mucosa-associated lymphoid tissues. As such, they are frequently studied for the development of mucosal vaccines. Unfortunately, the lack of a universal M cell marker hampers the progress in this field since researchers need species- and tissue-specific markers in order to isolate, identify or target M cells. Depicting the most appropriate M cell marker for the species and tissue under investigation might, however, be complicated by the numerous publications on this topic, often mentioning both positive and negative results for the tested marker. Therefore, this review gives an overview of the specific M cell markers in man and various domestic and laboratory animals. In addition, current state-of-the-art as regards the use of M cell models and markers in mucosal vaccine development is discussed.

How innate is locomotion in precocial animals? A study on the early development of spatio-temporal gait variables and gait symmetry in piglets

ABSTRACT Locomotion is one of the most important ecological functions in animals. Precocial animals, such as pigs, are capable of independent locomotion shortly after birth. This raises the question whether coordinated movement patterns and the underlying muscular control in these animals is fully innate or whether there still exists a rapid maturation. We addressed this question by studying gait development in neonatal pigs through the analysis of spatio-temporal gait characteristics during locomotion at self-selected speed. To this end, we made video recordings of piglets walking along a corridor at several time points (from 0 h to 96 h). After digitization of the footfalls, we analysed self-selected speed and spatio-temporal characteristics (e.g. stride and step lengths, stride frequency and duty factor) to study dynamic similarity, intralimb coordination and interlimb coordination. To assess the variability of the gait pattern, left–right asymmetry was studied. To distinguish neuromotor maturation from effects caused by growth, both absolute and normalized data (according to the dynamic similarity concept) were included in the analysis. All normalized spatio-temporal variables reached stable values within 4 h of birth, with most of them showing little change after the age of 2 h. Most asymmetry indices showed stable values, hovering around 10%, within 8 h of birth. These results indicate that coordinated movement patterns are not entirely innate, but that a rapid neuromotor maturation, potentially also the result of the rearrangement or recombination of existing motor modules, takes place in these precocial animals. Highlighted Article: Coordinated movement patterns in piglets are not entirely innate; instead, rapid neuromotor maturation also takes place in these precocial animals.

Age‐related Differences in CYP3A Abundance and Activity in the Liver of the Göttingen Minipig

In view of paediatric drug development, regulatory authorities often request safety studies in juvenile animals, including minipigs. Unfortunately, knowledge on the ontogeny of the biotransformation processes in animal models remains scarce and impedes a correct interpretation of the toxicity findings. CYP3A4 is one of the most important drug‐metabolizing enzymes in human beings and shows important similarities with CYP3A in the minipig. Therefore, the aim of this study was to assess the abundance and activity of CYP3A in liver microsomes from foetal, juvenile (days 1, 3, 7 and 28) and adult male and female Göttingen minipigs. CYP3A abundance was studied by an indirect enzyme‐linked immunosorbent assay (ELISA), whereas CYP3A activity was assessed by a biotransformation assay with Luciferin‐IPA. CYP3A abundance could not be detected until day 3. From day 7 onwards, a gradual increase in expression was noted, leading to the highest abundance in adult animals. CYP3A activity was not detectable in foetuses and 1‐day‐old animals. The CYP3A activity was detectable, but below the LLOQ in day 3 animals and increased gradually with age to reach the highest level in adults. The CYP3cide and ketoconazole inhibition, and testosterone and midazolam reduction of Luciferin‐IPA metabolism in minipig liver microsomes substantiate that Luciferin‐IPA is metabolized by CYP3A in minipigs. A positive correlation was found between CYP3A abundance and biotransformation of Luciferin‐IPA (Pearson r = 0.863; p < 0.0001). In conclusion, both abundance and activity of CYP3A increased gradually in juvenile minipigs, but remained below the levels observed in adult animals.

Interferon-γ modulates the functional profile of in-vitro-cultured porcine microglia.

Microglia are the most important immune cells within the highly specialized environment of the central nervous system. Upon activation, they transform from a resting ‘ramified’ into a fully functional ‘amoeboid’ phenotype with the ability to perform phagocytosis and generate free radicals. A combined flow cytometric assay for the simultaneous measurement of these two functions in porcine microglia in vitro is presented: reactive oxygen species are detected using hydroethidine; phagocytosis is assessed using fluorescein isothiocyanate-labeled latex beads. The combination of these two probes allowed us to distinguish four subpopulations within cultured porcine microglia on the basis of their functional activity. The effect of several exogenous stimuli [phorbol myristate acetate, conditioned medium, interferon &ggr; (IFN-&ggr;)] on the in-vitro functional properties of porcine microglia is investigated using this test. In particular for IFN-&ggr;, a significant modulatory effect on the intracellular reactive oxygen species production and phagocytic activity was observed. This result suggests an alternative role of IFN-&ggr; acting on cultured porcine microglia.

Evaluating Complex Mixtures in the Zebrafish Embryo by Reconstituting Field Water Samples: A Metal Pollution Case Study

Accurately assessing the toxicity of complex, environmentally relevant mixtures remains an important challenge in ecotoxicology. The goal was to identify biological effects after exposure to environmental water samples and to determine whether the observed effects could be explained by the waterborne metal mixture found in the samples. Zebrafish embryos were exposed to water samples of five different sites originating from two Flemish (Mol and Olen, Belgium) metal contaminated streams: “Scheppelijke Nete” (SN) and “Kneutersloop” (K), and a ditch (D), which is the contamination source of SN. Trace metal concentrations, and Na, K, Mg and Ca concentrations were measured using ICP-MS and were used to reconstitute site-specific water samples. We assessed whether the effects that were observed after exposure to environmental samples could be explained by metal mixture toxicity under standardized laboratory conditions. Exposure to “D” or “reconstituted D” water caused 100% mortality. SN and reconstituted SN water caused similar effects on hatching, swim bladder inflation, growth and swimming activity. A canonical discriminant analysis confirmed a high similarity between both exposure scenarios, indicating that the observed toxicity was indeed primarily caused by metals. The applied workflow could be a valuable approach to evaluate mixture toxicity that limits time and costs while maintaining biological relevance.

In Vitro Investigation of Six Antioxidants for Pig Diets

Oxidative stress in the small intestinal epithelium can lead to barrier malfunction. In this study, the effect of rosmarinic acid (RA), quercetin (Que), gallic acid (GA), lipoic acid (LA), ethoxyquin (ETQ) and Se-methionine (SeMet) pre-treatments using 2 mM Trolox as a control on the viability and the generation of intracellular reactive oxygen species (iROS) of oxidatively (H2O2) stressed intestinal porcine epithelial cells (IPEC-J2) was investigated. A neutral red assay showed that RA (50–400 µM), Que (12.5–200 µM), GA (50–400 µM), ETQ (6.25–100 µM), and SeMet (125–1000 µM) pre-treatments but not LA significantly increased the viability of H2O2-stressed IPEC-J2 cells (p < 0.05). A 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) fluorescent probe showed that RA (100–600 µM), Que (25–800 µM), ETQ (3.125–100 µM) and SeMet (500–2000 µM) pre-treatments significantly reduced iROS in IPEC-J2 monolayers (p < 0.05). Moreover, RA and Que were most effective in reducing iROS. Therefore, the effects of RA and Que on barrier functioning in vitro were examined. RA and Que pre-treatments significantly decreased fluorescein isothiocyanate (FITC)-conjugated dextran-4 (4 kDa) permeability and transepithelial electrical resistance (TEER) of an IPEC-J2 cell monolayer (p < 0.05). These in vitro results of RA and Que hold promise for their use as antioxidants in pig feed.

Lymph drainage from the ovine tonsils: an anatomical study of the tonsillar lymph vessels.

Although the tonsils of sheep have gained much attention during the last decade, only few data are available on their lymph vessel architecture. Tonsillar lymph vessels are immunologically important as they form the efferent routes for locally activated immune cells to reach the draining lymph nodes. To gain insight into the tonsillar lymph drainage in the sheep, Indian ink and a casting polymer were injected into the interstitium of the five tonsils present in the heads of slaughtered sheep. This enabled us to determine the draining lymph node and to examine the microscopic organization of lymph vessels using light and scanning electron microscopy. No lymph vessels were observed within the tonsillar lymphoid follicles. The corrosion casts demonstrated that the lymphoid follicles are surrounded by numerous sacculated lymph sinuses that drain into a dense interfollicular lymph vessel network. From here, the lymph flows into single small lymph vessels that in turn drain into larger lymph vessels extending towards the medial retropharyngeal lymph node. The presented results can be valuable for immunological studies, for example during oral or intranasal vaccine development.