Protocols for the Evaluation of Neurodevelopmental Alterations in Rabbit Models In Vitro and In Vivo

Pla, Laura; Kühne, Britta Anna; Guardia-Escote, Laia; Vázquez-Aristizabal, Paula; Loreiro, Carla; Flick, Burkhard; Gratacós, E.; Barenys, Marta; Illa, M.

doi:10.3389/ftox.2022.918520

Cited by 3 publications

(2 citation statements)

References 52 publications

(101 reference statements)

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“…The in vitro neurosphere culture was generated directly after decapitation at PND0, as described in Pla et al (2022) . Briefly, neural progenitor cells (NPCs) were isolated from rabbits’ whole brains by dissection, mechanical dissociation, digestion (20 min incubation with 20 U/ml papain [Worthington #LS003124] at 37°C), mechanical homogenization into a cell suspension, and centrifugation (10 min at 1200 rpm).…”

Section: Methodsmentioning

confidence: 99%

Lactoferrin/sialic acid prevents adverse effects of intrauterine growth restriction on neurite length: investigations in an in vitro rabbit neurosphere model

Kühne

Gutierrez-Vázquez

Lamelas

et al. 2023

Front. Cell. Neurosci.

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IntroductionIntrauterine growth restriction (IUGR) is a well-known cause of impaired neurodevelopment during life. In this study, we aimed to characterize alterations in neuronal development underlying IUGR and discover strategies to ameliorate adverse neurodevelopment effects by using a recently established rabbit in vitro neurosphere culture.MethodsIUGR was surgically induced in pregnant rabbits by ligation of placental vessels in one uterine horn, while the contralateral horn remained unaffected for normal growth (control). At this time point, rabbits were randomly assigned to receive either no treatment, docosahexaenoic acid (DHA), melatonin (MEL), or lactoferrin (LF) until c-section. Neurospheres consisting of neural progenitor cells were obtained from control and IUGR pup’s whole brain and comparatively analyzed for the ability to differentiate into neurons, extend neurite length, and form dendritic branching or pre-synapses. We established for the very first time a protocol to cultivate control and IUGR rabbit neurospheres not only for 5 days but under long-term conditions up to 14 days under differentiation conditions. Additionally, an in vitro evaluation of these therapies was evaluated by exposing neurospheres from non-treated rabbits to DHA, MEL, and SA (sialic acid, which is the major lactoferrin compound) and by assessing the ability to differentiate neurons, extend neurite length, and form dendritic branching or pre-synapses.ResultsWe revealed that IUGR significantly increased the neurite length after 5 days of cultivation in vitro, a result in good agreement with previous in vivo findings in IUGR rabbits presenting more complex dendritic arborization of neurons in the frontal cortex. MEL, DHA, and SA decreased the IUGR-induced length of primary dendrites in vitro, however, only SA was able to reduce the total neurite length to control level in IUGR neurospheres. After prenatal in vivo administration of SAs parent compound LF with subsequent evaluation in vitro, LF was able to prevent abnormal neurite extension.DiscussionWe established for the first time the maintenance of the rabbit neurosphere culture for 14 days under differentiation conditions with increasing complexity of neuronal length and branching up to pre-synaptic formation. From the therapies tested, LF or its major compound, SA, prevents abnormal neurite extension and was therefore identified as the most promising therapy against IUGR-induced changes in neuronal development.

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Section: Methodsmentioning

confidence: 99%

Lactoferrin/sialic acid prevents adverse effects of intrauterine growth restriction on neurite length: investigations in an in vitro rabbit neurosphere model

Kühne

Gutierrez-Vázquez

Lamelas

et al. 2023

Front. Cell. Neurosci.

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“…Представлены ожидаемые контрольные значения и их вариации, рассмотрены наиболее распространенные проблемы, связанные с каждым протоколом, и пути их решения. Таким образом, разработаны надежные экспериментальные подходы, которые можно применять для оценки нейротоксичности в процессе онтогенеза с использованием кроликов [54].…”

Section: нейротоксичностьunclassified

Rabbits in preclinical research

Makarova,

Makarov

2023

Lab Anim Sci Rus

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Rabbits are one of the most commonly used laboratory animal species in biomedical research. A large number of models of various human diseases have been developed on rabbits. Rabbits are widely used to assess the safety of substances and pharmaceuticals. The development of genetically modified rabbits has expanded their use for modeling various pathological conditions. Advantages of the rabbit include its relatively small size and relative ease of housing. Practically any experimental manipulations can be carried out on rabbits (any methods of administering the tested compounds are available, conducting various instrumental studies, multiple collection of blood samples, etc. is possible). In studies of reproductive toxicity, an important advantage of the rabbit relative to larger animals is a short gestation period, good reproductive ability. Rabbits are sensitive to human teratogens and are the preferred non-rodent species for embryofetal development studies. Rabbits are used in immunology for the production of diagnostic antibodies. An important point is that the scientific literature provides a sufficient amount of historical and reference materials on the anatomy, physiology, and pathophysiology of these animals. The disadvantages of rabbits include frequently developed disorders of the gastrointestinal tract. The functional activity of the rabbit’s digestive system is highly dependent on the diet. Changes in the diet should be done gradually. Rabbits need to be able to exercise daily. With the wrong manipulation technique, it is easy to cause injury to the rabbit. Since some diseases may spontaneously develop in laboratory rabbits, this may require either the use of therapy or the exclusion of the animal from the experiment. Therefore, in the case of planning long-term studies on rabbits, it is reasonable to include at least 8 males and 8 females in each group. If recovery groups are needed, the number of animals should be sufficient to evaluate possible long-term effects. When planning and conducting experiments, every effort should be made to implement the principles of 3Rs. Combination designs with pharmacological safety, immunotoxicity, reproductive toxicity, pharmacokinetics, and toxicokinetics studies as part of a toxicity study may help to reduce the number of rabbits in toxicological research. Studies should be designed taking into account the available scientific data on the test compound or on analogue compounds, on the basis of which scientific-based decision can be made to reduce the number of animals in the experiment without compromising the human safety.

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Application of the adverse outcome pathway to identify molecular changes in prenatal brain programming induced by IUGR: Discoveries after EGCG exposure

Kühne

Teixidó

Ettcheto

et al. 2022

Food and Chemical Toxicology

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Protocols for the Evaluation of Neurodevelopmental Alterations in Rabbit Models In Vitro and In Vivo

Cited by 3 publications

References 52 publications

Lactoferrin/sialic acid prevents adverse effects of intrauterine growth restriction on neurite length: investigations in an in vitro rabbit neurosphere model

Lactoferrin/sialic acid prevents adverse effects of intrauterine growth restriction on neurite length: investigations in an in vitro rabbit neurosphere model

Rabbits in preclinical research

Application of the adverse outcome pathway to identify molecular changes in prenatal brain programming induced by IUGR: Discoveries after EGCG exposure

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