Elsevier

Neurotoxicology and Teratology

Volume 30, Issue 5, September–October 2008, Pages 395-403
Neurotoxicology and Teratology

Gestational all-trans retinoic acid treatment in the rat: Neurofunctional changes and cerebellar phenotype

https://doi.org/10.1016/j.ntt.2008.03.064Get rights and content

Abstract

Neurofunctional effects produced by gestational all-trans retinoic acid (all-trans RA) treatment were investigated in the offspring of Sprague–Dawley rats. Reproduction data, onset of reflexive behavior, locomotor activity, motor coordination and motor learning were examined. Moreover, possible changes in size and morphology of the cerebellum were evaluated. The results show that all-trans RA treatment (2.5 mg/kg, by gavage) on gestational days (GD) 11–13 significantly increased postnatal mortality and decreased pup weight gain. Moreover, all-trans RA-treated rats showed a significant delay in eyes opening, hair growth as well as in the maturation of righting reflex, cliff aversion and pole grasping. All-trans RA treatment significantly impaired the ambulatory activity in adult rats without altering the number of rearings. All-trans RA-treated rats subjected to the rotarod/accelerod task showed significant impairment in both motor coordination and motor learning ability. The morphological analysis revealed a significant reduction in the cerebellar size and impairment in foliation profile, at PND 3 with subsequent recovery at PNDs 8 and 40. The evidence that functional alterations increase with age and persist in adulthood whereas the morphological changes decline with age, strongly supports the view that, besides the cerebellum morphology, the organization of the cerebellar circuitry, and in particular of cortico-cerebellar connections, are also affected by all-trans RA treatment.

Introduction

Retinoic acid (RA) is used as a therapeutic agent in a number of clinical disorders including leukaemia, psoriasis and acne, due to its influence on cell maturation [18]. An excess of retinoic acid is just as harmful as an insufficiency. Indeed, RA is a known human teratogen at very low doses. RA treatment during early stages of pregnancy may result in severe malformations of the foetus [15], [23]. Later stages of embryonic brain development are vulnerable as well, but abnormalities are more subtle. Surviving children of mothers treated with RA during pregnancy are often mentally retarded [2]. Therefore, the recommended vitamin A dietary allowance for non-pregnant and pregnant women is 800 µg which is equivalent to ≈ 2700 IU [20].

Retinoic acid and RA binding proteins also play a key role in the cerebellar development via interaction with key regulatory pathways [19]. Interestingly, the location of enzymes involved in the normal processing of retinoic acid in the embryo seems to play a critical role in determining which regions of the nervous system will be harmed by abnormal RA levels. High levels of these enzymes occur in the retina, spinal cord and cerebellum [1] which, as expected, are highly sensitive to the teratogenic effects of abnormal levels of retinoic acid. As far as the critical periods of brain development is concerned, the occasional accidental exposure of humans cannot shed much light on this question. Therefore, it is necessary to use laboratory animals.

As in humans [2], rats treated with retinoic acid exhibit a range of behavioral abnormalities. In this regard, previous studies have revealed significant effects of all-trans RA treatment on the cerebellar development both postnatally [28] and prenatally [8], [9], [10], [11], [12]. In particular, the gestational days (GD) 11–13 seem to be a highly sensitive developmental window for all-trans RA effects on the cerebellum [8], [10]. Surprisingly, although the GDs 11–13 all-trans RA (2.5 mg/kg) treatment has been shown to alter size and weight of the cerebellum, tasks such as horizontal activity in open field [9], motor coordination and motor learning [11], which are also under the control of this brain area, are unaffected.

In this article, the effects of GDs 11–13 all-trans RA (2.5 mg/kg) treatment on these behavioral tasks are further examined. Unlike previous studies [11], the performance on the accelerod was assessed using a higher rotation speed mode, thus requiring the animal to adapt motor coordination to a more complex task demand. It is well known that performance on a rotating rod requires an intact muscular strength [22]. Since muscular weakness could represent a confounding variable in the interpretation of the rotarod/accelerod results, this endpoint was also evaluated in prenatally all-trans RA-treated rats. To our knowledge, the RA effects on grip strength have not been described previously.

Moreover, locomotor activity was assessed by using the open field paradigm. In this regard, previous findings [9] have shown that all-trans RA treatment on GDs 11–13 does not affect horizontal activity in the open field arena. In the present study, vertical activity (rearings) was also analysed, taking into account that this endpoint may be influenced by changes in equilibrium and/or motivation to explore [6].

The effects of all-trans RA treatment on reproductive outcome and growth rate were also evaluated. Although there is no evidence for sex differences in the response to gestational all-trans RA treatment [8], [9], [10], [11], this aspect was further explored.

Finally, a morphological analysis was carried out in the attempt to establish a possible relationship between neurofunctional outcomes and changes in the cerebellar phenotype. In this regard, there is evidence that the GDs 11–13 all-trans RA treatment induces long-lasting changes in the gross morphology of the cerebellum [12].

Section snippets

Animals, animal husbandry, and dosing

The experiments have been conducted in accordance with guidelines released by the Italian Ministry of Health (D.L. 116/92), the Declaration of Helsinki and the “Guide for the Care and Use of Laboratory Animals” as adopted and promulgated by the National Institutes of Health. The minimum number of animals has been used. All animals have been treated humanely according to institutional guidelines, with due consideration to the alleviation of distress and discomfort.

Primiparous Sprague–Dawley

Reproduction data

General reproduction data are reported in Table 1. Statistical analysis (Student's t-test) indicated that dam weight gain, pregnancy length and litter size at birth were not significantly affected by prenatal treatment with all-trans RA. Moreover, Fisher's-exact test showed that prenatal treatment with all-trans RA did not influence the number of dams giving birth. Statistical analysis (Fisher's-exact test) revealed that all-trans RA significantly increased postnatal mortality of pups with

Discussion

The present findings show that the GDs 11–13 all-trans RA treatment induces, in rat offspring, long-term neurofunctional outcomes. In particular, locomotor activity, motor coordination and motor learning were found to be significantly impaired in young-adult all-trans RA-treated rats. Moreover, changes in the morphological phenotype of the cerebellum, consisting of size reduction mostly affecting folia IX–X, were observed in a developmental window spanning the first postnatal week with

Acknowledgements

This study was supported by grants from MiUR (PRIN-COFIN 2005) and Bari University (“Fondo Ateneo” 2007). Giovanni Boffoli is gratefully acknowledged for technical support.

References (28)

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