Impact of prenatal lipopolysaccharide exposure on the development of rats

The intrauterine environment is infl uenced by several factors, genetic or environmental, which are essential in understanding the pathophysiological mechanisms of some diseases. In this study, the aim was to investigate the impact of prenatal lipopolysaccharide exposure on the development of rats. Fifty pregnant rats received intraperitoneal administration of lipopolysaccharide (100 µg/kg), or saline at the same dose, on the 9.5th day of pregnancy. The offspring of these rats were analyzed for indicators of brain and somatic development and maturation of physical characteristics. Refl ex ontogenesis was also analyzed by vibrissae placement, negative geotaxis, palmar grasp, precipice aversion, decubitus recovery and acceleration reaction. Administration of lipopolysaccharide on the 9.5th gestational day caused delayed opening of the auditory pavilion, reduction in the length of the tail, body, cranial axes, and body weight. Thus, maternal infections can interfere in the intrauterine environment, impairing functional and structural aspects of the central nervous system, as well as the maturation of physical characteristics.


INTRODUCTION
During the ontogenic period, the central nervous system (CNS) is infl uenced by several factors, genetic and environmental, that modulate epigenetic mechanisms and, therefore, the functioning of these systems. The impairment of these molecular mechanisms, mainly in critical periods, can cause irreversible changes, associated with neuropsychiatric disorders (IZVOLSKAIA e colab., 2018; KEUNEN e colab., 2015; ZAKHAROVA, 2015). In this context, the concept of programming was developed to explain the process by which the organism adapts to environmental insults, which generate stable changes in the phenotype, through changes in the proliferation and differentiation process (DEIRÓ, Teresa Cristina Bomfim De Jesus e colab., 2008).
As adults, rodents exposed to adverse events during the intrauterine period -infections and pharmacological or nutritional manipulations -exhibit physiological and behavioral changes, including vulnerability to cognitive disabilities, reduced corticosterone response to stress and decreased social interaction (IZVOLSKAIA e colab., 2018). Further, the evidence indicates that these changes are not limited to animals exposed isolatedly, but also to their One of the ways to study the development of the CNS is to observe the ontogenesis of the reflex, since reflexes represent one of the behavioral expressions of brain function.
Reflex ontogenesis encompasses visual, auditory and motor maturation, and can be affected by any environmental or organic stimulus, generating consequences for the formation of the nervous system (SOARES e colab., 2014). They are the result of stimuli and appear in certain periods of development, following a pre-determined order, according to the age of the animals (DEIRÓ, Teresa Cristina Bomfim De Jesus e colab., 2008). The maturation of specific reflexes in rats has been well established, showing that disturbances at this stage of growth may point to insults in neurological development (LEITE e colab., 2002).
Therefore, to better understand the genesis of disorders related to neurodevelopment, which have a growing prevalence and important socioeconomic impact, this study has been proposed.

OBJECTIVES
Investigate whether an insult during a critical period of CNS development in rats reflects structural and functional changes in their offspring's somatic and motor-sensory development.

METHODS
This was an experimental study in which the animals were kept at controlled temperature (22 ± 1°C), artificial light cycle (12 hours light/ dark) and received standard commercial feed for laboratory rats and ad libitum water.
For the application of the prenatal inflammation protocol, 50 adults (60 days) virgin female Wistar rats (Rattus norvegicus) -obtained from the University of the Itajaí Valley vivarium -were used, weighing between 250 and 300g, to mate with 25 males of the same lineage and age.
For mating, at the end of the light period (7 p.m.) the females were placed in the males' cages, always using two females for each male. At 7 am the following day, pregnancy was diagnosed using biological material collected trough vaginal lavage, which consists of introducing solution of sterile 0.9% NaCl through a plastic pipette into the animal's vaginal canal.
Thus, secretion is obtained for analysis under an optical microscope in order to investigate the presence of sperm next to the animal's biological material (KIRSTEN e colab., 2010).
Once the presence of sperm was confirmed, gestational day (GD) zero was considered.
Next, the pregnant mothers were placed in individual cages and remained so throughout the pregnancy period.
On the 9.5th GD, the females were randomly subjected to two treatments: saline exposure (SAL group) and lipopolysaccharide exposure -obtained through phenolic extraction from Escherichia coli, serotype 0127: B8 (Sigma®) -at 100 μg/kg (LPS group). Both applications were intraperitoneal. LPS administration followed exposure protocol that mimics a bacterial infection (LUNARDELLI e colab., 2014). Births happened naturally. In total, 184 animals were used, male and female, divided according to the exposure protocol. Of these, 120 were used for the assessment of somatic growth and maturation of physical characteristics and 64 for post mortem brain assessments. Moreover, out of the 120 animals, 20 were also evaluated for reflex ontogenesis.

Parameters analyzed
Somatic development and reflex ontogenesis were analyzed in these groups, having as reference the following parameters:

Ethical aspects
The use of animals in this study followed the Laboratory Animal Care Principles and was approved by the Animal Use Ethics Committee of the University of South Santa Catarina (protocol number 15.016.2.01.IV).

Statistical analysis
GraphPad Prism 7 was used for statistical analysis, and p-value of < 0.05 was considered to be statistically significant. Parametric data were analyzed by analysis of variance for multiple comparisons between groups (ANOVA), followed by Tukey post-hoc. For nonparametric data, we used the Mann-Whitney test for two independent samples.

The anteroposterior axis of the skull
The results of the anteroposterior cranial axis growth parameter showed statistical significance from one-way ANOVA [F (21, 1298) = 828.9, p < 0.0001]. The offspring in the LPS group showed deficits in the parameter when compared to the offspring in the SAL group, on days 1 (p < 0.0001), 7 (p = 0.0407), 9 (p = 0.0004) and 13 (p = 0.0002) postnatal.
No significant differences (p > 0.05) were observed when comparing the groups on the other days analyzed ( Figure 1).  Effects of prenatal exposure to lipopolysaccharide (LPS), compared to the control group (SAL), on the development of rats: laterolateral axis of the skull (n=120). The evaluation was performed from the 1st to the 21st day of life. Data presented on average ± S.D. *p < 0.05.

Somatic development
Regarding somatic development, the one-way ANOVA test showed values of F (7, 472) = 974.6 and p < 0.0001. In Tukey's post-hoc, it was evidenced that the rats in the LPS group presented delayed opening of the auditory pavilion when compared to the SAL group (p < 0.0001). Regarding the Other parameters analyzed, no significant diferences were observed in the comparison between the two groups (p > 0.05) ( Figure 6).

Reflex ontogenesis
As for reflex ontogenesis, the one-way ANOVA test resulted in the values of F (9, 90) = 92.1 and p < 0.0001. In Tukey's post-hoc, when comparing the SAL and LPS group, it was observed that the maturation of the decubitus recovery (p = 0.0413) and precipice aversion (p < 0.0001) parameters occurred first in the SAL group and later in the LPS group. As to other reflexes, there was no statistically significant difference (p > 0.05) (Figure 7).

Post mortem evaluation
The one-way ANOVA result for brain post-death indicators were, on the 8th day F ( However, during the 8th (p < 0.0001), 15th (p < 0.0001) and 21st (p = 0.0443) post--mortem days, it was evidenced that the SAL group had a higher brain volume in comparison with the LPS group (p < 0.05) (Figures 8, 9 and 10).   Regarding the maturation of the structures that make up the head (opening of the auditory pavilion and auditory conduit, eye-opening and incisor eruption), it is known that its delay is related to somatic growth retardation (SILVA, 2008). It was observed that the offspring of the neuro-inflamed group presented a delayed opening of the auditory pavilion when compared to the control group. Because of this delay in neurodevelopment, hearing may be impaired. As well as motor development. This fact may be related to neurocognitive damage resulting from maternal immune activation.
Harmful effects of LPS exposure during pregnancy on fetal neurodevelopment, aside from impairing the sensorimotor reflex, may also lead to differences in brain volume as observed in the postmortem analyzes found in this study (SOMINSKY e colab., 2013). The brain volume observed at 8, 15 and 21 days of life of the SAL group was greater when compared to the group with maternal immune activation. Which is consistent with previous literature which says that there is a decrease in the overall neural cell population during the first 7 days of postnatal life in the offspring of mothers which received LPS application. Therefore, prenatal LPS exposure has specific detrimental effects on neuronal differentiation and affecting cell proliferation. After the first 7 days, it is suggested that prenatal exposure to LPS also triggers a neural proportion deficit (GRACIARENA e colab., 2010). In this study, the decrease in brain volume from the 8th postnatal day represents an affected neurogenesis. In this sense, it can be concluded that CNS changes resulting from prenatal immunological activation are dependent on the inflammation period as well as on the postnatal age of the offspring analyzed.
Age-related weight, height, and head circumference assessments are widely used in humans to assess growth and development, as well as nutritional status. Head circumference measurement, aside from assessing nutritional status, is an indicator of brain volume (WORLD HEALTH ORGANIZATION, 2006ORGANIZATION, , 2007. Maternal inflammation during pregnancy can cause differentiation in the offspring's body weight gain when compared to those of healthy mother (HARDING e colab., 2014). Body weight is considered a good indicator of physical development in rats. In the present study, the SAL group had a higher weight gain compared to the LPS group from the 17th postnatal day. The difference in weight between the groups is related to the fact that maternal immune stress is linked to the reduction in the offspring's body weight, especially in the first two weeks of life (BAKOS e colab., 2004; GOLAN e colab., 2004).
The insult imposed on the mothers during the prenatal period seems to have been decisive in causing a decrease in body weight and a delay in physical development. In the presente study, matrix-induced inflammation affected body growth deficit and tail length in neonatal rats. Similar results were found in previous studies, which showed delayed body growth and tail length, as well as a connection between tail length and malnutrition in neonatal rats (BARBOSA e SANTIAGO, 1994; GOLAN e colab., 2004).
There was also a decrease in the anteroposterior axis of the cranium. In rodents, the skull does not develop as a single unit but divides into two distinct regions (neurocranium and viscerocranium) (CHEVERUD, 1982). While the viscerocranium is used during feeding and breathing, the neurocranium houses the brain and its development is mainly influenced by its growth (CHEVERUD, 1982;HERRING, 1993;YOUNG, 1959). Thus, the anteroposterior axis of the brain represents the two regions, as it is measured from the anterior tip of the nasal bone to the posterior edge of the occipital bone (L.D. BARROS e colab., 2018; MILLER e GERMAN, 1999). The decrease in the animal's skull size may be associated with the decrease in brain volume observed in the LPS group.

CONCLUSION
Thereby, it is concluded that prenatal exposure to LPS in early pregnancy can interfere in the intrauterine environment, impairing functional and structural aspects of the central nervous system, as well as the maturation of physical characteristics. However, mechanisms related to immune responses need to be better clarified and there is also a need to investigate the effects of rodents'exposure to endotoxin in the adult life. It is expected that our study may help to understanding of the genesis of disorders related to neurodevelopment, which have an important socioeconomic impact and still have important therapeutic limitations.