Maternal pravastatin prevents altered fetal brain development in a preeclamptic CD-1 mouse model

Abstract

Objective: Using an animal model, we have previously shown that preeclampsia results in long-term adverse neuromotor outcomes in the offspring, and this phenotype was prevented by antenatal treatment with pravastatin. This study aims to localize the altered neuromotor programming in this animal model and to evaluate the role of pravastatin in its prevention.

Materials and methods: For the preeclampsia model, pregnant CD-1 mice were randomly allocated to injection of adenovirus carrying sFlt-1 or its control virus carrying mFc into the tail vein. Thereafter they received pravastatin (sFlt-1-pra "experimental group") or water (sFlt-1 "positive control") until weaning. The mFc group ("negative control") received water. Offspring at 6 months of age were sacrificed, and whole brains underwent magnetic resonance imaging (MRI). MRIs were performed using an 11.7 Tesla vertical bore MRI scanner. T2 weighted images were acquired to evaluate the volumes of 28 regions of interest, including areas involved in adaptation and motor, spatial and sensory function. Cytochemistry and cell quantification was performed using neuron-specific Nissl stain. One-way ANOVA with multiple comparison testing was used for statistical analysis.

Results: Compared with control offspring, male sFlt-1 offspring have decreased volumes in the fimbria, periaquaductal gray, stria medullaris, and ventricles and increased volumes in the lateral globus pallidus and neocortex; however, female sFlt-1 offspring showed increased volumes in the ventricles, stria medullaris, and fasciculus retroflexus and decreased volumes in the inferior colliculus, thalamus, and lateral globus pallidus. Neuronal quantification via Nissl staining exhibited decreased cell counts in sFlt-1 offspring neocortex, more pronounced in males. Prenatal pravastatin treatment prevented these changes.

Conclusion: Preeclampsia alters brain development in sex-specific patterns, and prenatal pravastatin therapy prevents altered neuroanatomic programming in this animal model.

Figure 1. Altered Brain Volumes in 6 Month Old Male Offspring Brain.

Regions of interest were analyzed by 1-way ANOVA in 6 month old male mice offspring born to pregnant CD1 mice injected either with an adenovirus carrying sFlt-1 or adenovirus carrying mFc fragment. Mice injected with sFlt-1 were treated with either pravastatin or water to result in three groups of offspring animals: mFc (n = 9), sFlt-1 (n = 3), and sFlt-1-pra (n = 6). Data are reported as mean ± SEM. The sFlt-1 group offspring showed decreased volumes at the fimbria (A; p = 0.04), periaqueductal grey (B; p<0.0001), and stria medullaris (C; p<0.0001) when compared to the mFc control offspring. Increased volumes were seen at the lateral globus pallidus (D; p = 0.01) and neocortex (E; p = 0.02). The overall ameliorative effect of pravastatin exposure in utero is seen. Not shown are brain regions with similar volumes between groups.

Figure 2. Altered Brain Volumes in 6 Month Old Female Offspring Brain.

Regions of interest were analyzed by 1-way ANOVA in 6 month old female mice offspring born to pregnant CD1 mice injected either with an adenovirus carrying sFlt-1 or adenovirus carrying mFc fragment. Mice injected with sFlt-1 were treated with either pravastatin or water to result in three groups of offspring animals: mFc (n = 6), sFlt-1 (n = 3), and sFlt-1-pra (n = 10). Data are reported as mean ± SEM. The sFlt-1 group offspring showed decreased volumes at the inferior colliculus (A; p = 0.04), thalamus (B; p<0.0001), and lateral globus pallidus (C; p = 0.02) when compared to the mFc control offspring. Increased volumes were seen at the stria medullaris (D; p = 0.01), and fasciculus retroflexus (E; p = 0.01). The overall ameliorative effect of pravastatin exposure in utero is seen. Not shown are brain regions with similar volumes between groups.

Figure 3. Magnetic Resonance Images of 6 Month Old Offspring Brains.

Three-dimensional T2-weighted images of whole brain were obtained using a vertical bore 11.7 Tesla MRI scanner. Here in these representative coronal slices, the ventricles and periventricular tissue of male (A) and female (B) offspring are outlined in coronal view, allowing comparison between treatment groups. A decrease in ventricular volume was seen in male sFlt-1 offspring (A: sFlt-1) when compared to controls (A: mFc). Pravastatin exposure (A: sFlt-1-pra) reversed this effect. An opposite trend was seen in the female sFlt-1 offspring brain (B: sFlt-1) when compared to control (B: mFc) and pravastatin-treatment offspring (B: sFlt-1-pra). Nissl staining of coronal sections at a single ventricle and its periventricular tissue in adult offspring brains at 40 X magnification is shown in panels C and D. In these representative images, male offspring showed a decrease in volume in sFlt-1 (C: sFlt-1) offspring when compared to control (C: mFc). In-utero pravastatin exposed male offspring (C: sFlt-1-pra) showed normalization of ventricular shape and size. An increase in ventricular volume is seen in female sFlt-1 offspring brains (D: sFlt-1) when compared to control (D: mFc). Though complete resolution is not seen, there is improvement in ventricle size in the pravastatin-exposed female offspring (D: sFlt-1-pra). These representative images correlate with MRI volumetric data for male (E: mFc (n = 9), sFlt-1 (n = 3), sFlt-1-pra (n = 6)) and female (F: mFc (n = 6), sFlt-1 (n = 3), sFlt-1-pra (n = 10)) offspring brains as analyzed by 1-way ANOVA. The male sFlt-1 offspring had significantly decreased volume when compared to mFc controls and the pravastatin treatment group in males (E: p = 0.02), whereas the opposite effect was seen in female offspring (F: p<0.05).

Figure 4. Altered Cortical Cell Counts in 6 Month Old Offspring Brain.

Nissl staining of neocortex of 6 month old mice offspring shown here at 40 X magnification in panels A (male) and C (female). Neurons were identified by dark-blue positive staining. Glial cells were smaller than neurons and had darker nuclei but no clearly visible Nissl bodies in the cytoplasm. A decrease in positive staining is seen male and female sFlt-1 offspring (A: sFlt-1, C: sFlt-1) when compared to controls (A: mFc, C: mFc) and pravastatin treatment group offspring (A: sFlt-1-pra, C: sFlt-1-pra). Total cell count as well as neuronal and glial cell counts were performed on randomly chosen 7-8 fields in the frontal cortex per animal and analyzed by 2-way ANOVA (n = 3 for all groups by gender). Data are reported as mean ± SEM. Male data are seen in Panel B, and female data in Panel D. Significant decreases in total cell count are seen in both male and female sFlt-1 offspring when compared to controls (p<0.0001) and sFlt-1-pra (male and female p<0.0001), though the female sFlt-1-pra group remained significantly different from mFc controls (p = 0.001). There is no difference in glial cell count between all three groups. Neuronal cell count appears to drive this total cell count difference. The male sFlt-1 offspring (B) show a significant decrease in neurons when compared to mFc controls (p = 0.0003) and sFlt-1-pra offspring (p = 0.001). Female sFlt-1 offspring (D) show a similar drop in neuronal count versus mFc controls and sFlt-1-pra offspring (p<0.0001), with pravastatin-exposed offspring remain significantly different from mFc controls (p = 0.03).