DataSheet_1_Maternal siRNA silencing of placental SAA2 mitigates preterm birth following intrauterine inflammation.pdf

The placental inflammatory processes induced maternally result in preterm birth (PTB). Serum amyloid A (SAA) is a well-known biomarker of inflammation. The objective of this study was to investigate whether murine placental SAA isoforms (SAA1–4) participate in the mechanism of spontaneous PTB and whether maternal regulation of SAA production may serve as a therapeutic approach. During the gestation, all isoforms of SAA were detectable except SAA2. The mouse model of intrauterine inflammation was established using LPS infusion to the uterus. Following intrauterine inflammation, placental SAA2 increased significantly. Inhibition of Saa2, using siSaa2, markedly decreased PTB. The increased placental expression of pro-inflammatory cytokines Il1β, Il6, and Tnfα were downregulated by siSaa2 treatment. Maternal inhibition of Saa2 did not change the expression of Saa1–4 in the fetal brain. Explant inflammatory culture of placentas with siSaa2 showed similar results to our in vivo experiments. This study demonstrates the highly expressed placental SAA2 as a novel therapeutic target, and maternal administration of siRNA as a promising approach to alleviate PTB.</p

Supplemental Material, Supplemenatary_figure_S1 - Maternal Glucose Supplementation in a Murine Model of Chorioamnionitis Alleviates Dysregulation of Autophagy in Fetal Brain

<p> Supplemental Material, Supplemenatary_figure_S1 for Maternal Glucose Supplementation in a Murine Model of Chorioamnionitis Alleviates Dysregulation of Autophagy in Fetal Brain by Jun Lei, Wenyu Zhong, Ahmad Almalki, Hongxi Zhao, Hattan Arif, Rayyan Rozzah, Ghada Al Yousif, Nader Alhejaily, Dan Wu, Michael McLane and Irina Burd in Reproductive Sciences </p

Altered Cortical Cell Counts in 6 Month Old Offspring Brain.

<p>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).</p

Altered Brain Volumes in 6 Month Old Female Offspring Brain.

<p>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.</p

Magnetic Resonance Images of 6 Month Old Offspring Brains.

<p>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).</p

Regions of Interest Imaged Via MRI in the Brains of 6-Month Old Male and Female Offspring.

<p>Regions of Interest Imaged Via MRI in the Brains of 6-Month Old Male and Female Offspring.</p

Functions of Brain Regions with Significant Volume Changes Seen in MRI Imaging of Adult Mouse Brains.

<p>Functions of Brain Regions with Significant Volume Changes Seen in MRI Imaging of Adult Mouse Brains.</p

Incidence rate ratio (95% confidence interval) of different activities for treatment group compared to control group: mixed effects poisson regression analysis that adjusted for offspring weight, gender, random selection, and testing time and day.

<p>P<0.008 was considered significant.</p

Cortical neuron densities in Nissl-stained frontal cortex of offspring between control and APAP groups.

<p>Whole brains were washed and processed for Nissl staining by immersing in 30% sucrose until saturation and cryosectioned at 20 μm thickness. Neurons were counted (field of view) based on randomly chosen 5 fields in frontal cortex per animal. The neurons were identified by a large cell body or perikaryon containing a large, pale nucleus with a prominent dark nucleolus. Experiments were performed in 3–5 repeats for all groups. There was no significant difference in cortical neuron density between control and treated groups (p < 0.05; Unpaired Student’s T-Test; n = 5 control, n = 3 treated). Data are presented as the mean±s.e.m. Statistical significance was determined using Student’s unpaired t-test.</p

Mean values of different activities according to group assignment.

<p>Mean values of different activities according to group assignment.</p