Prenatal administration of IL-1Ra attenuate the neurodevelopmental impacts following non-pathogenic inflammation during pregnancy

Abstract

Prenatal inflammation negatively affects placental function, subsequently altering fetal development. Pathogen-associated molecular patterns (PAMPs) are used to mimics infections in preclinical models but rarely detected during pregnancy. Our group previously developed an animal model of prenatal exposure to uric acid (endogenous mediator), leading to growth restriction alongside IL-1-driven placental inflammation (Brien et al. in J Immunol 198(1):443-451, 2017). Unlike PAMPs, the postnatal impact of prenatal non-pathogenic inflammation is still poorly understood. Therefore, we investigated the effects of prenatal uric acid exposure on postnatal neurodevelopment and the therapeutic potential of the IL-1 receptor antagonist; IL-1Ra. Uric acid induced growth restriction and placental inflammation, which IL-1Ra protected against. Postnatal evaluation of both structural and functional aspects of the brain revealed developmental changes. Both astrogliosis and microgliosis were observed in the hippocampus and white matter at postnatal day (PND)7 with IL-1Ra being protective. Decreased myelin density was observed at PND21, and reduced amount of neuronal precursor cells was observed in the Dentate Gyrus at PND35. Functionally, motor impairments were observed as evaluated with the increased time to fully turn upward (180 degrees) on the inclined plane and the pups were weaker on the grip strength test. Prenatal exposure to sterile inflammation, mimicking most clinical situation, induced growth restriction with negative impact on neurodevelopment. Targeted anti-inflammatory intervention prenatally could offer a strategy to protect brain development during pregnancy.

Figure 1

Placental inflammation and growth restriction induced by uric acid exposure during pregnancy. Sterile inflammation exposure led to increased placental inflammation and anti-inflammatory intervention decreased levels of IL-1β (A), IL-6 (B) and TNF-α (C) proteins at birth. N = 6–12 dams (i.e. Ctrl = 12; UA = 12; UA + Ra = 6), 3 placentas/dams. Quantification of postnatal weight at PND7 (D), PND14 (E), PND25 (F) and PND50 (G) after exposure to sterile inflammation. Treatment with UA led to decreased weight maintained in the early postnatal period. N = 6–10 litters (i.e. PBS = 8; IL-1Ra = 6; UA = 10; UA + Ra = 6) at PND7, 14 and 21 and N = 6 litters in each groups at PND50. *p < 0.05; **p < 0.01; ***p < 0.001; Results presented as mean ± SEM. Statistical analysis by one-way ANOVA and Tukey’s multiple comparisons test with with GraphPad Prism 9.2.0 (GraphPad Software, CA); www.graphpad.com.

Figure 2

Morphological brain alteration following uric acid exposure. Representative image of HE staining of the brain regions studied (A). Representative image of MBP staining at PND21 to measure the corpus callosum thickness (B) and quantification at different postnatal time (C). cc corpus callosum, cg cingulum, cx cortex, DG hippocampus-dentate gyrus, 3-CA 3 hippocampus-cornus amonis, ic internal capsule. N = 8–16 litters (i.e. PBS = 11; IL-1Ra = 8; UA = 16; UA + Ra = 12). *p < 0.05; **p < 0.01; ***p < 0.001. Results presented as mean ± SEM. Statistical analysis by one-way ANOVA with Tukey’s multiple comparisons test with GraphPad Prism 9.2.0 (GraphPad Software, CA); www.graphpad.com.

Figure 3

Histological analysis of Iba-1 and GFAP positive cell in the white matter following uric acid alone or with IL-1Ra prenatal treatment. Injection of uric acid during gestation increased the number of microglial cell and astrocytes in the corpus callosum and in the cingulum at PND7. Prenatal treatment with IL-1Ra reduced the number of microglia and astrocytes to a similar level than in the control group. Representative image shown in (A) and quantification in (B). N = 8–16 litters (i.e. PBS = 11; IL-1Ra = 8; UA = 16; UA + Ra = 12). *p < 0.05; **p < 0.01; ***p < 0.001 Results presented as mean ± SEM. Statistical analysis by one-way ANOVA with Tukey’s multiple comparisons test with GraphPad Prism 9.2.0 (GraphPad Software, CA); www.graphpad.com.

Figure 4

Histological analysis of Iba-1 and GFAP positive cell in the hippocampus following uric acid alone or with IL-1Ra prenatal treatment at PND7. Injection of uric acid during gestation increased the number of microglia and astrocytes in the hippocampus (CA3) at PND7. Prenatal treatment with IL-1Ra prevented the microgliosis and astrogliosis observed at PND7. Representative image shown in (A) and quantification in (B). N = 8–16 litters (i.e. PBS = 11; IL-1Ra = 8; UA = 16; UA + Ra = 12). *p < 0.05; ***p < 0.001 Results presented as mean ± SEM. Statistical analysis by one-way ANOVA with Tukey’s multiple comparisons test with GraphPad Prism 9.2.0 (GraphPad Software, CA); www.graphpad.com.

Figure 5

Myelin and neuronal precursor cell histological analysis following uric acid alone or with IL-1Ra prenatal treatment. Injection of uric acid during gestation decreased the myelin staining intensity in the internal capsule at PND21 (A) and decreased doublecortin staining in the hippocampus-DG at PND35 as opposed to the control group (B). Prenatal treatment with IL-1Ra did not statistically preserve these regions. N = 8–16 litters (i.e. PBS = 11; IL-1Ra = 8; UA = 16; UA + Ra = 12) for MBP and N = 6 litters/group for DCX. **p < 0.01. Results presented as mean ± SEM. Statistical analysis by one-way ANOVA with Tukey’s multiple comparisons test with GraphPad Prism 9.2.0 (GraphPad Software, CA); www.graphpad.com.

Figure 6

Impaired motricity following UA exposure during gestation. At PND7, prenatal inflammation induced a delay in turnaround time in the inclined plane in pups exposed to uric acid (A). At PND15, pups exposed in-utero to uric acid were weaker as evaluated by the grip strength test (B). N = 6 litters/group. *p < 0.05 Results presented as mean ± SEM. Statistical analysis by one-way ANOVA with Tukey’s multiple comparisons test with GraphPad Prism 9.2.0 (GraphPad Software, CA); www.graphpad.com.

Figure 7

Experimental schedule of the model. Pregnant dams were injected i.p. every 12 h with uric acid from gestational day (G)18 to 21. C-section were performed at G22 for placental protein analysis. Natural delivery also occurred in parallel to evaluate the pups in the postnatal period from postnatal day (PND)1 through PND50 (A). Behavioural assessment schedule of the model. Eight different behavior tests were performed from postnatal day (PND) 1 to PND50 to evaluate motricity and cognitive functions (B).