CXCR2 Blockade Mitigates Neural Cell Injury Following Preclinical Chorioamnionitis

Abstract

Minimizing central nervous system (CNS) injury from preterm birth depends upon identification of the critical pathways that underlie essential neurodevelopmental and CNS pathophysiology. While chorioamnionitis (CHORIO), is a leading cause of preterm birth, the precise mechanism linking prenatal brain injury and long-term CNS injury is unknown. The chemokine (C-X-C motif) ligand 1 (CXCL1) and its cognate receptor, CXCR2, are implicated in a variety of uterine and neuropathologies, however, their role in CNS injury associated with preterm birth is poorly defined. To evaluate the putative efficacy of CXCR2 blockade in neural repair secondary to CHORIO, we tested the hypothesis that transient postnatal CXCR2 antagonism would reduce neutrophil activation and mitigate cerebral microstructural injury in rats. To this end, a laparotomy was performed on embryonic day 18 (E18) in Sprague Dawley rats, with uterine arteries transiently occluded for 60 min, and lipopolysaccharide (LPS, 4 μg/sac) injected into each amniotic sac. SB225002, a CXCR2 antagonist (3 mg/kg), was administered intraperitoneally from postnatal day 1 (P1)-P5. Brains were collected on P7 and P21 and analyzed with western blot, immunohistochemistry and ex vivo diffusion tensor imaging (DTI). Results demonstrate that transient CXCR2 blockade reduced cerebral neutrophil activation (myeloperoxidase expression/MPO) and mitigated connexin43 expression, indicative of reduced neuroinflammation at P7 (p < 0.05 for all). CXCR2 blockade also reduced alpha II-spectrin calpain-mediated cleavage, improved pNF/NF ratio, and minimized Iba1 and GFAP expression consistent with improved neuronal and axonal health and reduced gliosis at P21. Importantly, DTI revealed diffuse white matter injury and decreased microstructural integrity following CHORIO as indicated by lower fractional anisotropy (FA) and elevated radial diffusivity (RD) in major white matter tracts (p < 0.05). Early postnatal CXCR2 blockade also reduced microstructural abnormalities in white matter and hippocampus at P21 (p < 0.05). Together, these data indicate that transient postnatal blockade of CXCR2 ameliorates perinatal abnormalities in inflammatory signaling, and facilitates neural repair following CHORIO. Further characterization of neuroinflammatory signaling, specifically via CXCL1/CXCR2 through the placental-fetal-brain axis, may clarify stratification of brain injury following preterm birth, and improve use of targeted interventions in this highly vulnerable patient population.

Keywords: CXCL1; alpha-II spectrin; chemokine; diffusion tensor imaging; neutrophil; preterm; white matter.

FIGURE 1

Experimental Design. On embryonic day 18 (E18), a prenatal chorioamnionitis insult was induced in pregnant Sprague Dawley rats. Rat pups were born on E22. SB225002, a CXCR2 antagonist, or vehicle, was administered during a critical neonatal window from postnatal day 1 (P1) through P5 (3 mg/kg i.p.). Pup brains were collected and analyzed on P7 or P21 for western blot analysis (WB), ex vivo diffusion tensor imaging (DTI), and immunohistochemistry (IHC).

FIGURE 2

CXCR2 Antagonism Reduces Neutrophil Activation and Connexin43 Expression on Postnatal Day 7 (P7). Following prenatal chorioamnionitis, transient postnatal administration of the CXCR2 antagonist, SB225002, reduced neutrophil reactivity (myeloperoxidase, MPO, A) and mitigated connexin43 expression (CX43, B) in the cortex on P7, consistent with reduced cellular neuroinflammation. At P7, neither CHORIO or CXCR2 antagonism changed cleaved alpha-II spectrin ratio, a marker of neuronal health (C). (n = 7–15, two-way ANOVA with Bonferroni’s post hoc correction, ^∗p < 0.05, ^∗∗p < 0.01).

FIGURE 3

CXCR2 Antagonism Improves Axonal and Neuronal Health Through Postnatal Day 21 (P21). Following prenatal chorioamnionitis (CHORIO), transient postnatal administration of the CXCR2 antagonist, SB225002, mitigated injury-induced alterations in cortical phosphoneurofilament to neurofilament ratio (pNF/NF, A), consistent with improved axonal health. Similarly, transient CXCR2 blockade also reduced pathological augmentation of alpha-II spectrin cleavage in the cortex (B), consistent with reduced calpain activity and improved neuronal health. Immunoreactivity for microglia (Iba1, C) and astrocytes (GFAP, D) is augmented at P21 in CHORIO fimbria compared to Sham, an effect that is ameliorated by CXCR2 antagonism. Scale bar = 200 μm. (n = 6–11/group for immunoblot, n = 3 for immunohistochemistry, two-way ANOVA with Bonferroni’s post hoc correction, ^∗p < 0.05, ^∗∗p < 0.01).

FIGURE 4

CXCR2 Antagonism Mitigates Abnormalities in Cerebral Diffusion. Following prenatal chorioamnionitis (CHORIO), transient postnatal administration of the CXCR2 antagonist, SB225002, attenuated losses of fractional anisotropy (FA) in major white matter tracts (left, black arrows) on postnatal day 21 (P21). Similarly, directionally encoded color maps (right) reveal loss of diffusion in white and gray matter, including the corpus callosum and anterior hippocampus (white arrows). Color coded FA legend (left) indicates the degree of anisotropy from 0 to 1, with 0 being unrestricted and 1 fully restricted. Directionally color encoded arrows (right) indicate horizontal diffusion (red), anterior to posterior diffusion (blue), and superior-inferior diffusion (green).

FIGURE 5

CXCR2 Antagonism Attenuates White Matter Microstructural Injury. Following prenatal chorioamnionitis (CHORIO), transient postnatal administration of the CXCR2 antagonist, SB225002, attenuated injury induced changes in fractional anisotropy (A) in the corpus callosum at postnatal day 21 (P21), together with mitigating changes in axial (AD, B) and radial (RD) diffusion (C). Similarly, CHORIO animals had reduced FA (D) and increased RD (F) in the external capsule. Treatment with a CXCR2 antagonist significantly improved both FA and RD. No changes were observed in AD (E). (n = 5–6, two-way ANOVA with Bonferroni’s post hoc correction, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001).

FIGURE 6

CXCR2 Antagonism Improves Gray Matter Microstructural Integrity. Following prenatal chorioamnionitis (CHORIO), transient postnatal administration of the CXCR2 antagonist, SB225002, attenuated injury induced changes in fractional anisotropy (FA) in the hippocampus (A) at postnatal day 21 (P21). While CHORIO reduced FA in the thalamus, no difference was observed with CXCR2 antagonism in the thalamus (B). (n = 5–6, two-way ANOVA with Bonferroni’s post hoc correction, ^∗p < 0.05, ^∗∗p < 0.01).