Cognitive impairments induced by necrotizing enterocolitis can be prevented by inhibiting microglial activation in mouse brain

Abstract

Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease of the premature infant. One of the most important long-term complications observed in children who survive NEC early in life is the development of profound neurological impairments. However, the pathways leading to NEC-associated neurological impairments remain unknown, thus limiting the development of prevention strategies. We have recently shown that NEC development is dependent on the expression of the lipopolysaccharide receptor Toll-like receptor 4 (TLR4) on the intestinal epithelium, whose activation by bacteria in the newborn gut leads to mucosal inflammation. Here, we hypothesized that damage-induced production of TLR4 endogenous ligands in the intestine might lead to activation of microglial cells in the brain and promote cognitive impairments. We identified a gut-brain signaling axis in an NEC mouse model in which activation of intestinal TLR4 signaling led to release of high-mobility group box 1 in the intestine that, in turn, promoted microglial activation in the brain and neurological dysfunction. We further demonstrated that an orally administered dendrimer-based nanotherapeutic approach to targeting activated microglia could prevent NEC-associated neurological dysfunction in neonatal mice. These findings shed light on the molecular pathways leading to the development of NEC-associated brain injury, provide a rationale for early removal of diseased intestine in NEC, and indicate the potential of targeted therapies that protect the developing brain in the treatment of NEC in early childhood.

Fig. 1.. NEC in humans and mice leads to impaired myelination and cognitive dysfunction.

(A) Representative micrographs of whole-brain midsagittal sections from P11 mice or preterm humans with NEC or controls [breastfed (BF)] stained for Mbp. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole. (B) Representative transmission EM images of midbrain sections from a mouse model of NEC in P11 mice or breastfed controls. Scale bars, 2 μm. (C) Myelin g-ratio (myelin sheath thickness; calculated as axon diameter/axon diameter with myelin) in the midbrain region obtained from a mouse model of NEC in P11 mice or breastfed controls (n = 5 per experimental group). (D) Representative horizontal and coronal T2-weighted images from P11 mice; white arrows indicate internal capsule, and arrowheads indicate corpus callosum/external capsule. (E) Volumetric and segmentation analysis of T2-weighted images from P11 mice; means ± SD (n = 5 breastfed controls and n = 4 NEC). (F) Representative confocal micrographs of the hippocampus (midsagittal section) from a mouse model of NEC in P11 mice or breastfed controls stained for NeuN; quantification in (G) (n = 3 per experimental group). NS, not significant. (H and I) Cognitive evaluation by Y-maze (H) and novel object recognition (I) (n = 13 breastfed controls and n = 9 NEC). Experiments were performed in triplicate with at least three mice per group per experiment. Statistical differences were determined using Student’s t test.

Fig. 2.. NEC leads to TLR4-dependent microglial activation and loss of OPCs in the newborn brain.

(A) Representative micrographs of Iba-1 3,3′-diaminobenzidine immunostaining of periventricular brain sections obtained from autopsy specimens of premature human infants with NEC or controls (n = 5 per group). Scale bars, 75 μm (top) and 25 μm (bottom). (B to G) Representative staining (B to D) and quantification (E to G) as indicated in whole-brain midsagittal sections from wild-type (Wt) or TLR4^ΔCx3cr1 P11 mice (n = 3 per experimental group); (H) representative confocal micrographs of Mbp immunostaining in whole-brain midsagittal sections obtained from a mouse model of NEC induced in oligodendrocyte lineage reporter Pdgfrα^GFP transgenic P11 mice or from breastfed controls. Scale bars, 50 μm (inset). Quantification in (I) (n = 4 per experimental group). Samples were obtained from three independent experiments with at least three to four animals per experimental condition. Image analysis and quantification were performed within the indicated region of interest (ROI) obtained from midsagittal whole-brain sections using the FIJI software. Statistical significance was determined by one-way analysis of variance (ANOVA), followed by post hoc Dunnett’s multiple comparisons test (E to G) and by two-tailed Student’s t test (I).

Fig. 3.. HMGB1 release from the intestinal epithelium leads to NEC-induced brain injury.

Representative micrographs from (A) hippocampus and periventricular regions, (B) periventricular region, and (C) whole-brain midsaggital section in a mouse model of NEC at P11 (stained as indicated) that were either wild type, lacking HMGB1 within the intestinal epithelium (HMGB1^ΔIEC), or received intranasal anti-HMGB1–neutralizing or intranasal nonspecific IgG. Scale bars, 100 μm (A) and 50 μm (B). (D to F) Quantification of the indicated cells within the ROI of the hippocampus and periventricular region in the indicated group corresponding to (A) to (C) [BF, n = 3; NEC, n = 5 (Wt), n = 3 (HMGB1Δ^IEC), n = 5 (IgG), and n = 3 (anti-HMGB1)]. (G to I) Representative confocal micrographs of the indicated stain in midsaggital brain sections from wild-type mice that received an intraperitoneal injection of either rHMGB1 or saline. Scale bars, 50 μm. In (I), midbrain, cerebellum, and brainstem are shown. (J to L) Quantification of the indicated cell within the ROI (hippocampus and periventricular region) in the indicated group corresponding to (G) and (H) (saline, n = 3; rHMGB1, n = 4). Samples from three to four animals were analyzed from three independent experiments. Image analysis and quantification were performed within the indicated ROI obtained from midsagittal whole-brain sections using the FIJI software. Statistical significance was determined by one-way ANOVA, followed by post hoc Dunnett’s multiple comparisons test (D to F) and two-tailed Student’s t test (J to L).

Fig. 4.. Orally administered dendrimer-based antioxidant reverses NEC-induced brain injury.

(A, C, E, and G) Representative micrographs showing brain sections from a mouse model of NEC in newborn mice (P11) treated or untreated with D-NAC and from breastfed controls stained as indicted and quantified in (B), (D), (F), and (H), respectively (n = 3 per experimental group). Scale bars, 100, 50, and 100 μm (A, C, and G, respectively) and 20 μm (inset). (I and J) Total amount of glutathione (GSH) (BF, n = 3; NEC, n = 6; NEC + NAC, n = 5; and NEC + D-NAC, n = 7) and 3-nitrotyrosine (NT-3) (BF, n = 6; NEC, n = 13; NEC + NAC, n = 9; and NEC + D-NAC, n = 7) within the hippocampus and periventricular region. All samples were obtained at the end of the NEC protocol (P11). Image analysis and quantification were performed in midsagittal whole-brain sections as described in Materials and Methods. Statistical significance was determined by one-way ANOVA, followed by post hoc Dunnett’s multiple comparisons test. (K and L) Effects of D-NAC on neurocognitive testing in (K) Y-maze (BF, n = 13; NEC, n = 9; BF + D-NAC, n = 5; and NEC + D-NAC, n = 5) and in (L) novel object recognition (BF, n = 14; NEC, n = 9; BF + D-NAC, n = 5; NEC + D-NAC, n = 5) analyzed by one-way ANOVA and post hoc Tukey’s test. Three independent experiments were performed with at least three mice per experimental group per experiment.

Fig. 5.. D-NAC administration in early life induces long-term protection from NEC-associated myelin loss.

(A) Micrographs showing the long-term effects (60 days after injury; P72) of D-NAC administration in a mouse model of NEC that received either saline of NAC in the newborn period stained for Mbp. (B) Quantification of Mbp expression (n = 3 per experimental condition). (C) Representative coronal T2-weighted images from 120-day-old (P120) mice; arrowheads indicate the corpus callosum. (D) Volumetric analysis of T2-weighted brain images from P120 mice (BF, n = 6; NEC, n = 4; BF + D-NAC, n = 7; NEC + D-NAC, n = 17). Two independent experiments were performed with at least three mice per experimental group per experiment. Statistical significance was determined by one-way ANOVA, followed by post hoc Tukey’s multiple comparisons test.