Intrauterine inflammation and postnatal intravenous dopamine alter the neurovascular unit in preterm newborn lambs

Abstract

Background: Intrauterine inflammation is considered a major cause of brain injury in preterm infants, leading to long-term neurodevelopmental deficits. A potential contributor to this brain injury is dysregulation of neurovascular coupling. We have shown that intrauterine inflammation induced by intra-amniotic lipopolysaccharide (LPS) in preterm lambs, and postnatal dopamine administration, disrupts neurovascular coupling and the functional cerebral haemodynamic responses, potentially leading to impaired brain development. In this study, we aimed to characterise the structural changes of the neurovascular unit following intrauterine LPS exposure and postnatal dopamine administration in the brain of preterm lambs using cellular and molecular analyses.

Methods: At 119-120 days of gestation (term = 147 days), LPS was administered into the amniotic sac in pregnant ewes. At 126-7 days of gestation, the LPS-exposed lambs were delivered, ventilated and given either a continuous intravenous infusion of dopamine at 10 µg/kg/min or isovolumetric vehicle solution for 90 min (LPS, n = 6; LPS_DA, n = 6). Control preterm lambs not exposed to LPS were also administered vehicle or dopamine (CTL, n = 9; CTL_DA, n = 7). Post-mortem brain tissue was collected 3-4 h after birth for immunohistochemistry and RT-qPCR analysis of components of the neurovascular unit.

Results: LPS exposure increased vascular leakage in the presence of increased vascular density and remodelling with increased astrocyte "end feet" vessel coverage, together with downregulated mRNA levels of the tight junction proteins Claudin-1 and Occludin. Dopamine administration decreased vessel density and size, decreased endothelial glucose transporter, reduced neuronal dendritic coverage, increased cell proliferation within vessel walls, and increased pericyte vascular coverage particularly within the cortical and deep grey matter. Dopamine also downregulated VEGFA and Occludin tight junction mRNA, and upregulated dopamine receptor DRD1 and oxidative protein (NOX1, SOD3) mRNA levels. Dopamine administration following LPS exposure did not exacerbate any effects induced by LPS.

Conclusion: LPS exposure and dopamine administration independently alters the neurovascular unit in the preterm brain. Alterations to the neurovascular unit may predispose the developing brain to further injury.

Keywords: Chorioamnionitis; Dopamine; Intrauterine inflammation; Neurovascular coupling; Neurovascular unit; Preterm brain.

Fig. 1

Sheep serum and MMP9 positive immunohistochemistry (A) Quantification of vessels with sheep serum immunopositive leakage and (B) quantification of MMP9 area coverage within vessels. Data are mean ± SD. Two-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.005. (C) Representative images of sheep serum extravasation from blood vessels into the brain parenchyma (black arrowhead), and sheep serum contained within the blood vessels (white arrowhead) in subcortical white matter (SCWM), periventricular white matter (PVWM) and caudate; and (D) representative images of MMP9 positive immunohistochemistry in the subcortical white matter (SCWM), periventricular white matter (PVWM). Scale bar represents 100 μm

Fig. 2

Laminin and GFAP positive immunohistochemistry (A) Quantification of laminin positive area coverage and (B) average vessel perimeter size as assessed using laminin positive immunolabelling. (C) Quantification of GFAP positive area coverage and (D) quantification of percentage of colocalization of GFAP with laminin positive immunolabelling. Data are mean ± SD. Two-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.005. (E) Representative images of laminin (green) and GFAP (red) positive immunofluorescent labellingand colocalization indicating astrocytic “end feet” covering of vessels in the cortical grey matter (cortex) and periventricular white matter (PVWM). Scale bar represents 50 μm

Fig. 3

Desmin and αSMA positive immunohistochemistry (A) Quantification of percentage of colocalization of desmin with αSMA positive immunolabelling. Data are mean ± SD. Two-way ANOVA. (E) Representative images of desmin (green) and αSMA (red) positive immunofluorescent labellingand colocalization indicating pericyte coverage of vessels in the cortical grey periventricular white matter (PVWM). Scale bar represents 50 μm

Fig. 4

Ki67 and GLUT1 positive immunohistochemistry (A) Quantification of Ki67 immunopositive cells within vessel walls and (B) quantification of GLUT1 area coverage within vessel walls. Data are mean ± SD. Two-way ANOVA, *P < 0.05, **P < 0.01. (C) Representative images of Ki67 immunopositive cells (black arrowhead) within the blood vessels in the subcortical white matter (SCWM) and caudate, and (D) representative images of GLUT1 positive immunohistochemistry in the cortical grey matter (cortex) and periventricular white matter (PVWM). Scale bar represents 100 μm

Fig. 5

NeuN and MAP2 positive immunohistochemistry (A) NeuN immunopositive cell density indicating mature neuronal population and (B) MAP2 immunopositive area coverage indicating coverage of neuronal dendrites. Data are mean ± SD. Two-way ANOVA, *P < 0.05. (C) Representative images of NeuN- and MAP2-positive cells in the cortical grey matter (cortex; Layer V) and caudate. Scale bar represents 50 μm

Fig. 6

Fold change of mRNA levels mRNA expression of genes relating to vasculature integrity, dopamine, and oxidative stress measured within the (A) cortex, (B) combined white matter and (C) caudate. mRNA expression expressed relative to CTL. Data are mean ± SD. Cortex and white matter data analysed using two-way ANOVA, Tukey’s post-hoc analyses. Note mRNA levels in the caudate of LPS lambs were not conducted and data analysed using a one-way ANOVA, Tukey’s post-hoc analyses. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001