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. 2014 May 16:11:89.
doi: 10.1186/1742-2094-11-89.

Synergistic white matter protection with acute-on-chronic endotoxin and subsequent asphyxia in preterm fetal sheep

Lotte G van den Heuij, Sam Mathai, Joanne O Davidson, Christopher A Lear, Lindsea C Booth, Mhoyra Fraser, Alistair J Gunn  1 , Laura Bennet
Affiliations

Synergistic white matter protection with acute-on-chronic endotoxin and subsequent asphyxia in preterm fetal sheep

Lotte G van den Heuij et al. J Neuroinflammation. .

Abstract

Background: Perinatal asphyxia and exposure to intrauterine infection are associated with impaired neurodevelopment in preterm infants. Acute exposure to non-injurious infection and/or inflammation can either protect or sensitize the brain to subsequent hypoxia-ischemia. However, the effects of subacute infection and/or inflammation are unclear. In this study we tested the hypothesis that acute-on-chronic exposure to lipopolysaccharide (LPS) would exacerbate white matter injury after subsequent asphyxia in preterm fetal sheep.

Methods: Fetal sheep at 0.7 gestational age received a continuous LPS infusion at 100 ng/kg for 24 hours, then 250 ng/kg/24 hours for 96 hours, plus 1 μg boluses of LPS at 48, 72, and 96 hours or the same volume of saline. Four hours after the last bolus, complete umbilical cord occlusion or sham occlusion was induced for 15 minutes. Sheep were sacrificed 10 days after the start of infusions.

Results: LPS exposure was associated with induction of microglia and astrocytes and loss of total and immature and mature oligodendrocytes (n = 9) compared to sham controls (n = 9). Umbilical cord occlusion with saline infusions was associated with induction of microglia, astrogliosis, and loss of immature and mature oligodendrocytes (n = 9). LPS exposure before asphyxia (n = 8) was associated with significantly reduced microglial activation and astrogliosis and improved numbers of immature and mature oligodendrocytes compared to either LPS exposure or asphyxia alone.

Conclusions: Contrary to our initial hypothesis, the combination of acute-on-chronic LPS with subsequent asphyxia reduced neuroinflammation and white matter injury compared with either intervention alone.

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Figures

Figure 1
Figure 1
Photomicrographs of coronal sections of fetal sheep brain showing the white matter regions analyzed in this study. 1) periventricular white matter, 2) intragyral white matter. Scale bar = 2.5 mm.
Figure 2
Figure 2
Changes in numbers of immunohistochemically labeled white matter cells. Bar graphs showing numbers of CNPase positive cells in the PVWM (A) and IGWM (B), Olig2 positive cells in the PVWM (C) and IGWM (D), Iba-1 positive cells (E) and GFAP positive cells in the PVWM (F), TNF-α positive cells in the PVWM, (G) and caspase-3 positive cells in the PVWM (H). *P <0.05, **P <0.01, ***P <0.001 versus Sal-Sham controls, # P <0.05 versus LPS-Sham, § P <0.05 versus Sal-Asp. Data are mean ± SEM. Asp, asphyxia; CNPase, 2′,-3′-Cyclic-nuceotide 3′-phosphodiesterase; GFAP, glial fibrillary acidic protein; Iba-1, ionized calcium-binding adapter molecule-1; IGWM, intragyral white matter; LPS, lipopolysaccharide; Olig2, Oligodendrocyte transcription factor-2; PVWM, periventricular white matter; TNF-α, tumor necrosis factor-α; Sal, saline.
Figure 3
Figure 3
Photomicrographs of immature and mature oligodendrocytes and caspase-3 labeled apoptotic cells in the PVWM. Photomicrographs showing CNPase immunolabeling (A-D), and cleaved caspase-3 (ASP175) immunolabeling (E-H) in the PVWM in Sal-Sham (row 1; A, E), LPS-Sham (row 2; B, F), Sal-Asp (row 3; C, G) and LPS-Asp (bottom; D, H). Arrows show examples of labeled cells. Note the reduction in CNPase positive cells and increase in activated caspase-3 labelling after LPS-Sham and Sal-Asp. Surviving CNPase positive cells show stunted processes. LPS-Asp treatment was associated with recovery of CNPase positive immature and mature oligodendrocytes to Sal-Sham values, and reduced numbers of activated caspase-3 positive cells compared with Sal-Asp but not LPS-Sham. Scale bar = 20 μm. Asp, asphyxia; CNPase, 2′,-3′-Cyclic-nuceotide 3′-phosphodiesterase; LPS, lipopolysaccharide; PVWM, periventricular white matter; Sal, saline.
Figure 4
Figure 4
Photomicrographs showing astrocytes, microglia and TNF-α labeled cells in the PVWM. Photomicrographs showing glial fibrillary acidic protein (GFAP) (First column; A-D), Iba-1 (Second column; E-H), and tumor necrosis factor (TNF)-α immunolabeling (Third column; I-L) in the PVWM in Sal-Sham (row 1; A, E, I), LPS-Sham (row 2; B, F, J), Sal-Asp (row 3; C, G, K) and LPS-Asp (bottom; D, H, L). Arrows show examples of labeled cells. Note the robust induction of Iba-1 positive reactive microglia and GFAP positive reactive astrocytes after LPS-Sham treatment and Sal-Asp treatment, which was significantly attenuated in the LPS-Asp group. The LPS-Asp group showed astrocytes with thinner bodies and fewer processes than LPS-Sham and Sal-Asp. Scale bar = 20 μm. Asp, asphyxia; GFAP, glial fibrillary acidic protein; Iba-1, ionized calcium-binding adapter molecule-1; LPS, lipopolysaccharide; PVWM, periventricular white matter; Sal, saline; TNF-α: tumor necrosis factor-α.
Figure 5
Figure 5
Time sequence of changes in fetal plasma IL-6, IL-10 and cortisol concentrations. The horizontal bars at the top of the figure show the chronic infusions of LPS or saline. The arrows represent the times of the three LPS bolus doses (B1, B2 and B3). The vertical dashed line shows the time of the 15 minute period of umbilical cord occlusion in the asphyxia groups. Data were not available in the LPS-Sham group for the 11 am time point on day five. Data are mean ± SEM. *P <0.05. A) Sal-Sham versus LPS-Sham; B) Sal-Sham versus LPS-Asp; C) LPS-Sham versus Sal-Asp; D) LPS-Sham versus LPS-Asp; E) Sal-Asp versus LPS-Asp. Asp, asphyxia; IL, interleukin; LPS, lipopolysaccharide; Sal, saline.
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