Low-dose lipopolysaccharide selectively sensitizes hypoxic ischemia-induced white matter injury in the immature brain

Abstract

Little is known about roles of inflammation and hypoxic ischemia (HI) in the generation of neuroinflammation and damage of blood-brain barrier (BBB) in the white matter (WM) that displays regional vulnerability in preterm infants. We investigated whether low-dose lipopolysaccharide (LPS) sensitizes HI-induced WM injury in postpartum (P) day 2 rat pups by selectively increasing neuroinflammation and BBB damage in the WM. Pups received LPS (0.05 mg/kg) (LPS + HI) or normal saline (NS + HI) followed by 90-min HI. LPS and NS group were the pups that had LPS or NS only. Myelin basic protein immunohistochemistry on P11 showed WM injury in LPS + HI group, but not in NS + HI, LPS, and NS groups. In contrast, no gray matter injury was found in the four groups. LPS + HI group also showed decreased number of oligodendrocytes in the WM 72-h postinsult. In the same brain region, increases of activated microglia, TNF-alpha expression, BBB leakage, and cleaved caspase-3 positive cells were much more prominent in LPS + HI group than in the other three groups 24-h postinsult. The oligodendrocytes were the major cells with cleaved caspase-3 expression. We concluded that low-dose LPS sensitized HI-induced WM injury in the immature brain by selectively up-regulating neuroinflammation and BBB damage in the WM.

Figure 1

Nissl staining on P11 (9 days post-insult) (A) showed no significant injury in the gray matter in the NS (n=5), LPS (n=4), NS+HI (n=4) and LPS+HI (n=8) groups (gross pictures in the upper panel, microscopic pictures in the lower panel). Quantitative analysis (B) showed no significant differences in the ratios of the ipsilateral to contralateral areas between the 4 groups in the cortex, striatum and hippocampus. Scale bar = 200 μm in (A). NS, normal saline; LPS, lipopolysaccharide; HI, hypoxic ischemia. Values are means ± SEM.

Figure 2

Immunohistochemistry (A) and quantitative analysis (B) for myelin basic protein (MBP) staining on P11 (9 days post-insult). The LPS+HI group (n=8) had markedly decreased MBP expression in the ipsilateral hemisphere than the NS (n=5), LPS (n=4) and NS+HI (n=4) groups (A). The four groups did not differ in the MBP expression in the contralateral hemispheres. Scale bar = 100 μm in (A). Values are means ± SEM. *p< 0.05; **p < 0.01.

Figure 3

Immunohistochemistry (A) and quantitative analysis (B) for O4-postive oligodendrocyte progenitors at 72 hours post-insult. (A) The LPS+HI group (n=6) had significantly decreased numbers of O4-postive oligodendrocyte progenitors in the ipsilateral white matter than the NS (n=5), LPS (n=4) and NS+HI (n=6) group. The oligodendroglial numbers in the contralateral white matter showed no differences between the 4 groups. Scale bar = 50 μm in (A). Values are means ± SEM. **p< 0.01; ***p< 0.001

Figure 4

CD-11b immunohistochemistry and its quantitative analyses at 24 hours post-insult showed very few microglia (arrows) in the cortices of the LPS (n=4), NS (n=5), NS+HI (n=7) and LPS+HI (n=9) groups (A). The LPS+HI group had significant increases of activated microglia (arrows) in the white matter of the ipsilateral hemisphere than the other 3 groups (B). The 4 groups did not differ in the microglia in the white matter of the contralateral hemispheres. Scale bar = 50 μm in (A, B). Values are means ± SEM. ***p < 0.001.

Figure 4

CD-11b immunohistochemistry and its quantitative analyses at 24 hours post-insult showed very few microglia (arrows) in the cortices of the LPS (n=4), NS (n=5), NS+HI (n=7) and LPS+HI (n=9) groups (A). The LPS+HI group had significant increases of activated microglia (arrows) in the white matter of the ipsilateral hemisphere than the other 3 groups (B). The 4 groups did not differ in the microglia in the white matter of the contralateral hemispheres. Scale bar = 50 μm in (A, B). Values are means ± SEM. ***p < 0.001.

Figure 5

TNF-α immunohistochemistry and its semi-quantitative analyses by integrated optical density at 24 hours post-insult showed no significant differences of TNF-α levels in the cortices of the ipsilateral and the contralateral hemispheres between the 4 groups (A). The LPS+HI group (n=9) had significantly higher TNF-α levels in the white matter of the ipsilateral hemisphere than the NS (n=5), LPS (n=4) and NS+HI (n=7) group (B). The 4 groups did not differ in the TNF-α immunoreactivity in the white matter of the contralateral hemisphere. (C) Immunofluorescence revealed that the CD-11b-positive microglia (arrows) in the white matter of the LPS+HI group co-expressed TNF-α. Scale bar = 100 μm in (A, B), 50 μm in (C). Values are means ± SEM. ***p < 0.001.

Figure 5

TNF-α immunohistochemistry and its semi-quantitative analyses by integrated optical density at 24 hours post-insult showed no significant differences of TNF-α levels in the cortices of the ipsilateral and the contralateral hemispheres between the 4 groups (A). The LPS+HI group (n=9) had significantly higher TNF-α levels in the white matter of the ipsilateral hemisphere than the NS (n=5), LPS (n=4) and NS+HI (n=7) group (B). The 4 groups did not differ in the TNF-α immunoreactivity in the white matter of the contralateral hemisphere. (C) Immunofluorescence revealed that the CD-11b-positive microglia (arrows) in the white matter of the LPS+HI group co-expressed TNF-α. Scale bar = 100 μm in (A, B), 50 μm in (C). Values are means ± SEM. ***p < 0.001.

Figure 5

TNF-α immunohistochemistry and its semi-quantitative analyses by integrated optical density at 24 hours post-insult showed no significant differences of TNF-α levels in the cortices of the ipsilateral and the contralateral hemispheres between the 4 groups (A). The LPS+HI group (n=9) had significantly higher TNF-α levels in the white matter of the ipsilateral hemisphere than the NS (n=5), LPS (n=4) and NS+HI (n=7) group (B). The 4 groups did not differ in the TNF-α immunoreactivity in the white matter of the contralateral hemisphere. (C) Immunofluorescence revealed that the CD-11b-positive microglia (arrows) in the white matter of the LPS+HI group co-expressed TNF-α. Scale bar = 100 μm in (A, B), 50 μm in (C). Values are means ± SEM. ***p < 0.001.

Figure 6

IgG immunohistochemistry and its semi-quantitative analyses by integrated optical density at 24 hours post-insult showed very few IgG extravasation in the ipsilateral cortices of the NS+HI and LPS+HI group (arrows), but the 4 groups showed no significant differences in the cortices of the ipsilateral and contralateral hemispheres (A). The LPS+HI (n= 9) group had significantly higher levels of IgG extravasation in the white matter of the ipsilateral hemisphere than the NS (n=5), LPS (n=4) and NS+HI (n=7) groups (B); while the 4 groups did not differ in the white matter of the contralateral hemispheres. Scale bar = 100 μm in (A, B). Values are means ± SEM. ***p < 0.001.

Figure 6

IgG immunohistochemistry and its semi-quantitative analyses by integrated optical density at 24 hours post-insult showed very few IgG extravasation in the ipsilateral cortices of the NS+HI and LPS+HI group (arrows), but the 4 groups showed no significant differences in the cortices of the ipsilateral and contralateral hemispheres (A). The LPS+HI (n= 9) group had significantly higher levels of IgG extravasation in the white matter of the ipsilateral hemisphere than the NS (n=5), LPS (n=4) and NS+HI (n=7) groups (B); while the 4 groups did not differ in the white matter of the contralateral hemispheres. Scale bar = 100 μm in (A, B). Values are means ± SEM. ***p < 0.001.

Figure 7

Immunohistochemistry and quantitative analysis for cleaved caspase-3-positive cells at 24 hours post-insult showed no significant difference in the cleaved caspase-3-positive cells in the cortices of the ipsilateral and the contralateral hemispheres between the 4 groups (A). The LPS+HI group (n=9) had significantly increased numbers of cleaved caspase-3-positive cells in the white matter of the ipsilateral hemisphere than the NS (n=5), LPS (n=4) and NS+HI (n=7) groups (B). The 4 groups did not differ in the cleaved caspase-3-positive cells in the white matter of the contralateral hemispheres. Immunofluorescence of the ipsilateral white matter of the LPS+HI group showed the cleaved caspase-3 cells were O4-postive oligodendrocyte progenitors rather than GFAP-positive astrocytes (C). Scale bar = 50μm in (A, B) and 25μm in (C). Values are means ± SEM. ***p < 0.001.

Figure 7

Immunohistochemistry and quantitative analysis for cleaved caspase-3-positive cells at 24 hours post-insult showed no significant difference in the cleaved caspase-3-positive cells in the cortices of the ipsilateral and the contralateral hemispheres between the 4 groups (A). The LPS+HI group (n=9) had significantly increased numbers of cleaved caspase-3-positive cells in the white matter of the ipsilateral hemisphere than the NS (n=5), LPS (n=4) and NS+HI (n=7) groups (B). The 4 groups did not differ in the cleaved caspase-3-positive cells in the white matter of the contralateral hemispheres. Immunofluorescence of the ipsilateral white matter of the LPS+HI group showed the cleaved caspase-3 cells were O4-postive oligodendrocyte progenitors rather than GFAP-positive astrocytes (C). Scale bar = 50μm in (A, B) and 25μm in (C). Values are means ± SEM. ***p < 0.001.

Figure 7

Immunohistochemistry and quantitative analysis for cleaved caspase-3-positive cells at 24 hours post-insult showed no significant difference in the cleaved caspase-3-positive cells in the cortices of the ipsilateral and the contralateral hemispheres between the 4 groups (A). The LPS+HI group (n=9) had significantly increased numbers of cleaved caspase-3-positive cells in the white matter of the ipsilateral hemisphere than the NS (n=5), LPS (n=4) and NS+HI (n=7) groups (B). The 4 groups did not differ in the cleaved caspase-3-positive cells in the white matter of the contralateral hemispheres. Immunofluorescence of the ipsilateral white matter of the LPS+HI group showed the cleaved caspase-3 cells were O4-postive oligodendrocyte progenitors rather than GFAP-positive astrocytes (C). Scale bar = 50μm in (A, B) and 25μm in (C). Values are means ± SEM. ***p < 0.001.