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. Author manuscript; available in PMC: 2013 Mar 26.
Published in final edited form as: Pediatr Res. 2010 Jul;68(1):41–47. doi: 10.1203/PDR.0b013e3181df5f6b

Low-dose Lipopolysaccharide Selectively Sensitizes Hypoxic-Ischemia White Matter Injury in the Immature Brain

LAN-WAN WANG 1, YING-CHAO CHANG 1, CHANG-YI LIN 1, JAU-SHYONG HONG 1, CHAO-CHING HUANG 1,★
PMCID: PMC3608684  NIHMSID: NIHMS451865  PMID: 20351655

Abstract

Little is known about the effects of inflammation and hypoxic ischemia (HI), the two important risk factors for white matter (WM) injury in preterm infants, on neuroinflammation and blood-brain barrier (BBB) damage in the WM that displays selective vulnerability in preterm infants. We investigated whether low-dose lipopolysaccharide (LPS) selectively sensitizes HI WM injury in postpartum (P) day 2 pups by selectively increasing neuroinflammation and BBB damage in the WM. P2 pups received LPS (0.05 mg/kg) (LPS+HI) or normal saline (NS+HI) followed by 90-minute HI. LPS and NS group were the pups that had LPS or NS but without HI. Neuropathological examinations on P11 showed no gray matter injury in LPS+HI, NS+HI, LPS and NS groups, but WM injury manifested as decreases of myelin basic protein in LPS+HI group. The LPS+HI group also had significant decreases of oligodendrocyte progenitors 72 hours post-insult, and increases of activated microglia, TNF-α expression, BBB leakage and cleaved caspase-3-positive cells in the WM than the other 3 groups 24 hours post-insult. The oligodendrocytes were the major cells with cleaved caspase-3 expression. We concluded that low-dose LPS sensitized HI WM injury in the immature brain by selectively up-regulating neuroinflammation and BBB damage in the WM.

INTRODUCTION

Despite the increase in the survival of very-low-birth-weight (VLBW) preterm infants in recent years, cerebral palsy still occurs in 10% and cognitive/behavioral deficits in 25–50% of the very preterm survivors (1,2). Periventricular white mater (WM) injury is the major brain injury and accounts for the most prominent determinant of neurological deficits in VLBW infants (1). Epidemiological observations show that hypoxic ischemia (HI) (2,3) and inflammation (4) are the two major risk factors for WM injury or cerebral palsy in very preterm infants. Inflammation might predispose to or act in concert with HI in premature infants. Increased systemic cytokines in premature infants with chorioamnionitis were associated with hemodynamic disturbance leading to cerebral HI (3,5), and concurrent chorioamnionitis and placental perfusion defect placed preterm infants at a higher risk of abnormal neurological outcomes than either insult alone (6). Although most HI and inflammatory episodes are not life-threatening and treatable clinically, the combined effects of HI and inflammation during the perinatal period may have a significant impact on the immature brain.

Experimental studies have shown that pre-exposure to systemic bacterial lipopolysaccharide (LPS) sensitized neonatal brain to HI injury in the gray matter and WM of rodent pups (7–8). Either LPS or HI insult alone could result in significant gray matter and WM injury in the immature brain (9–12). Therefore, when the combined insults of LPS and HI are given, it remains unclear whether the injury is induced by the effect of LPS or HI or both. Furthermore, whether low-dose LPS, which mimics less severe infection, is sufficient to sensitize the developing WM to HI injury also remains unknown. Pre-myelinating oligodendrocytes are the target cells of damage during the developmental window of vulnerability for WM injury in premature infants at 23–32 weeks of gestation (13). Comparing the timing of human and rodent oligodendroglial lineage progression, the predominance of pre-myelinating oligodendrocytes in P2 pups (equivalent to human 20–28 weeks’ gestation) coincides with the high-risk period of WM injury in VLBW infants (14). However, very few studies have examined the effects of low-dose LPS and HI on WM injury in P2 pups.

Inflammatory responses and vascular factors may account for the relative susceptibility of the developing WM to HI injury (1). Animal studies showed that LPS or HI increased microglial activation and upregulated TNF-α expression throughout the gray and WM in the neonatal rat brain (10,15–17). Little is known about the combined effects of low-dose LPS and HI on neuroinflammation and blood-brain barrier (BBB) damage selectively in the WM that displays regional vulnerability to HI. In this study, we hypothesized that low-dose LPS selectively sensitizes HI WM injury in P2 rat pups in association with increases of neuroinflammation and BBB damage in the WM.

MATERIALS AND METHODS

A rat-pup model of cerebral white matter injury

This study was approved by the Animal Care Committee at National Cheng Kung University. Rat pups were kept under standard condition with a 12/12-hour light/dark cycle. We injected P2 Sprague-Dawley pups with 0.05 mg/kg LPS (Escherichia coli 0111:B4; Sigma-Aldrich, St Louis, MO) or pyrogen-free normal saline (NS) (i.p.). The pups were randomly assigned to four different groups: NS (NS injected without HI), LPS (LPS injected without HI), NS+HI (NS injected 2 hours before HI) and LPS+HI (LPS injected 2 hours before HI). HI was induced by right carotid artery ligation followed by hypoxia, as previously described (18). The right common carotid artery was permanently ligated under 2.5% halothane anesthesia. After surgery, the pups were returned to an incubator for a 1-hour recovery. They were then placed in airtight 500-mL containers partially submerged in a 36°C water bath, and humidified 6.5% oxygen was kept at a flow rate of 3 L/minute for 90 minutes. Following hypoxia, the pups were returned to their dam. The rats were sacrificed for examinations in cryosections on P3 (24 hours post-insult) and P5 (72 hours post-insult), and in paraffin sections on P11 (9 days post-insult).

Assessment of gray matter and white matter injury

The rats were sacrificed on P11 after pentobarbital anesthesia. After the brains had been removed and post-fixed in 4% paraformaldehyde at room temperatures for 48 hours, they were dehydrated through graded alcohols and embedded in paraffin, and then coronally sectioned (10-μm thick) from the genu of the corpus callosum to the end of the dorsal hippocampus. Nissl and myelin basic protein (MBP) staining were performed in four sections per brain. The cross-sectional areas, two at the level of the striatum (Plate 18 and Plate 24) and another two at the dorsal hippocampus (Plate 30 and Plate 36), were selected according to the reference planes in a rat brain atlas (19).

Nissl staining

Images of Nissl-stained sections were scanned and analyzed by a computerized software (ImagePro Plus 6.0; Media Cybernetics, Bethesda, MD) linked to a Nikon E400 microscope to calculate the percentage of area loss in the cortex, hippocampus and striatum in the ipsilateral versus the contralateral hemisphere (20).

Myelin basic protein staining

For the evaluation of WM injury, paraffin-embedded sections were deparaffinized and hydrated through graded alcohols. Endogenous peroxidase was eradicated for 30 minutes in 0.3% H2O2 in methanol. Heat-induced antigen retrieval was subsequently performed using 10 mmol/L citrate buffer (pH = 6.0) for 10 minutes in a microwave oven. After permealization and blocking of non-specific binding, sections were first incubated at 4°C overnight with the primary anti-MBP monoclonal antibody (1: 100; Chemicon), rinsed, and then incubated for 1 hour at room temperature with biotinylated goat anti-rat IgG (1:200; Santa Cruz). Positively-stained cells were visualized using avidin-biotin-peroxidase complex amplification (Pierce Biotechnology) with diaminobenzidine tetrahydrochloride detection (Millipore, Chemicon).

MBP expression was assessed in three regions (medial, middle and lateral) along the corpus callosum in each hemisphere of each section, and graded using a modified 4-point scoring system (21): 0, immunohistochemical staining hardly visible; 1, faint staining of the corpus callosum with rarefaction of the periventricular WM and loss of fibrillar features; 2, thinning of the corpus callosum with broken fibrillar processes; 3, few cortical processes or supracallosal fibers without cortical processes; 4, thick corpus callosum with dense and extended cortical processes. The scores of each region were summed up to obtain a total score (range, 0–12) for each hemisphere. Each section had a total MBP score in the ipsilateral and contralateral hemisphere, respectively. Two independent observers, blind to the treatment conditions, measured the degrees of gray matter and WM injury.

Evaluation of neuroinflammation

Rats pups were sacrificed on P3, and the brains were post-fixed in ice-cold 4% paraformaldehyde overnight, dehydrated using 30% (w/v) sucrose in PBS for 2 days, and coronally sectioned (20-μm thick) from the genu of the corpus callosum to the end of the dorsal hippocampus. Four coronal sections, as described above, were assessed for each brain. The immunohistochemistry for microglial cell activation (CD-11b) and TNF-α were done 24 hours post-insult. The specific primary antibodies included monoclonal anti-CD-11b (1:100; AbD Serotec, Oxford, UK) and polyclonal anti- TNF-α (1:100; Bender MedSystems, Vienna, Austria). Biotinylated secondary antibodies included anti-mouse IgG and anti-rabbit IgG (all 1:200). Biotin-peroxidase signals were detected using 0.5 mg/mL 3′3′-diaminobenzidine (DAB)/0.003% H2O2 as a substrate. Results were recorded using a microscope (BX51; Olympus, Tokyo, Japan). The number of CD-11b-positive cells and the integrated optical density (IOD) of TNF-α signals were analyzed as described previously (22), using imaging software (ImagePro Plus 6.0; Media Cybernetics, Bethesda, MD) at 400× magnification per visual field (one visual field = 0.0356 mm2) for CD-11b and at 200× magnification per visual field (one visual field = 0.145 mm2) for TNF-α. Three visual fields within the medial, middle and lateral areas in the cortex and the subcortical WM were analyzed and averaged, respectively. The mean IOD values in the WM and the cortex in the ipsilateral and contralateral hemisphere of each experimental group were compared to those of the NS group to obtain the relative IOD ratios.

Evaluation of blood-brain barrier permeability

IgG extravasation was used as an indicator of BBB permeability (12), and the IgG immunoreactivity (HRP-conjugated anti-rat IgG 1:300; Chemicon) was measured 24 hours post-insult with IOD using the image analysis software as described above. The relative IOD ratios in the ipsilateral and contralateral WM and cortex were compared among the four groups.

Evaluation of oligodendroglial cell death

To assess the extent of damage to the oligodendrocyte progenitors, the numbers of O4-positive cells (anti-O4 IgM 1:100; Chemicon) in the WM were compared among the four groups 72 hours post-insult. Cleaved caspased-3 (1:100; Cell Signaling) was used as an apoptotic marker 24 hours post-insult, and the numbers of cleaved caspased-3-positive cells in the WM and the cortex were compared among the four groups.

Immunofluorescence

After blocking (1× PBS, 2% normal goat serum and 0.1% Triton X-100) for 1 hour, the sections were incubated overnight at 4°C with a mixture of two of the following primary antibodies: anti-CD-11b (1:100; AbD Serotec, Oxford, UK), anti- TNF-α (1:100; Bender MedSystems, Vienna, Austria), anti-O4 IgM (1:100; Chemicon), anti-GFAP (1:100; Chemicon) and anti-cleaved caspase-3 (1:100; Cell Signaling). The sections were washed three times with 0.1 M PBS and then incubated with Alexa Fluor 594 anti-mouse IgG/IgM or Alexa Fluor 488 anti-rabbit IgG (1:400 in blocking reagents; Invitrogen Molecular Probes) for 1 hour at room temperature. The fluorescence signals were detected and the results were recorded using a microscope (E400; Nikon Instech, Kawasaki, Japan) at excitation-emission wavelengths of 596–615 nm (Alexa Fluor 594, red) and 470–505 nm (Alexa Fluor 488, green).

Statistical analysis

Statistical significance (p < 0.05) was determined using one-way ANOVA, and Tukey’s method was used for post-hoc comparisons. Continuous data are means ± SEM.

RESULTS

Low-dose LPS selectively sensitized hypoxic-ischemia white matter injury

First, we examined whether WM injury could be selectively induced by low-dose LPS followed by HI in P2 rat pups. Neuropathological examinations were performed on P11 in the NS, LPS, NS+HI and LPS+HI group. Nissl staining showed no gross gray matter injury in the four groups (Fig. 1A), and further quantitative analysis demonstrated no significant brain area loss in the cortex, striatum and hippocampus of the four groups (Fig. 1B). For WM injury, the LPS+HI group had significantly decreased MBP expression in the WM of the ipsilateral hemisphere than the NS, LPS and NS+HI group, whereas the four groups had similar MBP expression in the contralateral hemispheres (Fig. 2A, B).

Figure 1.

Figure 1

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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.

Figure 2

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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.

We next examined whether there were changes in the numbers of oligodendrocyte progenitors, the target cells of WM injury in preterm infants, 72 hour post-insult. Immunohistochemistry demonstrated that the LPS+HI group had significantly decreased number of oligodendrocyte progenitors in the WM of the ipsilateral hemisphere than the NS, LPS and NS+HI group, while no significant differences were found in the WM of the contralateral hemispheres between the four groups (Fig. 3A, B).

Figure 3.

Figure 3

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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

Low-dose LPS followed by hypoxic ischemia selectively induced neuroinflammation in the white matter

Microglia activation

At 24 hours post-insult, the four groups had very few CD-11b-positive microglia in the bilateral cerebral cortices (Fig. 4A). In contrast, the LPS+HI group had significantly increased number of microglia in the WM of the ipsilateral hemisphere than the LPS, NS, and NS+HI group (Fig. 4B). The four groups had similar numbers of activated microglia in the WM of the contralateral hemispheres.

Figure 4.

Figure 4

Figure 4

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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.

TNF-α expression

The four groups had similar TNF-α expression in the bilateral cerebral cortices 24 hours post-insult (Fig 5A). In contrast, the LPS+HI group had significant increases of TNF-α immunoreactivities in the WM of the ipsilateral hemisphere than the NS, LPS and NS+HI group (Fig. 5B). The TNF-α expression in the WM of the contralateral hemispheres did not differ between the four groups. Immunofluorescence study in the LPS+HI group confirmed that the microglial cells co-expressed TNF-α (Fig. 5C).

Figure 5.

Figure 5

Figure 5

Figure 5

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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.

Low-dose LPS followed by hypoxic ischemia selectively induced blood-brain barrier damage in the white matter

Using IgG extravasation as a marker of BBB disruption, we found little or no IgG extravasation in the bilateral cerebral cortices or in the WM of the contralateral hemisphere of the four groups (Fig. 6A); while the LPS+HI group had markedly increased IgG immunoreactivities throughout the WM in the ipsilateral hemisphere than the NS, LPS and NS+HI group (Fig. 6B).

Figure 6.

Figure 6

Figure 6

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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.

Low-dose LPS followed by hypoxic ischemia caused apoptosis of oligodendrocyte progenitors in the white matter

To determine the target cells of injury in response to microglial activation and BBB breakdown, cleaved caspase-3 was used as a marker of apoptosis. The four groups had no significant differences in the numbers of cleaved caspase-3-positive cells in the bilateral cortices, but the LPS+HI group had significantly increases of cleaved caspase-3-positive cells in the WM of the ipsilateral hemisphere than the other three groups. The four groups showed no differences in the numbers of cleaved caspase-3-positive cells in the WM of the contralateral hemispheres (Fig. 7A, B). The immunofluorescent pictures in the LPS+HI group demonstrated that the O4-positive oligodendrocyte progenitors, but not the GFAP-positive astrocytes, were the major cells that co-expressed cleaved caspase-3 (Fig. 7C).

Figure 7.

Figure 7

Figure 7

Figure 7

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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.

DISCUSSION

HI and infection/inflammation are the two major risk factors for WM injury and cerebral palsy in very preterm infants (1–4). Clinically stable preterm infants may suffer from prolonged episodes of hypoxemia without apparent apnea/bradycardia (23), and may be potentially vulnerable to impaired cerebral perfusion with small decreases in systemic blood pressure (1). The recurrent or chronic physiological instability in VLBW infants was associated with neurodevelopmental morbidity in early childhood (24). Moreover, culture-negative clinical infection, though may be less toxic than culture-positive sepsis, still imposed similar risk of neurodevelopmental impairment in very preterm infants as sepsis did (4). These observations raise the possibility that less severe HI and inflammation may jointly exert negative impact on the immature brain even in the absence of overt cardiorespiratory distress. Studies have shown that systemic LPS (doses ranged from 0.3 to 1 mg/kg for pregnant rats) (9, 25) or HI (duration ranged from 50 minutes for P9 and up to 4 hours for P2 rodent pups) alone (11–12, 26) induced gray matter and WM injury in the immature brain. The combined effect of LPS (0.1–0.3 mg/kg) and HI (duration ranged from 30–40 minutes) in pups at P7 to P10 also caused injury in the gray matter and WM injury (7–8, 27). We studied the effect of low-dose LPS (0.05 mg/kg) and 90-minute HI (a sub-threshold HI duration for P2 pups) on WM injury in the immature brain. We demonstrated that low-dose LPS or 90-minute HI alone caused no significant injury in the gray matter or WM, and that selective WM injury could only be induced by the combination of the two. WM injury induced by low-dose LPS and sub-threshold HI was associated with regional increases of microglial activation, TNF-α expression, BBB damage, and oligodendrocyte progenitor apoptosis in the WM. Our study suggests that mild inflammation and sub-clinical HI, though not life-threatening, may jointly cause WM injury in VLBW infants.

Activated microglia are the hallmark of neuroinflammation and exacerbate brain injury through cytokine production (28). Previous studies in neonatal rats showed that intracerebral LPS (1 mg/kg for P5 pups) or HI (duration ranged from 70 minutes to 3 hours for P7 pups) increased microglia activation (10,15) and TNF-α levels in the immature brain (16–17). We found that low-dose LPS or sub-threshold HI did not elicit obvious microglia activation and TNF-α production, and also did not induce significant damage in the gray matter and WM. In contrast, low-dose LPS followed by sub-threshold HI increased microglia activation and TNF-α production selectively in the WM and caused significant WM injury. The selective increases of TNF-α immunoreactivities in the WM corresponded to the region-specific response of microglia in this P2 rat-pup model. Human study showed increased activated microglia in the cerebral WM of the preterm newborn relative to the term newborn and to the overlying cortex of either group. The finding of a developmental-dependent abundance of activated microglia in the cerebral WM of the preterm newborns suggests a potential vulnerability of this area for brain insults characterized by activation of microglia (29).

The BBB plays an important role in brain injury induced by central- or peripheral-derived insults. In neonatal mice, moderate to severe HI resulted in extensive BBB disruption with a maximum IgG immunoreactivity at 24 hours, followed by significant gray matter and WM injury at 7 days post-insult (12). While in neonatal rats suffering from inflammation induced by repetitive injections of LPS, increased BBB permeability was confined to the WM and associated with a substantial decrease in the WM volume (30). When both the peripheral and central insults occurred in adult mice, systemic inflammation caused sustained BBB breakdown and exacerbated ischemic brain injury (31). Similarly, our study demonstrated that low-dose LPS complicated with sub-threshold HI induced BBB disruption selectively in the WM and subsequently caused WM injury. The regional vulnerability of BBB in the WM may be related to the region-specific activation of microglia, which may contribute to BBB disruption through matrix protease generation (32). In addition, the vascularized terminal areas and the BBB in the periventricular WM of preterm infants are particularly susceptible to hypoperfusion due to HI (1, 33). The increase of BBB permeability selectively in the WM may act in concert with microglia activation to accentuate WM injury through leukocyte recruitment into the brain (34).

Maturational vulnerability of oligodendrocytes plays another important role in the pathogenesis of WM injury. Pre-myelinating oligodendrocytes display greater susceptibility to oxidative damage and glutamate excitotoxicity than mature oligodendrocytes (1). In this model of low-dose LPS followed by sub-threshold HI, apoptosis occurred predominantly in the WM, and the O4-positive oligodendrocyte progenitors, the mainly pre-myelinating oligodendrocytes in P2 rat brain, were the major cells that showed apoptosis. The decreased O4-positive cells corresponded to reduced MBP-expressing mature oligodendrocytes at an older age. These findings again support the maturation-dependent vulnerability to WM injury in P2 rat pups at the age equivalent to human VLBW infants (13).

Our findings is consistent with the autopsy studies in preterm infants with periventricular leukomalacia showing that activated microglia were selectively localized in the WM (35), and that infants with history of systemic infection and asphyxia had significant increases of TNF-α immunoreactivities than those with asphyxia alone (36). Our finding suggests that low-dose LPS selectively sensitizes HI WM injury by regionally up-regulating neuroinflammation and BBB damage in the WM. Establishing a rat pup model of selectively acquiring WM injury through low-dose LPS and sub-threshold HI may have clinical implications for perinatal medicine.

Acknowledgments

Statement of financial support: This study was supported by grants from the Taiwan National Health Research Institute (NHRI-EX 97–9414NI), the National Science Counsel (NSC: 97–2811-B-006–014), Chi Mei Medical Center (CMNCKU 9802) and the Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University.

We thank Chien-Jung Ho for her skillful technical assistance with animal preparations.

Abbreviations

VLBW

very-low-birth-weight

WM

white matter

HI

hypoxic ischemia

LPS

lipopolysaccharide

BBB

blood-brain barrier

NS

normal saline

MBP

myelin basic protein

IOD

integrated optical density

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