White matter injury following prolonged free radical formation in the 0.65 gestation fetal sheep brain

Free radicals seem to be involved in the development of cerebral white matter damage after asphyxia in the premature infant. The immature brain may be at increased risk of free radical mediated injury, as particularly the preterm infant has a relative deficiency in brain antioxidants systems, such as superoxide dismutase and glutathione peroxidase. In vitro studies show that immature oligodendrocytes express an intrinsic vulnerability to reactive oxygen species and free radical scavengers are able to protect immature oligodendrocytes from injury. The aim of this study was to examine the formation of ascorbyl radicals as a marker of oxidative stress in the preterm brain in association with cerebral white matter injury after intrauterine asphyxia. Fetal sheep at 0.65 gestation were chronically instrumented with vascular catheters and an occluder cuff around the umbilical cord. A microdialysis probe was placed in the periventricular white matter. Fetal asphyxia was induced by occlusion of the umbilical cord for 25 min (n = 10). Microdialysis samples were collected for 72 h and analyzed for ascorbyl radicals using electron spin resonance. Five instrumented fetuses served as controls. Three days after the insult, fetal brains were examined for morphologic injury. Umbilical cord occlusion resulted in prolonged and marked increase in ascorbyl radical production in the brain in connection with white matter injury, with activation of microglia cells in periventricular white matter and axonal injury. These data suggest that reperfusion injury following asphyxia in the immature brain is associated with marked free radical production.     

Fetal and neonatal origins of altered brain development

Abnormal development of the brain during fetal life is now thought to contribute to the aetiology of many neurological disorders that manifest throughout life. Many factors are likely to underlie such abnormal development including genetic makeup and an adverse intrauterine environment. This review will focus on prenatal hypoxic/ischaemic injury, inflammatory/infective insults and preterm birth. A range of experimental models have been used to characterize lesions formed in response to these insults and to determine mechanisms of damage resulting from such events. Relatively brief periods of fetal hypoxia result in neuronal death (cerebellum, hippocampus, and cerebral cortex), white matter damage and reduced growth of neural processes. These effects are more profound at mid than late gestation. Chronic mild placental insufficiency can result in fetal growth restriction and deficits in neural connectivity and myelination. Exposure of the preterm fetus to inflammatory agents causes brain damage particularly in the white matter and this is exacerbated by hypoxia. Premature birth without potentiating factors can result in subtle neuropathologies including cerebral white matter gliosis, hippocampal sclerosis and subarachnoid haemorrhage; the extent of the damage appears to be related to the regimen of ventilatory support. These studies show that the timing, severity and nature of specific insults are critical in determining the pattern of injury and thus the extent to which neurological function will be affected postnatally. Defining the causes, patterns and mechanisms of brain injury is crucial if we are to develop rational neuroprotective strategies to reduce the burden of altered brain growth and poor functional and behavioural outcomes.     

Lipopolysaccharide-induced inflammation and perinatal brain injury

Both energy failure and infections are important risk factors for brain injury in term and preterm infants. In this review we focus on recent experimental studies that have examined the effects of lipopolysaccharide (LPS) exposure to the fetus or neonate and the interaction of LPS with other events. Intracerebral LPS injections induce a marked cerebral cytokine response and prominent white matter lesions. LPS administered intravenously to the fetus also induces gross lesions, which are mainly localised to the white matter and are accompanied by activation of inflammatory cells. Cerebral effects following fetal LPS exposure via more distant routes, such as intracervical, intrauterine or maternal LPS administration, are characterised by reductions in oligodendrocyte or myelin markers without macroscopic lesions being evident. Both antenatal and neonatal LPS exposures increase the sensitivity of the brain to subsequent hypoxic/ischaemic events, even in adulthood. These studies suggest that fetal inflammation is the strongest predictor of brain lesions.     

Effects of antenatal steroids on ischemic brain injury in near-term ovine fetuses

BACKGROUND: Hypoxia/ischemia in utero can result in brain damage to the fetus and newborn. Antenatal steroids are a routine part of the management of women who develop premature labor. Pretreatment of young postnatal rats with dexamethasone before hypoxic/ischemic insults has been reported to attenuate brain injury. However, the effects of antenatal steroids on ischemic brain injury in fetuses have not been investigated. OBJECTIVE: We examined the effects of maternally administered antenatal corticosteroids on ischemic brain injury in near-term ovine fetuses. METHODS: Chronically instrumented fetuses at 122 days of gestation were studied 12 h after the last of four 4 mg dexamethasone, or placebo injections were given over 48 h to the ewes. Groups were dexamethasone/ischemic, placebo/ischemic and sham-treated control. Fetuses were exposed to 30 min of carotid occlusion (ischemia) or no occlusion (control) and 72 h of reperfusion. Whole brain coronal sections stained with Luxol fast blue-hematoxylin-eosin were scored for white matter and cerebral cortical lesions. Both areas received pathological scores of 0 to 5 reflecting the degree of injury (0=0%, 1=1-10%, 2=11-50%, 3=51-90%, 4=91-99% and 5=100%). Bilateral carotid blood flow also was measured before, during and after brain ischemia in the dexamethasone/ischemic and placebo/ischemic groups. RESULTS: White matter (WM) and cerebral cortical scores did not differ between the dexamethasone/ischemic and placebo/ischemic (WM: 3.0+/-1.9 and 2.9+/-1.7; cortex: 3.1+/-1.7 and 2.6+/-1.8, mean+/-S.D.) groups. White matter and cerebral cortical scores were higher in the dexamethasone/ischemic (WM: 3.0+/-1.9, P<0.02; cortex: 3.1+/-1.7, P<0.005) and placebo/ischemic (WM: 2.9+/-1.7, P<0.006; cortex: 2.6+/-1.8, P<0.007) than control (WM: 0.2+/-0.4; cortex: 0.2+/-0.4) group. Carotid blood flow was relatively higher (P<0.05) after 24, 48 and 72 h of reperfusion in the dexamethasone/ischemic than placebo/ischemic group. CONCLUSIONS: We conclude that maternal pretreatment with antenatal dexamethasone did not attenuate ischemic brain injury in the fetus, and that carotid blood flow was higher during reperfusion in fetuses of dexamethasone than placebo-treated ewes, most likely secondary to decreases in arterial oxygen tension.     

Intrauterine infection induces programmed cell death in rabbit periventricular white matter

An association between chorioamnionitis and periventricular leukomalacia has been reported in human preterm infants. However, whether this link is causal has not been convincingly established, and the underlying molecular mechanisms remain unclear. The objective of this study was to establish a reproducible model of cerebral white matter disease in preterm rabbits after intrauterine infection. Escherichia coli was inoculated into both uterine horns of laparotomized pregnant rabbits when gestation was 80% complete. The fetuses were delivered by cesarean section and killed 12, 24, or 48 h after the inoculation. Programmed cell death in the white matter was evaluated by hematoxylin-eosin-saffron staining and in situ fragmented DNA labeling (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling). In a first group of 14 pregnant rabbits not treated with antibiotics, all fetuses delivered 48 h after inoculation were stillborn, whereas fetuses extracted 12 or 24 h after inoculation were alive. No significant cell death was detected in the live fetuses compared with the control noninfected rabbits. In a second group of five pregnant rabbits treated with ceftriaxone initiated 24 h after the inoculation and continued until cesarean section was performed 48 h after inoculation, 13 fetuses were alive, but all showed evidence of extensive programmed cell death in the white matter by hematoxylin-eosin-saffron staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. White matter damage became histologically detectable only 48 h after inoculation. Three of the 13 brains displayed periventricular white matter cysts mimicking human cystic periventricular leukomalacia. The high reproducibility of white matter damage in our model should permit further studies aimed at unraveling the molecular mechanisms of periventricular leukomalacia.     

早产儿暂时性低甲状腺素血症与脑损伤及神经行为学相关性研究

目的 通过对早产儿甲状腺素水平测定及脑、神经行为发育测评,分析甲状腺素水平与脑损伤、神经行为学的相关性.方法 选取2009年11月至2010年4月,上海交通大学附属上海市儿童医院新生儿科收治的早产儿52例,生后6 h内留取血清样本,放射免疫法测定T3、T4、TSH值.所有患儿出生后3 d行头颅B超检查,每周复查1次,出院前行头颅MRI检查.根据头颅MRI结果将患儿分为3组:无脑损伤组(33例)、脑室内出血组(10例)、脑白质损伤组(9例).所有患儿于纠正胎龄40±2周时行新生儿20项行为神经测定.结果 3组患儿TSH均正常,排除先天性甲状腺功能减低症;共8例早产儿甲状腺功能正常,占15.4%(8/52);另44例早产儿甲状腺功能均低下,占84.6%(44/52).无脑损伤组T3、T4水平高于脑室内出血组及脑白质损伤组,并以脑白质损伤组T3、T4水平最为低下,3组间比较差异有统计学意义(P＜0.05).无脑损伤组患儿行为能力、被动肌张力、主动肌张力及总分4项得分显著高于有脑损伤的两组患儿,且脑室内出血组患儿得分又高于脑白质损伤组患儿,3组间比较差异有统计学意义(P＜0.05).结论 早产儿脑损伤越严重,甲状腺素水平越低.有脑损伤的早产儿神经行为学评分较无脑损伤的早产儿低.     

Melatonin reduces inflammation and cell death in white matter in the mid-gestation fetal sheep following umbilical cord occlusion

The premature infant is at increased risk of cerebral white matter injury. Melatonin is neuroprotective in adult models of focal cerebral ischemia and attenuates ibotenate-induced white matter cysts in neonatal mice. Clinically, melatonin has been used to treat sleep disorders in children without major side effects. The aim of this study was to investigate the protective and anti-inflammatory effects of melatonin in the immature brain following intrauterine asphyxia. Fetal sheep at 90 d of gestation were subjected to umbilical cord occlusion. Melatonin (20 mg/kg, n = 9) or vehicle (n = 10) was administered IV to the fetus, starting 10 min after the start of reperfusion and continued for 6 h. Melatonin treatment resulted in a slower recovery of fetal blood pressure following umbilical cord occlusion, but without changes in fetal heart rate, acid base status or mortality. The production of 8-isoprostanes following umbilical cord occlusion was attenuated and there was a reduction in the number of activated microglia cells and TUNEL-positive cells in melatonin treated fetuses, suggesting a protective effect of melatonin. In conclusion, this study shows that melatonin attenuates cell death in the fetal brain in association with a reduced inflammatory response in the blood and the brain following intrauterine asphyxia in mid-gestation fetal sheep.     

早产儿脑病的研究进展

随着低出生体重儿存活率的提高,早产儿脑损伤成为热点问题,损伤部位不仅局限于脑白质,灰质区域也有受累,故提出了“早产儿脑病”的概念。早产儿脑病包括脑白质损伤和神经元轴突病变。在缺血缺氧、围生期感染等致病因素作用下,脑组织发生直接或间接的神经细胞死亡或者是成熟障碍,最终导致脑性瘫痪、认知、语言、行为等能力障碍。该文总结了早产儿脑病的病理、发病机制、治疗及预后等情况。     

The sequelae of neonatal brain injury

The neonatal brain is very vulnerable to injury due to its relatively large size, rapid rate of development and immature immunological systems. Injury at this time often results in lifelong neuro-developmental sequelae such as cerebral palsy, learning difficulties and sensory deficits. In the term brain injury is most commonly due to hypoxia–ischaemia during labour, but hyperbilirubinaemia, trauma, thrombosis and infections remain important causes.In the preterm infant, because of immaturity, the pattern of injury is different with forms of white matter damage predominating. Germinal matrix haemorrhage, parenchymal infarction and forms of periventricular leucomalacia predominate. Preterm white matter damage often leads to altered or reduced development of cortical grey matter subsequently. All forms of cerebral palsy are seen in preterm children, but spastic cerebral diplegia is the commonest. Minor motor impairments in childhood are also very common as are behavioural disorders.Exact prognoses for infants with neonatal brain lesions are difficult to make owing to the fact that more than one lesion may co-exist in the same infant.     

Intra-amniotic endotoxin accelerates lung maturation in fetal rabbits

The hypothesis that endotoxin in amniotic fluid accelerates fetal lung maturation was tested. On day 25 of gestation, LPS (5 microg/fetus) was injected intra-amniotically into one uterine horn of eight New Zealand white rabbits, whereas the contralateral amniotic sacs were injected with saline vehicle. The fetuses were delivered 48 h after LPS administration and their lungs were studied. One dam went into premature labor prior to the 48 h time point and was excluded from the study. Mean white cell counts in amniotic fluid and bronchoalveolar lavage fluid from LPS-treated fetuses were increased 3.2-fold (p = 0.04) and 9.9-fold (p = 0.04), respectively. Fetal weights and lung weights were not affected by LPS. Surfactant protein SP-A and SP-B mRNA expressions in LPS-treated fetuses were increased 2.3-fold (p = 0.03) and 1.4-fold (p = 0.04), respectively. Static lung compliance was increased in animals treated with LPS (p = 0.001). Lungs from LPS-treated animals had better aeration than those of controls. Mean volume of inflation-fixed lungs of LPS-treated fetuses was 1.7 times greater than that of controls (p = 0.03). CONCLUSION: Intra-uterine exposure to LPS increases surfactant protein expression and improves lung stability and aeration in preterm animals.     

High-field diffusion tensor imaging characterization of cerebral white matter injury in lipopolysaccharide-exposed fetal sheep

Background:In gyrencephalic species such as sheep, precise anatomical and microstructural characterization of the consequences of fetal inflammation remains scarce. The goal of this study was to characterize changes in white matter (WM) structure using advanced magnetic resonance imaging (MRI) following lipopolysaccharide (LPS) exposure in the preterm-equivalent fetal sheep.Methods:Preterm (0.7 gestation) fetal sheep received vehicle (Sham group) or LPS (LPS group), and fetal brains were collected 10 d later for subsequent ex vivo MRI. T(1)-weighted (T(1)W), T(2)-weighted (T(2)W), and diffusion tensor imaging (DTI) data were collected.Results:Fetuses exposed to LPS exhibited reductions in WM volume and corpus callosum thickness at 10 d recovery. Characteristic patterns of diffuse and focal WM lesions (necrosis or cysts) could be identified by various T(1), T(2), and DTI signal changes.Conclusion:Fetal LPS exposure induces a pattern of injury characterized by diffuse and focal WM injury that closely reproduces that observed clinically in preterm infants. This work provides anatomical and microstructural MRI assessment, as well as histopathological correlates, of the consequences of LPS exposure in an animal model with a WM structure similar to that of the human brain. This work will help to further our understanding of MRI changes in preterm infants.     

Neuropathologic documentation of prenatal brain damage

Neuropathologic evidence of prenatal brain damage, chiefly in cerebral white matter, was found in 25% of infants who died at 7 days of age or less, with a total of ten preterm (16%) and 12 term (48%) infants among the 89 subjects studied. Few clinical features distinguished infants with prenatal injury from those without such injuries. Apgar scores were low, seizures were rare, and acute intracranial hemorrhage occurred equally often in both groups. Few pregnancies were entirely normal, but hydramnios was the only factor that occurred more often in prenatally injured infants, a statistically significant difference only among term infants. Oligohydramnios was not associated with prenatal brain injury. Unless fetal/maternal abnormalities in late gestation are identified and corrected, improved neonatal care will increase survival for prenatally damaged infants and the incidence of cerebral palsy may rise.     

高危晚期早产儿脑损伤病因学及其磁共振发现

目的 探讨以弥散加权成像(DWI)结合常规磁共振成像(T1WI-T2WI)诊断的高危晚期早产儿脑损伤的相关危险因素及临床特点,并分析不同时间MRI序列的信号特点及DWI的早期诊断价值。方法 首先对符合纳入标准的649例晚期早产儿的MRI片重新阅片,按照脑损伤评估标准得出诊断,其次收集相关的临床资料,分析不同类型脑损伤的危险因素和临床特点,并对其中271例确诊脑白质损伤（CWMD）的MRI序列进行分析,探讨不同类型CWMD的信号特点、损伤部位及结局。结果 ①晚期早产儿发生脑损伤332例（51.2%）,其中CWMD 271例（41.8%）,以局灶性CWMD为主（62.7％,170例）;颅内出血112例（17.3%）,主要为蛛网膜下腔出血55.4%（62/112）。②非出血性脑损伤的危险因素是男性（OR=1.510,95％CI：1.067～2.136,P=0.020）、阴道分娩（OR=2.367,95％CI：0.251～22.294 ,P=0.000）、早发型败血症（OR=2.194,95％CI：1.159～4.155,P=0.016）及抢救复苏史（OR=3.784,95％CI：1.908～7.506,P=0.000）。出血性脑损伤的危险因素是阴道分娩（OR=7.195,95％CI：4.249～12.184 ,P=0.000）和早发型败血症（OR=2.692,95％CI：1.185～6.117,P=0.018）。低钙血症（OR=2.593,95％CI：1.343～5.005,P=0.005）、晚发型败血症（OR=1.533,95％CI：1.012～2.323,P=0.044）和抽搐（OR=4.006,95％CI：1.790～8.970,P=0.001）是非出血性脑损伤组的主要临床特点。出血性脑损伤组主要表现为高血糖和抽搐。③局灶性CWMD 65.3％仅累及一处损伤,主要集中在侧脑室后脚（53.5%）,有97.1%病灶消失或病灶范围减少;广泛性CWMD 79.2%累及胼胝体和内囊;弥漫性CWMD 50％合并灰质损伤,全部发生软化。④生后2周内,DWI具有较高的敏感性,98.0%表现为高信号,T1WI信号无变化或稍高信号,伴或不伴T2WI低信号。局灶性CWMD DWI高信号持续时间长达3周以上,弥漫性CWMD DWI高信号持续时间2周以内。结论 晚期早产儿仍然容易受产前产时因素影响而发生不同类型的脑损伤。对有高危因素,或早期出现临床表现或电解质紊乱的患儿应选择生后2周内（1周内最佳）进行DWI和常规MRI检查,以早期发现病变。局灶性CWMD预后较好,合并有灰质损伤或弥漫性CWMD预后极差,需要动态随访,并进行早期康复训练。     

Electroencephalography and brain damage in preterm infants

Electroencephalography (EEG) is a sensitive method for detection of brain injury in preterm infants. Although the acute and chronic EEG changes are mainly non-specific regarding type of damage, they correlate with later neurological and cognitive function. In infants developing brain white matter damage, acute EEG findings include depression of background activity and presence of epileptic seizure activity. The chronic EEG changes associated with white matter injury and abnormal neurological development include delayed maturation, and presence of abundant Rolandic sharp waves. Cognitive limitations in preterm infants have been associated with changes in various sleep measures in EEG's recorded at full term. Continuous EEG-monitoring during neonatal intensive care shows that cerebral electrical activity during this vulnerable period can be affected by several extracerebral factors, e.g. cerebral blood flow, acidosis and some commonly used medications. For diagnosis of brain damage in preterm infants with neurophysiological methods, a combination of early continuous EEG monitoring during the initial intensive care period and full EEG, performed at later stages, is probably optimal.     

脂多糖诱导小胶质细胞依赖的早产鼠脑白质损伤机制研究

目的:研究中枢神经系统小胶质细胞（MG）正常发育尤其是少突胶质细胞前体细胞（OPCs) 最易受损阶段的发育,探讨宫内感染早产鼠MG依赖的OPCs损伤机制。方法:①观察正常C57B/L鼠不同胎龄(孕10、15 d )和生后(0、5、10 d)MG和OPCs在脑白质的发育分布情况,明确两者在发育和分布上的关联。②建立脂多糖（LPS）宫内感染新生鼠模型（宫内分别接种LPS 5、10和20 μg·mL-1为感染A～C组）,以PBS溶液接种为对照组。以Tomato lectin作为静息状态MG标志,CD68作为活化MG的特殊抗体,O4+作为OPCs抗体,抗体浮片法进行免疫组化染色并计数分析。③ Western blot法检测各组脑室周围白质组织Toll样受体-4 (TLR-4)蛋白表达。④ 采用ELISA法检测各组MG活化后IL-2、TNF-α和SOD水平变化。结果:①MG在孕10 d胎鼠Tomato lectin表达低下,孕15 d胎鼠表达显著增高,MG主要分布在脑室周围白质区域,灰质皮质几乎不表达。出生后,脑室周围白质区域MG的表达有所下降,灰质皮质的表达逐渐增高。②感染A~C组CD68+细胞数量均显著增加,与对照组差异有统计学意义(P<0.01),但感染C组与B组CD68+细胞数量差异无统计学意义(P>0.05)。与对照组比较,感染A~C组均可见O4+细胞数量显著性下降(P<0.01),其中以感染C组下降最为明显。③对照组未检测到TLR-4蛋白表达, 感染A~C组均可见LPS剂量依赖的TLR-4蛋白表达增加,与对照组差异有统计学意义(P<0.05)。④随接种LPS剂量增大,IL-2和TNF-α水平较对照组呈显著增加趋势, SOD水平较对照组呈显著降低趋势。结论:新生鼠发育依赖的MG在脑白质受损区域过度表达,表明活化MG起到本底激活效应,是早产儿脑白质损伤的物质基础。     

Repetitive prenatal glucocorticoids increase lung endothelial nitric oxide synthase expression in ovine fetuses delivered at term

Antenatal administration of glucocorticoids has been shown to improve postnatal lung function after preterm birth in the ovine fetus. Mechanisms of steroid-induced lung maturation include increased surfactant production and altered parenchymal lung structure. Whether steroid treatment also affects lung vascular function is unclear. Because nitric oxide contributes to the fall in pulmonary vascular resistance at birth, we hypothesized that the improvement of postnatal lung function of preterm lambs after treatment with prenatal glucocorticoids may be in part caused by an increase in endothelial nitric oxide synthase (eNOS) activity. To determine whether glucocorticoid treatment increases lung eNOS expression, we measured eNOS protein content by Western blot analysis of distal lung homogenates and immunostaining of formalin-fixed lungs from ovine fetuses delivered at preterm and term gestation after prenatal administration of glucocorticoids. Treatment protocols were followed in which ewes were treated with intramuscular betamethasone (0.5 mg/kg) at single or multiple doses at weekly intervals, and fetuses were delivered at 125, 135, or 145 d gestation. All groups were compared with saline-treated controls. Western blot analysis of whole lung homogenates demonstrated a 4-fold increase in eNOS protein content in lambs treated with repetitive doses of glucocorticoids and delivery at term (145 d; p < 0.002). In addition, a small increase in lung eNOS protein content was seen in lambs treated with a single dose of betamethasone at 128 d gestation with delivery at 135 d gestation. In comparison with control animals, there were no differences in lung eNOS content from the remaining lambs treated with glucocorticoids when delivery occurred at preterm ages (125 and 135 d). Immunostaining showed eNOS predominantly in the vascular endothelium in all vessel sizes. Pattern of staining was not altered by treatment with antenatal glucocorticoids. We conclude that maternal treatment with glucocorticoids increases lung eNOS content after multiple doses and delivery at term gestation. We speculate that antenatal glucocorticoids may up-regulate eNOS but that the timing and duration of steroid administration appears to be critical to this response.     

Intracranial haemorrhage in the preterm sheep fetus

The germinal layer in the brain of the sheep fetus at 58--85 days of gestation was found to resemble that of the human infant at 28--30 wk of gestation. Experiments were done on 65 exteriorized fetuses to explore the effect of various combinations of asphyxia and raised intravascular pressures in causing bleeding into the germinal layer, ventricles and other parts of the brain. Asphyxia by itself did not produce an increase in the incidence of intracranial hemorrhages when compared with control fetuses. The combination of asphyxia with intermittent increases in arterial or venous pressure, or both, did cause haemorrhages. Large increases in arterial pressure without asphyxia also caused intracranial haemorrhages, whereas increases in venous pressure without asphyxia did not. The types of haemorrhage observed closely resembled those seen in the preterm human infant, although massive intraventricular haemorrhages (IVHs) were rare. We conclude that: (1) the sheep fetus can be used for investigating factors associated with intracranial haemorrhage in the preterm brain; (2) the most effective method of producing haemorrhages into the germinal layer was by a combination of asphyxia with intermittent increases in cerebral intravascular pressure. Similar mechanisms may be at work in the newborn human infant, and could lead to IVH.     

Fetal MRI: obstetrical and neurological perspectives

Despite major advances in the understanding and in the genetics of several diseases of the developing brain, early prediction of the neurological prognosis of brain abnormality discovered in utero or of white matter damage discovered in a preterm neonate remains particularly difficult. Advances in prenatal diagnosis and the increased rate of survival of extremely preterm infants who are at higher risk of developing white matter damage underline the critical and urgent need for reliable predictive techniques. New imaging techniques such as diffusion-weighted imaging, magnetic resonance spectroscopy or functional MRI applied to the fetus represent promising tools in this perspective.     

White matter injury after cerebral ischemia in ovine fetuses

The effects of cerebral ischemia on white matter changes in ovine fetuses were examined after exposure to bilateral carotid artery occlusion. Fetal sheep were exposed to 30 min of ischemia followed by 48 (I/R-48, n = 8) or 72 (I/R-72, n = 10) h of reperfusion or control sham treatment (control, n = 4). Serial coronal sections stained with Luxol fast blue/hematoxylin and eosin were scored for white matter, cerebral cortical, and hippocampal lesions. All areas received graded pathologic scores of 0 to 5, reflecting the degree of injury where 0 = 0%, 1 = 1% to 25%, 2 = 26% to 50%, 3 = 51% to 75%, 4 = 76% to 95%, and 5 = 96% to 100% of the area damaged. Dual-label immunofluorescence using antibodies against glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) were used to characterize white matter lesions. Basic fibroblast growth factor (FGF-2) was measured in the frontal cortex by ELISA. Results of the pathologic scores showed that the white matter of the I/R-72 (2.74 +/- 0.53, mean +/- SEM) was more (p < 0.05) damaged when compared with the control (0.80 +/- 0.33) group. Cortical lesions were greater (p < 0.05) in the I/R-48 (2.12 +/- 0.35) than the control (0.93 +/- 0.09) group. White matter lesions were characterized by reactive GFAP-positive astrocytes and a loss of MBP in oligodendrocytes. The ratio of MBP to GFAP decreased (p < 0.05) as a function of ischemia, indicative of a proportionally greater loss of MBP than GFAP. FGF-2 concentrations were higher (p < 0.05) in the I/R-72 than the control group and there was a direct correlation between the pathologic scores (PS) and FGF-2 concentrations (FGF-2 = e((1.6 PS-0.90)) + 743, n = 17, r = 0.73, p < 0.001). We conclude that carotid artery occlusion results in quantifiable white matter lesions that are associated with a loss of MBP from myelin, and that FGF-2, a purported mediator of recovery from brain injury in adult subjects, increases in concentration in proportion to the severity of brain damage in the fetus.