The cerebral cortex overlying periventricular leukomalacia: analysis of pyramidal neurons.

The role of the cerebral cortex in the cognitive deficits in preterm survivors is poorly understood. Periventricular leukomalacia (PVL), the key feature of encephalopathy of prematurity, is characterized by periventricular necrotic foci and diffuse gliosis in the surrounding cerebral white matter. Here, we tested the hypothesis that reductions in the density of layer I neurons and/or pyramidal neurons in layers III and/or V are associated with PVL, indicating cortical pathology potentially associated with cognitive deficits in long-term survivors. In controls (23 gestational weeks to 18 postnatal months) (n = 15), a lack of significant differences in pyramidal density among incipient Brodmann areas suggested that cytoarchitectonic differences across functional areas are not fully mature in the fetal and infant periods. There was a marked reduction (38%) in the density of layer V neurons in all areas sampled in the PVL cases (n = 17) compared to controls (n = 12) adjusted for postconceptional age at or greater than 30 weeks, when the six-layer cortex is visually distinct (P < 0.024). This may reflect a dying-back loss of somata complicating transection of layer V axons projecting through the necrosis in the underlying white matter. This study underscores the potential role of secondary cortical injury in the encephalopathy of prematurity.

Diffuse axonal injury in periventricular leukomalacia as determined by apoptotic marker fractin.

Periventricular leukomalacia (PVL), the major substrate of neurologic deficits in premature infants, is associated with reduced white matter volume. Using immunomarkers of axonal pathology [beta-amyloid precursor protein (beta-APP) and apoptotic marker fractin], we tested the hypothesis that widespread (diffuse) axonal injury occurs in the gliotic white matter beyond the foci of necrosis in PVL, thus contributing to the white matter volume reduction. In a cohort of 17 control cases and 13 PVL cases with lesions of different chronological ages, diffuse axonal damage in PVL was detected by fractin in white matter sites surrounding and distant from acute and organizing foci of necrosis. Using beta-APP, axonal spheroids were detected within necrotic foci in the acute and organizing (subacute) stages, a finding consistent with others. Interestingly, GAP-43 expression was also detected in spheroids in the necrotic foci, suggesting attempts at axonal regeneration. Thirty-one percent of the PVL cases had thalamic damage and 15% neuronal injury in the cerebral cortex overlying PVL. We conclude that diffuse axonal injury, as determined by apoptotic marker fractin, occurs in PVL and that its cause likely includes primary ischemia and trophic degeneration secondary to corticothalamic neuronal damage.

Glutamate transporter EAAT2 expression is up-regulated in reactive astrocytes in human periventricular leukomalacia.

The major neuropathological correlate of cerebral palsy in premature infants is periventricular leukomalacia (PVL), a disorder of the immature cerebral white matter. Cerebral ischemia leading to excitotoxicity is thought to be important in the pathogenesis of this disorder, implying a critical role for glutamate transporters, the major determinants of extracellular glutamate concentration. Previously, we found that EAAT2 expression is limited primarily to premyelinating oligodendrocytes early in development and is rarely observed in astrocytes until >40 weeks. In this study, we analyzed the expression of EAAT2 in cerebral white matter from PVL and control cases. Western blot analysis suggested an up-regulation of EAAT2 in PVL compared with control cases. Single- and double-label immunocytochemistry showed a significantly higher percentage of EAAT2-immunopositive astrocytes in PVL (51.8% +/- 5.6%) compared with control white matter (21.4% +/- 5.6%; P = 0.004). Macrophages in the necrotic foci in PVL also expressed EAAT2. Premyelinating oligodendrocytes in both PVL and control cases expressed EAAT2, without qualitative difference in expression. The previously unrecognized up-regulation of EAAT2 in reactive astrocytes and its presence in macrophages in PVL reported here may reflect a response to either hypoxic-ischemic injury or inflammation.

Myelin abnormalities without oligodendrocyte loss in periventricular leukomalacia.

The cellular basis of myelin deficits detected by neuroimaging in long-term survivors of periventricular leukomalacia (PVL) is poorly understood. We tested the hypothesis that oligodendrocyte lineage (OL) cell density is reduced in PVL, thereby contributing to subsequent myelin deficits. Using computer-based methods, we determined OL cell density in sections from 18 PVL and 18 age-adjusted control cases, immunostained with the OL-lineage marker Olig2. Myelination was assessed with myelin basic protein (MBP) immunostaining. We found no significant difference between PVL and control cases in Olig2 cell density in the periventricular or intragyral white matter. We did find, however, a significant increase in Olig2 cell density at the necrotic foci, compared with distant areas. Although no significant difference was found in the degree of MBP immunostaining, we observed qualitative abnormalities of MBP immunostaining in both the diffuse and necrotic components of PVL. Abnormal MBP immunostaining in PVL despite preserved Olig2 cell density may be secondary to arrested OL maturation, damage to OL processes, and/or impaired axonal-OL signaling. OL migration toward the "core" of injury may occur to replenish OL cell number. This study provides new insight into the cellular basis of the myelin deficits observed in survivors of PVL.

Neuronal cell death in the arcuate nucleus of the medulla oblongata in stillbirth.

The hypothesis that unexplained stillbirth arises in a similar manner as the sudden infant death syndrome (SIDS) is based in part on shared neuropathologic features between the two entities, including hypoxic-ischemic lesions such as white matter and brainstem gliosis, as well as aplasia or hypoplasia of the arcuate nucleus on the ventral surface of the medulla. The arcuate nucleus is the putative homologue of the respiratory chemosensory region at the ventral medullary surface in animals that is involved in central chemosensitivity. To determine arcuate nucleus pathology in stillbirth, and its co-occurrence with evidence of hypoxia-ischemia, we reviewed brain specimens from the archives of our hospitals from 22 consecutive stillbirths from 22 to 41 gestational weeks. Explained causes of death (n=17) included nuchal cord, acute chorioamnionitis, placental abruption, and fetal glomerulosclerosis; 5 cases were unexplained. In 12 brains, we observed nuclear karyorrhexis and/or pyknosis with cytoplasmic hypereosinophilia in neurons in the arcuate nucleus in both explained (n=8) and unexplained (n=4) cases (54.5% of total cases). Three additional cases had arcuate aplasia (n=1) or hypoplasia (n=2) (13.6% of total cases); one of the latter cases also had neuronal necrosis in the hypoplastic arcuate. The degree of gliosis in the region of the arcuate nucleus was variable across all cases, without statistically significant differences between groups with and without arcuate nucleus necrosis. Other lesions in association with (n=14) and without (n=8) arcuate nucleus abnormalities were diffuse cerebral white matter gliosis, periventricular leukomalacia (PVL), and neuronal necrosis in the hippocampus, basal ganglia, thalamus, basis pontis, and brainstem tegmentum. In 16/20 (80.0%) cases (with or without histologic necrosis of the arcuate), immunostaining with caspase-3 demonstrated positive neurons. Our findings suggest that neuronal pathology in the arcuate nucleus may be both developmental (13.6%) and acquired (54.5%). The association of neuronal necrosis and apoptosis in the arcuate nucleus with systemic entities involving fetal ischemia, and with other brain lesions consistent with ischemia, e.g., cerebral white matter gliosis, suggests that ischemia plays a role in the arcuate nucleus damage as well. Thus, the underpopulation of arcuate neurons detected postnatally in some SIDS infants may be secondary to an acquired insult in mid- or late gestation, and in other cases, a primary developmental lesion in early gestation, or both. The role of arcuate nucleus pathology in the pathogenesis of fetal demise remains to be determined.

Gray matter injury associated with periventricular leukomalacia in the premature infant.

Neuroimaging studies indicate reduced volumes of certain gray matter regions in survivors of prematurity with periventricular leukomalacia (PVL). We hypothesized that subacute and/or chronic gray matter lesions are increased in incidence and severity in PVL cases compared to non-PVL cases at autopsy. Forty-one cases of premature infants were divided based on cerebral white matter histology: PVL (n = 17) with cerebral white matter gliosis and focal periventricular necrosis; diffuse white matter gliosis (DWMG) (n = 17) without necrosis; and "Negative" group (n = 7) with no abnormalities. Neuronal loss was found almost exclusively in PVL, with significantly increased incidence and severity in the thalamus (38%), globus pallidus (33%), and cerebellar dentate nucleus (29%) compared to DWMG cases. The incidence of gliosis was significantly increased in PVL compared to DWMG cases in the deep gray nuclei (thalamus/basal ganglia; 50-60% of PVL cases), and basis pontis (100% of PVL cases). Thalamic and basal ganglionic lesions occur almost exclusively in infants with PVL. Gray matter lesions occur in a third or more of PVL cases suggesting that white matter injury generally does not occur in isolation, and that the term "perinatal panencephalopathy" may better describe the scope of the neuropathology.

Is the late preterm infant more vulnerable to gray matter injury than the term infant?

This article addresses the issue of whether the late preterm infant is more susceptible to gray matter injury induced by hypoxia-ischemia than the term infant. Although different gray matter regions display varying patterns of neuronal injury in the face of hypoxia-ischemia during advancing gestational development, little is known about the specific patterns of injury faced by the late preterm infant. This changing pattern of neuronal vulnerability with age likely reflects developmental changes of susceptibility and protective factors essential for responding to energy deprivation at the molecular, cellular, biochemical, and vascular levels. Future research involving closer examination of the late preterm period is essential to provide a greater understanding of the neuronal vulnerability in the face of hypoxic-ischemic injury.

Hypoxia potentiates endotoxin-induced allopregnanolone concentrations in the newborn brain.

BACKGROUND: Allopregnanolone is a neurosteroid produced in the brain that can alter the excitability of the CNS. Neurosteroids have neuroprotective properties, and their elevation in response to stress may protect the newborn brain following infection or hypoxia. Infection, particularly of the respiratory tract, may lead to episodes of hypoxia. Infection and hypoxia have been identified as factors contributing to neonatal morbidity and mortality. OBJECTIVES: To determine the effect of acute episodes of hypoxia alone or in combination with lipopolysaccharide (LPS) exposure on plasma and brain allopregnanolone concentrations in lambs 10-21 days old. Also, to examine plasma levels of cortisol and the cytokines, tumour necrosis factor-alpha and interleutkin-6 after these challenges. RESULTS: Allopregnanolone concentrations in the brain were markedly increased after hypoxia. Hypoxia following prior LPS treatment resulted in greater increases in brain allopregnanolone concentrations compared to either the LPS or hypoxia treatment alone. Importantly, brain regions unaffected by LPS or hypoxia alone (thalamus/hypothalamus, cerebellum) showed significant increases of allopregnanolone content following the combined LPS and hypoxia treatments. Plasma tumour necrosis factor-alpha and interleukin-6 concentrations were increased after LPS treatment with and without hypoxia, but not by hypoxia alone. In contrast, plasma cortisol concentrations were increased after both stressors. CONCLUSIONS: These results show that the brain of young lambs readily responds to physiological stress by increased production of allopregnanolone. This response may protect the developing brain from the cytotoxicity following hypoxic and infectious episodes.

Development of microglia in the cerebral white matter of the human fetus and infant.

Although microglial activation may be an initial beneficial response to a variety of insults, prolonged activation can release toxic substances and lead to cell death. Microglial activation secondary to hypoxia-ischemia and/or infection in immature cerebral white matter is important in the pathogenesis of periventricular leukomalacia (PVL), the major pathological substrate of cerebral palsy in the premature infant. We hypothesize that a transient overexpression in activated microglial density occurs normally in the cerebral white matter of the human fetus during the peak window of vulnerability for PVL. Such an increase could render this region susceptible to insults that cause prolonged microglial activation, as conceptualized in PVL. To examine the developmental profile of microglia in the human fetus and infant brain, immunocytochemistry with microglial specific markers were used in 23 control (non-PVL) cases ranging from 20 to 183 postconceptional (PC) weeks. Tomato lectin, used to identify microglial morphology, revealed that the cerebral white matter of the human fetus and infant is densely populated with intermediate and amoeboid microglia; the latter is indicative of an activated state. Quantitative analysis with CD68 showed increased density of activated microglia in the cerebral white matter of the fetus (<37 PC weeks) relative to the neonate/infant (> or =37 PC weeks) and to the overlying cortex of either age group (P = 0.01). The primary finding of a transient, developmental-dependent overabundance of CD68-activated microglia in the cerebral white matter of the fetus suggests a potential "priming" of this area for diverse brain insults characterized by activation of microglia, particularly PVL. J.

Changes in 5alpha-pregnane steroids and neurosteroidogenic enzyme expression in fetal sheep with umbilicoplacental embolization.

Pregnane steroids have sedative and neuroprotective effects on the brain, due to interactions with the steroid-binding site of the GABAA receptor. In the adult brain, synthesis of the pregnane steroids is increased in response to stress. Therefore, we have used umbilicoplacental embolization to mimic chronic placental insufficiency during late gestation in sheep, to investigate the expression of the steroidogenic enzymes p450scc, 5alpha-reductase type I (5alphaRI), 5alpha-reductase type II (5alphaRII), and allopregnanolone (AP) content in the fetal brain. Umbilicoplacental embolization was induced from 114 d gestation (term approximately 147 d) by daily injection of inert microspheres into the umbilical artery and continued for 17-23 d. Fetal arterial oxygen saturation was reduced to approximately 60% of the preembolization value in each fetus, with a significant reduction in blood arterial Po2, pH, and plasma glucose concentrations (p < 0.05) and a significant increase in blood arterial Pco2 and plasma lactate concentrations (p < 0.05). At postmortem at 131-137 d gestation, embolized fetuses were growth-restricted (2.10 +/- 0.14 kg, n = 5) compared with age-matched controls (4.43 +/- 0.56 kg, n = 7, p < 0.05). Umbilicoplacental embolized fetuses showed increased P450scc expression in the primary motor cortex; 5alphaRI expression was not changed in any of the regions examined, whereas 5alphaRII expression was markedly increased in all brain regions. Brain AP content did not significantly change, whereas plasma concentrations were increased. These findings suggest that the increased expression of p450scc and 5alphaRII may be a response that maintains AP concentration in the fetal brain after compromised placental function and/or intrauterine stress.

Changes in 5alpha-pregnane steroids and neurosteroidogenic enzyme expression in the perinatal sheep.

Pregnane steroids have sedative and neuroprotective effects on the brain as a result of interactions with the steroid-binding site of the GABAA receptor. To determine whether the fetal brain is able to synthesize pregnane steroids de novo from cholesterol, we measured the expression of cytochrome P450 side-chain cleavage (P450scc) and 5alpha-reductase type II (5alphaRII) enzymes in fetal sheep from 72 to 144 d gestation (term approximately 147 d) and in newborn lambs at 3 and 19-26 d of age. Both P450scc and 5alphaRII expression was detectable by 90 d gestation in the major regions of the brain and also in the adrenal glands. Expression increased with advancing gestation and was either maintained at fetal levels or increased further after birth. In contrast, the relatively high content (200-400 pmol/g) of allopregnanolone (5alpha-pregnan-3alpha-ol-20-one), a major sedative 5alpha-pregnane steroid, present throughout the brain from 90 d gestation to term, was reduced significantly (<50 pmol/g) immediately after birth. These results suggest that although the perinatal brain has the enzymes potentially to synthesize pregnane steroids de novo from cholesterol, either the placenta is a major source of these steroids to the brain or other factors associated with intrauterine life may be responsible for high levels of allopregnanolone production in the fetal brain until birth.

Endotoxin increases sleep and brain allopregnanolone concentrations in newborn lambs.

Infection has been identified as a risk factor for sudden infant death syndrome (SIDS). Synthesis of allopregnanolone, a neuroactive steroid with potent sedative properties, is increased in response to stress. In this study, we investigated the effect of endotoxin (lipopolysaccharide, LPS) on brain and plasma allopregnanolone concentrations and behavior in newborn lambs. LPS was given intravenously (0.7 micro g/kg) at 12 and 15 d of age (n = 7), and resulted in a biphasic febrile response (p < 0.001), hypoglycemia, lactic acidemia (p < 0.05), a reduction in the incidence of wakefulness, and increased nonrapid eye movement sleep and drowsiness (p < 0.05) compared with saline-treated lambs (n = 5). Plasma allopregnanolone and cortisol were significantly (p < 0.05) increased after LPS treatment. These responses to LPS lasted 6-8 h, and were similar at 12 and 15 d of age. Each lamb was then given LPS at 20 d of age and killed 3 h posttreatment to obtain samples of the brain. Allopregnanolone concentrations were increased (p < 0.05) in all brain areas except the cerebellum and diencephalon. We suggest that LPS-induced increase of allopregnanolone in the brain may contribute to somnolence in the newborn, and may be responsible for the reduced arousal thought to contribute to the risk of SIDS in human infants.