The glutamate transporter EAAT2 is transiently expressed in developing human cerebral white matter

The major brain abnormality underlying cerebral palsy in premature infants is periventricular leukomalacia (PVL), a lesion of the immature cerebral white matter. Oligodendrocyte precursors (pre-OLs; O4(+)O1(-)) predominate in human cerebral white matter during the peak time frame for PVL (24-32 gestational weeks) and are vulnerable to excitotoxicity. We hypothesize that PVL reflects, in part, excitotoxicity to pre-OLs resulting from cerebral ischemia/reperfusion. Reversal of glutamate transport in the setting of energy failure is a major source of pathologic accumulation of extracellular glutamate. Here, we identify and localize the glutamate transporters in human cerebral white matter during the age range of PVL. In situ hybridization was performed with digoxigenin-labeled probes directed against the full-length coding regions of EAAT1, EAAT2, and EAAT3. EAAT2 mRNA was abundant in human fetal white matter during the period of peak incidence of PVL and virtually disappeared by 2 postnatal months. Its developmental profile differed significantly from that of both EAAT1 and EAAT3 mRNA. Immunoblotting demonstrated that EAAT2 protein was highly expressed in early development relative to adult values. Double-label immunocytochemistry detected EAAT2 in OLs but not astrocytes or axons in the human fetal white matter. We conclude that transient expression of EAAT2 occurs during the window of peak vulnerability for PVL, suggesting that this developmentally up-regulated transporter may be a major source of extracellular glutamate in ischemic injury to the cerebral white matter of the preterm infant.     

Correlation between clinical and ultrasound findings in preterm infants with cystic periventricular leukomalacia

Cystic periventricular Leukomalacia (CPVL), a hypoxic-ischemic lesion of the neonatal brain, which can now be diagnosed in life thanks to ultrasound brain scanning, is considered to be one of the main causes of cerebral palsy (CP), especially in preterm infants. The purpose of our study was to verify this assumption in a population of 337 of gestational age ≥32 weeks. The frequency of CPVL proved to be 5.4% for lesions with a diameter of ≥3 mm or 9.3% including those of smaller diameter. The development of CPVL infants was favorable in 29% and adverse in 71% of cases. In the latter cases neuromotor sequelae (CP in 62.5% and motor retardation in 8.5%) were accompanied by various other neuropsychic deficits. Prognosis depends on the site and size of the cysts, being harsher for posterior lesions and those exceeding 1 cm in diameter.

---

Sommario La leucomalacia periventricolare cistica (CPVL), lesione ipossico-ischemica del cervello neonatale diagnosticabile in vita grazie all'ecografia cerebrale, viene considerata una delle principali cause della paralisi cerebrale infantile (P.C.I.), in particolare nei nati pretermine. Scopo della nostra ricerca è verificare tale ipotesi con una popolazione di 337 soggetti con E.G.≤32 settimane. L'incidenza della CPVL da noi rilevata è del 5.4% o del 9.3% considerando rispettivamente le forme con diametro ≥3 mm od anche quelle microcistiche (diametro <3 mm). L'evoluzione dei soggetti affetti da CPVL è favorevole nel 29% dei casi e sfavorevole nel 71%, con lo sviluppo di una sequela neuromotoria (PCI nel 62.5% e ritardo motorio nell'8.5%) variamente associata ad altri deficit neuropsichici. La prognosi è legata alla sede e alle dimensioni della cisti ed è tanto più grave per lesioni localizzate in sede posteriore od estese (di diametro ≥1 cm).

     

Mature myelin basic protein-expressing oligodendrocytes are insensitive to kainate toxicity

We examined the vulnerability to excitotoxicity of rat oligodendrocytes in dissociated cell culture at different developmental stages. Mature oligodendrocytes that express myelin basic protein were resistant to excitotoxic injury produced by kainate, whereas earlier stages in the oligodendrocyte lineage were vulnerable to this insult. To test the hypothesis that the sensitivity of immature oligodendrocytes and the resistance of mature oligodendrocytes to kainate toxicity were due to differences in membrane responsiveness to kainate, we used whole-cell patch-clamp recording. Oligodendrocyte precursors in cultures vulnerable to kainate toxicity responded to 500 microM kainate with large inward currents, whereas mature myelin basic protein-expressing oligodendrocytes in cultures resistant to kainate toxicity showed no clear response to application of this agonist. We assayed expression of glutamate receptor subunits (GluR) -2, -4, -6, -7, and KA2 using immunoblot analysis and found that expression of all of these glutamate receptors was significantly down-regulated in mature oligodendrocytes. These results suggest a striking developmental regulation of glutamate receptors in oligodendrocytes and suggest that the vulnerability of oligodendrocytes to non- N-methyl-D-aspartate receptor-mediated excitotoxicity might be much greater in developing oligodendrocytes than after the completion of myelination.     

Neuronal and non-neuronal catechol-O-methyltransferase in primary cultures of rat brain cells

Previous biochemical and histochemical studies have suggested that catechol-O-methyltransferase (COMT) is a predominantly glial enzyme in the brain. The aim of this work was to study its localization and molecular forms in primary cultures, where cell types can be easily distinguished with specific markers. COMT immunoreactivity was studied in primary astrocytic cultures from newborn rat cerebral cortex, and in neuronal cultures from rat brain from 18-day-old rat embryos using antisera against rat recombinant COMT made in guinea pig. Double-staining studies with specific cell markers to distinguish astrocytes, neurons and oligodendrocytes were performed. COMT immunoreactivity colocalized with a specific oligodendrocyte marker galactocerebroside in cells displaying oligodendrocyte morphology, flat cells displaying type-1 astrocyte morphology and glial fibrillary acidic protein, in branched cells displaying type-2 astrocyte morphology and in cell bodies of neurons, the processes of which displayed neurofilament immunoreactivity. Western blots detected both soluble 24 kDa and membrane-bound 28-kDa COMT proteins in neuronal and astrocyte cultures. The results suggest that COMT is synthesized by cultured astrocytes, oligodendrocytes and neurons.     

Cellular phenotype of androgen receptor-immunoreactive nuclei in the developing and adult rat brain

Androgen exposure during development and adulthood promotes cell-to-cell communication, modulates the size of specific brain nuclei, and influences hormone-dependent behavioral and neuroendocrine functions. Androgen action involves the activation of androgen receptors (AR). To elucidate the mechanisms involved in AR-mediated effects on forebrain development, double-label fluorescent immunohistochemistry and confocal microscopy were employed to identify the cellular phenotype of AR-immunoreactive (AR(+)) cells in the developing (embryonic day 20, postnatal days 0, 4, 10) and adult male rat forebrain. Sections were doubly labeled with antibodies directed against AR and one of the following: neurons (immature, nestin; mature, NeuN) or astrocytes [immature, vimentin; mature, glial fibrillary acidic protein (GFAP)] or mature oligodendrocytes (mGalC). In all brain regions examined, by far the majority of AR(+) cells were neurons. In addition, small subsets of AR(+) cells were identified as mature astrocytes (GFAP(+)) but only in specific brain regions at specific ages. AR(+)/GFAP(+) cells were observed in the cerebral cortex but only in postnatal day 10 rats and in the arcuate nucleus of the hypothalamus but only in adult rats. Immature neurons, immature astrocytes, and oligodendrocytes were not AR(+) at any age, in any region. Thus, both neurons and astrocytes in the male rat forebrain contain ARs, suggesting that androgens, via ARs, may exert effects on both cell types in an age- and region-dependent manner.     

Axonal development in the cerebral white matter of the human fetus and infant

After completion of neuronal migration to form the cerebral cortex, axons undergo rapid elongation to their intra- and subcortical targets, from midgestation through infancy. We define axonal development in the human parietal white matter in this critical period. Immunocytochemistry and Western blot analysis were performed on 46 normative cases from 20-183 postconceptional (PC) weeks. Anti-SMI 312, a pan-marker of neurofilaments, stained axons as early as 23 weeks. Anti-SMI 32, a marker for nonphosphorylated neurofilament high molecular weight (NFH), primarily stained neuronal cell bodies (cortical, subcortical, and Cajal-Retzius). Anti-SMI 31, which stains phosphorylated NFH, was used as a marker of axonal maturity, and showed relatively low levels of staining (approximately one-fourth of adult levels) from 24-34 PC weeks. GAP-43, a marker of axonal growth and elongation, showed high levels of expression in the white matter from 21-64 PC weeks and lower, adult-like levels beyond 17 postnatal months. The onset of myelination, as seen by myelin basic protein expression, was approximately 54 weeks, with progression to "adult-like" staining by 72-92 PC weeks. This study provides major insight into axonal maturation during a critical period of growth, over an age range not previously examined and one coinciding with the peak period of periventricular leukomalacia (PVL), the major disorder underlying cerebral palsy in premature infants. These data suggest that immature axons are susceptible to damage in PVL and that the timing of axonal maturation must be considered toward establishing its pathology relative to the oligodendrocyte/myelin/axonal unit.     

Ethyl pyruvate protects against lipopolysaccharide-induced white matter injury in the developing rat brain

The neuroprotective effects of ethyl pyruvate (EP) have been proved in several brain injury models, yet very little is known about its action on neonatal white matter injury. To investigate the effect of EP on white matte damage, a stereotactic intracerebral injection of lipopolysaccharide (LPS, 1 mg/kg) was performed on postnatal day 5 Sprague–Dawley rat pups, and EP was administrated intraperitoneally at a dose of 40 mg/kg immediately, 1 h and 12 h after LPS exposure. Significantly, treatment with EP reduced LPS-induced ventricle dilation, loss of O4+ and O1+ oligodendrocytes, apoptosis of oligodendrocytes, and hypomyelination. The protective effect of EP was associated with suppressed inflammatory responses, indicated by the inhibition of activation of microglia and astrocytes, as well as the decreased expression of tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) in rat brains. Also, EP prevented the elevation of cleaved caspase-3 in periventricular white matter tissue after LPS insult. Taken together, these results suggest that EP confers potent protection against LPS-induced white matter injury via its anti-inflammatory and anti-apoptotic properties.     

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.     

Expression of plasmolipin in the developing rat brain.

Plasmolipin is an hydrophobic plasma membrane proteolipid present in both kidney and brain. The protein consists of two subunits of 17-18.5 kD, which together form K+ selective voltage-dependent channels. In this report, we define the embryonic and postnatal expression of plasmolipin in the developing rat brain. Plasmolipin was found to be essentially restricted to the postnatal period increasing eight-fold between the first to fourth week after birth. A fetal plasmolipin immunoreactive protein (FPIP) was identified in embryonic brain and also during the early postnatal development of the cerebellum. The expression of FPIP was biphasic with an initial transient increase between E15-E20 followed by a decrease in its levels. FPIP was not detected in the developed rat CNS. FPIP was found in a variety of dividing and immature cells including cultured astrocytes and embryonic neurons, neuroblastoma cells, and rat thymus. In contrast, plasmolipin was restricted to oligodendrocytes of the neural cells tested and to renal tubular epithelial cells.     

Lentiviral-mediated gene transfer of brain-derived neurotrophic factor is neuroprotective in a mouse model of neonatal excitotoxic challenge

Excitotoxicity may be a critical factor in the formation of brain lesions associated with cerebral palsy. When injected into the murine neopallium at postnatal day 5, the glutamatergic analog N-methyl-D-aspartate (NMDA) produces transcortical neuronal death and periventricular white matter cysts, which mimic brain damage observed in human term and preterm neonates at risk for developing cerebral palsy. We previously showed that intracerebral injection of brain-derived neurotrophic factor (BDNF) was neuroprotective in this model. Because BDNF does not easily cross the blood-brain barrier, alternative strategies to avoid repeated intracerebral injections of BDNF should be tested, particularly when the goal of such translational research is ultimately to achieve clinical application. The goal of the present study was to assess the protective role of lentiviral-mediated gene transfer of BDNF against excitotoxic lesions induced by NMDA in newborn mice. We first assessed the biological activity of BDNF gene transfer in vitro and determined the efficiency of gene transfer in our in vivo model. We next administered the BDNF-expressing vector by intracerebral injection in neonatal mice, 3 days before inducing NMDA lesions. When compared with a control green fluorescent protein-expressing lentiviral vector, administration of BDNF-expressing vector induced a significant protection of the periventricular white matter and cortical plate against the NMDA-mediated insult. Intraventricular delivery of the BDNF-expressing lentiviral vector was more efficient in terms of neuroprotection than the intraparenchymal route. Altogether, the present study shows that viral-mediated gene transfer of BDNF to newborn mouse brain is feasible and affords significant neuroprotection against an excitotoxic insult.     

Using the Alberta Infant Motor Scale to early identify very low-birth-weight infants with cystic periventricular leukomalacia

We examined whether the Alberta Infant Motor Scale (AIMS) is able to identify very low-birth-weight (VLBW) preterm infants with cystic periventricular leukomalacia (PVL) as early as 6 months of corrected age. Longitudinal follow-up AIMS assessments were done at 6, 12, and 18 months old for 35 VLBW infants with cystic PVL (cPVL⁺), 70 VLBW infants without cystic PVL (cPVL⁻), and 76 term infants (healthy controls: HC). Corrected age was used for the preterm infants. The cPVL⁺ group had significantly lower prone, supine and sitting subscales at age 6, 12, and 18 months than the cPVL⁻ group (all p < 0.05). The cPVL⁻ group showed significantly lower supine, prone, sitting, and standing subscales than the HC group only at age 6 months. At age 6 months, the areas under the receiver operator curve used to discriminate the cPVL⁺ infants from cPVL⁻ infants were 0.82 ± 0.04 for prone, 0.93 ± 0.02 for supine, 0.83 ± 0.05 for sitting, and 0.62 ± 0.07 for standing. The AIMS may help early identify VLBW infants with cystic PVL at age 6 months old.     

Expression of EAAT2 in neurons and protoplasmic astrocytes during human cortical development

The major regulators of synaptic glutamate in the cerebral cortex are the excitatory amino acid transporters 1–3 (EAAT1–3). In this study, we determined the cellular and temporal expression of EAAT1–3 in the developing human cerebral cortex. We applied single‐ and double‐label immunocytochemistry to normative frontal or parietal (associative) cortex samples from 14 cases ranging in age from 23 gestational weeks to 2.5 postnatal years. The most striking finding was the transient expression of EAAT2 in layer V pyramidal neuronal cell bodies up until 8 postnatal months prior to its expression in protoplasmic astrocytes at 41 postconceptional weeks onward. EAAT2 was also expressed in neurons in layer I (presumed Cajal–Retzius cells), and white matter (interstitial) neurons. This expression in neurons in the developing human cortex contrasts with findings by others of transient expression exclusively in axon tracts in the developing sheep and rodent brain. With western blotting, we found that EAAT2 was expressed as a single band until 2 postnatal months, after which it was expressed as two bands. The expression of EAAT2 in pyramidal neurons during human brain development may contribute to cortical vulnerability to excitotoxicity during the critical period for perinatal hypoxic–ischemic encephalopathy. In addition, by studying the expression of EAAT1 and EAAT2 glutamate transporters, it was possible to document the development of protoplasmic astrocytes. J. Comp. Neurol. 520:3912–3932, 2012. © 2012 Wiley Periodicals, Inc.     

产前接触脂多糖合并缺氧制作围产期脑性瘫痪动物模型：模拟人类实际状况的可能性

目的 探讨孕鼠宫内感染合并缺氧致幼鼠脑瘫的模型制作及评价方法。 方法 实验动物分为三组：（1）脂多糖＋缺氧组:选用孕16～18天健康Wistar雌性大鼠脂多糖腹腔注射（0.4mg/kg）,12小时后置于缺氧环境中缺氧2.5小时,4小时后再次腹腔注射脂多糖(0.4mg/kg);（2）颈动脉结扎＋缺氧组：组出生后第7天Wistar幼鼠结扎右侧颈动脉结合缺氧环境的方法制作幼鼠脑瘫模型;（3）对照组：仅行颈正中切口,然后缝合切口。于术后4周对三组实验动物进行足印分析试验、股四头肌复合肌肉运动诱发电位及病理学检测。 结果 （1）足印分析试验：脂多糖＋缺氧组和颈动脉结扎＋缺氧组实验鼠前后肢足印重复性差,表现为间距较大,且间距不稳定,明显大于对照组（P<0.05）;（2）两实验组动物后肢股四头肌CMAP与对照组相比,引出最大波幅所需的刺激强度增大,引出波幅降低（P<0.05）; （3）HE染色光镜观察两实验组脑室旁正常组织结构破坏,细胞排列紊乱,局部大小不等圆形软化灶,在颈动脉结扎＋缺氧组以损伤侧脑室周围变化明显。 结论 应用孕鼠宫内感染合并缺氧的方法制作幼鼠脑瘫模型稳定可靠,此模型与成熟的单侧颈动脉结扎缺氧模型相比具有微创的、简单易行、逼真的模拟人类在怀孕晚期宫内感染缺氧致胎儿脑部损伤的优点,值得推广。     

An animal model of cerebral palsy induced by prenatal exposure to lipopolysaccharide and hypoxia

BACKGROUND:Neonatal cerebral palsy is mainly caused by prenatal factors.At present,an animal model of prenatal infection and early postnatal hypoxia does not exist.OBJECTIVE:To observe morphology and motor performance following prenatal infection and hypoxic insult-induced brain damage of neonatal rats to verify the feasibility to establish a model of cerebral palsy.DESIGN,TIME AND SETTING:A randomized,controlled,animal experiment was performed at the Laboratories of Xinjiang Center for Disease Control and Prevention from September 2007 to June2008.MATERIALS:The hypoxic incubator was purchased from Shanghai Pediatric Medical Institute,China.Lipopolysaccharide(LPS,Escherichia coli,055:B5)was purchased from Sigma-Aldrich(St.Louis,MO,USA).METHODS:A total of 27 Wistar rats,aged 7 days,were randomly assigned to sham-surgery group(n = 15)with no carotid artery incision or hypoxia treatment,hypoxia/ischemia(H/I)group(n = 12)undergoing ligature of the right common carotid artery followed by exposure to hypoxia at postnatal day 7(P7),and LPS/H group(n = 19),in which pregnant rats were exposed in utero to LPS followed by prenatal hypoxia at embryonic day 16.MAIN OUTCOME MEASURES:Behavior,compound muscle action potential,and pathological changes were observed in 28-day-old rats.RESULTS:The footprint repeat space showed that left limb footprint repeatability in the H/I and LPS/H groups was lower than in the sham-surgery group(P< 0.05).The space between the footprints was larger and unstable.Hind limb quadricep compound muscle action potential in the H/I and LPS/H groups showed lower wave amplitude compared with the sham-surgery group(P< 0.05).Hematoxylin and eosin staining showed irregular cells around the ventricle,as well as periventricular leukomalacia.CONCLUSION:An animal model of cerebral palsy was established,which simulated the human condition most likely associated with occurrence of this disease.This model could be used for experimental studies related to cerebral patsy.     

Cerebral palsy

Cerebral palsy (CP) is a group of disorders of movement and posture resulting from non-progressive disturbances of the fetal or neonatal brain. More than 80% of cases of CP in term infants originate in the prenatal period; in premature infants, both prenatal or postnatal causes contribute. The most prevalent pathological lesion seen in CP is periventricular white matter injury (PWMI) resulting from vulnerability of the immature oligodendrocytes (pre-OLs) before 32 wk of gestation. PWMI is responsible for the spastic diplegia form of CP and a spectrum of cognitive and behavioral disorders. Oxidative stress and excitotoxicity resulting from excessive stimulation of ionotropic glutamate receptors on preOLs are the most prominent molecular mechanisms for PWMI. Asphyxia around the time of birth in term infants accounts for less than 15% of CP in developed countries but the incidence is higher in underdeveloped areas Asphyxia causes a different pattern of brain injury and CP than is seen after preterm injuries. This type of CP is associated with the clinical syndrome of hypoxic-ischemic encephalopathy shortly after the insult, and the cortex, basal ganglia, and brainstem are selectively vulnerable to injury. Experimental models indicate that neurons in the neonatal brain are more likely to die by delayed apoptosis extending over days to weeks than those in the adult brain. Neurons die by glutamate-mediated excitotoxicity involving downstream caspase-dependent and caspase-independent cell death pathways. Recent reports indicate that males and females preferentially utilize different pathways. Clinical trials indicate that mild hypothermia reduces death or disability in term infants following asphyxia and basic research suggests that this approach might be combined with pharmacological strategies in the future.     

17β‐estradiol protects against hypoxic/ischemic white matter damage in the neonatal rat brain

Developing oligodendrocytes (pre‐OLs) are highly vulnerable to hypoxic‐ischemic injury and associated excitotoxicity and oxidative stress. 17β‐Estradiol plays an important role in the development and function of the CNS and is neuroprotective. The sudden drop in circulating estrogen after birth may enhance the susceptibility of developing OLs to injury. Estrogen receptor (ER)–α and ER‐β are both expressed in OLs. We examined the effect of 17β‐estradiol on oxygen‐glucose deprivation and oxidative stress–induced cell death in rat pre‐OLs in vitro and on hypoxic‐ischemic brain injury in vivo. Pre‐OLs in culture were subjected to oxygen‐glucose deprivation (OGD) or glutathione depletion in the presence or absence of 17β‐estradiol. LDH release, the Alamar blue assay, and phase‐contrast microscopy were used to assess cell viability. Hypoxic‐ischemic injury was generated in 6‐day‐old rats (P6) by unilateral carotid ligation and hypoxia (6% O₂ for 1 hr). Rat pups received one intraperitoneal injection of 300 or 600 μg/kg 17β‐estradiol or vehicle 12 hr prior to the surgical procedure. Injury was assessed by myelin basic protein (MBP) immunocytochemistry at P10. 17β‐Estradiol produced significant protection against OGD‐induced cell death in primary OLs (EC₅₀ = 1.3 ± 0.46 × 10^?9 M) and against oxidative stress. Moreover, 17β‐estradiol attenuated the loss of MBP labeling in P10 pups ipsilateral to the carotid ligation. These results suggest a potential role for estrogens in attenuation of hypoxic‐ischemic and oxidative injury to developing OLs and in the prevention of periventricular leukomalacia. © 2009 Wiley‐Liss, Inc.     

Selective toxicity of the general anesthetic propofol for GABAergic neurons in rat brain cell cultures

The intravenous, short-acting general anesthetic propofol was applied to three-dimensional (aggregating) cell cultures of fetal rat telencephalon. Both the clinically used formulation (Disoprivan®, ICI Pharmaceuticals, Cheshire, England) and the pure form (2,6-diisopropylphenol) were tested at two different periods of brain development: immature brain cell cultures prior to synaptogenesis and at the time of intense synapses and myelin formation. At both time periods and for clinically relevant concentrations and time of exposure (i.e., concentrations ≤2.0 μg/ml for 8 hr), propofol caused a significant decrease of glutamic acid decarboxylase activity. This effect persisted after removal of the drug, suggesting irreversible structural changes in GABAergic neurons. The gamma-aminobutyric acid type A (GABA_A) blocking agents bicuculline and picrotoxin partially attenuated the neurotoxic effect of propofol in cultures treated at the more mature phase of development. This protective effect was not observed in the immature brain cells. The present data suggest that propofol may cause irreversible lesions to GABAergic neurons when given at a critical phase of brain development. In contrast, glial cells and myelin appeared resistant even to high doses of propofol. © 1996 Wiley-Liss, Inc.     

Activity-dependent neurotrophic factor-14 requires protein kinase C and mitogen-associated protein kinase kinase activation to protect the developing mouse brain against excitotoxicity

Activity-dependent neurotrophic factor (ADNF) is a newly identified compound that prevents in vitro neuronal death when present in fentomolar concentrations. ADNF-14, a 14 amino acid peptide derived from ADNF, has the same effects on growth as the parent molecule. However, the transduction pathways and target cells for these highly potent trophic factors are still unknown. We previously described a mouse model of excitotoxic lesions of the developing neocortex mimicking several hypoxic or hypoxic-like brain lesions observed in human fetuses and neonates. In this model, cotreatment with the excitotixin ibotenate and ADNF-14 prevented both neuronal death in pups injected on the day of birth and white matter cystic lesions in pups treated 5 d after birth. In the present study, coadministration of ibotenate, ADNF-14, and selective transduction pathway inhibitors showed that activation of protein kinase C (PKC) and mitogen-associated protein kinase kinase was critical for neuroprotection. Immunocytochemistry revealed that ADNF-14 activated PKC and mitogen-associated protein kinase in cortical neurons on the day of birth and in white matter astrocytes on the fifth postnatal day. Taken in concert, these data identify PKC and mitogen-associated protein kinase pathways as critical to ADNF-14-induced neuroprotection of the developing brain against excitotoxic damage.     

Chronic ischemia preferentially causes white matter injury in the neonatal rat brain

Chronic ischemic brain injuries were studied in 7- and 14-day-old rat pups, which were subjected to bilateral carotid artery occlusion (BCAO) on postnatal day 1. BCAO preferentially injured white matter in the corpus callosum, subcortex and internal capsule areas while largely spared cortical neurons. White matter rarefaction in the corpus callosum was observed in 12 out of the 17 BCAO rat brains and significantly enlarged lateral ventricles were found in five out of seven P14 BCAO rat brains. These white matter changes were similar to injuries found in newborn infants with periventricular leukomalacia (PVL). White matter injuries in the 7-day-old BCAO rat brain were accompanied with increased activation of microglia/macrophages, as indicated by ED1 and OX42 positive immunostaining. Immature oligodendrocytes in the 7-day-old BCAO rat brain, as indicated by O4+/O1+ staining, were much fewer than in the sham-operated rat brain. Immunostaining for myelin basic protein (MBP) at the fimbria hippocampus and the internal capsule areas in the 7-day-old BACO rat brain was also much less than in the control rat brain. Consistent with the immunostaining data, MBP mRNA expression in the 7-day-old, but not in the 14-day-old, BCAO rat brain was significantly less than in the control rat brain. The overall results suggest that pre-oligodendrocytes and immature oligodendrocytes might be major targets for chronic ischemic insults and activated microglia/macrophages are possibly involved in the process of white matter injury.