Preterm White Matter Injury Research Articles

Profiling analysis reveals the potential contribution of long non-coding RNAs to preterm white matter injury

AimsThe aim of this study was to investigate the molecular mechanism underlying preterm white matter injury (WMI) via the identification and functional analysis of differentially expressed long non-coding RNAs (lncRNAs) and mRNAs. Main methodsA neonatal rat model of preterm WMI was established by ligating the common carotid artery and hypoxia induction. RNA sequencing was performed to analyze gene expression profiles of brain samples. Gene Ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) analyses were performed to evaluate functions of target mRNAs. A co-expression network was generated to explore regulatory mechanisms. Key findingsIn total, 210 lncRNAs and 619 mRNAs were differentially expressed between the preterm WMI group and the sham group. Based on GO and KEGG analyses, enriched pathways included the apoptotic signaling pathway, vascular endothelial growth factor (VEGF) signaling pathway, natural killer cell-mediated cytotoxicity pathway, and the autophagy pathway. SignificanceDifferentially expressed lncRNAs and mRNAs in the brain tissues of preterm WMI model were identified, and the biological processes were closely associated with the development of preterm WMI, thus being considered potential targets for future studies.

Expression of triggering receptor expressed on myeloid cells in periventricular leukomalacia of neonatal rat model

Objective To investigate the expression of triggering receptor expressed on myeloid cells(TREMs)in periventricular leukomalacia(PVL) of the neonatal rat model. Methods Thirty-two 3-day-old neonatal rats were double-blinded randomly divided into Sham group and Model group.The PVL rat model was established by ligating right carotid artery and oxygen deprivation.Hematoxylin-eosin (HE) was adopted to compare pathological changes of brain tissue between the two groups, and immunofluorescence was adopted to detect the expression of myline basic protein (MBP) in the right hemisphere of the two groups.Western blot was performed to detect the expression of TREM1 and TREM2 in the right hemisphere of the two groups. Results The results of HE staining showed that the brain tissues of Model group were significantly damaged compared with that of Sham group, and the mean fluorescence intensity of MBP in Model group(26.629±2.317) was significantly lower than that in Sham group(33.579±2.824), with statistically significant differences(t=9.124, P<0.05). The expression of TREM1 in Model group(0.789 ±0.120) was higher than that in Sham group(0.567±0.093), with statistically significant differences(t=-3.891, P<0.05). The expression of TREM2 in Model group(0.544±0.133) was lower than that in Sham group(0.791±0.118), with statistically significant differences(t=3.667, P<0.05). Conclusion The expressions of TREM1 and TREM2 in the neonatal rat model of PVL change abnormally, suggesting that TREMs may be involved in the pathological process of preterm white matter injury. Key words: Periventricular leukomalacia; White matter injury; Brain injury; Triggering receptor expressed on myeloid cells; Rat

Oligogenesis in the "oligovascular unit" involves PI3K/AKT/mTOR signaling in hypoxic-ischemic neonatal mice

The "oligovascular unit" is a dynamic structural complex composed of endothelial cells (ECs) and oligodendrocyte progenitor cells (OPCs)/oligodendrocytes. By improving the microenvironment of OPCs in the "oligovascular unit" and promoting the proliferation and differentiation of OPCs, both myelination and white matter injury can be repaired. However, it is unclear what characteristic changes occur in the microenvironment of the "oligovascular unit" after preterm white matter injury (PWMI). Here, we demonstrate the changes in the "oligovascular unit" induced by hypoxia-ischemia (HI) and its underlying mechanism in PWMI mice. White matter injury and inhibited production of myelin from OPCs were observed by histopathological staining in HI neonatal mice. We further observed that the proliferation of OPCs and angiogenesis were increased after HI, which is considered the response of the body and cells to HI. HI-induced oligogenesis occurs around the vessels, indicating that "oligovascular units" exist and promote the proliferation and differentiation of OPCs after HI in the short term. We also determined that angiogenesis and oligogenesis induced by HI in the white matter are related to the PI3K/AKT/mTOR pathway. Furthermore, the myelin sheaths were shown to be disordered on the side of the surgery, and the myelin-dense layer was poorly developed at P14 and P28. Different degrees of damage to the vascular ECs and basement membrane on the surgical side were detected beginning at P4, indicating that EC injury is an early phenomenon that subsequently affects oligogenesis. Taken together, our findings indicate that the proliferation of OPCs and angiogenesis in white matter are increased in the early stage of HI involving PI3K/AKT/mTOR pathway activation. Promoting vascular endothelial function and angiogenesis may increase the proliferation and survival of OPCs via the "oligovascular unit," which suggests a potential method to repair injured white matter in the early stage of PWMI.

Synaptic Injury in the Thalamus Accompanies White Matter Injury in Hypoxia/Ischemia-Mediated Brain Injury in Neonatal Rats.

The broad spectrum of disabilities caused by white matter injury (WMI) cannot be explained simply by hypomyelination. Synaptic injury in the thalamus may be related to disabilities in WMI survivors. Neuronal injury in the thalamus has been found most commonly in autopsy cases of preterm WMI. We hypothesized that hypoxia/ischemia (HI) in neonatal rats results in synaptic abnormalities in the thalamus that contribute to disabilities in WMI survivors. We examined changes in synapses in a neonatal rat model of HI-induced WMI. Right common carotid artery ligation and hypoxia (8% oxygen for 2.5 hours (h)) were performed in three-day-old Sprague-Dawley rats. We found HI rats performed worse in the Morris water maze test than sham rats, suggesting long-term cognition impairment after HI injury. A loss of synapses in the thalamus accompanied by hypomyelination and oligodendrocytes (OLs) reduction was observed. At the ultrastructural level, reductions in active zone (AZ) length and postsynaptic density (PSD) thickness were detected at 2 weeks after HI exposure. Furthermore, increased expression of synaptophysin and PSD-95 in both groups was observed from 3 days (d) to 21 d after hypoxic/ischemic (HI) injury. PSD-95 expression was significantly lower in HI rats than in sham rats from 14 d to 21 d after HI injury, and synaptophysin expression was significantly lower in HI rats from 7 d to 14 d after HI injury. However, no significant difference in synaptophysin expression was observed between HI rats and sham rats at 21 d after HI injury. The results demonstrated synaptic abnormalities in the thalamus accompanied by hypomyelination in WMI in response to HI exposure, which may contribute to the diverse neurological defects observed in WMI patients. Although synaptic reorganization occurred as a compensatory response to HI injury, the impairments in synaptic transmission were not reversed.

Neurodevelopmental Outcomes in Preterm Infants with White Matter Injury Using a New MRI Classification

Objective: The aim of this study was to evaluate whether a new MRI scoring system for preterm non-haemorrhagic white matter injury (WMI), derived from the analysis of the natural evolution of WMI throughout the neonatal period until term-equivalent age, can be used for outcome prediction. Methods: Eighty-two infants <36 weeks gestation with WMI diagnosed from sequential cranial ultrasound and confirmed on neonatal MRI were retrospectively included. WMI was classified in four grades of severity. Neurodevelopmental data at a median age of 24 months were analysed. Results: In 74 surviving children WMI severity was strongly associated with the presence and severity of cerebral palsy (CP) and other neurodevelopmental impairments (Spearman’s rank correlation 0.88, p < 0.001). Only 3 children with grade I WMI (9%) developed CP (all ambulant) and their developmental scores were not different to those from the controls, although they started walking significantly later (p = 0.036). Of the 6 children with grade II, 83% developed CP (mild in most), whereas 91% of the 34 children with grade III had CP (moderate-severe in 76%) and all had some degree of neurodevelopmental impairment. Three children with grade III WMI did not develop CP; their imaging showed, in contrast to children who developed CP, that the cysts did not affect the corticospinal tracts; also, myelin in the posterior limb of the internal capsule appeared normal in 2 children and suboptimal in 1. Conclusions: This MRI scoring system for preterm WMI can be used to predict neurodevelopmental outcomes. Individualized assessment of the site of lesions and the progression of myelination improves prognostic accuracy.

Myelination induction by a histamine H3 receptor antagonist in a mouse model of preterm white matter injury

Fifteen million babies are born preterm every year and a significant number suffer from permanent neurological injuries linked to white matter injury (WMI). A chief cause of preterm birth itself and predictor of the severity of WMI is exposure to maternal-fetal infection-inflammation such as chorioamnionitis. There are no neurotherapeutics for this WMI. To affect this healthcare need, the repurposing of drugs with efficacy in other white matter injury models is an attractive strategy. As such, we tested the efficacy of GSK247246, an H3R antagonist/inverse agonist, in a model of inflammation-mediated WMI of the preterm born infant recapitulating the main clinical hallmarks of human brain injury, which are oligodendrocyte maturation arrest, microglial reactivity, and hypomyelination. WMI is induced by mimicking the effects of maternal-fetal infection-inflammation and setting up neuroinflammation. We induce this process at the time in the mouse when brain development is equivalent to the human third trimester; postnatal day (P)1 through to P5 with i.p. interleukin-1β (IL-1β) injections. We initiated GSK247246 treatment (i.p at 7 mg/kg or 20 mg/kg) after neuroinflammation was well established (on P6) and it was administered twice daily through to P10. Outcomes were assessed at P10 and P30 with gene and protein analysis. A low dose of GSK247246 (7 mg/kg) lead to a recovery in protein expression of markers of myelin (density of Myelin Basic Protein, MBP & Proteolipid Proteins, PLP) and a reduction in macro- and microgliosis (density of ionising adaptor protein, IBA1 & glial fibrillary acid protein, GFAP). Our results confirm the neurotherapeutic efficacy of targeting the H3R for WMI seen in a cuprizone model of multiple sclerosis and a recently reported clinical trial in relapsing-remitting multiple sclerosis patients. Further work is needed to develop a slow release strategy for this agent and test its efficacy in large animal models of preterm infant WMI.

White matter injury in term neonates with congenital heart diseases: Topology & comparison with preterm newborns

BackgroundNeonates with congenital heart disease (CHD) are at high risk of punctate white matter injury (WMI) and impaired brain development. We hypothesized that WMI in CHD neonates occurs in a characteristic distribution that shares topology with preterm WMI and that lower birth gestational age (GA) is associated with larger WMI volume. Objective(1) To quantitatively assess the volume and location of WMI in CHD neonates across three centres. (2) To compare the volume and spatial distribution of WMI between term CHD neonates and preterm neonates using lesion mapping. MethodsIn 216 term born CHD neonates from three prospective cohorts (mean birth GA: 39 weeks), WMI was identified in 86 neonates (UBC: 29; UCSF: 43; UCZ: 14) on pre- and/or post-operative T1 weighted MRI. WMI was manually segmented and volumes were calculated. A standard brain template was generated. Probabilistic WMI maps (total, pre- and post-operative) were developed in this common space. Using these maps, WMI in the term CHD neonates was compared with that in preterm neonates: 58 at early-in-life (mean postmenstrual age at scan 32.2 weeks); 41 at term-equivalent age (mean postmenstrual age at scan 40.1 weeks). ResultsThe total WMI volumes of CHD neonates across centres did not differ (p = 0.068): UBC (median = 84.6 mm3, IQR = 26–174.7 mm3); UCSF (median = 104 mm3, IQR = 44–243 mm3); UCZ (median = 121 mm3, IQR = 68–200.8 mm3). The spatial distribution of WMI in CHD neonates showed strong concordance across centres with predilection for anterior and posterior rather than central lesions. Predominance of anterior lesions was apparent on the post-operative WMI map relative to the pre-operative map. Lower GA at birth predicted an increasing volume of WMI across the full cohort (41.1 mm3 increase of WMI per week decrease in gestational age; 95% CI 11.5–70.8; p = 0.007), when accounting for centre and heart lesion. While WMI in term CHD and preterm neonates occurs most commonly in the intermediate zone/outer subventricular zone there is a paucity of central lesions in the CHD neonates relative to preterms. ConclusionsWMI in term neonates with CHD occurs in a characteristic topology. The spatial distribution of WMI in term neonates with CHD reflects the expected maturation of pre-oligodendrocytes such that the central regions are less vulnerable than in the preterm neonates.

\u03b3\u03b4T cells but not \u03b1\u03b2T cells contribute to sepsis-induced white matter injury and motor abnormalities in mice

BackgroundInfection and sepsis are associated with brain white matter injury in preterm infants and the subsequent development of cerebral palsy.MethodsIn the present study, we used a neonatal mouse sepsis-induced white matter injury model to determine the contribution of different T cell subsets (αβT cells and γδT cells) to white matter injury and consequent behavioral changes. C57BL/6J wild-type (WT), T cell receptor (TCR) δ-deficient (Tcrd−/−, lacking γδT cells), and TCRα-deficient (Tcra−/−, lacking αβT cells) mice were administered with lipopolysaccharide (LPS) at postnatal day (PND) 2. Brain myelination was examined at PNDs 12, 26, and 60. Motor function and anxiety-like behavior were evaluated at PND 26 or 30 using DigiGait analysis and an elevated plus maze.ResultsWhite matter development was normal in Tcrd−/− and Tcrα−/− compared to WT mice. LPS exposure induced reductions in white matter tissue volume in WT and Tcrα−/− mice, but not in the Tcrd−/− mice, compared with the saline-treated groups. Neither LPS administration nor the T cell deficiency affected anxiety behavior in these mice as determined with the elevated plus maze. DigiGait analysis revealed motor function deficiency after LPS-induced sepsis in both WT and Tcrα−/− mice, but no such effect was observed in Tcrd−/− mice.ConclusionsOur results suggest that γδT cells but not αβT cells contribute to sepsis-induced white matter injury and subsequent motor function abnormalities in early life. Modulating the activity of γδT cells in the early stages of preterm white matter injury might represent a novel therapeutic strategy for the treatment of perinatal brain injury.

Exploring the multiple-hit hypothesis of preterm white matter damage using diffusion MRI.

BackgroundPreterm infants are at high risk of diffuse white matter injury and adverse neurodevelopmental outcome. The multiple hit hypothesis suggests that the risk of white matter injury increases with cumulative exposure to multiple perinatal risk factors. Our aim was to test this hypothesis in a large cohort of preterm infants using diffusion weighted magnetic resonance imaging (dMRI).MethodsWe studied 491 infants (52% male) without focal destructive brain lesions born at < 34 weeks, who underwent structural and dMRI at a specialist Neonatal Imaging Centre. The median (range) gestational age (GA) at birth was 30+ 1 (23+ 2–33+ 5) weeks and median postmenstrual age at scan was 42+ 1 (38–45) weeks. dMRI data were analyzed using tract based spatial statistics and the relationship between dMRI measures in white matter and individual perinatal risk factors was assessed. We tested the hypothesis that increased exposure to perinatal risk factors was associated with lower fractional anisotropy (FA), and higher radial, axial and mean diffusivity (RD, AD, MD) in white matter. Neurodevelopmental performance was investigated using the Bayley Scales of Infant and Toddler Development, Third Edition (BSITD-III) in a subset of 381 infants at 20 months corrected age. We tested the hypothesis that lower FA and higher RD, AD and MD in white matter were associated with poorer neurodevelopmental performance.ResultsIdentified risk factors for diffuse white matter injury were lower GA at birth, fetal growth restriction, increased number of days requiring ventilation and parenteral nutrition, necrotizing enterocolitis and male sex. Clinical chorioamnionitis and patent ductus arteriosus were not associated with white matter injury. Multivariate analysis demonstrated that fetal growth restriction, increased number of days requiring ventilation and parenteral nutrition were independently associated with lower FA values. Exposure to cumulative risk factors was associated with reduced white matter FA and FA values at term equivalent age were associated with subsequent neurodevelopmental performance.ConclusionThis study suggests multiple perinatal risk factors have an independent association with diffuse white matter injury at term equivalent age and exposure to multiple perinatal risk factors exacerbates dMRI defined, clinically significant white matter injury. Our findings support the multiple hit hypothesis for preterm white matter injury.

Intranasal Delivery of Umbilical Cord-Derived Mesenchymal Stem Cells Preserves Myelination in Perinatal Brain Damage

Preterm white matter injury (WMI) is an important cause for long-term disability. Stem cell transplantation has been proposed as a novel therapeutic approach. However, intracerebral transplantation is not feasible for clinical purpose in newborns. Intranasal delivery of cells to the brain might be a promising, noninvasive therapeutic approach to restore the damaged brain. Therefore, our goal is to study the remyelinating potential of human Wharton's jelly mesenchymal stem cells (hWJ-MSCs) after intranasal delivery. Wistar rat pups, previously brain-damaged by a combined hypoxic-ischemic and inflammatory insult, received hWJ-MSC (150,000 cells in 3 μL) that were intranasally delivered twice to each nostril (600,000 cells total). WMI was assessed by immunohistochemistry and western blot for myelination, astrogliosis, and microgliosis. The expression of preoligodendrocyte markers, and neurotrophic factors, was analyzed by real-time polymerase chain reaction. Animals treated with intranasally delivered hWJ-MSC showed increased myelination and decreased gliosis compared to untreated animals. hWJ-MSC may, therefore, modulate the activation of microglia and astrocytes, resulting in a change of the brain microenvironment, which facilitates the maturation of oligodendrocyte lineage cells. This is the first study to show that intranasal delivery of hWJ-MSC in rats prevented hypomyelination and microgliosis in a model of WMI in the premature rat brain. Further studies should address the dose and frequency of administration.

Preterm white matter brain injury is prevented by early administration of umbilical cord blood cells

Infants born very preterm are at high risk for neurological deficits including cerebral palsy. In this study we assessed the neuroprotective effects of umbilical cord blood cells (UCBCs) and optimal administration timing in a fetal sheep model of preterm brain injury. 50 million allogeneic UCBCs were intravenously administered to fetal sheep (0.7 gestation) at 12h or 5d after acute hypoxia-ischemia (HI) induced by umbilical cord occlusion. The fetal brains were collected at 10d after HI. HI (n=7) was associated with reduced number of oligodendrocytes (Olig2+) and myelin density (CNPase+), and increased density of activated microglia (Iba-1+) in cerebral white matter compared to control fetuses (P<0.05). UCBCs administered at 12h, but not 5d after HI, significantly protected white matter structures and suppressed cerebral inflammation. Activated microglial density showed a correlation with decreasing oligodendrocyte number (P<0.001). HI caused cell death (TUNEL+) in the internal capsule and cell proliferation (Ki-67+) in the subventricular zone compared to control (P<0.05), while UCBCs at 12h or 5d ameliorated these effects. Additionally, UCBCs at 12h induced a significant systemic increase in interleukin-10 at 10d, and reduced oxidative stress (malondialdehyde) following HI (P<0.05). UCBC administration at 12h after HI reduces preterm white matter injury, via anti-inflammatory and antioxidant actions.

MRI Based Preterm White Matter Injury Classification: The Importance of Sequential Imaging in Determining Severity of Injury.

BackgroundThe evolution of non-hemorrhagic white matter injury (WMI) based on sequential magnetic resonance imaging (MRI) has not been well studied. Our aim was to describe sequential MRI findings in preterm infants with non-hemorrhagic WMI and to develop an MRI classification system for preterm WMI based on these findings.MethodsEighty-two preterm infants (gestation ≤35 weeks) were retrospectively included. WMI was diagnosed and classified based on sequential cranial ultrasound (cUS) and confirmed on MRI.Results138 MRIs were obtained at three time-points: early (<2 weeks; n = 32), mid (2–6 weeks; n = 30) and term equivalent age (TEA; n = 76). 63 infants (77%) had 2 MRIs during the neonatal period. WMI was non-cystic in 35 and cystic in 47 infants. In infants with cystic-WMI early MRI showed extensive restricted diffusion abnormalities, cysts were already present in 3 infants; mid MRI showed focal or extensive cysts, without acute diffusion changes. A significant reduction in the size and/or extent of the cysts was observed in 32% of the infants between early/mid and TEA MRI. In 4/9 infants previously seen focal cysts were no longer identified at TEA. All infants with cystic WMI showed ≥2 additional findings at TEA: significant reduction in WM volume, mild-moderate irregular ventriculomegaly, several areas of increased signal intensity on T1-weighted-images, abnormal myelination of the PLIC, small thalami.ConclusionIn infants with extensive WM cysts at 2–6 weeks, cysts may be reduced in number or may even no longer be seen at TEA. A single MRI at TEA, without taking sequential cUS data and pre-TEA MRI findings into account, may underestimate the extent of WMI; based on these results we propose a new MRI classification for preterm non-hemorrhagic WMI.

Role of recurrent hypoxia-ischemia in preterm white matter injury severity.

ObjectiveAlthough the spectrum of white matter injury (WMI) in preterm infants is shifting from cystic necrotic lesions to milder forms, the factors that contribute to this changing spectrum are unclear. We hypothesized that recurrent hypoxia-ischemia (rHI) will exacerbate the spectrum of WMI defined by markers of inflammation and molecules related to the extracellular matrix (hyaluronan (HA) and the PH20 hyaluronidase) that regulate maturation of the oligodendrocyte (OL) lineage after WMI.MethodsWe employed a preterm fetal sheep model of in utero moderate hypoxemia and global severe but not complete cerebral ischemia that reproduces the spectrum of human WMI. The response to rHI was compared against corresponding early or later single episodes of HI. An ordinal rating scale of WMI was compared against an unbiased quantitative image analysis protocol that provided continuous histo-pathological outcome measures for astrogliosis and microglial activation. Late oligodendrocyte progenitors (preOLs) were quantified by stereology. Analysis of hyaluronan and the hyaluronidase PH20 defined the progressive response of the extracellular matrix to WMI.ResultsrHI resulted in a more severe spectrum of WMI with a greater burden of necrosis, but an expanded population of preOLs that displayed reduced susceptibility to cell death. WMI from single episodes of HI or rHI was accompanied by elevated HA levels and increased labeling for PH20. Expression of PH20 in fetal ovine WMI was confirmed by RT-PCR and RNA-sequencing.ConclusionsrHI is associated with an increased risk for more severe WMI with necrosis, but reduced risk for preOL degeneration compared to single episodes of HI. Expansion of the preOL pool may be linked to elevated hyaluronan and PH20.

Microglia toxicity in preterm brain injury

Microglia are the resident phagocytic cells of the central nervous system. During brain development they are also imperative for apoptosis of excessive neurons, synaptic pruning, phagocytosis of debris and maintaining brain homeostasis. Brain damage results in a fast and dynamic microglia reaction, which can influence the extent and distribution of subsequent neuronal dysfunction. As a consequence, microglia responses can promote tissue protection and repair following brain injury, or become detrimental for the tissue integrity and functionality. In this review, we will describe microglia responses in the human developing brain in association with injury, with particular focus on the preterm infant. We also explore microglia responses and mechanisms of microglia toxicity in animal models of preterm white matter injury and in vitro primary microglia cell culture experiments.

Neuromonitoring of Neonatal Extracorporeal Membrane Oxygenation Patients Using Serial Cranial Ultrasounds*

Neuromonitoring of Neonatal Extracorporeal Membrane Oxygenation Patients Using Serial Cranial Ultrasounds*

Diffusion magnetic resonance imaging in preterm brain injury.

White matter injury and abnormal maturation are thought to be major contributors to the neurodevelopmental disabilities observed in children and adolescents who were born preterm. Early detection of abnormal white matter maturation is important in the design of preventive, protective, and rehabilitative strategies for the management of the preterm infant. Diffusion-weighted magnetic resonance imaging (d-MRI) has become a valuable tool in assessing white matter maturation and injury in survivors of preterm birth. In this review, we aim to (1) describe the basic concepts of d-MRI; (2) evaluate the methods that are currently used to analyse d-MRI; (3) discuss neuroimaging correlates of preterm brain injury observed at term corrected age; during infancy, adolescence and in early adulthood; and (4) explore the relationship between d-MRI measures and subsequent neurodevelopmental performance. References for this review were identified through searches of PubMed and Google Scholar before March 2013. The impact of premature birth on cerebral white matter can be observed from term-equivalent age through to adulthood. Disruptions to white matter development, identified by d-MRI, are related to diminished performance in functional domains including motor performance, cognition and behaviour in early childhood and in later life. d-MRI is an effective tool for investigating preterm white matter injury. With advances in image acquisition and analysis approaches, d-MRI has the potential to be a biomarker of subsequent outcome and to evaluate efficacy of clinical interventions in this population.

Effects of white matter injury on resting state fMRI measures in prematurely born infants.

The cerebral white matter is vulnerable to injury in very preterm infants (born prior to 30 weeks gestation), resulting in a spectrum of lesions. These range from severe forms, including cystic periventricular leukomalacia and periventricular hemorrhagic infarction, to minor focal punctate lesions. Moderate to severe white matter injury in preterm infants has been shown to predict later neurodevelopmental disability, although outcomes can vary widely in infants with qualitatively comparable lesions. Resting state functional connectivity magnetic resonance imaging has been increasingly utilized in neurodevelopmental investigations and may provide complementary information regarding the impact of white matter injury on the developing brain. We performed resting state functional connectivity magnetic resonance imaging at term equivalent postmenstrual age in fourteen preterm infants with moderate to severe white matter injury secondary to periventricular hemorrhagic infarction. In these subjects, resting state networks were identifiable throughout the brain. Patterns of aberrant functional connectivity were observed and depended upon injury severity. Comparisons were performed against data obtained from prematurely-born infants with mild white matter injury and healthy, term-born infants and demonstrated group differences. These results reveal structural-functional correlates of preterm white matter injury and carry implications for future investigations of neurodevelopmental disability.

12/15-Lipoxygenase Expression Is Increased in Oligodendrocytes and Microglia of Periventricular Leukomalacia

Oxidative stress involving premyelinating oligodendrocytes (OLs) is a major factor in the pathogenesis of preterm white matter injury. In animal and cell culture studies, activation of the lipid-oxidizing enzyme 12/15-lipoxygenase (12/15-LOX) plays a central role as an inflammatory mediator in the pathology of oxidative stress and OL cell death, as well as ischemia and neuronal death. The role of 12/15-LOX, however, is unclear in the developing human brain. The mechanism of 12/15-LOX involves the production of reactive oxygen species through the metabolism of arachidonic acid, as well as direct detrimental effects on organelle membranes. Here we tested the hypothesis that the density of 12/15-LOX-expressing cells is increased in periventricular leukomalacia (PVL). Using immunocytochemistry (ICC) in human paraffin-embedded tissue, 12/15-LOX expression was seen in macrophages of the focally necrotic lesions in the periventricular white matter, as well as in glial cells throughout the surrounding white matter with reactive gliosis. Interestingly, no significant 12/15-LOX expression was detected in neurons in the cerebral cortex overlying the damaged white matter. Using a scoring system from 0 to 3, we assessed the density of 12/15-LOX-expressing cells in diffusely gliotic white matter from 20 to 43 postconceptional (PC) weeks in 19 PVL cases (median = 36 PC weeks) and 10 control (non-PVL) cases (median = 34 PC weeks). The density of 12/15-LOX-positive cells was significantly increased in the diffuse component of PVL (score = 1.17 ± 0.15) compared to controls (score = 0.48 ± 0.21; p = 0.014). Using double-label ICC, 12/15-LOX was observed in PVL in OLs of the O4 and O1 premyelinating stages, as well as in mature OLs as determined with the mature OL marker adenomatous polyposis coli (APC). In addition, 12/15-LOX expression was present in a population of CD68-positive activated microglia. There was no 12/15-LOX expression in reactive astrocytes. Finally we observed terminal deoxynucleotide transferase dUTP nick end-labeling-positive cells within the white matter of PVL that expressed 12/15-LOX and/or within close proximity of 12/15-LOX-positive cells. Our data support a role for 12/15-LOX activation as an inflammatory mediator of injury in PVL, with a contribution of 12/15-LOX to PVL-induced damage to or cell death of OLs, including those at the O1 and O4 stages.

Altered Glutamatergic Metabolism Associated with Punctate White Matter Lesions in Preterm Infants

Preterm infants (∼10% of all births) are at high-risk for long-term neurodevelopmental disabilities, most often resulting from white matter injury sustained during the neonatal period. Glutamate excitotoxicity is hypothesized to be a key mechanism in the pathogenesis of white matter injury; however, there has been no in vivo demonstration of glutamate excitotoxicity in preterm infants. Using magnetic resonance spectroscopy (MRS), we tested the hypothesis that glutamate and glutamine, i.e., markers of glutamatergic metabolism, are altered in association with punctate white matter lesions and “diffuse excessive high signal intensity” (DEHSI), the predominant patterns of preterm white matter injury. We reviewed all clinically-indicated MRS studies conducted on preterm infants at a single institution during a six-year period and determined the absolute concentration of glutamate, glutamine, and four other key metabolites in the parietal white matter in 108 of those infants after two investigators independently evaluated the studies for punctate white matter lesions and DEHSI. Punctate white matter lesions were associated with a 29% increase in glutamine concentration (p = 0.002). In contrast, there were no differences in glutamatergic metabolism in association with DEHSI. Severe DEHSI, however, was associated with increased lactate concentration (p = 0.001), a marker of tissue acidosis. Findings from this study support glutamate excitotoxicity in the pathogenesis of punctate white matter lesions, but not necessarily in DEHSI, and suggest that MRS provides a useful biomarker for determining the pathogenesis of white matter injury in preterm infants during a period when neuroprotective agents may be especially effective.

Evaluation of the Role of Ceacam1 in the Developing Neonatal Brain and its Impact on Encephalopathy of Prematurity

Encephalopathy of prematurity subsumes the two main neuropathological disorders of preterm white matter injury and neuronal/axonal disease. Factors such as hypoxia-ischemia, drug exposure, hyperoxia and maternal/neonatal inflammation are causal. Particularly with regards to infection/inflammation a significant role of CEACAM1 in different diseases is described. CEACAM1, a member of the carcinoembryonic antigen family of cell adhesion molecules, is a multifunctional molecule that contributes i.e. to morphogenesis of new blood vessels, cell proliferation, apoptosis, insulin metabolism, infection and inflammation. Because of its important role in inflammation-associated signaling we hypothesize that CEACAM1 might contribute to inflammation-induced perinatal brain injury. The aim of the current study is to evaluate the physiological regulation of CEACAM1 in the developing brain in newborn rats, which is yet unknown. Brains of Wistar rats were obtained at day 1 to 15. CEACAM1 protein expression was analysed in serial brain sections by immunohistochemistry including double labeling for markers such as MPB, GFAP, Neurofilament and Nestin to identify the CEACAM1-positive cell types using confocal microscopy. In addition immunostaining for CEACAM1 was performed on cultured pre-oligodendrocytes and mixed glia cells. CEACAM1 expression was detected in broad areas of the brain beginning on day 5 in brain stem and afterwards in the forebrain. CEACAM1 immunostaining on cultivated cells revealed its expression in oligodendrocytes. Our results show for the first time an expression of CEACAM1 in oligodendrocytes. This is of special importance since white matter injury is a myelination disorder. Alterations of CEACAM1 expression under pathologic conditions are subject of current investigations