Comparative utility of MRI and EEG for early detection of cortical dysmaturation after postnatal systemic inflammation in the neonatal rat.

BACKGROUND: Exposure to postnatal systemic inflammation is associated with increased risk of brain injury in preterm infants, leading to impaired maturation of the cerebral cortex and adverse neurodevelopmental outcomes. However, the optimal method for identifying cortical dysmaturation is unclear. Herein, we compared the utility of electroencephalography (EEG), diffusion tensor imaging (DTI), and neurite orientation dispersion and density imaging (NODDI) at different recovery times after systemic inflammation in newborn rats. METHODS: Sprague Dawley rat pups of both sexes received single-daily lipopolysaccharide (LPS; 0.3 mg/kg i.p.; n = 51) or saline (n = 55) injections on postnatal days (P)1, 2, and 3. A subset of these animals were implanted with EEG electrodes. Cortical EEG was recorded for 30 min from unanesthetized, unrestrained pups at P7, P14, and P21, and in separate groups, brain tissues were collected at these ages for ex-vivo MRI analysis (9.4 T) and Golgi-Cox staining (to assess neuronal morphology) in the motor cortex. RESULTS: Postnatal inflammation was associated with reduced cortical pyramidal neuron arborization from P7, P14, and P21. These changes were associated with dysmature EEG features (e.g., persistence of delta waveforms, higher EEG amplitude, reduced spectral edge frequency) at P7 and P14, and higher EEG power in the theta and alpha ranges at P21. By contrast, there were no changes in cortical DTI or NODDI in LPS rats at P7 or P14, while there was an increase in cortical fractional anisotropy (FA) and decrease in orientation dispersion index (ODI) at P21. CONCLUSIONS: EEG may be useful for identifying the early evolution of impaired cortical development after early life postnatal systemic inflammation, while DTI and NODDI seem to be more suited to assessing established cortical changes.

Early intensive rehabilitation reverses locomotor disruption, decrease brain inflammation and induces neuroplasticity following experimental Cerebral Palsy.

BACKGROUND: Cerebral Palsy (CP) is a major cause of motor and cognitive disability in children due to injury to the developing brain. Early intensive sensorimotor rehabilitation has been shown to change brain structure and reduce CP symptoms severity. We combined environmental enrichment (EE) and treadmill training (TT) to observe the effects of a one-week program of sensorimotor stimulation (EETT) in animals exposed to a CP model and explored possible mechanisms involved in the functional recovery. METHODS: Pregnant Wistar rats were injected with Lipopolysaccharide (LPS - 200 µg/kg) intraperitoneally at embryonic days 18 and 19. At P0, pups of both sexes were exposed to 20' anoxia at 37 °C. From P2 to P21, hindlimbs were restricted for 16 h/day during the dark cycle. EETT lasted from P21 to P27. TT - 15 min/day at 7 cm/s. EE - 7 days in enriched cages with sensorimotor stimulus. Functional 3D kinematic gait analysis and locomotion were analyzed. At P28, brains were collected for ex-vivo MRI and histological assessment. Neurotrophins and key proteins involved in CNS function were assessed by western blotting. RESULTS: CP model caused gross and skilled locomotor disruption and altered CNS neurochemistry. EETT reversed locomotor dysfunction with minor effects over gait kinematics. EETT also decreased brain inflammation and glial activation, preserved myelination, upregulated BDNF signaling and modulated the expression of proteins involved in excitatory synaptic function in the brain and spinal cord. CONCLUSIONS: Using this translational approach based on intensive sensorimotor rehabilitation, we highlight pathways engaged in the early developmental processes improving neurological recovery observed in CP.

Deleterious effect of sustained neuroinflammation in pediatric traumatic brain injury.

INTRODUCTION: Despite improved management of traumatic brain injury (TBI), it still leads to lifelong sequelae and disability, particularly in children. Chronic neuroinflammation (the so-called tertiary phase), in particular, microglia/macrophage and astrocyte reactivity, is among the main mechanisms suspected of playing a role in the generation of lesions associated with TBI. The role of acute neuroinflammation is now well understood, but its persistent effect and impact on the brain, particularly during development, are not. Here, we investigated the long-term effects of pediatric TBI on the brain in a mouse model. METHODS: Pediatric TBI was induced in mice on postnatal day (P) 7 by weight-drop trauma. The time course of neuroinflammation and myelination was examined in the TBI mice. They were also assessed by magnetic resonance, functional ultrasound, and behavioral tests at P45. RESULTS: TBI induced robust neuroinflammation, characterized by acute microglia/macrophage and astrocyte reactivity. The long-term consequences of pediatric TBI studied on P45 involved localized scarring astrogliosis, persistent microgliosis associated with a specific transcriptomic signature, and a long-lasting myelination defect consisting of the loss of myelinated axons, a decreased level of myelin binding protein, and severe thinning of the corpus callosum. These results were confirmed by reduced fractional anisotropy, measured by diffusion tensor imaging, and altered inter- and intra-hemispheric connectivity, measured by functional ultrasound imaging. In addition, adolescent mice with pediatric TBI showed persistent social interaction deficits and signs of anxiety and depressive behaviors. CONCLUSIONS: We show that pediatric TBI induces tertiary neuroinflammatory processes associated with white matter lesions and altered behavior. These results support our model as a model for preclinical studies for tertiary lesions following TBI.

Early Neuroprotective Effects of Bovine Lactoferrin Associated with Hypothermia after Neonatal Brain Hypoxia-Ischemia in Rats.

Neonatal hypoxic-ischemic (HI) encephalopathy (HIE) in term newborns is a leading cause of mortality and chronic disability. Hypothermia (HT) is the only clinically available therapeutic intervention; however, its neuroprotective effects are limited. Lactoferrin (LF) is the major whey protein in milk presenting iron-binding, anti-inflammatory and anti-apoptotic properties and has been shown to protect very immature brains against HI damage. We hypothesized that combining early oral administration of LF with whole body hypothermia could enhance neuroprotection in a HIE rat model. Pregnant Wistar rats were fed an LF-supplemented diet (1 mg/kg) or a control diet from (P6). At P7, the male and female pups had the right common carotid artery occluded followed by hypoxia (8% O2 for 60') (HI). Immediately after hypoxia, hypothermia (target temperature of 32.5-33.5 °C) was performed (5 h duration) using Criticool®. The animals were divided according to diet, injury and thermal condition. At P8 (24 h after HI), the brain neurochemical profile was assessed using magnetic resonance spectroscopy (1H-MRS) and a hyperintense T2W signal was used to measure the brain lesions. The mRNA levels of the genes related to glutamatergic excitotoxicity, energy metabolism and inflammation were assessed in the right hippocampus. The cell markers and apoptosis expression were assessed using immunofluorescence in the right hippocampus. HI decreased the energy metabolites and increased lactate. The neuronal-astrocytic coupling impairments observed in the HI groups were reversed mainly by HT. LF had an important effect on astrocyte function, decreasing the levels of the genes related to glutamatergic excitotoxicity and restoring the mRNA levels of the genes related to metabolic support. When combined, LF and HT presented a synergistic effect and prevented lactate accumulation, decreased inflammation and reduced brain damage, pointing out the benefits of combining these therapies. Overall, we showed that through distinct mechanisms lactoferrin can enhance neuroprotection induced by HT following neonatal brain hypoxia-ischemia.

Long-term coordinated microstructural disruptions of the developing neocortex and subcortical white matter after early postnatal systemic inflammation.

Severe postnatal systemic infection is highly associated with persistent disturbances in brain development and neurobehavioral outcomes in survivors of preterm birth. However, the contribution of less severe but prolonged postnatal infection and inflammation to such disturbances is unclear. Further, the ability of modern imaging techniques to detect the underlying changes in cellular microstructure of the brain in these infants remains to be validated. We used high-field ex-vivo MRI, neurohistopathology, and behavioral tests in newborn rats to demonstrate that prolonged postnatal systemic inflammation causes subtle, persisting disturbances in brain development, with neurodevelopmental delays and mild motor impairments. Diffusion-tensor MRI and neurite orientation dispersion and density imaging (NODDI) revealed delayed maturation of neocortical and subcortical white matter microstructure. Analysis of pyramidal neurons showed that the cortical deficits involved impaired dendritic arborization and spine formation. Analysis of oligodendrocytes showed that the white matter deficits involved impaired oligodendrocyte maturation and axonal myelination. These findings indicate that prolonged postnatal inflammation, without severe infection, may critically contribute to the diffuse spectrum of brain pathology and subtle long-term disability in preterm infants, with a cellular mechanism involving oligodendrocyte and neuronal dysmaturation. NODDI may be useful for clinical detection of these microstructural deficits.

Genetic and microstructural differences in the cortical plate of gyri and sulci during gyrification in fetal sheep.

Gyrification of the cerebral cortex is a developmentally important process, but the mechanisms that drive cortical folding are not fully known. Theories propose that changes within the cortical plate (CP) cause gyrification, yet differences between the CP below gyri and sulci have not been investigated. Here we report genetic and microstructural differences in the CP below gyri and sulci assessed before (at 70 days of gestational age [GA] 70), during (GA 90), and after (GA 110) gyrification in fetal sheep. The areal density of BDNF, CDK5, and NeuroD6 immunopositive cells were increased, and HDAC5 and MeCP2 mRNA levels were decreased in the CP below gyri compared with sulci during gyrification, but not before. Only the areal density of BDNF-immunopositive cells remained increased after gyrification. MAP2 immunoreactivity and neurite outgrowth were also increased in the CP below gyri compared with sulci at GA 90, and this was associated with microstructural changes assessed via diffusion tensor imaging and neurite orientation dispersion and density imaging at GA 98. Differential neurite outgrowth may therefore explain the localized changes in CP architecture that result in gyrification.

Magnetic Resonance Imaging Correlates of White Matter Gliosis and Injury in Preterm Fetal Sheep Exposed to Progressive Systemic Inflammation.

Progressive fetal infection/inflammation is strongly associated with neural injury after preterm birth. We aimed to test the hypotheses that progressively developing fetal inflammation leads to neuroinflammation and impaired white matter development and that the histopathological changes can be detected using high-field diffusion tensor magnetic resonance imaging (MRI). Chronically instrumented preterm fetal sheep at 0.7 of gestation were randomly assigned to receive intravenous saline (control; n = 6) or a progressive infusion of lipopolysaccharide (LPS, 200 ng intravenous over 24 h then doubled every 24 h for 5 days to induce fetal inflammation, n = 7). Sheep were killed 10 days after starting the infusions, for histology and high-field diffusion tensor MRI. Progressive LPS infusion was associated with increased circulating interleukin (IL)-6 concentrations and moderate increases in carotid artery perfusion and the frequency of electroencephalogram (EEG) activity (p < 0.05 vs. control). In the periventricular white matter, fractional anisotropy (FA) was increased, and orientation dispersion index (ODI) was reduced (p < 0.05 vs. control for both). Histologically, in the same brain region, LPS infusion increased microglial activation and astrocyte numbers and reduced the total number of oligodendrocytes with no change in myelination or numbers of immature/mature oligodendrocytes. Numbers of astrocytes in the periventricular white matter were correlated with increased FA and reduced ODI signal intensities. Astrocyte coherence was associated with increased FA. Moderate astrogliosis, but not loss of total oligodendrocytes, after progressive fetal inflammation can be detected with high-field diffusion tensor MRI.

Synbiotics Combined with Glutamine Stimulate Brain Development and the Immune System in Preterm Pigs.

Background: Preterm infants are born with an immature gut, brain, and immune system, predisposing them to short- and long-term complications. Objective: We hypothesized that a milk diet supplemented with pre- and probiotics (i.e. synbiotics) and glutamine would improve gut, brain, and immune maturation in preterm neonates, using preterm pigs as a model. Methods: Preterm pigs (Landrace x Yorkshire x Duroc, n = 40, delivered by c-section at 90% of gestation) were reared individually until day 23 after birth under highly standardized conditions. Piglets in the intervention group (PPG, n = 20) were fed increasing volumes of bovine milk supplemented with prebiotics (short-chain galacto- and long chain fructo-oligosaccharides 9:1, 4-12 g/L), probiotics (Bifidobacterium breve M16-V, 3 × 109 CFU/d) and l-glutamine [0.15-0.30 g/(kg · d)], and compared with pigs fed bovine milk with added placebo compounds as control (CON, n = 20). Clinical, gastrointestinal, immunological, cognitive, and neurological endpoints were measured. Results: The PPG pigs showed more diarrhea but weight gain, body composition, and gut parameters were similar between the groups. Cognitive performance, assessed in a T-maze, was significantly higher in PPG pigs (P < 0.01), whereas motor function and exploratory interest were similar between the groups. Using ex vivo diffusion imaging, the orientation dispersion index in brain cortical gray matter was 50% higher (P = 0.04), and fractional anisotropy value was 7% lower (P = 0.05) in PPG pigs compared with CON pigs, consistent with increased dendritic branching in PPG. In associative fibers, radial diffusivity was lower and fractional anisotropy was higher in PPG pigs compared with CON pigs (all P < 0.05), while measures in the internal capsule showed a tendency towards reduced radial diffusivity and mean diffusivity (both P = 0.09). On day 23 pigs in the PPG group showed higher blood leukocyte numbers (+43%), neutrophil counts (+100%), and phagocytic rates (+24%), relative to CON, all P < 0.05. Conclusion: Preterm pigs supplemented with Bifidobacterium breve, galacto- and fructo-oligosaccharides, and l-glutamine showed enhanced neuronal and immunological development. The findings indicate the potential for targeted nutritional interventions after preterm birth, to support development of important systems such as immunity and brain.

Multimodal MRI Imaging of Apoptosis-Triggered Microstructural Alterations in the Postnatal Cerebral Cortex.

Prematurely born children often develop neurodevelopmental delay that has been correlated with reduced growth and microstructural alterations in the cerebral cortex. Much research has focused on apoptotic neuronal cell death as a key neuropathological features following preterm brain injuries. How scattered apoptotic death of neurons may contribute to microstructural alterations remains unknown. The present study investigated in a rat model the effects of targeted neuronal apoptosis on cortical microstructure using in vivo MRI imaging combined with neuronal reconstruction and histological analysis. We describe that mild, targeted death of layer IV neurons in the developing rat cortex induces MRI-defined metabolic and microstructural alterations including increased cortical fractional anisotropy. Delayed architectural modifications in cortical gray matter and myelin abnormalities in the subcortical white matter such as hypomyelination and microglia activation follow the acute phase of neuronal death and axonal degeneration. These results establish the link between mild cortical apoptosis and MRI-defined microstructure changes that are reminiscent to those previously observed in preterm babies.

Mesenchymal stem cell-derived extracellular vesicles ameliorate inflammation-induced preterm brain injury.

OBJECTIVE: Preterm brain injury is a major cause of disability in later life, and may result in motor, cognitive and behavioural impairment for which no treatment is currently available. The aetiology is considered as multifactorial, and one underlying key player is inflammation leading to white and grey matter injury. Extracellular vesicles secreted by mesenchymal stem/stromal cells (MSC-EVs) have shown therapeutic potential in regenerative medicine. Here, we investigated the effects of MSC-EV treatment on brain microstructure and maturation, inflammatory processes and long-time outcome in a rodent model of inflammation-induced brain injury. METHODS: 3-Day-old Wistar rats (P3) were intraperitoneally injected with 0.25mg/kg lipopolysaccharide or saline and treated with two repetitive doses of 1×108 cell equivalents of MSC-EVs per kg bodyweight. Cellular degeneration and reactive gliosis at P5 and myelination at P11 were evaluated by immunohistochemistry and western blot. Long-term cognitive and motor function was assessed by behavioural testing. Diffusion tensor imaging at P125 evaluated long-term microstructural white matter alterations. RESULTS: MSC-EV treatment significantly ameliorated inflammation-induced neuronal cellular degeneration reduced microgliosis and prevented reactive astrogliosis. Short-term myelination deficits and long-term microstructural abnormalities of the white matter were restored by MSC-EV administration. Morphological effects of MSC-EV treatment resulted in improved long-lasting cognitive functions INTERPRETATION: MSC-EVs ameliorate inflammation-induced cellular damage in a rat model of preterm brain injury. MSC-EVs may serve as a novel therapeutic option by prevention of neuronal cell death, restoration of white matter microstructure, reduction of gliosis and long-term functional improvement.

Transcriptomic regulations in oligodendroglial and microglial cells related to brain damage following fetal growth restriction.

Fetal growth restriction (FGR) is a major complication of human pregnancy, frequently resulting from placental vascular diseases and prenatal malnutrition, and is associated with adverse neurocognitive outcomes throughout life. However, the mechanisms linking poor fetal growth and neurocognitive impairment are unclear. Here, we aimed to correlate changes in gene expression induced by FGR in rats and abnormal cerebral white matter maturation, brain microstructure, and cortical connectivity in vivo. We investigated a model of FGR induced by low-protein-diet malnutrition between embryonic day 0 and birth using an interdisciplinary approach combining advanced brain imaging, in vivo connectivity, microarray analysis of sorted oligodendroglial and microglial cells and histology. We show that myelination and brain function are both significantly altered in our model of FGR. These alterations, detected first in the white matter on magnetic resonance imaging significantly reduced cortical connectivity as assessed by ultrafast ultrasound imaging. Fetal growth retardation was found associated with white matter dysmaturation as shown by the immunohistochemical profiles and microarrays analyses. Strikingly, transcriptomic and gene network analyses reveal not only a myelination deficit in growth-restricted pups, but also the extensive deregulation of genes controlling neuroinflammation and the cell cycle in both oligodendrocytes and microglia. Our findings shed new light on the cellular and gene regulatory mechanisms mediating brain structural and functional defects in malnutrition-induced FGR, and suggest, for the first time, a neuroinflammatory basis for the poor neurocognitive outcome observed in growth-restricted human infants. GLIA 2016;64:2306-2320.

Fingolimod protects against neonatal white matter damage and long-term cognitive deficits caused by hyperoxia.

Cerebral white matter injury is a leading cause of adverse neurodevelopmental outcome in prematurely born infants involving cognitive deficits in later life. Despite increasing knowledge about the pathophysiology of perinatal brain injury, therapeutic options are limited. In the adult demyelinating disease multiple sclerosis the sphingosine-1-phosphate (S1P) receptor modulating substance fingolimod (FTY720) has beneficial effects. Herein, we evaluated the neuroprotective potential of FTY720 in a neonatal model of oxygen-toxicity, which is associated with hypomyelination and impaired neuro-cognitive outcome. A single dose of FTY720 (1mg/kg) at the onset of neonatal hyperoxia (24h 80% oxygen on postnatal day 6) resulted in improvement of neuro-cognitive development persisting into adulthood. This was associated with reduced microstructural white matter abnormalities 4 months after the insult. In search of the underlying mechanisms potential non-classical (i.e. lymphocyte-independent) pathways were analysed shortly after the insult, comprising modulation of oxidative stress and local inflammatory responses as well as myelination, oligodendrocyte degeneration and maturation. Treatment with FTY720 reduced hyperoxia-induced oxidative stress, microglia activation and associated pro-inflammatory cytokine expression. In vivo and in vitro analyses further revealed that oxygen-induced hypomyelination is restored to control levels, which was accompanied by reduced oligodendrocyte degeneration and enhanced maturation. Furthermore, hyperoxia-induced elevation of S1P receptor 1 (S1P1) protein expression on in vitro cultured oligodendrocyte precursor cells was reduced by activated FTY720 and protection from degeneration is abrogated after selective S1P1 blockade. Finally, FTY720s' classical mode of action (i.e. retention of immune cells within peripheral lymphoid organs) was analysed demonstrating that FTY720 diminished circulating lymphocyte counts independent from hyperoxia. Cerebral immune cell counts remained unchanged by hyperoxia and by FTY720 treatment. Taken together, these results suggest that beneficial effects of FTY720 in neonatal oxygen-induced brain injury may be rather attributed to its anti-oxidative and anti-inflammatory capacity acting in concert with a direct protection of developing oligodendrocytes than to a modulation of peripheral lymphocyte trafficking. Thus, FTY720 might be a potential new therapeutic option for the treatment of neonatal brain injury through reduction of white matter damage.

Glutathione Deficit Affects the Integrity and Function of the Fimbria/Fornix and Anterior Commissure in Mice: Relevance for Schizophrenia.

BACKGROUND: Structural anomalies of white matter are found in various brain regions of patients with schizophrenia and bipolar and other psychiatric disorders, but the causes at the cellular and molecular levels remain unclear. Oxidative stress and redox dysregulation have been proposed to play a role in the pathophysiology of several psychiatric conditions, but their anatomical and functional consequences are poorly understood. The aim of this study was to investigate white matter throughout the brain in a preclinical model of redox dysregulation. METHODS: In a mouse model with impaired glutathione synthesis (Gclm KO), a state-of-the-art multimodal magnetic resonance protocol at high field (14.1 T) was used to assess longitudinally the white matter structure, prefrontal neurochemical profile, and ventricular volume. Electrophysiological recordings in the abnormal white matter tracts identified by diffusion tensor imaging were performed to characterize the functional consequences of fractional anisotropy alterations. RESULTS: Structural alterations observed at peri-pubertal age and adulthood in Gclm KO mice were restricted to the anterior commissure and fornix-fimbria. Reduced fractional anisotropy in the anterior commissure (-7.5% ± 1.9, P<.01) and fornix-fimbria (-4.5% ± 1.3, P<.05) were accompanied by reduced conduction velocity in fast-conducting fibers of the posterior limb of the anterior commissure (-14.3% ± 5.1, P<.05) and slow-conducting fibers of the fornix-fimbria (-8.6% ± 2.6, P<.05). Ventricular enlargement was found at peri-puberty (+25% ± 8 P<.05) but not in adult Gclm KO mice. CONCLUSIONS: Glutathione deficit in Gclm KO mice affects ventricular size and the integrity of the fornix-fimbria and anterior commissure. This suggests that redox dysregulation could contribute during neurodevelopment to the impaired white matter and ventricle enlargement observed in schizophrenia and other psychiatric disorders.

MRI of animal models of developmental disorders and translation to human imaging.

PURPOSE OF REVIEW: In order to understand the pathophysiological mechanisms leading to the specific brain alterations observed in immature newborn babies, preclinical studies on animal models mimicking clinical reality are mandatory and are ideally coupled with imaging modalities transferable to the human scenario. The availability of MRI techniques on both clinical and animal scanners allows this methodological transfer from bench to bedside. The aim of this review is to give an overview of the recent findings in MRI of animal models of developmental disorders and emphasize what we can learn from MRI on these models. RECENT FINDINGS: Progress in newborn medicine has allowed the survival of increasingly immature newborns that is often associated with specific morbidities. The brain in particular shows developmentally linked vulnerability leading to specific brain injury and subsequent developmental disorders. MRI delivers a large amount of anatomical, microstructural and functional information and has been widely used to monitor cerebral development and characterize the specificity of brain lesions in the immature brain in humans and animal models. SUMMARY: In this review, we will present the different animal models assessed by magnetic resonance techniques and the histopathological correlations observed, as well as the implications for human imaging.

Protective effects of maternal nutritional supplementation with lactoferrin on growth and brain metabolism.

BACKGROUND: Intrauterine growth restriction (IUGR) is a major risk factor for both perinatal and long-term morbidity. Bovine lactoferrin (bLf) is a major milk glycoprotein considered as a pleiotropic functional nutrient. The impact of maternal supplementation with bLf on IUGR-induced sequelae, including inadequate growth and altered cerebral development, remains unknown. METHODS: IUGR was induced through maternal dexamethasone infusion (100 µg/kg during last gestational week) in rats. Maternal supplementation with bLf (0.85% in food pellet) was provided during both gestation and lactation. Pup growth was monitored, and Pup brain metabolism and gene expression were studied using in vivo (1)H NMR spectroscopy, quantitative PCR, and microarray in the hippocampus at postnatal day (PND)7. RESULTS: Maternal bLf supplementation did not change gestational weight but increased the birth body weight of control pups (4%) with no effect on the IUGR pups. Maternal bLf supplementation allowed IUGR pups to recover a normalized weight at PND21 (weaning) improving catch-up growth. Significantly altered levels of brain metabolites (γ-aminobutyric acid, glutamate, N-acetylaspartate, and N-acetylaspartylglutamate) and transcripts (brain-derived neurotrophic factor (BDNF), divalent metal transporter 1 (DMT-1), and glutamate receptors) in IUGR pups were normalized with maternal bLf supplementation. CONCLUSION: Our data suggest that maternal bLf supplementation is a beneficial nutritional intervention able to revert some of the IUGR-induced sequelae, including brain hippocampal changes.

Mesenchymal stem cells restore cortical rewiring after neonatal ischemia in mice.

OBJECTIVE: A study was undertaken to investigate the effect of neonatal hypoxic-ischemic (HI) brain damage and mesenchymal stem cell (MSC) treatment on the structure and contralesional connectivity of motor function-related cerebral areas. METHODS: Brain remodeling after HI±MSC treatment in neonatal mice was analyzed using diffusion tensor magnetic resonance imaging, immunohistochemistry, anterograde tracing with biotinylated dextran amine (BDA), and retrograde tracing with fluorescent pseudorabies virus (PRV). RESULTS: MSC treatment after HI reduced contralesional rewiring taking place after HI. Following MSC treatment, fractional anisotropy values, which were increased in both ipsi- and contralesional cortices and decreased in the corpus callosum (CC) after HI, were normalized to the level observed in sham-operated mice. These results were corroborated by myelin basic protein intensity and staining pattern in these areas. Anterograde tracing of ipsilesional motor neurons showed that after MSC treatment, fewer BDA-positive fibers crossed the CC and extended into the contralesional motor cortex compared to HI mice. This remodeling was functional, because retrograde labeling showed increased connectivity between impaired (left) forepaw and the contralesional (left) motor cortex after HI, whereas MSC treatment reduced this connection and increased the connection between the impaired (left) forepaw and the ipsilesional (right) motor cortex. Finally, the extent of contralesional rewiring measured with BDA and PRV tracing was related to sensorimotor dysfunction. INTERPRETATION: This is the first study to describe MSC treatment after neonatal HI markedly reducing contralesional axonal remodeling induced by HI brain injury.

Investigation of field and diffusion time dependence of the diffusion-weighted signal at ultrahigh magnetic fields.

Over the last decade, there has been a significant increase in the number of high-magnetic-field MRI magnets. However, the exact effect of a high magnetic field strength (B0 ) on diffusion-weighted MR signals is not yet fully understood. The goal of this study was to investigate the influence of different high magnetic field strengths (9.4 T and 14.1 T) and diffusion times (9, 11, 13, 15, 17 and 24 ms) on the diffusion-weighted signal in rat brain white matter. At a short diffusion time (9 ms), fractional anisotropy values were found to be lower at 14.1 T than at 9.4 T, but this difference disappeared at longer diffusion times. A simple two-pool model was used to explain these findings. The model describes the white matter as a first hindered compartment (often associated with the extra-axonal space), characterized by a faster orthogonal diffusion and a lower fractional anisotropy, and a second restricted compartment (often associated with the intra-axonal space), characterized by a slower orthogonal diffusion (i.e. orthogonal to the axon direction) and a higher fractional anisotropy. Apparent T2 relaxation time measurements of the hindered and restricted pools were performed. The shortening of the pseudo-T2 value from the restricted compartment with B0 is likely to be more pronounced than the apparent T2 changes in the hindered compartment. This study suggests that the observed differences in diffusion tensor imaging parameters between the two magnetic field strengths at short diffusion time may be related to differences in the apparent T2 values between the pools.

Low-molecular weight sulfated marine polysaccharides: Promising molecules to prevent neurodegeneration in mucopolysaccharidosis IIIA?

Mucopolysaccharidosis IIIA is a hereditary disease caused by mutations in the sulfamidase enzyme that participates in catabolism of heparan sulfate (HS), leading to HS fragment accumulation and multisystemic failure. No cure exists and death occurs around the second decade of life. Two low molecular weight highly sulfated compounds derived from marine diabolican and infernan exopolysaccharides (A5_3 and A5_4, respectively) with heparanase inhibiting properties were tested in a MPSIIIA cell line model, resulting in limited degradation of intracellular HS. Next, we observed the effects of intraperitoneal injections of the diabolican derivative A5_3 from 4 to 12 weeks of age on MPSIIIA mice. Brain metabolism and microstructure, levels of proteins and genes involved in MPSIIIA brain pathophysiology were also investigated. 1H-Magnetic Resonance Spectroscopy (MRS) indicated deficits in energetic metabolism, tissue integrity and neurotransmission at both 4 and 12 weeks in MPSIIIA mice, with partial protective effects of A5_3. Ex-vivo Diffusion Tensor Imaging (DTI) showed white matter microstructural damage in MPSIIIA, with noticeable protective effects of A5_3. Protein and gene expression assessments displayed both pro-inflammatory and pro-apoptotic profiles in MPSIIIA mice, with benefits of A5_3 counteracting neuroinflammation. Overall, derivative A5_3 was well tolerated and was shown to be efficient in preventing brain metabolism failure and inflammation, resulting in preserved brain microstructure in the context of MPSIIIA.

Delayed cortical impairment following lipopolysaccharide exposure in preterm fetal sheep.

OBJECTIVE: Preterm infants exhibit chronic deficits in white matter (WM) and cortical maturation. Although fetal infection/inflammation may contribute to WM pathology, the factors contributing to cortical changes are largely unknown. We examined the effect of fetal lipopolysaccharide (LPS) exposure on WM and cortical development as assessed by magnetic resonance imaging (MRI), electroencephalography (EEG), and histopathology in fetal sheep at preterm human equivalent age. METHODS: LPS was administered to fetal sheep at 102.5 ± 0.5 days of gestation. Continuous biophysical recordings were analyzed for 10 days after LPS. At postmortem, measurement of cerebral WM and cortical tissue volumes was achieved by stereological techniques. Specific effects of LPS on MRI-assessed T(1)-weighted and T(2)-weighted images, and immunohistochemical expression of oligodendrocytes, proliferating cells, cortical NeuN-positive and Nurr1-positive neurons (subplate marker), and cell death mechanisms were examined. RESULTS: We observed reductions in WM (~21%; LPS, 1.19 ± 0.04 vs control, 1.51 ± 0.07 cm(3); p < 0.001) and cortical (~18%; LPS, 2.34 ± 0.10 vs control, 2.85 ± 0.07 cm(3); p < 0.001) volumes, associated with overt and diffuse WM injury, T(1)-/T(2) -weighted signal alterations, and reduced numbers of WM oligodendrocytes (LPS, 485 ± 31 vs control, 699 ± 69 cells/mm(2); p = 0.0189) and NeuN-positive (LPS, 421 ± 71 vs control 718 ± 92 cells/mm(2); p = 0.04) and Nurr1-positive (control, 2.5 ± 0.6 vs LPS, 0.6 ± 0.1 cells/mm(2); p = 0.007) cortical neurons after LPS. Moreover, there was loss of the normal maturational increase in cortical EEG amplitude, which correlated with reduced cortical volumes. INTERPRETATION: Fetal exposure to LPS prior to myelination onset can impair both white matter and cortical development in a preclinical large animal model, supporting a role for maternal/fetal infection in the pathogenesis of preterm brain injury.

Systemic inflammation disrupts the developmental program of white matter.

OBJECTIVE: Perinatal inflammation is a major risk factor for neurological deficits in preterm infants. Several experimental studies have shown that systemic inflammation can alter the programming of the developing brain. However, these studies do not offer detailed pathophysiological mechanisms, and they rely on relatively severe infectious or inflammatory stimuli that most likely do not reflect the levels of systemic inflammation observed in many human preterm infants. The goal of the present study was to test the hypothesis that moderate systemic inflammation is sufficient to alter white matter development. METHODS: Newborn mice received twice-daily intraperitoneal injections of interleukin-1ß (IL-1ß) over 5 days and were studied for myelination, oligodendrogenesis, and behavior and with magnetic resonance imaging (MRI). RESULTS: Mice exposed to IL-1ß had a long-lasting myelination defect that was characterized by an increased number of nonmyelinated axons. They also displayed a reduction of the diameter of the myelinated axons. In addition, IL-1ß induced a significant reduction of the density of myelinating oligodendrocytes accompanied by an increased density of oligodendrocyte progenitors, suggesting a partial blockade in the oligodendrocyte maturation process. Accordingly, IL-1ß disrupted the coordinated expression of several transcription factors known to control oligodendrocyte maturation. These cellular and molecular abnormalities were correlated with a reduced white matter fractional anisotropy on diffusion tensor imaging and with memory deficits. INTERPRETATION: Moderate perinatal systemic inflammation alters the developmental program of the white matter. This insult induces a long-lasting myelination deficit accompanied by cognitive defects and MRI abnormalities, further supporting the clinical relevance of the present data.