Experimental cerebral palsy causes microstructural brain damage in areas associated to motor deficits but no spatial memory impairments in the developing rat.

INTRODUCTION: Cerebral palsy (CP) is the major cause of motor and cognitive impairments during childhood. CP can result from direct or indirect structural injury to the developing brain. In this study, we aimed to describe brain damage and behavioural alterations during early adult life in a CP model using the combination of maternal inflammation, perinatal anoxia and postnatal sensorimotor restriction. METHODS: Pregnant Wistar rats were injected intraperitoneally with 200 µg/kg LPS at embryonic days E18 and E19. Between 3 and 6 h after birth (postnatal day 0 - PND0), pups of both sexes were exposed to anoxia for 20 min. From postnatal day 2 to 21, hindlimbs of animals were immobilized for 16 h daily during their active phase. From PND40, locomotor and cognitive tests were performed using Rota-Rod, Ladder Walking and Morris water Maze. Ex-vivo MRI Diffusion Tensor Imaging (DTI) and Neurite Orientation Dispersion and Density Imaging (NODDI) were used to assess macro and microstructural damage and brain volume alterations induced by the model. Myelination and expression of neuronal, astroglial and microglial markers, as well as apoptotic cell death were evaluated by immunofluorescence. RESULTS: CP animals showed decreased body weight, deficits in gross (rota-rod) and fine (ladder walking) motor tasks compared to Controls. No cognitive impairments were observed. Ex-vivo MRI showed decreased brain volumes and impaired microstructure in the cingulate gyrus and sensory cortex in CP brains. Histological analysis showed increased cell death, astrocytic reactivity and decreased thickness of the corpus callosum and altered myelination in CP animals. Hindlimb primary motor cortex analysis showed increased apoptosis in CP animals. Despite the increase in NeuN and GFAP, no differences between groups were observed as well as no co-localization with the apoptotic marker. However, an increase in Iba-1+ microglia with co-localization to cleaved caspase 3 was observed. CONCLUSION: Our results suggest that experimental CP induces long-term brain microstructural alterations in myelinated structures, cell death in the hindlimb primary motor cortex and locomotor impairments. Such new evidence of brain damage could help to better understand CP pathophysiological mechanisms and guide further research for neuroprotective and neurorehabilitative strategies for CP patients.

Functional, neuroplastic and biomechanical changes induced by early Hand-Arm Bimanual Intensive Therapy Including Lower Extremities (e-HABIT-ILE) in pre-school children with unilateral cerebral palsy: study protocol of a randomized control trial.

BACKGROUND: Cerebral palsy (CP) causes motor, cognitive and sensory impairment at different extents. Many recent rehabilitation developments (therapies) have focused solely on the upper extremities (UE), although the lower extremities (LE) are commonly affected. Hand-arm Bimanual Intensive Therapy Including Lower Extremities (HABIT-ILE) applies the concepts of motor skill learning and intensive training to both the UE and LE. It involves constant stimulation of the UE and LE, for several hours each day over a 2-week period. The effects of HABIT-ILE have never been evaluated in a large sample of young children. Furthermore, understanding of functional, neuroplastic and biomechanical changes in infants with CP is lacking. The aim of this study is to carry out a multi-center randomized controlled trial (RCT) to evaluate the effects of HABIT-ILE in pre-school children with unilateral CP on functional, neuroplastic and biomechanical parameters. METHODS: This multi-center, 3-country study will include 50 pre-school children with CP aged 1-4 years. The RCT will compare the effect of 50 h (two weeks) of HABIT-ILE versus usual motor activity, including regular rehabilitation. HABIT-ILE will be delivered in a day-camp setting, with structured activities and functional tasks that will be continuously progressed in terms of difficulty. Assessments will be performed at 3 intervals: baseline (T0), two weeks later and 3 months later. Primary outcomes will be the Assisting Hand Assessment; secondary outcomes include the Melbourne Assessment-2, executive function assessments, questionnaires ACTIVLIM-CP, Pediatric Evaluation of Disability Inventory, Young Children's Participation and Environment Measure, Measure of the Process of Care, Canadian Occupational Performance Measure, as well as neuroimaging and kinematics measures. DISCUSSION: We expect that HABIT-ILE will induce functional, neuroplastic and biomechanical changes as a result of the intense, activity-based rehabilitation process and these changes will impact the whole developmental curve of each child, improving functional ability, activity and participation in the short-, mid- and long-term. Name of the registry: Changes Induced by Early HABIT-ILE in Pre-school Children With Uni- and Bilateral Cerebral Palsy (EarlyHABIT-ILE). TRIAL REGISTRATION: Trial registration number: NCT04020354-Registration date on the International Clinical Trials Registry Platform (ICTRP): November 20th, 2018; Registration date on NIH Clinical Trials Registry: July 16th, 2019.

Structural brain maturation differs between preterm and term piglets, whereas brain activity does not.

AIM: The aim of the study was to investigate whether amplitude-integrated electroencephalography (aEEG) and cerebral magnetic resonance imaging (MRI) in preterm piglets would provide measures of cerebral functional, microstructural and anatomical maturation, which might reflect the signs of functional brain immaturity, documented in preterm piglets. METHODS: During July-October 2013 at the NEOMUNE Centre, Copenhagen University, Denmark, 31 preterm (90% gestation) and 10 term piglets underwent aEEG on days 1, 2, 4 and 11, and MRI on day 25. Physical activity levels were recorded. RESULTS: Preterm showed delayed neonatal arousal and physical activity, relative to term piglets. Preterm piglets had lower growth rates and brain volume than term piglets, but aEEG patterns were similar. MRI mean diffusivity was also similar, but fractional anisotropy (FA) was lower in preterm piglets (p < 0.001). CONCLUSION: Functional brain maturation, as assessed by aEEG, was relatively advanced in preterm piglets. Conversely, the low FA in the preterm piglets suggests that the white matter microstructure remains less mature in preterm compared to term piglets at postnatal day 25. The results might be utilised to define whether and how preterm piglets may contribute to preclinical models for brain development in preterm infants.

Pregnancy as a valuable period for preventing hypoxia-ischemia brain damage.

Neonatal brain Hypoxia-Ischemia (HI) is one of the major causes of infant mortality and lifelong neurological disabilities. The knowledge about the physiopathological mechanisms involved in HI lesion have increased in recent years, however these findings have not been translated into clinical practice. Current therapeutic approaches remain limited; hypothermia, used only in term or near-term infants, is the golden standard. Epidemiological evidence shows a link between adverse prenatal conditions and increased risk for diseases, health problems, and psychological outcomes later in life, what makes pregnancy a relevant period for preventing future brain injury. Here, we review experimental literature regarding preventive interventions used during pregnancy, i.e., previous to the HI injury, encompassing pharmacological, nutritional and/or behavioral strategies. Literature review used PubMed database. A total of forty one studies reported protective properties of maternal treatments preventing perinatal hypoxia-ischemia injury in rodents. Pharmacological agents and dietary supplementation showed mainly anti-excitotoxicity, anti-oxidant or anti-apoptotic properties. Interestingly, maternal preconditioning, physical exercise and environmental enrichment seem to engage the same referred mechanisms in order to protect neonatal brain against injury. This construct must be challenged by further studies to clearly define the main mechanisms responsible for neuroprotection to be explored in experimental context, as well as to test their potential in clinical settings.

[Neurodevelopmental follow-up of premature children in Lausanne and Geneva]. / Suivi neurodéveloppemental de l'enfant né prématuré dans l'Arc lémanique.

Preterm children born before 32 weeks of gestation represent 1% of the annual births in Switzerland, and are the most at risk of neurodevelopmental disabilities. A neurological surveillance is thus implemented in the neonatal units, and multidisciplinary neurodevelopmental follow-up is offered to all our preterm patients. The follow-up clinics of the University hospitals in Lausanne and Geneva follow the Swiss guidelines for follow-up. An extended history and neurological examination is taken at each appointment, and a standardized test of development is performed. These examinations, which take place between the ages of 3 months and 9 years old, allow the early identification and treatment of developmental disorders frequent in this population, such as motor, cognitive or behavioral disorders, as well as the monitoring of the quality of neonatal care.

[Erythropoietin and neuroprotection]. / Erythropoïétine et neuroprotection.

Erythropoietin (Epo) has long been recognised for its role in the control of erythropoiesis and therefore in the treatment of anemia including anemia of prematurity. The erythropoietin receptor (Epo-R) though is expressed in many other organs including the CNS. This review focuses on the role of erythropoietin during the development of the CNS and its potential role as a neuroprotective agent. Epo-R is expressed in many different cellules of the CNS during development including neural progenitor cells, neurons, astrocytes and oligodendrocytes. In the event of hypoxia CNS cells respond with increase of erythropoietin release with subsequent stimulation of neurogenesis through Epo-R on neural progenitor cells. In an Epo-R knock-out model therefore cerebral development is severely impaired. In models of hypoxia-ischemia exogenous Epo has been shown to reduce lesion size and improve structural and functional recovery. Human studies are emerging using Epo as a neuroprotective agent both for the term infant with hypoxia-ischemia as well as for the extremely preterm infant.

Diffusion-weighted spectroscopy: a novel approach to determine macromolecule resonances in short-echo time 1H-MRS.

Quantification of short-echo time proton magnetic resonance spectroscopy results in >18 metabolite concentrations (neurochemical profile). Their quantification accuracy depends on the assessment of the contribution of macromolecule (MM) resonances, previously experimentally achieved by exploiting the several fold difference in T(1). To minimize effects of heterogeneities in metabolites T(1), the aim of the study was to assess MM signal contributions by combining inversion recovery (IR) and diffusion-weighted proton spectroscopy at high-magnetic field (14.1 T) and short echo time (= 8 msec) in the rat brain. IR combined with diffusion weighting experiments (with δ/Δ = 1.5/200 msec and b-value = 11.8 msec/µm(2)) showed that the metabolite nulled spectrum (inversion time = 740 msec) was affected by residuals attributed to creatine, inositol, taurine, choline, N-acetylaspartate as well as glutamine and glutamate. While the metabolite residuals were significantly attenuated by 50%, the MM signals were almost not affected (< 8%). The combination of metabolite-nulled IR spectra with diffusion weighting allows a specific characterization of MM resonances with minimal metabolite signal contributions and is expected to lead to a more precise quantification of the neurochemical profile.

Primary cortical folding in the human newborn: an early marker of later functional development.

In the human brain, the morphology of cortical gyri and sulci is complex and variable among individuals, and it may reflect pathological functioning with specific abnormalities observed in certain developmental and neuropsychiatric disorders. Since cortical folding occurs early during brain development, these structural abnormalities might be present long before the appearance of functional symptoms. So far, the precise mechanisms responsible for such alteration in the convolution pattern during intra-uterine or post-natal development are still poorly understood. Here we compared anatomical and functional brain development in vivo among 45 premature newborns who experienced different intra-uterine environments: 22 normal singletons, 12 twins and 11 newborns with intrauterine growth restriction (IUGR). Using magnetic resonance imaging (MRI) and dedicated post-processing tools, we investigated early disturbances in cortical formation at birth, over the developmental period critical for the emergence of convolutions (26-36 weeks of gestational age), and defined early 'endophenotypes' of sulcal development. We demonstrated that twins have a delayed but harmonious maturation, with reduced surface and sulcation index compared to singletons, whereas the gyrification of IUGR newborns is discordant to the normal developmental trajectory, with a more pronounced reduction of surface in relation to the sulcation index compared to normal newborns. Furthermore, we showed that these structural measurements of the brain at birth are predictors of infants' outcome at term equivalent age, for MRI-based cerebral volumes and neurobehavioural development evaluated with the assessment of preterm infant's behaviour (APIB).

[Intrauterine growth restriction: impact on brain development and function]. / Retard de croissance intra-utérin: impact sur le développement et tla fonction cérébrale.

Evidence exists that the developing organism adapts to the environment it finds itself. Short and long-term adjustments take place and will initially induce intrauterine growth retardation but will also have consequences that will appear later in life. These adjustments are referred as "programming". The use of advanced magnetic resonance imaging techniques in IUGR babies has delineated changes in the development of the central nervous system that correlate with altered neurodevelopment and could be implicated in the development of neuropsychiatric disorders in adult life. In this review, we will delineate some modifications of CNS development and functions that occur after exposition to adverse environment and that can now be studied in vivo with advanced imaging technology.

Mapping the early cortical folding process in the preterm newborn brain.

In the developing human brain, the cortical sulci formation is a complex process starting from 14 weeks of gestation onward. The potential influence of underlying mechanisms (genetic, epigenetic, mechanical or environmental) is still poorly understood, because reliable quantification in vivo of the early folding is lacking. In this study, we investigate the sulcal emergence noninvasively in 35 preterm newborns, by applying dedicated postprocessing tools to magnetic resonance images acquired shortly after birth over a developmental period critical for the human cortex maturation (26-36 weeks of age). Through the original three-dimensional reconstruction of the interface between developing cortex and white matter and correlation with volumetric measurements, we document early sulcation in vivo, and quantify changes with age, gender, and the presence of small white matter lesions. We observe a trend towards lower cortical surface, smaller cortex, and white matter volumes, but equivalent sulcation in females compared with males. By precisely mapping the sulci, we highlight interindividual variability in time appearance and interhemispherical asymmetries, with a larger right superior temporal sulcus than the left. Thus, such an approach, included in a longitudinal follow-up, may provide early indicators on the structural basis of cortical functional specialization and abnormalities induced by genetic and environmental factors.

Neuroprotective effects of the N-terminal tripeptide of IGF-1, glycine-proline-glutamate, in the immature rat brain after hypoxic-ischemic injury.

Insulin growth factor 1 (IGF-1) has an important role in brain development and is strongly expressed during recovery after a hypoxic-ischemic injury. Some of its central actions could be mediated through the N-terminal tripeptide fragment of IGF-1: Gly-Pro-Glu (GPE). The neuroprotective properties of GPE given after a moderate injury in the developing rat brain were evaluated and the binding sites of [(3)H]GPE characterised by autoradiography. After right unilateral injury, GPE or vehicle (V) was injected in the right lateral ventricle (i.c.v.) or in the peritoneal cavity (i.p.) of 21-day-old rats. The percentage of surviving neurons in CA1-2 of the hippocampus was higher in the animals treated with 30 microg of GPE i.c.v. (V: 7.7+/-4.9%, GPE: 26.4+/-7.5%, P=0.02) and 300 microg i.p. (V: 30.2+/-9.1%, GPE: 68.8+/-10.6%, P=0.02) than in animals receiving vehicle. I.p. injection of 300 microg of GPE (V: 78.4+/-7.5%, GPE: 88.4+/-3.2%, P=0.04) was also neuroprotective in the lateral cortex. I.c.v. injection of [(3)H]GPE suggested binding to glial cells in the white matter tracts, the cortex and striatum as opposed to neurons. Although the precise mode of action of GPE is unknown, this study suggests that local administration of GPE is neuroprotective after brain HI injury via glial cells. In addition, systemic administration of GPE showed a more widespread neuroprotective effect. GPE may represent a complementary pathway for central and systemic IGF-1's antiapoptotic effects.

The transition from fetus to neonate--an endocrine perspective.

The transition from fetus to neonate involves three phases: late gestation, parturition and the processes needed to establish independent homoeostatic regulation after separation from the placenta. These phases are regulated by a series of fetal and placental endocrine events. Glucocorticoids have an important role in the preparation for birth, including involvement in lung and cardiac development, and the maturation of enzymes in a variety of pathways. Fetal cortisol production is, in turn, also under hormonal control. Parturition is a complex process, which is still poorly understood in humans. The final steps are largely dependent on the effect of prostaglandin F2 alpha on the myometrium associated with increased oxytocin activity. The transition to birth is accompanied by changes in respiration, circulation, glucose homoeostasis, and the onset of independent oral feeding and thermoregulation. Several examples of endocrine components of the transition from fetal to neonatal life are reviewed here: the role of prostanoids, the onset of thermogenesis, and changes in the thyroid hormone and growth hormone axes. The effects of hormone levels on prematurity and growth retardation are also discussed.

Slow cleavage at the proinsulin B-chain/connecting peptide junction associated with low levels of endoprotease PC1/3 in transformed beta cells.

Conversion of rat proinsulins I and II was slower in transformed INS cells than in primary (islet) beta cells, with accumulation of des-64,65 but no detectable des-31,32-split proinsulin, indicating slow cleavage at the B-chain/connecting peptide (C-peptide) junction. Western blot analysis showed lower levels of the endoprotease PC1/3 in INS cells than in beta cells, as well as a 4-fold reduction in the ratio of PC1/3 to PC2, thus supporting the hypothesis that PC1/3 is the endoprotease responsible for cleavage at the B-chain/C-peptide junction.

Kinetics of proinsulin conversion in human islets.

Islets isolated from human cadaver pancreas were pulse-labeled (10 min with [3H]leucine) and then incubated for a 180-min chase. Islets and chase medium were collected every 15 min and analyzed by reversed-phase HPLC to quantify the percentage of radioactively labeled proinsulin, conversion intermediates, and fully processed insulin. Release of proinsulin-related labeled products into the chase medium was < 10% of total. Whereas 50% of labeled proinsulin had been lost by conversion within 45 min, fully processed insulin only appeared with a half-time of 100 min. This discrepancy is attributable to accumulation of radioactive conversion intermediates. Des 64.65 split proinsulin was a minor component, reaching a maximum of 5.2 +/- 1.7% (n = 4) at 60 min of chase. By contrast, des 31.32 split proinsulin--and a truncated form lacking the first three residues of C-peptide--rose progressively to 29.3 +/- 1.4% by 75 min, and declined thereafter. The accumulation of des 31.32 split proinsulin rather than the des 64.65 split form during the conversion of human proinsulin reflects slower conversion at the C-peptide/A-chain than at the B-chain/C-peptide junction, and is consistent with the appearance of this particular conversion intermediate in the circulation.

Differential rates of conversion of rat proinsulins I and II. Evidence for slow cleavage at the B-chain/C-peptide junction of proinsulin II.

Rat proinsulin I is converted into insulin more rapidly than is proinsulin II. To study this further, rat islets were labelled (10 min) and conversion kinetics of the labelled proinsulins were monitored during a 120 min chase. Proinsulins, conversion intermediates and both insulins were separated by h.p.l.c. The accumulation of des-64,65-(split proinsulin II) during the chase suggests that the B-chain/C-peptide junction of proinsulin II is cleaved more slowly than the equivalent site on proinsulin I. This accounts for the differential kinetics of conversion of proinsulins I and II, and is presumed to be caused by one (or more) of the amino acid replacements which distinguish the two proinsulins.

Cultured human epidermis cells produce cell-associated interleukin 1-like prostaglandin E2- and collagenase-stimulating factors.

In order to identify factors which may regulate the functions of dermal fibroblasts, cell lysates and conditioned media of cultured human epidermal cells were tested on dermal fibroblasts for the stimulation of prostaglandin E2- and collagenase-production. Both prostaglandin E2- and collagenase-stimulating activities appeared during epidermal cell culture: after 2 d they were detected in the cell lysate, and after 4 d of culture they were found also in the conditioned media. Molecular sieving chromatography of epidermal cell lysates led to the detection of two main peaks showing concomitant prostaglandin E2- and collagenase-stimulating activities at Mr approximately equal to 18 000 and Mr approximately equal to 10 000. A single peak of concomitant prostaglandin E2- and collagenase-stimulating activities were seen at Mr approximately equal to 10 000 in the epidermal cell conditioned media. This suggests that the cell-associated concomitant prostaglandin E2- and collagenase-stimulating activities are processed from a common precursor molecule and released. Collagenase-stimulating activity without accompanying prostaglandin E2 was also detected in the range of Mr approximately equal to 30 000-45 000.