Maternal anxiety-driven modulation of fetal limbic connectivity designs a backbone linking neonatal brain functional topology to socio-emotional development in early childhood.

A link between maternal anxiety during pregnancy and adverse socio-emotional outcomes in childhood has been consistently sustained on the very early neurodevelopmental alteration of structural pathways between fetal limbic and cortical brain regions. In this study, we provide follow-up evidence for a feed-forward model linking (i) maternal anxiety, (ii) fetal functional neurodevelopment, (iii) neonatal functional network organization with (iv) socio-emotional neurobehavioral development in early childhood. Namely, we investigate a sample of 16 mother-fetus dyads and show how a maternal state-trait anxiety profile with pregnancy-specific worries can significantly influence functional synchronization patterns between regions of the fetal limbic system (i.e., hippocampus and amygdala) and the neocortex, as assessed through resting-state functional magnetic resonance imaging. Generalization of the findings was supported by leave-one-out cross-validation. We further show how this maternal-fetal cross-talk propagates to functional network topology in the neonate, specifically targeting connector hubs, and further maps onto socio-emotional profiles, assessed through Bayley-III socio-emotional scale in early childhood (i.e., in the 12-24 months range). Based on this evidence, we put forward the hypothesis of a "Maternal-Fetal-Neonatal Anxiety Backbone", through which neurobiological changes driven by maternal anxiety could trigger a divergence in the establishment of a cognitive-emotional development blueprint, in terms of the nascent functional homeostasis between bottom-up limbic and top-down higher-order neuronal circuitry.

Transient structural MRI patterns correlate with the motor functions in preterm infants.

AIM: To explore the relationships between transient structural brain patterns on MRI at preterm and at term-equivalent age (TEA) as a predictor of general movements (GMs) and motor development at 1-year corrected age (CA) in very preterm infants. METHODS: In this prospective study, 30 very preterm infants (median = 28wks; 16 males) had structural magnetic resonance imaging (MRI) at preterm (median = 31wks + 6d) and at TEA (median = 40wks) and neuromotor assessments. The quality of GMs was assessed by Prechtl's general movements assessment and a detailed analysis of the motor repertoire was performed by calculating a motor optimality score (MOS), both at term age and at 3 months post-term. Motor development at 1-year CA was evaluated with the Infant Motor Profile (IMP). Associations between qualitative MRI findings and neuromotor scores were investigated. RESULTS: Abnormal GMs and low motor performance at 1-year CA were associated with the poor visibility of transient structural pattern, that is with sagittal strata. INTERPRETATION: Transient structural MRI pattern, sagittal strata, at preterm age is related to the quality of GMs and later motor development in preterm infants. This transient fetal brain compartment may be considered as a component of neurobiological basis for early neuromotor behavior, as expressed by GMs.

Brain tissue volumes at term-equivalent age are associated with early motor behavior in very preterm infants.

Preterm birth is associated with a wide range of adverse developmental outcomes, including sensory, motor, cognitive and language impairments, and behavioral or attention problems. Subtle motor deficits that might emerge in premature infants with no evident or with mild brain injury encompass qualitative and quantitative aspects of motor behavior. This prospective cohort study provided an evaluation of the relationship between brain tissue volumes revealed by magnetic resonance imaging (MRI) at term-equivalent age and motor behavior in infancy in very preterm infants (total number = 40; mean gestational age = 28 weeks + 4 days; mean birth weight = 1190 g) without evident or with mild brain injury. Infants were recruited at birth and assessed at 12 months corrected age using the tool for qualitative and quantitative assessment of motor behavior, infant motor profile. The brain tissue was segmented first using advanced segmentation techniques and the volumes were measured by summing the volumes of all voxels belonging to a particular tissue class. The associations between volumetric brain MRI measures with motor behavior were explored using linear regression analyses. Results showed that larger total brain volumes were associated with higher motor score. Similar relationships were documented for parietal lobe, deep gray matter, and cerebellum volumes. Volumetric quantitative data of brain structures may serve as biomarkers for subtle motor deficits described in very preterm born infants without or with mild brain lesions apparent on MRI.

Impact of 5-azacytidine on rat decidual cell proliferation.

The DNA demethylating agent 5-azacytidine (5-azaC) has a teratogenic influence during rat development influencing both the embryo and the placenta. Our aim was to investigate its impact on early decidual cell proliferation before the formation of placenta. Thus, female Fischer rats received 5-azaC (5 mg/kg, i.p.) on the 2nd, 5th or 8th day of gestation and the decidual tissues were harvested on gestation day 9. They were then analysed immunohistochemically for expression of cell proliferation marker proliferating cell nuclear antigen (PCNA) in decidual cells and for global DNA methylation using the coupled restriction enzyme digestion, random amplification and pyrosequencing assays. We found that 5-azaC administered on the 5th and 8th (but not on 2nd) day of gestation led to increased PCNA expression in decidual cells compared with untreated controls. No significant changes in DNA methylation were detected, with either method, in any of the treated rat groups compared with untreated controls. Thus, we conclude that 5-azaC can stimulate decidual cell proliferation without simultaneously changing global DNA methylation level in treated cells.

Visual stimulations' critical period in infants with perinatal brain damage.

BACKGROUND: Children with perinatal brain damage have a high prevalence of visual impairment. Stimulation of vision at a critical period can encourage brain plasticity and the recovery of impaired function. OBJECTIVE: The aim of our study was to investigate when is the critical period for visual stimulation in children with perinatal brain damage. METHODS: We compared 35 children within the first eight months of life (median age = 4 months) to 35 children aged between eight and thirty months (median age = 15 months), all with perinatal brain damage. All the children were attending an early intervention program at Mali dom, Zagreb, a rehabilitation centre for children with visual impairment. We compared the results from baseline and follow-up assessments of visual functions (grating acuity and contrast sensitivity). We also compared differences in change scores between the two groups. RESULTS: Our results have shown that children who commence a visual stimulation program within the first eight months of life had more improvement in both visual functions. This improvement is statistically significant in visual acuity (p = 0.048). CONCLUSIONS: These results indicate the importance of starting a visual stimulation program within the first eight months after birth.

The effect of vibration therapy on spasticity and motor function in children with cerebral palsy: a randomized controlled trial.

As the motor system relies heavily on deep sensory stimulation, recent studies have investigated the effect of vibration stimuli. Although research suggests a positive influence of vibration on motor performance in individuals with neurological disorders, there are very limited numbers of studies in children with cerebral palsy (CP). The objective of the present study was to evaluate the effects of sound wave vibration therapy on spasticity and motor function in children with CP. In this 3-month trial, 89 children with spastic CP were randomized to either continue their physiotherapy treatment (PT) or to receive vibration therapy twice a week in addition to their PT program. The randomization was stratified according to the Gross Motor Function Classification System (GMFCS) level to ensure similar functional ability. Children were assessed at baseline and after the 12-week intervention period. The outcomes measured were spasticity level as assessed by Modified Modified Ashworth Scale (MMAS) and gross motor function as assessed by Gross Motor Function Measurement (GMFM-88). Subgroup analysis was performed for the GMFCS. Significant differences between groups were detected for changes in spasticity level and gross motor function after the three months intervention. In conclusion, vibration therapy may decrease spasticity and improve motor performance in children with CP. The results of the present trial serve as valuable input for evidence-based treatments in paediatric neurorehabilitation.

The relationship between spasticity and gross motor capability in nonambulatory children with spastic cerebral palsy.

Spasticity has been considered as a major impairment in cerebral palsy (CP), but the relationship between this impairment and motor functions is still unclear, especially in the same group of patients with CP. The aim of this investigation is to determine the relationship between spasticity and gross motor capability in nonambulatory children with spastic CP. Seventy-one children (30 boys, 41 girls) with bilateral spastic cerebral palsy and with Gross Motor Function Classification System (GMFCS) levels IV (n=34) and V (n=37) were included in the study. The spasticity level in lower limbs was evaluated using the Modified Modified Ashworth Scale and the gross motor function with the Gross Motor Function Measure (GMFM-88). Spearman's correlation analysis was used to determine the nature and the strength of the relationship. The results showed a moderate correlation between spasticity and gross motor skills (ρ=0.52 for the GMFCS level; ρ=0.57 for the GMFM-88), accounting for less than 30% of the explained variance. It seems that spasticity is just one factor among many others that could interfere with gross motor skills, even in children with severe forms of spastic CP. Knowledge of the impact of spasticity on motor skills may be useful in the setting of adequate rehabilitation strategies for nonambulatory children with spastic CP.

Effects of vibrotactile stimulation on the control of muscle tone and movement facilitation in children with cerebral injury.

Afferent signals from the muscle's proprioceptors play important role in the control of muscle tone and in the facilitation of movements. Peripheral afferent pathway enables the restoration of connections with supraspinal structures and so includes mechanism of synaptic inhibition in the performance of normal movement. Different sensory stimuli, as vibrotactile stimulation, excite muscle's proprioceptors which then send sensorimotor information via spinal cord. In this way afferent signals promote cortical control and modulation of movements. The goal of this study is to evaluate the effects of vibrotactile stimulation on the spasticity and motor performance in children with cerebral injury. Subjects included in this study were 13 children who were developing the classification of spastic cerebral palsy. For all children perinatal brain damage was documented by medical reports and neonatal brain ultrasound scan. At the mean age of 3 years and 6 months subject underwent the assessment of motor development by Gross Motor Function Measurement (GMFM-88). Gross Motor Classification System (GMFCS) has been used to classify functions of lower extremities. Therapeutic intervention was conducted once a week during 3 months. All subjects were stimulated with vibrotactile stimuli of 40Hz in duration of 20 minutes in order to reduce spasticity. After the ending of the treatment subjects underwent second assessment of motor performance and the classification of lower extremities functions. The results have shown that there was a significant improvement in motor performance, what has been seen in the facilitation of rotations, better postural trunk stability and head control and in greater selectivity of movements. Further randomized, control trial investigations with bigger sample and included spasm scale are needed to gain better insight in the role of vibrotactile stimulation in the facilitation of normal movements.

5-azacytidine enhances proliferation in transplanted rat fetal epiglottis.

Fetal rat epiglottis and its developmental potential in ectopic transplants under the influence of the epigenetic drug was investigated. Epiglottises from 17-days-old rat embryos were transplanted under kidney capsules of adult rats for 14 days. 5-azacytidine (5 mg/kg) was injected intraperitoneally during first three days and controls were sham treated. TEM, immunohistochemical detection and quantitative stereological analysis of the Proliferating Cell Nuclear Antigen (PCNA) expression (numerical density N(v)) were performed. Typical chondroblasts with long surface processes and sparse lipid droplets were found in fetal epiglottis and chondrocytes with shorter processes, numerous lipid droplets and elastic fibers in adult. PCNA was expressed in almost all cells of the fetal epiglottis while in the adult it was expressed in minority of cells. In transplants, differentiation progressed towards the differentiation found in the adult. Application of 5-azacytidine increased the capacity for proliferation (N(v PCNA)) in comparison to controls but no difference in differentiation was observed. Data about the developmental potential and induction of proliferation in mammalian epiglottis by epigenetic modulation is of importance for regenerative medicine.

Epigenetic deregulation through DNA demethylation seems not to interfere with the differentiation of epithelia from pre-gastrulating rat embryos in vitro.

One of the epigenetic mechanisms controlling differentiation during mammalian development is the process of DNA methylation. The differentiation of tissues in pre-gastrulating rat embryos cultivated in vitro under the influence of the demethylating agent 5-azacytidine (5azaC) was investigated. Eight-day-old Fisher rat embryos consisting of epiblast and hypoblast (primitive ectoderm and primitive endoderm) were isolated and cultivated in serum-supplemented medium by air-lifting method in vitro. A single dose of 5azaC (30 microM) was added to the culture medium on day 5 of cultivation. After 14 days, teratoma-like structures developed and were processed by routine histology. When compared to controls, the explants treated with 5azaC showed a statistically significant higher incidence of neuroblasts, myotubes, cartilage, and blood islands. On the other hand, the incidence of stratified squamous, columnar and glandular epithelium was not statistically different from controls. It seems that differentiation of epithelia was not sensitive to DNA demethylation caused by 5azaC like differentiation of other tissues, especially mesodermal derivatives.

Development of epithelia in experimental teratomas derived from rodent embryos.

Investigation of developmental potential of various embryonal tissues is important for design of new approaches to regenerative medicine aimed at supplementing tissues damaged by trauma or disease. Rodent embryos have been extensively used in experiments designed for investigation of developmental potential to give rise to various types of epithelia such as superficial epithelia, neuroepithelium and sometimes even malignantly transformed epithelium in teratoma-like structures. These experiments have been done in vitro, in transplants in vivo and by combined in vitro-in vivo methods.

Development of epithelia in the ectopic transplant of the fetal rat epiglottis.

Embryonic in situ development is strictly regulated within the specific microenvironment of developing tissues. However, for regenerative medicine purposes (supplementation of damaged tissues/organs), transplantation to ectopic sites has been considered. To investigate developmental potential of fetal epiglottic epithelia at an ectopic site, fetal epiglottis was transplanted under the kidney capsule and its development compared to fetal and adult epiglottis. Seventeen-day-old Fischer rat epiglottides were microsurgically isolated under a dissecting microscope and transplanted under the kidney capsule of adult males. After 14 days, classic histology and immunohistochemical detection of the Proliferating Cell Nuclear Antigen (PCNA) were done in isolated and accordingly fixed transplants. The 17-day-old fetal epiglottis and adult epiglottis were processed in the same way. The 17-day-old fetal epiglottides were covered with immature epithelium expressing PCNA in almost all cells. Adult epiglottis was covered with two types of epithelia (stratified squamous epithelium and ciliated pseudostratified epithelium). In the stratified squamous epithelium PCNA was abundantly expressed in the basal cell layer and absent from more superficial and more differentiated cells. Transplants survived well during the experimental period. On their surface ciliated pseudostratified epithelium could be easily recognized, but squamous epithelium was almost absent. PCNA was expressed in basal cells of the ciliated pseudostratified epithelium and was absent from the more differentiated superficial cells. It seems that at this ectopic site further differentiation of the epiglottic epithelia can proceed but differentiation of squamous epithelium seems not to be favored. It seems that this ectopic site is optimal for further differentiation of the epiglottic epithelium towards ciliated pseudostratified epithelium.

Development of the fetal neural retina in vitro and in ectopic transplants in vivo.

Investigation of the developmental potential of immature tissues is important for novel approaches to human regenerative medicine. Development of the fetal neural retina has therefore been investigated in two experimental systems. Retinas were microsurgically isolated from 20-days-old rat fetuses and cultivated in vitro for 12 days or transplanted in vivo under the kidney capsule of adult males for as long as 6 months. Shedding of the photoreceptor outer segment which is a process occurring at the terminal stage of photoreceptor differentiation was observed in culture by transmission electron microscopy (TEM). In transplants, no photoreceptors were found although markers of terminal neural and glial differentiation (e,g. synaptophysin, chromogranin and glial fibrilary acidic protein--GFAP) along with the molecules involved in the process of differentiation (guidance molecule semaphorin IIIA and chondroitin sulfate proteoglycan) were expressed. Semaphorin was differentially expressed being absent from older transplants. Proliferating cell nuclear antigen and nestin (marker of undifferentiated neural cells) were still weakly expressed even in six-months-old transplants. We could conclude that in both our experimental systems fetal neural retina proceeded to differentiate further on. However, even in long-term ectopic transplants a small population of cells still retained the potential for proliferation and has not yet reached the stage of terminal differentiation.

Differentiation of epiglottal epithelia during prenatal and postnatal human development.

Differentiation of epiglottal epithelia during human development was for the first time investigated by the light microscopy and documented in celoidine collection of human embryos from the Archive of the Department of Histology and Embryology, School of Medicine University of Zagreb, Croatia. At 6 weeks epiglottal swelling was found to be covered by a simple squamous epithelium consisting of a single layer of cells. At 8 weeks epithelium changed to a two-layered cuboidal epithelium which at the end of the 8th week transformed to multilayered columnar epithelium without cilia and goblet cells. In the one-day-old newborn, the majority of epiglottis was found to be covered by the mature ciliated columnar pseudostratified epithelium with goblet cells while only a minor part of the oral surface next to the tongue by the stratified squamous epithelium. This unexpected finding is in contrast to the domination of the stratified squamous epithelium found at the age of 13 years and in 35-years-old adult. Reversal of proportion covered by different types of epithelia between birth and puberty /adulthood is probably connected to the establishment of the air-flow which could be stimulating for differentiation of stratified squamous epithelium.

Of mice and men: teratomas and teratocarcinomas.

Teratomas and teratocarcinomas are tumors containing tissue derivatives of all three germ-layers. They can be induced by transplantation of animal embryos to ectopic microenvironment. Development of malignant teratocarcinomas depends on embryonic stage, species-specificity and immunological competence of the host. In the man, teratomas and teratocarcinomas usually represent a subtype of germ-cell tumors but sacrococcygeal teratomas arise from the remnants of the pluripotent primitive streak. Undifferentiated embryonal carcinoma (EC) cells are responsible for the malignancy of experimental mouse teratocarcinomas. Mouse EC cells injected to the adult give rise to tumors and upon injection to early embryos to differentiated tissues--thus resembling normal mouse embryonic stem cells (mESC). Epigenetic changes rather than mutations are associated with transformation of mESC to EC cells. Human EC and ES cell-lines (hESC) contain chromosomal abnormalities and can form teratocarcinoma after transplantation. ES cells are among those proposed for cell replacement therapy in the man. Suicide gene introduction should be recommended prior to their use in vivo to ablate them in case of malignant transformation.