Head circumference at birth and postnatal growth trajectory in vulnerable groups from Argentina.

OBJECTIVES: To investigate the association between the anthropometric status at birth and brain and bone growth during the first year of life. According to the brain-sparing hypothesis, we expect catch-up to be faster in head circumference (HC) than in body length. METHODS: This is a longitudinal design that included Argentinian infants under 12 months of age with at least three anthropometric records. We classified study participants into four growth status categories according to z-scores for HC (HCZ) and length (LAZ) at birth, with z-score = -2 as a threshold. We used the Count model to describe growth trajectories in HC and length in the first year of life according to the growth status at birth. Recovery indicator for HC and length was taken as the time until the predicted growth trajectory surpassed the threshold curve predicted by z-score = -2 for age. RESULTS: Growth models included 3399 infants. There were significant differences in the growth parameters between groups in all cases (p < 0.05). Within the group with a low HCZ and a low LAZ at birth, HC recovery was faster than length. In the case of a low z-score for only one of the variables, newborns with a low HCZ recovered faster than individuals born with a low LAZ. CONCLUSIONS: The postnatal growth pattern in HC and length is associated with the growth status of HC and length at birth. As we hypothesized, the fastest postnatal recovery occurs for HC in cases of intrauterine delayed growth.

Micro-CT Imaging and Morphometric Analysis of Mouse Neonatal Brains.

Neuroimages are a valuable tool for studying brain morphology in experiments using animal models. Magnetic resonance imaging (MRI) has become the standard method for soft tissues, although its low spatial resolution poses some limits for small animals. Here, we describe a protocol for obtaining high-resolution three-dimensional (3D) information on mouse neonate brains and skulls using micro-computed tomography (micro-CT). The protocol includes those steps needed to dissect the samples, stain and scan the brain, and obtain morphometric measurements of the whole organ and regions of interest (ROIs). Image analysis includes the segmentation of structures and the digitization of point coordinates. In sum, this work shows that the combination of micro-CT and Lugol's solution as a contrast agent is a suitable alternative for imaging the perinatal brains of small animals. This imaging workflow has applications in developmental biology, biomedicine, and other sciences interested in assessing the effect of diverse genetic and environmental factors on brain development.

Morphological correspondence between brain and endocranial surfaces in mice exposed to undernutrition during development.

The morphological changes of the brain and the skull are highly integrated as a result of shared developmental pathways and different types of interactions between them. Shared developmental trajectories between these two structures might be influenced by genetic and environmental factors. Although the effect of environmental factors on neural and craniofacial traits has been extensively studied, less is known about the specific impact of stressful conditions on the coordinated variation between these structures. Here, we test the effect of early nutrient restriction on morphological correspondence between the brain and the endocast. For this purpose, mice exposed to protein or calorie-protein restriction during gestation and lactation were compared with a control group in which dams were fed standard food ad libitum. High-resolution images were obtained after weaning to describe brain and endocranial morphology. By magnetic resonance imaging (MRI), brain volumes were obtained and endocasts were segmented from skull reconstructions derived from micro-computed tomography (microCT). Brain and endocranial volumes were compared to assess the correspondence in size. Shape changes were analyzed using a set of landmarks and semilandmarks on 3D surfaces. Results indicated that brain volume is relatively less affected by undernutrition during development than endocast volume. Shape covariation between the brain and the endocast was found to be quite singular for protein-restricted animals. Procrustes distances were larger between the brain and the endocast of the same specimens than between brains or endocasts of different animals, which means that the greatest similarity is by type of structure and suggests that the use of the endocast as a direct proxy of the brain at this intraspecific scale could have some limitations. In the same line, patterns of brain shape asymmetry were not directly estimated from endocranial surfaces. In sum, our findings indicate that morphological variation and association between the brain and the endocast is modulated by environmental factors and support the idea that head morphogenesis results from complex processes that are sensitive to the pervasive influence of nutrient intake.

VBM sensitivity to localization and extent of mouse brain lesions: A simulation approach.

BACKGROUND AND OBJECTIVES: Voxel-based morphometry (VBM) is a popular neuroimaging technique, used to detect and quantify morphological differences in brain tissues between groups. Widely used in human studies, VBM approaches have tremendous potential for neuroimaging studies in animal models. A significant challenge for applying VBM to small animal studies is the poor understanding of how the design of preprocessing pipelines impacts quantitative results. This is important because the large differences in size, resolution, and imaging parameters implies that human imaging preprocessing pipelines cannot be uncritically applied to small animal studies. In this work, we assessed and validated the performance of different VBM pipelines for the study of the mouse brain. METHODS: We applied two pipelines -namely DARTEL VBM and Optimized VBM- by varying spatial normalization used during preprocessing. Using an automatic method, we simulated varying levels of volumetric gray matter (GM) loss and sizes of tissue atrophy on specific areas of the mouse brain. We evaluated the performance of each pipeline by comparing location and extent of the differences detected by them with the simulated ones. Finally, we applied both pipelines on magnetic resonance (MR) images of the brain derived from an experimental model of growth restriction on mice. RESULTS: Our results demonstrated that some subtle atrophies were detected by the Optimized workflow but not by the DARTEL VBM workflow. Detection of less subtle atrophies was similar for the two workflows, but DARTEL VBM performed better at estimating their size and anatomical location. Both VBM pipelines had difficulties at finding atrophies with a very small level of volumetric loss and, in general, they underestimated the magnitudes of difference between groups. These results also varied across brain regions, with better performance on brain cortex than other regions such as the cerebellum. CONCLUSIONS: The analysis and quantification of VBM pipelines on different areas of the mouse brain allows a better understanding of the advantages and limitations of their results. We performed a controlled and quantitative analysis of the method providing robust evidence to interpret changes in real contexts.

Modeling the effect of brain growth on cranial bones using finite-element analysis and geometric morphometrics.

PURPOSE: Brain expansion during ontogeny has been identified as a key factor for explaining the growth pattern of neurocranial bones. However, the dynamics of this relation are only partially understood and a detailed characterization of integrated morphological changes of the brain and the neurocranium along ontogeny is still lacking. The aim of this study was to model the effect of brain growth on cranial bones by means of finite-element analysis (FEA) and geometric morphometric techniques. METHODS: First, we described the postnatal changes in brain size and shape by digitizing coordinates of 3D semilandmarks on cranial endocasts, as a proxy of brain, segmented from CT-scans of an ontogenetic sample. Then, two scenarios of brain growth were simulated: one in which brain volume increases with the same magnitude in all directions, and other that includes the information on the relative expansion of brain regions obtained from morphometric analysis. RESULTS: Results indicate that in the first model, in which a uniform pressure is applied, the largest displacements were localized in the sutures, especially in the anterior and posterior fontanels, as well as the metopic suture. When information of brain relative growth was introduced into the model, displacements were also concentrated in the lambda region although the values along both sides of the neurocranium (parietal and temporal bones) were larger than under the first scenario. CONCLUSION: In sum, we propose a realistic approach to the use of FEA based on morphometric data that offered different results to more simplified models.

The cyanobacterial saxitoxin exacerbates neural cell death and brain malformations induced by Zika virus.

The northeast (NE) region of Brazil commonly goes through drought periods, which favor cyanobacterial blooms, capable of producing neurotoxins with implications for human and animal health. The most severe dry spell in the history of Brazil occurred between 2012 and 2016. Coincidently, the highest incidence of microcephaly associated with the Zika virus (ZIKV) outbreak took place in the NE region of Brazil during the same years. In this work, we tested the hypothesis that saxitoxin (STX), a neurotoxin produced in South America by the freshwater cyanobacteria Raphidiopsis raciborskii, could have contributed to the most severe Congenital Zika Syndrome (CZS) profile described worldwide. Quality surveillance showed higher cyanobacteria amounts and STX occurrence in human drinking water supplies of NE compared to other regions of Brazil. Experimentally, we described that STX doubled the quantity of ZIKV-induced neural cell death in progenitor areas of human brain organoids, while the chronic ingestion of water contaminated with STX before and during gestation caused brain abnormalities in offspring of ZIKV-infected immunocompetent C57BL/6J mice. Our data indicate that saxitoxin-producing cyanobacteria is overspread in water reservoirs of the NE and might have acted as a co-insult to ZIKV infection in Brazil. These results raise a public health concern regarding the consequences of arbovirus outbreaks happening in areas with droughts and/or frequent freshwater cyanobacterial blooms.

Micromorphology of osteoderms in Dasypodidae (Cingulata, Mammalia): Characterization and 3D-reconstructions.

Osteoderms are present in a variety of extinct and extant vertebrates, but among mammals, the presence of osteoderms is essentially restricted to armadillos (Cingulata, Dasypodidae). Osteoderms have been proposed to exhibit a variety of functionalities in Dasypodidae, mainly protection and thermoregulation, and they have been considered as one of the synapomorphies of this group. In this study, we use high-resolution microcomputed tomography to describe the osteoderm micromorphology of several extant species of Dasypodidae in a comparative context. This study allowed the identification, 3D-reconstruction and volume quantification of different internal structures of osteoderms as well as their interrelations. This detailed characterization of the internal osteoderm morphology was compared in a phylogenetic context to assess the evolutionary trends of the species involved. This enables the identification of distinctive patterns for the most widely recognized clades, the Dasypodinae and Euphractinae with a morphological homogeneity in the microstructure of their osteoderms, in comparison with Tolypeutinae where it has not been possible to establish a common morphological pattern. The most important features for linage differentiation is the degree of compaction of the osteoderms, the number of cavities and the development of hairs. It is likely that the differential development of the various structures occurred as adaptive response to climate changes.

Region-specific changes in Mus musculus brain size and cell composition under chronic nutrient restriction.

Nutrition is one of the most influential environmental factors affecting the development of different tissues and organs. It is suggested that under nutrient restriction the growth of the brain is spared as a result of the differential allocation of resources from other organs. However, it is not clear whether this sparing occurs brain-wide. Here, we analyzed morphological changes and cell composition in different regions of the offspring mouse brain after maternal exposure to nutrient restriction during pregnancy and lactation. Using high-resolution magnetic resonance imaging, we found that brain regions were differentially sensitive to maternal protein restriction and exhibited particular patterns of volume reduction. The cerebellum was reduced in absolute and relative volume, while cortex volume was relatively preserved. Alterations in cell composition (examined by the isotropic fractionator method) and organization of white matter (measured by diffusor tensor images) were also region specific. These changes were not related to the metabolic rate of the regions and were only partially explained by their specific growth trajectories. This study is a first step towards understanding the mechanisms of regional brain sparing at microstructural and macrostructural levels resulting from undernutrition.

Why is congenital Zika syndrome asymmetrically distributed among human populations?

Zika virus (ZIKV) is a health burden due to the severe neurological abnormalities that arise after congenital infection. Although multiple experimental studies have linked ZIKV with neural birth defects, the scientific community has not been able to fully explain why Congenital Zika Syndrome (CZS) was only apparent after the virus entered the Americas and why these occurrences have an asymmetric geographic distribution. Here, we review the impact of ZIKV infection on human populations by exploring evolutionary changes in the virus' genome as well as examining the diverse genetic and environmental cofactors of the human hosts.

Brain Structural Networks in Mouse Exposed to Chronic Maternal Undernutrition.

Brain structural connectivity is known to be altered in cases of intrauterine growth restriction and premature birth, although the specific effect of maternal nutritional restriction, a common burden in human populations, has not been assessed yet. Here we analyze the effects of maternal undernutrition during pregnancy and lactation by establishing three experimental groups of female mice divided according to their diet: control (Co), moderate calorie-protein restriction (MCP) and severe protein restriction (SP). Nutritionally restricted dams gained relatively less weight during pregnancy and the body weight of the offspring was also affected by maternal undernutrition, showing global growth restriction. We performed magnetic resonance imaging (MRI) of the offspring's brains after weaning and analyzed their connectivity patterns using complex graph theory. In general, changes observed in the MCP group were more subtle than in SP. Results indicated that brain structures were not homogeneously affected by early nutritional stress. In particular, the growth of central brain regions, such as the temporo-parietal cortex, and long integrative myelinated tracts were relatively preserved, while the frequency of short tracts was relatively reduced. We also found a differential effect on network parameters: network degree, clustering, characteristic path length and small-worldness remained mainly unchanged, while the rich-club index was lower in nutritionally restricted animals. Rich-club decrease reflects an impairment in the structure by which brain regions with large number of connections tend to be more densely linked among themselves. Overall, the findings presented here support the hypothesis that chronic nutritional stress produces long-term changes in brain structural connectivity.

Morphological asymmetries of mouse brain assessed by geometric morphometric analysis of MRI data.

Mammalian brain has repeated structures at both sides of the median plane, although some asymmetries have been described even under normal conditions. Characterizing normal patterns of asymmetry in mouse brain is important to recognize features that depart from expected ranges in the most widely used mammalian model. Analyses on brain morphology based on magnetic resonance image (MRI) have largely focused on volumes while less is known about shape asymmetry. We introduce a flexible protocol based on geometric morphometrics to assess patterns of asymmetry in shape and size of mouse brain from microMRI scans. After systematic digitization of landmarks and semilandmarks, we combine multivariate methods for statistical analyses with visualization tools to display the results. No preliminary treatment of the images (e.g. space normalization) is needed to collect data on MRI slices and visual representations improve the interpretation of the results. Results indicated that the protocol is highly repeatable. Asymmetry was more evident for shape than for size. Particularly, fluctuating asymmetry accounted for more variation than directional asymmetry in all brain regions. Since this approach can detect subtle shape variation between sides, it is a promising methodology to explore morphological changes in the brain of model organisms and can be applied in future studies addressing the effect of genetic and environmental factors on brain morphology.

Chronic Protein Restriction in Mice Impacts Placental Function and Maternal Body Weight before Fetal Growth.

Mechanisms of resource allocation are essential for maternal and fetal survival, particularly when the availability of nutrients is limited. We investigated the responses of feto-placental development to maternal chronic protein malnutrition to test the hypothesis that maternal low protein diet produces differential growth restriction of placental and fetal tissues, and adaptive changes in the placenta that may mitigate impacts on fetal growth. C57BL/6J female mice were fed either a low-protein diet (6% protein) or control isocaloric diet (20% protein). On embryonic days E10.5, 17.5 and 18.5 tissue samples were prepared for morphometric, histological and quantitative RT-PCR analyses, which included markers of trophoblast cell subtypes. Potential endocrine adaptations were assessed by the expression of Prolactin-related hormone genes. In the low protein group, placenta weight was significantly lower at E10.5, followed by reduction of maternal weight at E17.5, while the fetuses became significantly lighter no earlier than at E18.5. Fetal head at E18.5 in the low protein group, though smaller than controls, was larger than expected for body size. The relative size and shape of the cranial vault and the flexion of the cranial base was affected by E17.5 and more severely by E18.5. The junctional zone, a placenta layer rich in endocrine and energy storing glycogen cells, was smaller in low protein placentas as well as the expression of Pcdh12, a marker of glycogen trophoblast cells. Placental hormone gene Prl3a1 was altered in response to low protein diet: expression was elevated at E17.5 when fetuses were still growing normally, but dropped sharply by E18.5 in parallel with the slowing of fetal growth. This model suggests that nutrients are preferentially allocated to sustain fetal and brain growth and suggests the placenta as a nutrient sensor in early gestation with a role in mitigating impacts of poor maternal nutrition on fetal growth.

Technical note: Performance of semi and fully automated approaches for registration of 3D surface coordinates in geometric morphometric studies.

OBJECTIVES: One of the biggest challenges in the study of complex morphologies is to adequately describe shape variation. Here, we assess how the random sampling of surface points automatically obtained performs, when compared with observer-guided sampling procedures, and also evaluate the effect of sliding surface points by bending energy and minimum Procrustes distance. MATERIAL AND METHODS: Three datasets comprising structures with disparate levels of complexity and intrasample variation are as follows: mouse molars, mouse brains, and primate endocasts. Different configurations of 3D coordinates on curves and surfaces were digitized from MRI images and CT scans using semi and fully automated procedures. Shape variables were obtained by Generalized Procrustes Superpositions before and after sliding the pseudolandmarks. Multivariate analyses were used to summarize and compare shape variation. RESULTS: For the primate endocast, the semiautomated and automated strategies yield similar ordinations of specimens. Conversely, the semiautomated strategy better discriminates molar shapes between mouse groups. Shape differences among specimens are not adequately represented by the PCs calculated with surface pseudolandmarks. This is improved when the points are converted into semilandmarks by a sliding criterion. DISCUSSION: Surface semilandmarks automatically obtained from 3D models are promising although they should be used with some caution in complex structures. This approach can be taken as complementary of semiautomated procedures which perform better for assessing shape variation in localized traits previously selected while automated procedures are suitable in studies aimed at comparing overall variation in shape and when there is no previous information about highly variable anatomical regions.

Morphological Integration of the Orbital Region in a Human Ontogenetic Sample.

Most studies on craniofacial morphology have focused on adult individuals, but patterns of variation are the outcome of genetic and epigenetic variables that interact throughout ontogeny. Among cranial regions, the orbits exhibit morphological variation and occupy an intermediate position between neurocranial and facial structures. The main objective of this work was to analyze postnatal ontogenetic variation and covariation in the morphology of the orbital region in a cross-sectional series of humans from 0 to 31 years old. Landmarks and semilandmarks were digitized on the orbital rim, as well as in neighboring neural and facial structures. Data were analyzed using geometric morphometrics. Results indicated that orbital size increases during the first years of postnatal life, while the shape of the orbital aperture does not change significantly with age. In general, the pattern and magnitude of shape covariation do not vary markedly during postnatal life although some subtle shifts were documented. Additionally, the shape of the orbital aperture is more related to the anterior neurocranium than to zygomatic structures, even when the allometry is adjusted. Although we expected some influence from postnatal craniofacial growth and from some functional factors, such as mastication, on the development of the orbits, this assumption was not completely supported by our results. As a whole, our findings are in line with the prediction of an early influence of the eyes and extraocular tissues on orbital morphology, and could be interpreted in relation to processes promoting early neural development that coordinately affects orbital traits and the neurocranial skeleton.

Ontogenetic changes in cranial vault thickness in a modern sample of Homo sapiens.

OBJECTIVES: This work assesses cranial vault thickness (CVT) ontogenetic changes using a computed tomography database to register thickness across multiple regions. METHODS: Vault images of 143 individuals from 0 to 31 years old were analyzed by thickness semiautomatic measurements. For each individual, we obtained a thickness mean measure (TMM) and its coefficient of variation, a measure of endocranial volume (EV), the distribution of relative frequencies of thickness-relative frequency polygon, and a topographic mapping that shows the thickness arrangement through a chromatic scale. Ontogenetic changes of these variables were evaluated by different regression models (TMM vs. age, EV vs. age, TMM vs. EV) and visual comparisons between the age groups. RESULTS: TMM increased during ontogeny until the onset of adulthood without sex differences, but the most accelerated growth rates occur during the first 6 years of postnatal life. TMM variations were associated with EV only in infants and children, but not in later periods. The polygons showed a flattening during ontogeny, probably due to an increase in thickness variation within individuals. However, the adult pattern of thickness arrangement, with the lateral region thinner than the regions near sagittal plane, was detected from infancy. CONCLUSION: The pattern of thickness arrangement is established early in ontogeny but CVT increases and changes in distribution until adolescence. Several factors may influence CVT, such as the brain, muscles, vessels, and sutures.

Developmental covariation of human vault and base throughout postnatal ontogeny.

In the present study, we analyzed postnatal ontogenetic integration among morphological traits of the human neurocranium. Particularly, the covariation between the vault and the base during postnatal life was assessed. Since the association between these regions may depend on the generalized change produced by allometry, we tested its effect on their covariation. On a sample of adults and subadults ranging from 0 to 31 years, 3D coordinates of neurocranial landmarks and semilandmarks were digitized and geometric morphometric technics were applied. Main aspects of shape variation were examined using Principal Components analysis. Covariation between the vault and the base was examined by Partial Least Squares analysis. According to our results, the vault and the base covary strongly during postnatal ontogeny and their relation depends largely on allometry. Two size variables were studied: centroid size, which was obtained from the recorded morphometric points, and endocranial volume, taken as an estimation of brain size. Although growing brain was found to be a developmental process that contributes to covariation among neurocranial traits, there would be other factors that exert their influence during ontogeny. These results lead to reconsider cranial morphological evolution taking into account the developmental constraints given by ontogenetic patterns of integration and reinforcing the idea that in human evolution a suite of relevant characters may be fuelled by few developmental processes.

Ontogenetic patterns of morphological variation in the ectocranial human vault.

The skull is considered a modular structure in which different parts are influenced by different factors and, as a result, achieve adult shape at different ages. Previous studies have suggested that the basicranium presents a modular pattern that distinguishes sagittal and lateral parts, probably affected by the brain and masticatory structures, respectively. The vault of modern humans, in contrast, has been considered as a highly integrated system mainly influenced by brain growth. Here, we explored developmental shape variation in sagittal and lateral ectocranial vault in humans in order to assess if both regions are ontogenetically dissociated. We used a sample of 135 cranial computed tomography images from 0 to 31 ages. Landmarks and semilandmarks were collected on sagittal and lateral regions and geometric morphometric techniques were applied separately for each region. On the shape coordinates, we used Goodall's F-test in order to assess the age when the adult configuration is attained. Principal component analysis enabled us to evaluate shape variation during ontogeny. Results indicated that both sagittal and lateral structures attain adult shape at early adolescence. Both regions express coordinated shape modifications probably due to shared developmental factors. It is concluded that masticatory muscles may not exert a strong enough influence to produce independent variation in the lateral traits. Thus, it is likely that the brain integrates sagittal and lateral parts of the vault across human ontogeny.

Measurement error of 3D cranial landmarks of an ontogenetic sample using Computed Tomography.

BACKGROUND/AIM: Computed Tomography (CT) is a powerful tool in craniofacial research that focuses on morphological variation. In this field, an ontogenetic approach has been taken to study the developmental sources of variation and to understand the basis of morphological evolution. This work aimed to determine measurement error (ME) in cranial CT in diverse developmental stages and to characterize how this error relates to different types of landmarks. MATERIAL AND METHODS: We used a sample of fifteen skulls ranging from 0 to 31 years. Two observers placed landmarks in each image three times. Measurement error was assessed before and after Generalized Procrustes Analysis. RESULTS: The results indicated that ME is larger in neurocranial structures, which are described mainly by type III landmarks and semilandmarks. In addition, adult and infant specimens showed the same level of ME. These results are specially relevant in the context of craniofacial growth research. CONCLUSION: CT images have become a frequent evidence to study cranial variation. Evaluation of ME gives insight into the potential source of error in interpreting results. Neural structures present higher ME which is mainly associated to landmark localization. However, this error is irrespective of age. If landmarks are correctly selected, they can be analyzed with the same level of reliability in adults and subadults.