Virtual histological assessment of the prenatal life history and age at death of the Upper Paleolithic fetus from Ostuni (Italy)

The fetal remains from the Ostuni 1 burial (Italy, ca 27 ka) represent a unique opportunity to explore the prenatal biological parameters, and to reconstruct the possible patho-biography, of a fetus (and its mother) in an Upper Paleolithic context. Phase-contrast synchrotron X-ray microtomography imaging of two deciduous tooth crowns and microfocus CT measurements of the right hemimandible of the Ostuni 1b fetus were performed at the SYRMEP beamline and at the TomoLab station of the Elettra - Sincrotrone laboratory (Trieste, Italy) in order to refne age at death and to report the enamel developmental history and dental tissue volumes for this fetal individual. The virtual histology allowed to estimate the age at death of the fetus at 31–33 gestational weeks. Three severe physiological stress episodes were also identifed in the prenatal enamel. These stress episodes occurred during the last two months and half of pregnancy and may relate to the death of both individuals. Compared with modern prenatal standards, Os1b’s skeletal development was advanced. This cautions against the use of modern skeletal and dental references for archaeological fnds and emphasizes the need for more studies on prenatal archaeological skeletal samples

The study of renal function and toxicity using zebrafish (Danio rerio) larvae as a vertebrate model

Zebrafish (Danio rerio) is a powerful model in biomedical and pharmaceutical sciences. The zebrafish model was introduced to toxicological sciences in 1960, followed by its use in biomedical sciences to investigate vertebrate gene functions. As a consequence of many research projects in this field, the study of human genetic diseases became instantly feasible. Consequently, zebrafish have been intensively used in developmental biology and associated disciplines. Due to the simple administration of medicines and the high number of offspring, zebrafish larvae became widely more popular in pharmacological studies in the following years. In the past decade, zebrafish larvae were further established as a vertebrate model in the field of pharmacokinetics and nanomedicines. In this PhD thesis, zebrafish larvae were investigated as an earlystage in vivo vertebrate model to study renal function, toxicity, and were applied in drug-targeting projects using nanomedicines. The first part focused on the characterization of the renal function of three-to four-dayold zebrafish larvae. Non-renal elimination processes were additionally described. Moreover, injection techniques, imaging parameters, and post-image processing scripts were established to serve as a toolbox for follow-up projects. The second part analyzed the impact of gentamicin (a nephrotoxin) on the morphology of the pronephros of zebrafish larvae. Imaging methodologies such as fluorescent-based laser scanning microscopy and X-ray-based microtomography were applied. A profound comparison study of specimens acquired with different laboratory X-ray-based microtomography devices and a radiation facility was done to promote the use of X-ray-based microtomography for broader biomedical applications. In the third part, the toxicity of nephrotoxins on mitochondria in renal epithelial cells of proximal tubules was assessed using the zebrafish larva model. Findings were compared with other teleost models such as isolated renal tubules of killifish (Fundulus heteroclitus). In view of the usefulness and high predictability of the zebrafish model, it was applied to study the pharmacokinetics of novel nanoparticles in the fourth part. Various in vivo pharmacokinetic parameters such as drug release, transfection of mRNA/pDNA plasmids, macrophage clearance, and the characterization of novel drug carriers that were manipulated with ultrasound were assessed in multiple collaborative projects. Altogether, the presented zebrafish model showed to be a reliable in vivo vertebrate model to assess renal function, toxicity, and pharmacokinetics of nanoparticles. The application of the presented model will hopefully encourage others to reduce animal experiments in preliminary studies by fostering the use of zebrafish larvae

X-ray microtomography–based atlas of mouse cranial development

X-ray microtomography (CT) has become an invaluable tool for non-destructive analysis of biological samples in the field of developmental biology. Mouse embryos are a typical model for investigation of human developmental diseases. By obtaining 3D high-resolution scans of the mouse embryo heads, we gain valuable morphological information about the structures prominent in the development of future face, brain, and sensory organs. The development of facial skeleton tracked in these CT data provides a valuable background for further studies of congenital craniofacial diseases and normal development. In this work, reusable tomographic data from 7 full 3D scans of mouse embryo heads are presented and made publicly available. The ages of these embryos range from E12.5 to E18.5. The samples were stained by phosphotungstic acid prior to scanning, which greatly enhanced the contrast of various tissues in the reconstructed images and enabled precise segmentation. The images were obtained on a laboratory-based CT system. Furthermore, we provide manually segmented masks of mesenchymal condensations (for E12.5 and E13.5) and cartilage present in the nasal capsule of the scanned embryos. We present a comprehensive dataset of X-ray 3D computed tomography images of the developing mouse head with high-quality manual segmentation masks of cartilaginous nasal capsules. The provided CT images can be used for studying any other major structure within the developing mouse heads. The high quality of the manually segmented models of nasal capsules may be instrumental to understanding the complex process of the development of the face in a mouse model

Showing their true colors: a practical approach to volume rendering from serial sections

<p>Abstract</p> <p>Background</p> <p>In comparison to more modern imaging methods, conventional light microscopy still offers a range of substantial advantages with regard to contrast options, accessible specimen size, and resolution. Currently, tomographic image data in particular is most commonly visualized in three dimensions using volume rendering. To date, this method has only very rarely been applied to image stacks taken from serial sections, whereas surface rendering is still the most prevalent method for presenting such data sets three-dimensionally. The aim of this study was to develop standard protocols for volume rendering of image stacks of serial sections, while retaining the benefits of light microscopy such as resolution and color information.</p> <p>Results</p> <p>Here we provide a set of protocols for acquiring high-resolution 3D images of diverse microscopic samples through volume rendering based on serial light microscopical sections using the 3D reconstruction software Amira (Visage Imaging Inc.). We overcome several technical obstacles and show that these renderings are comparable in quality and resolution to 3D visualizations using other methods. This practical approach for visualizing 3D micro-morphology in full color takes advantage of both the sub-micron resolution of light microscopy and the specificity of histological stains, by combining conventional histological sectioning techniques, digital image acquisition, three-dimensional image filtering, and 3D image manipulation and visualization technologies.</p> <p>Conclusions</p> <p>We show that this method can yield "true"-colored high-resolution 3D views of tissues as well as cellular and sub-cellular structures and thus represents a powerful tool for morphological, developmental, and comparative investigations. We conclude that the presented approach fills an important gap in the field of micro-anatomical 3D imaging and visualization methods by combining histological resolution and differentiation of details with 3D rendering of whole tissue samples. We demonstrate the method on selected invertebrate and vertebrate specimens, and propose that reinvestigation of historical serial section material may be regarded as a special benefit.</p

New regression formula to estimate the prenatal crown formation time of human deciduous central incisors derived from a Roman Imperial sample (Velia, Salerno, Italy, I-II cent. CE)

The characterization and quantification of human dental enamel microstructure, in both permanent and deciduous teeth, allows us to document crucial growth parameters and to identify stressful events, thus contributing to the reconstruction of the past life history of an individual. Most studies to date have focused on the more accessible post-natal portion of the deciduous dental enamel, even though the analysis of prenatal enamel is pivotal in understanding fetal growth, and reveals information about the mother’s health status during pregnancy. This contribution reports new data describing the prenatal enamel development of 18 central deciduous incisors from the Imperial Roman necropolis of Velia (I-II century CE, Salerno, Italy). Histomorphometrical analysis was performed to collect data on prenatal crown formation times, daily secretion rates and enamel extension rates. Results for the Velia sample allowed us to derive a new regression formula, using a robust statistical approach, that describes the average rates of deciduous enamel formation. This can now be used as a reference for pre-industrial populations. The same regression formula, even when daily incremental markings are difficult to visualize, may provide a clue to predicting the proportion of infants born full term and pre-term in an archaeological series

Growth and development of the third permanent molar in Paranthropus robustus from Swartkrans, South Africa

Third permanent molars (M3s) are the last tooth to form but have not been used to estimate age at dental maturation in early fossil hominins because direct histological evidence for the timing of their growth has been lacking. We investigated an isolated maxillary M3 (SK 835) from the 1.5 to 1.8-million-year-old (Mya) site of Swartkrans, South Africa, attributed to Paranthropus robustus. Tissue proportions of this specimen were assessed using 3D X-ray micro-tomography. Thin ground sections were used to image daily growth increments in enamel and dentine. Transmitted light microscopy and synchrotron X-ray fluorescence imaging revealed fluctuations in Ca concentration that coincide with daily growth increments. We used regional daily secretion rates and Sr marker-lines to reconstruct tooth growth along the enamel/dentine and then cementum/dentine boundaries. Cumulative growth curves for increasing enamel thickness and tooth height and age-of-attainment estimates for fractional stages of tooth formation differed from those in modern humans. These now provide additional means for assessing late maturation in early hominins. M3 formation took ≥ 7 years in SK 835 and completion of the roots would have occurred between 11 and 14 years of age. Estimated age at dental maturation in this fossil hominin compares well with what is known for living great apes

A geometric morphometric approach to the study of variation of shovel-shaped incisors

Objectives: The scoring and analysis of dental nonmetric traits are predominantly accomplished by using the Arizona State University Dental Anthropology System (ASUDAS), a standard protocol based on strict definitions and three‐dimensional dental plaques. However, visual scoring, even when controlled by strict definitions of features, visual reference, and the experience of the observer, includes an unavoidable part of subjectivity. In this methodological contribution, we propose a new quantitative geometric morphometric approach to quickly and efficiently assess the variation of shoveling in modern human maxillary central incisors (UI1). Materials and methods: We analyzed 87 modern human UI1s by means of virtual imaging and the ASU‐UI1 dental plaque grades using geometric morphometrics by placing semilandmarks on the labial crown aspect. The modern human sample was composed of individuals from Europe, Africa, and Asia and included representatives of all seven grades defined by the ASUDAS method. Results: Our results highlighted some limitations in the use of the current UI1 ASUDAS plaque, indicating that it did not necessarily represent an objective gradient of expression of a nonmetric tooth feature. Rating of shoveling tended to be more prone to intra‐ and interobserver bias for the highest grades. In addition, our analyses suggest that the observers were strongly influenced by the depth of the lingual crown aspect when assessing the shoveling. Discussion: In this context, our results provide a reliable and reproducible framework reinforced by statistical results supporting the fact that open scale numerical measurements can complement the ASUDAS method

Analog vs digital: Testing the comparability and compatibility of diceCT and gross dissection, with special emphasis on muscle tissue

Diffusible iodine-based contrast-enhanced computed tomography (diceCT) is a new tool in the study of anatomy. With diceCT, researchers can visualize in situ soft tissue, in three dimensional space. The relationship of these results to traditional gross dissection is unknown. Despite this, it has begun to be used for quantitative comparisons. This approach requires more research to determine the comparability of diceCT and gross dissection. To study the relationship of these two methods, the head of a common marmoset, Callithrix jacchus, was stained in 2.5% Lugol’s solution (I2KI) for 37 days. The head CT scanned for digital dissection prior to physical dissection. Amira 5.6 was used for digital segmentation to reconstruct connective, epithelial, muscle, and nervous tissues. Masses of muscle were taken for muscle density comparisons to the previously established mammalian muscle density constants. Based on Bland-Altman analyses, gross dissection and diceCT do not produce comparable measurements in all circumstances. Muscle and epithelial tissue, as well as volumetric measurements are significantly different between gross dissection and diceCT. Muscle densities were also found to be significantly different than previously established constants, through the use of one sample t tests. New, diceCT-calibrated constants are proposed for use with specimens that cannot be dissected. Muscle density is not constant, and should not be treated as such. This process is still widely beneficial when traditional destructive dissection is not possible. It allows for three dimensional views of structures that are not otherwise visible due to size and/or morphology, however, comparisons between gross dissection and diceCT should be approached with caution