Micro-CT Imaging of Tracheal Development in Down Syndrome and Non-Down Syndrome Fetuses

Objectives: Down syndrome (DS) is associated with airway abnormalities including a narrowed trachea. It is uncertain whether this narrowed trachea in DS is a consequence of deviant fetal development or an acquired disorder following endotracheal intubation after birth. This study aimed to compare the tracheal morphology in DS and non-DS fetuses using microfocus computed tomography (micro-CT). Methods: Twenty fetal samples were obtained from the Dutch Fetal Biobank and divided into groups based on gestational age. Micro-CT images were processed to analyze tracheal length, volume, and cross-sectional area (CSA). Results: Mean tracheal length and tracheal volume were similar in DS and non-DS fetuses for all gestational age groups. Mean, minimum, and maximal tracheal CSA were statistically significantly increased in the single DS fetus in the group of 21–24 weeks of gestation, but not in other gestational age groups. In 90% of all studied fetuses, the minimum tracheal CSA was located in the middle third of the trachea. Conclusion: Tracheal development in DS fetuses was similar to non-DS fetuses between 13 and 21 weeks of gestation. This suggests that the narrowed tracheal diameter in DS children may occur later in fetal development or results from postnatal intubation trauma. The narrowest part of the trachea is in majority of DS and non-DS fetuses the middle third. Level of Evidence: Level 3 Laryngoscope, 2024.</p

Micro-CT Imaging of Tracheal Development in Down Syndrome and Non-Down Syndrome Fetuses

Objectives: Down syndrome (DS) is associated with airway abnormalities including a narrowed trachea. It is uncertain whether this narrowed trachea in DS is a consequence of deviant fetal development or an acquired disorder following endotracheal intubation after birth. This study aimed to compare the tracheal morphology in DS and non-DS fetuses using microfocus computed tomography (micro-CT). Methods: Twenty fetal samples were obtained from the Dutch Fetal Biobank and divided into groups based on gestational age. Micro-CT images were processed to analyze tracheal length, volume, and cross-sectional area (CSA). Results: Mean tracheal length and tracheal volume were similar in DS and non-DS fetuses for all gestational age groups. Mean, minimum, and maximal tracheal CSA were statistically significantly increased in the single DS fetus in the group of 21–24 weeks of gestation, but not in other gestational age groups. In 90% of all studied fetuses, the minimum tracheal CSA was located in the middle third of the trachea. Conclusion: Tracheal development in DS fetuses was similar to non-DS fetuses between 13 and 21 weeks of gestation. This suggests that the narrowed tracheal diameter in DS children may occur later in fetal development or results from postnatal intubation trauma. The narrowest part of the trachea is in majority of DS and non-DS fetuses the middle third. Level of Evidence: Level 3 Laryngoscope, 2024.</p

Scaling up contrast-enhanced micro-CT imaging:Optimizing contrast and acquisition for large ex-vivo human samples

Microfocus Computed Tomography (Micro-CT) is a novel method for non-destructive 3D imaging of samples, reaching microscale resolutions. While initially prominent in material sciences for small samples, micro-CT now gains significance in biological and medical studies. Here we present our utilization of micro-CT for imaging large ex-vivo human samples for anatomical and forensic research in three recent experiments and discuss the fundamentals of micro-CT imaging. For pelvic anatomical research, whole human pelvises were imaged to explore nerve anatomy around the prostate using various concentrations of buffered lugol (B-lugol). Advanced acquisition protocols were essential due to X-ray attenuation properties of the sample, which required higher energy for sufficient photon transmission. For fetal research, B-lugol stained fetuses of 20–24 gestational weeks underwent full body imaging. However, this led to challenging acquisition parameters and images of insufficient quality. Subsequent destaining yielded less dense, yet contrast-maintaining samples allowing higher quality images. Refined acquisition protocols with reduced energy improved image quality. For forensic research, explanted hyoid-larynx complexes were imaged. Micro-CT imaging showed potential in visualizing micro-fractures. The addition of B-lugol allowed for excellent soft tissue contrast and promising possibilities for forensic evaluation. In conclusion, micro-CT imaging accommodates a diversity of large ex-vivo human samples for anatomical and forensic purposes, though challenges arise with optimal soft tissue staining and acquisition protocols. We describe partial destaining as a new possibility to alleviate scanning issues to improve scan quality and highlight topics for future research. Micro-CT imaging is a promising new avenue for medical research and forensic evaluation.</p

Scaling up contrast-enhanced micro-CT imaging:Optimizing contrast and acquisition for large ex-vivo human samples

Microfocus Computed Tomography (Micro-CT) is a novel method for non-destructive 3D imaging of samples, reaching microscale resolutions. While initially prominent in material sciences for small samples, micro-CT now gains significance in biological and medical studies. Here we present our utilization of micro-CT for imaging large ex-vivo human samples for anatomical and forensic research in three recent experiments and discuss the fundamentals of micro-CT imaging. For pelvic anatomical research, whole human pelvises were imaged to explore nerve anatomy around the prostate using various concentrations of buffered lugol (B-lugol). Advanced acquisition protocols were essential due to X-ray attenuation properties of the sample, which required higher energy for sufficient photon transmission. For fetal research, B-lugol stained fetuses of 20–24 gestational weeks underwent full body imaging. However, this led to challenging acquisition parameters and images of insufficient quality. Subsequent destaining yielded less dense, yet contrast-maintaining samples allowing higher quality images. Refined acquisition protocols with reduced energy improved image quality. For forensic research, explanted hyoid-larynx complexes were imaged. Micro-CT imaging showed potential in visualizing micro-fractures. The addition of B-lugol allowed for excellent soft tissue contrast and promising possibilities for forensic evaluation. In conclusion, micro-CT imaging accommodates a diversity of large ex-vivo human samples for anatomical and forensic purposes, though challenges arise with optimal soft tissue staining and acquisition protocols. We describe partial destaining as a new possibility to alleviate scanning issues to improve scan quality and highlight topics for future research. Micro-CT imaging is a promising new avenue for medical research and forensic evaluation.</p

CS23_0950_Muscles_v2017-01.pdf

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Stereological measurement of porto-central gradients in gene expression in mouse liver

The liver is thought to consist of lobules, numerous repeating, randomly oriented units. Within these lobules, genes are expressed in gradients along the porto‐central axis, which spans the distance between portal and central veins. We have developed a robust stereological method to map all points in an image to their position on this porto‐central axis. This approach is based on the distribution of well‐characterized periportal and pericentral enzymes, which are visualized on sections preceding and following the section of interest. Because expression of the model genes phosphoenolpyruvate carboxykinase and ornithine aminotransferase declines gradually with increasing distance from the portal vein and central vein, respectively, these genes can be used to prepare images with topographical information without any assumption about the shape of the hepatic unit, or about the direction or shape of the gradient to be determined. The “relative distance” image is a 2‐dimensional image that accurately maps the relative position of hepatocytes on the porto‐central axis in 3‐dimensional space. It is superimposed on the serial section under investigation to relate local staining density to position on the porto‐central axis and obtain the gene expression gradient. The method was used to determine the expression gradient of 2 periportal and 2 pericentral enzymes and their response to fasting. The “total distance” image was used to measure the length of the porto‐central axis, which was approximately 210 μm in mice and found to decrease 13% after 1 day of starvation. The method can be applied to any tissue component that can be stained quantitatively

CALIBRATION OF DENSITOMETRY IN RADIO-ISOTOPIC IN SITU HYBRIDIZATION

Densitometry on autoradiographs of sections processed for in situ hybridization provides a direct measure for the in situ quantification of mRNA. Gelatin spots, containing different concentrations of the radioisotope, and processed in parallel with the tissue sections, can be used as a sensitive model to calibrate the densitometric measurements. The shape of the gelatin spots was shown to be circular with a parabolic crosssectional profile. This simple shape allows the subdivision of the spot into a series of concentric rings, which enables an unbiased measurement of the optical density - radioactivity relation. This spot measurement is also applicable to DNA arrays spotted on glass or membranes. A new model, explaining the optical density of autoradiographs, was derived and fitted to the calibration points. The use of this calibration method is crucial for the correct interpretation of autoradiograph

Calibration of densitometry in radio-isotopic in situ hybridization

Densitometry on autoradiographs of sections processed for in situ hybridization provides a direct measure for the in situ quantification of mRNA. Gelatin spots, containing different concentrations of the radioisotope, and processed in parallel with the tissue sections, can be used as a sensitive model to calibrate the densitometric measurements. The shape of the gelatin spots was shown to be circular with a parabolic crosssectional profile. This simple shape allows the subdivision of the spot into a series of concentric rings, which enables an unbiased measurement of the optical density - radioactivity relation. This spot measurement is also applicable to DNA arrays spotted on glass or membranes. A new model, explaining the optical density of autoradiographs, was derived and fitted to the calibration points. The use of this calibration method is crucial for the correct interpretation of autoradiographs

Molecular imaging of the embryonic heart: Fables and facts on 3D imaging of gene expression patterns

Molecular imaging, which is the three-dimensional (3D) visualization of gene expression patterns, is indispensable for the study of the function of genes in cardiac development. The instrumentation, as well as the development of specific contrast agents for molecular imaging, has shown spectacular advances in the last decade. In this review, the spatial resolutions, contrast agents, and applications of these imaging methods in the field of cardiac embryology are discussed. Apart from 3D reconstructions from histological sections, not many of these methods have been applied in embryological research. This review shows that, for most methods, neither the spatial resolutions nor the specificity and applicability of the contrast agents are adequate for the reliable imaging of specific gene expression at the microscopic resolution required for embryological studies of small organs like the developing heart. Although a 3D reconstruction from sections will always suffer from imperfections, the resulting reconstructions meet the aim of most biological studies, especially since the original microscopic images are linked. With respect to imaging of gene expression, only histological sections and laser scanning microscopy provide the required resolution and specificity at the tissue and cellular level. Episcopic fluorescence image capturing and optical projection tomography are being used for microscopic phenotyping and lineage analysis, and both show potential for detailed molecular imaging. Other methods can be used very efficiently in rapid evaluation of biological experiments and high-throughput screens of large-scale gene expression profiling efforts when high spatial resolution is not require

Quantified growth of the human embryonic heart

The size and growth patterns of the components of the human embryonic heart have remained largely undefined. To provide these data, three-dimensional heart models were generated from immunohistochemically stained sections of ten human embryonic hearts ranging from Carnegie stage 10 to 23. Fifty-eight key structures were annotated and volumetrically assessed. Sizes of the septal foramina and atrioventricular canal opening were also measured. The heart grows exponentially throughout embryonic development. There was consistently less left thanrightatrialmyocardium, andless right than left ventricular myocardium. We observed a later onset of trabeculation in the left atrium compared to the right. Morphometry showed that the rightward expansion of the atrioventricular canal starts in week 5. The septal foramina are less than 0.1 mm2 and are, therefore,much smaller than postnatal septal defects. This chronological, graphical atlas of the growth patterns of cardiac components in the human embryo provides quantified references for normal heart development. Thereby, this atlas may support early detection of cardiac malformations in the foetus