The salt marsh harvest mouse (Reithrodontomys raviventris) is an endangered species, endemic to the San Francisco Bay Estuary, that co-occurs with the more broadly distributed species, the western harvest mouse (Reithrodontomys megalotis). Despite their considerable external morphological similarities, the northern subspecies of salt marsh harvest mice have relatively longer and thicker tails than do western harvest mice, which may be related to their abilities to climb emergent marsh vegetation to avoid tidal inundation. We used micro-CT to compare post-cranial skeletal anatomy between the salt marsh and western harvest mouse, to examine whether the salt marsh harvest mouse\'s restriction to brackish marshes is associated with skeletal adaptations for scansorial locomotion. We found that salt marsh harvest mice exhibited a deeper 3rd caudal vertebra, a more caudally located longest tail vertebra, craniocaudally longer tail vertebrae, and a longer digit III proximal phalanx than western harvest mice. These phalangeal and vertebral characteristics are known to decrease body rotations during climbing, increase contact with substrates, and decrease fall susceptibility in arboreal mammals, suggesting that the salt marsh harvest mouse may be morphologically specialized for scansorial locomotion, adaptive for its dynamic wetland environment.

Comparative anatomy is an important tool for investigating evolutionary relationships among species, but the lack of scalable imaging tools and stains for rapidly mapping the microscale anatomies of related species poses a major impediment to using comparative anatomy approaches for identifying evolutionary adaptations. We describe a method using synchrotron source micro-x-ray computed tomography (syn-μXCT) combined with machine learning algorithms for high-throughput imaging of Lepidoptera (i.e., butterfly and moth) eyes. Our pipeline allows for imaging at rates of ~15 min/mm3 at 600 nm3 resolution. Image contrast is generated using standard electron microscopy labeling approaches (e.g., osmium tetroxide) that unbiasedly labels all cellular membranes in a species-independent manner thus removing any barrier to imaging any species of interest. To demonstrate the power of the method, we analyzed the 3D morphologies of butterfly crystalline cones, a part of the visual system associated with acuity and sensitivity and found significant variation within six butterfly individuals. Despite this variation, a classic measure of optimization, the ratio of interommatidial angle to resolving power of ommatidia, largely agrees with early work on eye geometry across species. We show that this method can successfully be used to determine compound eye organization and crystalline cone morphology. Our novel pipeline provides for fast, scalable visualization and analysis of eye anatomies that can be applied to any arthropod species, enabling new questions about evolutionary adaptations of compound eyes and beyond.

UNASSIGNED: Imaging technologies that capture three-dimensional (3D) variation in floral morphology at micro- and nano-resolutions are increasingly accessible. In herkogamous flowers, such as those of Theobroma cacao, structural barriers between anthers and stigmas represent bottlenecks that restrict pollinator size and access to reproductive organs. To study the unresolved pollination biology of cacao, we present a novel application of micro-computed tomography (micro-CT) using floral dimensions to quantify pollinator functional size limits.
UNASSIGNED: We generated micro-CT data sets from field-collected flowers and museum specimens of potential pollinators. To compare floral variation, we used 3D Slicer to place landmarks on the surface models and performed a geometric morphometric (GMM) analysis using geomorph R. We identified the petal side door (an opening between the petal hoods and filament) as the main bottleneck for pollinator access. We compared its mean dimensions with proposed pollinators to identify viable candidates.
UNASSIGNED: We identified three levels of likelihood for putative pollinators based on the number of morphological (body) dimensions that fit through the petal side door. We also found floral reward microstructures whose presence and location were previously unclear.
UNASSIGNED: Using micro-CT and GMM to study the 3D pollination biology of cacao provides new evidence for predicting unknown pollinators. Incorporating geometry and floral rewards will strengthen plant-pollinator trait matching models for cacao and other species.
UNASSIGNED: Las tecnologías de imagen que capturan la variación tridimensional (3D) en la morfología floral en resoluciones a nivel micro y nano son cada vez más accesibles. En las flores hercógamas, tal como las de cacao (Theobroma cacao L.), las barreras estructurales entre las anteras y los estigmas representan cuellos de botella que restringen el tamaño de los polinizadores y el acceso a los órganos reproductivos. Para estudiar la biología de polinización del cacao que hasta estos momentos no ha sido completamente explicada, presentamos una nueva aplicación de microtomografía computarizada (microTC) que utiliza dimensiones florales para cuantificar los límites de tamaño funcional de los polinizadores.
UNASSIGNED: Generamos conjuntos de datos microTC de flores recolectadas en el campo, así como especímenes de museo de potenciales polinizadores. Para comparar la variación floral, usamos el programa 3D Slicer para colocar puntos de referencia en los modelos de superficie y realizamos un análisis morfométrico geométrico (MGM) usando el programa geomorph R. Identificamos la parte lateral de los pétalos (abertura entre las capuchas de los pétalos y el filamento) como el cuello de botella principal para el acceso de los polinizadores. Comparamos las medias de sus dimensiones con las de los polinizadores propuestos para identificar candidatos viables.
UNASSIGNED: Identificamos tres niveles de probabilidad para los supuestos polinizadores basado en el número de dimensiones morfológicas (de cuerpo) que caben a través la parte lateral de los pétalos. Encontramos microestructuras de recompensa floral a lo largo de las rutas de los polinizadores cuya presencia y ubicación no estaban muy claras anteriormente.
UNASSIGNED: El uso de microTC y GMM para estudiar la biología de polinización tridimensional (3D) del cacao proporciona nueva evidencia para predecir el tamaño de polinizadores desconocidos. La incorporación del análisis geométrico y de las microestructuras de recompensa floral fortalecerá los modelos de combinación de características de plantas y polinizadores para el cacao y otras especies.
UNASSIGNED: Tecnologias que usam imagem para capturar a variação tridimensional (3D) na morfologia floral em resoluções micro e nano estão cada vez mais acessíveis. Em flores hercogâmicas, tal como as do cacao (Theobroma cacao), barreiras estruturais entre as anteras e estigmas representam gargalos que restringem o tamanho do polinizador e o acesso aos órgãos reprodutivos. Para estudar a polinização do cacau, apresentamos uma nova aplicação de microtomografia computadorizada (microTC) usando dimensões florais para quantificar os limites de tamanho funcional de polinizadores.
UNASSIGNED: Nós geramos conjuntos de dados de microTC para flores coletadas em campo e para espécimes de museus de potenciais espécies polinizadoras. Para comparar a variação floral, usamos o software 3D Slicer para determinar pontos de referência na superfície dos modelos. Realizamos também análises de morfometria geométrica (MGM) usando o pacote geomorph no software R. Nós identificamos a abertura lateral da pétala (entre a pétala cuculada e os filamentos) como o principal gargalo para o acesso do polinizador. Por fim, comparamos as dimensões médias das flores com a dos polinizadores propostos para identificar candidatos viáveis.
UNASSIGNED: Identificamos três níveis de probabilidade para os supostos polinizadores baseados no número de dimensões morfológicas (corporais) que se ajustam à parte lateral das pétalas. Também encontramos microestruturas presentes nas flores que servem como recompensas aos polinizadores, cuja presença e localização não eram claras anteriormente.
UNASSIGNED: O uso de microTC e MGM para estudar a biologia da polinização do cacau em 3D fornece novas evidências para prevermos polinizadores desconhecidos. A incorporação de análises geométricas e microestruturas de recompensa floral poderá fortalecer modelos que combinam características da associação planta – polinizador tanto para o cacau, quanto para outras espécies.

Spiders evolved a distinctive sperm transfer system, with the male copulatory organs located on the tarsus of the pedipalps. In entelegyne spiders, these organs are usually very complex and consist of various sclerites that not only allow the transfer of the sperm themselves but also provide a mechanical interlock between the male and female genitalia. This interlocking can also involve elements that are not part of the copulatory organ such as the retrolateral tibial apophysis (RTA)-a characteristic of the most diverse group of spiders (RTA clade). The RTA is frequently used for primary locking i.e., the first mechanical engagement between male and female genitalia. Despite its functional importance, some diverse spider lineages have lost the RTA, but evolved an apophysis on the femur instead. It can be hypothesized that this femoral apophysis is a functional surrogate of the RTA during primary locking or possibly serves another function, such as self-bracing, which involves mechanical interaction between male genital structures themselves to stabilize the inserted pedipalp. We tested these hypotheses using ghost spiders of the genus Josa (Anyphaenidae). Our micro-computed tomography data of cryofixed mating pairs show that the primary locking occurs through elements of the copulatory organ itself and that the femoral apophysis does not contact the female genitalia, but hooks to a projection of the copulatory bulb, representing a newly documented self-bracing mechanism for entelegyne spiders. Additionally, we show that the femoral self-bracing apophysis is rather uniform within the genus Josa. This is in contrast to the male genital structures that interact with the female, indicating that the male genital structures of Josa are subject to different selective regimes.

Patients with type 1 diabetes mellitus (T1DM) exhibit reduced BMD and significant increases in fracture risk. Changes in BMD are attributed to blunted osteoblast activity and inhibited bone remodeling, but these cannot fully explain the impaired bone integrity in T1DM. The goal of this study was to determine the cellular mechanisms that contribute to impaired bone morphology and composition in T1DM. Nonobese diabetic (NOD) mice were used, along with μCT, histomorphometry, histology, Raman spectroscopy, and RNAseq analyses of several skeletal sites in response to naturally occurring hyperglycemia and insulin treatment. The bone volume in the axial skeleton was found to be severely reduced in diabetic NOD mice and was not completely resolved with insulin treatment. Decreased bone volume in diabetic mice was associated with increased sclerostin expression in osteocytes and attenuation of bone formation indices without changes in bone resorption. In the face of blunted bone remodeling, decreases in the mineral:matrix ratio were found in cortical bones of diabetic mice by Raman microspectroscopy, suggesting that T1DM did not affect the bone mineralization process per se, but rather resulted in microenvironmental alterations that favored mineral loss. Bone transcriptome analysis indicated metabolic shifts in response to T1DM. Dysregulation of genes involved in fatty acid oxidation, transport, and synthesis was found in diabetic NOD mice. Specifically, pyruvate dehydrogenase kinase isoenzyme 4 and glucose transporter 1 levels were increased, whereas phosphorylated-AKT levels were significantly reduced in diabetic NOD mice. In conclusion, in addition to the blunted bone formation, osteoblasts and osteocytes undergo metabolic shifts in response to T1DM that may alter the microenvironment and contribute to mineral loss from the bone matrix. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Understanding of normal fetal organ development is crucial for the evaluation of the pathogenesis of congenital anomalies. Various techniques have been used to generate imaging of fetal rat organogenesis, such as histological dissection with 3-dimensional reconstruction and scanning electron microscopy. However, these techniques did not imply quantitative measurements of developing organs (volumes, surface areas of organs). Furthermore, a partial or total destruction of the embryos prior to analysis was inevitable. Recently, micro-computed tomography (micro-CT) has been established as a novel tool to investigate embryonic development in non-dissected embryos of rodents. In this study, we used the micro-CT technique to generate 4D datasets of rat embryos aged between embryonic day 15-22 and newborns. Lungs, hearts, diaphragms, and livers were digitally segmented in order to measure organ volumes and analyze organ development as well as generate high-resolution 3D images. These data provide objective values compiling a 4D atlas of pulmonary, cardiac, diaphragmatic, and hepatic development in the fetal rat.

BACKGROUND: Hemophilic arthropathy is a debilitating morbidity of hemophilia caused by recurrent joint bleeds. We investigated if the joint bleed volume, before initiation of treatment, was linked to the subsequent degree of histopathological changes and the development of bone pathology in a mouse model of hemophilic arthropathy.
METHODS: FVIII knock-out (F8-KO) mice were dosed with a micro-CT blood pool agent prior to induction of hemarthrosis. Eight hours after induction, the bleed volume was quantified with micro computed tomography (micro-CT) and recombinant FVIII treatment initiated. On Day 8, inflammation in the knees was characterized by fluorescence molecular tomography. On Day 14, knee pathology was characterized by micro-CT and histopathology. In a second study, contrast agent was injected into the knee of wild-type (WT) mice, followed by histopathological evaluation on Day 14.
RESULTS: The average joint bleed volume before treatment was 3.9 mm3. The inflammation-related fluorescent intensities in the injured knees were significantly increased on Day 8. The injured knees had significantly increased synovitis scores, vessel counts, and areas of hemosiderin compared to un-injured knees. However, no cartilage- or bone pathology was observed. The bleed volume before initiation of treatment correlated with the degree of synovitis and was associated with high fluorescent intensity on Day 8. In F8-KO and WT mice, persistence of contrast agent in the joint elicited morphological changes.
CONCLUSIONS: When applying a delayed on-demand treatment regimen to hemophilic mice subjected to an induced knee hemarthrosis, the degree of histopathological changes on Day 14 reflected the bleed volume prior to initiation of treatment.

Congenital heart disease (CHD) is the major cause of morbidity/mortality in infancy and childhood. Using a mouse model to uncover the mechanism of CHD is essential to understand its pathogenesis. However, conventional 2D phenotyping methods cannot comprehensively exhibit and accurately distinguish various 3D cardiac malformations for the complicated structure of heart cavity. Here, a new automated tool based on microcomputed tomography (micro-CT) image data sets known as computer-assisted cardiac cavity tracking (CACCT) is presented, which can detect the connections between cardiac cavities and identify complicated cardiac malformations in mouse hearts automatically. With CACCT, researchers, even those without expert training or diagnostic experience of CHD, can identify complicated cardiac malformations in mice conveniently and precisely, including transposition of the great arteries, double-outlet right ventricle and atypical ventricular septal defect, whose accuracy is equivalent to senior fetal cardiologists. CACCT provides an effective approach to accurately identify heterogeneous cardiac malformations, which will facilitate the mechanistic studies into CHD and heart development.

Although administration of simvastatin has been reported to promote bone formation, the effect of short-term simvastatin administration is not well known. Following implant installation, 10-week-old male Wistar rats (n = 24) were divided into two groups randomly. The experimental group received 10 mg/kg of simvastatin daily for seven days. Then simvastatin administration was discontinued, and the animals were observed up to 28 days. Animals in the control group underwent the same procedure but received saline instead of simvastatin. All animals were analyzed by micro-computed tomography. Samples at days 14 and 21 were subjected to histological analyses. After seven days of simvastatin administration, more new bone formation around the implant was observed in the simvastatin group compared with the control group. Seven days after simvastatin discontinuation, however, the amount of peri-implant trabecular bone began to decrease. Results from morphometric analysis also showed a reduction in new bone area after day 7, which was lowest at day 14. These results were confirmed by histological analyses. In contrast, both the peri-implant trabecular bone and new bone area were maintained in the control group. Short-term administration of simvastatin may affect implant stability owing to a rebound phenomenon and an immediate loss of peri-implant bone.

OBJECTIVE: There is an impending need for noninvasive biomarkers of breast cancer angiogenesis to evaluate the efficacy of new anti-angiogenic therapies in vivo. The purpose of this study was to systematically evaluate the sensitivity of in vivo steady-state susceptibility contrast-MRI biomarkers of angiogenesis in a human breast cancer model.
METHODS: Orthotopic MDA-MB-231 human breast cancer xenografts were imaged by steady-state susceptibility contrast-MRI at post-inoculation week 3 and post-inoculation week 5, followed by ex vivo whole tumor 3D micro-CT angiography. \"Absolute\" (i.e., measures of vascular morphology in appropriate units) and \"relative\" (i.e., proportional to measures of vascular morphology) MRI biomarkers of tumor blood volume, vessel size, and vessel density were computed and their ability to predict the corresponding micro-CT analogs assessed using cross-validation analysis.
RESULTS: All MRI biomarkers significantly correlated with their micro-CT analogs and were sensitive to the micro-CT-measured decreases in tumor blood volume and vessel density from post-inoculation week 3 to post-inoculation week 5. However, cross-validation analysis revealed there was no significant difference between the predictive accuracy of \"absolute\" and \"relative\" biomarkers.
CONCLUSIONS: As \"relative\" biomarkers are more easily computed from steady-state susceptibility contrast-MRI (i.e., without additional MRI measurements) than \"absolute\" biomarkers, it makes them promising candidates for assessing breast cancer angiogenesis in vivo.