Histopathological heterogeneity in the human pancreas is well documented; however, functional evidence at the tissue level is scarce. Herein, we investigate in situ glucose-stimulated islet and carbachol-stimulated acinar cell secretion across the pancreas head (PH), body (PB), and tail (PT) regions in donors without diabetes (ND; n = 15), positive for one islet autoantibody (1AAb+; n = 7), and with type 1 diabetes (T1D; <14 months duration, n = 5). Insulin, glucagon, pancreatic amylase, lipase, and trypsinogen secretion along with 3D tissue morphometrical features are comparable across regions in ND. In T1D, insulin secretion and beta-cell volume are significantly reduced within all regions, while glucagon and enzymes are unaltered. Beta-cell volume is lower despite normal insulin secretion in 1AAb+, resulting in increased volume-adjusted insulin secretion versus ND. Islet and acinar cell secretion in 1AAb+ are consistent across the PH, PB, and PT. This study supports low inter-regional variation in pancreas slice function and, potentially, increased metabolic demand in 1AAb+.

Cluster analyzes of facial models of autistic patients aim to clarify whether it is possible to diagnose autism on the basis of facial features and further to stratify the autism spectrum disorder. We performed a cluster analysis of sets of 3D scans of ASD patients (116) and controls (157) using Euclidean and geodesic distances in order to recapitulate the published results on the Czech population. In the presented work, we show that the major factor determining the clustering structure and consequently also the correlation of resulting clusters with autism severity degree is body mass index corrected for age (BMIFA). After removing the BMIFA effect from the data in two independent ways, both the cluster structure and autism severity correlations disappeared. Despite the fact that the influence of body mass index (BMI) on facial dimensions was studied many times, this is the first time to our knowledge when BMI was incorporated into the faces clustering study and it thereby casts doubt on previous results. We also performed correlation analysis which showed that the only correction used in the existing clustering studies-dividing the facial distance by the average value within the face-is not eliminating correlation between facial distances and BMIFA within the facial cohort.

During aging, muscle gradually undergoes sarcopenia, the loss of function associated with loss of mass, strength, endurance, and oxidative capacity. However, the 3D structural alterations of mitochondria associated with aging in skeletal muscle and cardiac tissues are not well described. Although mitochondrial aging is associated with decreased mitochondrial capacity, the genes responsible for the morphological changes in mitochondria during aging are poorly characterized. We measured changes in mitochondrial morphology in aged murine gastrocnemius, soleus, and cardiac tissues using serial block-face scanning electron microscopy and 3D reconstructions. We also used reverse transcriptase-quantitative PCR, transmission electron microscopy quantification, Seahorse analysis, and metabolomics and lipidomics to measure changes in mitochondrial morphology and function after loss of mitochondria contact site and cristae organizing system (MICOS) complex genes, Chchd3, Chchd6, and Mitofilin. We identified significant changes in mitochondrial size in aged murine gastrocnemius, soleus, and cardiac tissues. We found that both age-related loss of the MICOS complex and knockouts of MICOS genes in mice altered mitochondrial morphology. Given the critical role of mitochondria in maintaining cellular metabolism, we characterized the metabolomes and lipidomes of young and aged mouse tissues, which showed profound alterations consistent with changes in membrane integrity, supporting our observations of age-related changes in muscle tissues. We found a relationship between changes in the MICOS complex and aging. Thus, it is important to understand the mechanisms that underlie the tissue-dependent 3D mitochondrial phenotypic changes that occur in aging and the evolutionary conservation of these mechanisms between Drosophila and mammals.

Cardioprotection by preventing or repairing mitochondrial damage is an unmet therapeutic need. To understand the role of cardiomyocyte mitochondria in physiopathology, the reliable characterization of the mitochondrial morphology and compartment is pivotal. Previous studies mostly relied on two-dimensional (2D) routine transmission electron microscopy (TEM), thereby neglecting the real three-dimensional (3D) mitochondrial organization. This study aimed to determine whether classical 2D TEM analysis of the cardiomyocyte ultrastructure is sufficient to comprehensively describe the mitochondrial compartment and to reflect mitochondrial number, size, dispersion, distribution, and morphology.
Spatial distribution of the complex mitochondrial network and morphology, number, and size heterogeneity of cardiac mitochondria in isolated adult mouse cardiomyocytes and adult wild-type left ventricular tissues (C57BL/6) were assessed using a comparative 3D imaging system based on focused ion beam-scanning electron microscopy (FIB-SEM) nanotomography. For comparison of 2D vs. 3D data sets, analytical strategies and mathematical comparative approaches were performed. To confirm the value of 3D data for mitochondrial changes, we compared the obtained values for number, coverage area, size heterogeneity, and complexity of wild-type cardiomyocyte mitochondria with data sets from mice lacking the cytosolic and mitochondrial protein BNIP3 (BCL-2/adenovirus E1B 19-kDa interacting protein 3; Bnip3-/- ) using FIB-SEM. Mitochondrial respiration was assessed on isolated mitochondria using the Seahorse XF analyser. A cardiac biopsy was obtained from a male patient (48 years) suffering from myocarditis.
The FIB-SEM nanotomographic analysis revealed that no linear relationship exists for mitochondrial number (r = 0.02; P = 0.9511), dispersion (r = -0.03; P = 0.9188), and shape (roundness: r = 0.15, P = 0.6397; elongation: r = -0.09, P = 0.7804) between 3D and 2D results. Cumulative frequency distribution analysis showed a diverse abundance of mitochondria with different sizes in 3D and 2D. Qualitatively, 2D data could not reflect mitochondrial distribution and dynamics existing in 3D tissue. 3D analyses enabled the discovery that BNIP3 deletion resulted in more smaller, less complex cardiomyocyte mitochondria (number: P < 0.01; heterogeneity: C.V. wild-type 89% vs. Bnip3-/- 68%; complexity: P < 0.001) forming large myofibril-distorting clusters, as seen in human myocarditis with disturbed mitochondrial dynamics. Bnip3-/- mice also show a higher respiration rate (P < 0.01).
Here, we demonstrate the need of 3D analyses for the characterization of mitochondrial features in cardiac tissue samples. Hence, we observed that BNIP3 deletion physiologically acts as a molecular brake on mitochondrial number, suggesting a role in mitochondrial fusion/fission processes and thereby regulating the homeostasis of cardiac bioenergetics.

Microvascularization of domestic fowl kidneys was studied using scanning electron microscopy (SEM) of vascular corrosion casts (VCCs). Two types of nephrons, mammalian-type (MT) and reptilian-type (RT) nephrons and their glomerular structure were analysed quantitatively by 3D morphometry. A significant difference in shape and size between the MT and RT glomeruli was found. The mean diameter of the RT glomeruli was about 56 µm, while that of MT glomeruli was significantly larger, namely about 80 µm. The afferent arterioles in mammalian-type glomeruli usually bifurcated into two lobular branches and formed a complex glomerular capillary network with numerous loops. Reptilian-type glomeruli consisted of a single capillary forming few loops and leaving the glomerulus as efferent arteriole. Diameters of afferent and efferent arteriolar replicas were similar in all three kidney divisions of MT and RT nephrons. The absence of the interconnecting branches between the MT nephron capillaries at the gross inspection suggests that the mammalian-type nephron glomeruli, although more complex than the reptilian type, are not equivalent to those in mammalian kidneys.

Liver fibrosis is a dynamic condition caused by wound-healing in which scar tissue replaces the liver parenchyma following repetitive injuries. It is hypothesized that α-mangostin (AM), the major constituent of the xanthone fraction in extracts of Garcinia mangostana L., may protect the hepatic microvascular bed from thioacetamide (TAA)-induced fibrosis. In the present study, rats were divided into 4 groups: Control rats received no treatment; TAA-treated rats received 150 mg/kg TAA 3 times per week intraperitoneally; AM-treated rats received 75 mg/kg AM twice per week intraperitoneally; and TAA+AM-treated rats received both TAA and AM as described above. Rat livers were processed either for light microscopy or for vascular corrosion casting after 30 and 60 days of treatment. Vascular parameters were measured by 3D morphometry analysis of scanning electron micrographs. AM attenuated hepatocellular injuries and delayed both periportal and pericentral fibrosis in the TAA-treated rats. The comparison of findings at day 30 and 60 showed that TAA-induced fibrotic changes were progressive in time, and that the beneficial effects of AM only became apparent after prolonged treatment. The livers of rats treated with both TAA and AM had less space surrounding the portal vessels, improved preservation of the hepatic microvascular pattern, and minimally altered sinusoidal patterns with few signs of terminal portal venule remodeling. AM therefore partially protected the liver against hepatotoxin-induced fibrosis and the associated microvascular changes. The mechanism of the protective effect of AM on the liver remains to be investigated.

How complicated cell activities produce characteristic tissue and organ morphologies is an important question in plant morphogenesis. To address this question, 3D morphometry of plant organs on multiscales is indispensable. In recent years, advances in confocal microscopy with fluorescent probes that mark the cell wall or plasma membrane enable the visualization of organ morphology with submicron precision. In parallel, new quantitative and correlative imaging pipelines realize 3D image processing on 2D curved surface, facilitating the study of cell and tissue behaviors in plant organogenesis. Here, we describe methods for 3D morphometry of Arabidopsis sepals, focusing on live imaging coupled with MorphoGraphX-based 3D image processing for cellular growth analysis.

The ovarian bursa is a small peritoneal cavity enclosed by the mesovarium and mesosalpinx, which surrounds the ovaries and oviductal infundibulum in mammals. The ovarian bursa is considered as the structure facilitating the transport of ovulated oocytes into the oviduct. Our previous study revealed reduced oocyte pick-up function in the oviduct of lupus-prone MRL/MpJ-Faslpr/lpr mouse, suggesting the possibility of an escape of ovulated oocytes into the peritoneal cavity, despite the presence of an almost complete ovarian bursa in the mouse. In this study, we revealed anatomical and histological characteristics of the ovarian bursa in C57BL/6 N, MRL/MpJ, and MRL/MpJ-Faslpr/lpr mice. All strains had the foramen of ovarian bursa (FOB), with a size of approximately 0.04 to 0.12 cm2 , surrounded by the ligament of ovarian bursa (LOB), which is part of the mesosalpinx. The LOB was partially lined with the cuboidal mesothelial cells and consisted of a thick smooth muscle layer in all strains. In 6-month-old MRL/MpJ-Faslpr/lpr mice, in which the systemic autoimmune abnormality deteriorated and oocyte pick-up function was impaired, the size of the FOB tended to be larger than that of other strains. Additionally, in MRL/MpJ-Faslpr/lpr mice at 6 months of age, there was infiltration by numerous immune cells in the mesosalpinx suspending the isthmus; however, the LOB prevented severe inflammation and showed deposition of collagen fibers. These results not only indicate that the FOB is a common structure within mice, but also imply the physiological function of the LOB and its role in maintaining the microenvironment around the ovary, as well as regulating healthy reproduction.

Genetic and biochemical defects of mitochondrial function are a major cause of human disease, but their link to mitochondrial morphology in situ has not been defined. Here, we develop a quantitative three-dimensional approach to map mitochondrial network organization in human muscle at electron microscopy resolution. We establish morphological differences between human and mouse and among patients with mitochondrial DNA (mtDNA) diseases compared to healthy controls. We also define the ultrastructure and prevalence of mitochondrial nanotunnels, which exist as either free-ended or connecting membrane protrusions across non-adjacent mitochondria. A multivariate model integrating mitochondrial volume, morphological complexity, and branching anisotropy computed across individual mitochondria and mitochondrial populations identifies increased proportion of simple mitochondria and nanotunnels as a discriminant signature of mitochondrial stress. Overall, these data define the nature of the mitochondrial network in human muscle, quantify human-mouse differences, and suggest potential morphological markers of mitochondrial dysfunction in human tissues.

Subdivision of cloaca into urogenital and anorectal passages has remained controversial because of disagreements about the identity and role of the septum developing between both passages. This study aimed to clarify the development of the cloaca using a quantitative 3D morphological approach in human embryos of 4-10 post-fertilisation weeks. Embryos were visualised with Amira 3D-reconstruction and Cinema 4D-remodelling software. Distances between landmarks were computed with Amira3D software. Our main finding was a pronounced difference in growth between rapidly expanding central and ventral parts, and slowly or non-growing cranial and dorsal parts. The entrance of the Wolffian duct into the cloaca proved a stable landmark that remained linked to the position of vertebra S3. Suppressed growth in the cranial cloaca resulted in an apparent craniodorsal migration of the entrance of the Wolffian duct, while suppressed growth in the dorsal cloaca changed the entrance of the hindgut from cranial to dorsal on the cloaca. Transformation of this \'end-to-end\' into an \'end-to-side\' junction produced temporary \'lateral (Rathke\'s) folds\'. The persistent difference in dorsoventral growth straightened the embryonic caudal body axis and concomitantly extended the frontally oriented \'urorectal (Tourneux\'s) septum\' caudally between the ventral urogenital and dorsal anorectal parts of the cloaca. The dorsoventral growth difference also divided the cloacal membrane into a well-developed ventral urethral plate and a thin dorsal cloacal membrane proper, which ruptured at 6.5 weeks. The expansion of the pericloacal mesenchyme followed the dorsoventral growth difference and produced the genital tubercle. Dysregulation of dorsal cloacal development is probably an important cause of anorectal malformations: too little regressive development may result in anorectal agenesis, and too much regression in stenosis or atresia of the remaining part of the dorsal cloaca.