Three-Dimensional Architecture of Glomerular Endothelial Cells Revealed by FIB-SEM Tomography.

Focused-ion beam-scanning electron microscopic (FIB-SEM) tomography enables easier acquisition of a series of ultrastructural, sectional images directly from resin-embedded biological samples. In this study, to clarify the three-dimensional (3D) architecture of glomerular endothelial cells (GEnCs) in adult rats, we manually extracted GEnCs from serial FIB-SEM images and reconstructed them on an Amira reconstruction software. The luminal and basal surface structures were clearly visualized in the reconstructed GEnCs, although only the luminal surface structures could be observed by conventional SEM. The luminal surface visualized via the reconstructed GEnCs was quite similar to that observed through conventional SEM, indicating that 3D reconstruction could be performed with high accuracy. Thus, we successfully described the 3D architecture of normal GEnCs in adult rats more clearly and precisely than ever before. The GEnCs were found to consist of three major subcellular compartments, namely, the cell body, cytoplasmic ridges, and sieve plates, in addition to two associated subcellular compartments, namely, the globular protrusions and reticular porous structures. Furthermore, most individual GEnCs made up a "seamless" tubular shape, and some of them formed an autocellular junction to make up a tubular shape. FIB-SEM tomography with reconstruction is a powerful approach to better understand the 3D architecture of GEnCs. Moreover, the morphological information revealed in this study will be valuable for the 3D pathologic evaluation of GEnCs in animal and human glomerular diseases and the structural analysis of developmental processes in the glomerular capillary system.

Anatomic characterization of the tibial and fibular nutrient arteries in humans.

The location of nutrient foramina has been extensively studied in long bones; however, accurate information on the origin and extra-osseous course of the nutrient artery remains clearly defined in some long bones, although it is crucial to protect the nutrient arteries during operative procedures. In this study, we elucidated the origin and extra-osseous course of tibial and fibular nutrient arteries based on the 54 cadaveric legs. The tibial nutrient artery typically arose from the posterior tibial artery. Some of the tibial nutrient arteries arose from the anterior tibial, popliteal, and fibular arteries. The tibial nutrient artery arose from these parent arteries as a long descending branch. It penetrated the most proximal portion of the tibialis posterior or flexor digitorum longus to enter the tibial nutrient foramen. The fibular nutrient artery arose from the fibular artery as a short descending branch in all the cases. The fibular nutrient artery penetrated the flexor hallucis longus to enter the fibular nutrient foramen. Our present and previous findings provide new insight into the anatomical characteristics for the nutrient arteries in the long bones of upper and lower extremities. Namely, the nutrient arteries of the long bones go away from the elbow or knee to enter the nutrient foramina.

Anatomical study of the soleus: Application to improved imaging diagnoses.

INTRODUCTION: Strains of the soleus are widely found both in amateur and professional athletes. For their accurate regional diagnoses, understanding the anatomy of the spatial relationship between muscular fibers and tendinous structures is important because their interfaces are susceptible sites to muscle strains. Therefore, this study evaluated the precise architecture of the soleus. MATERIALS AND METHODS: We evaluated the precise anatomical architecture of the soleus in 87 formaldehyde-fixed soleus muscles. To calculate mean relative physiological cross-sectional area of each muscular fiber compartment, we measured the fiber length, volume, and pennation angle in isolated compartments. RESULTS: The posterior soleus surface was covered by a broad aponeurotic posterior insertion tendon (PIT), which continued inferiorly to the insertion tendon. The anterior surface had three aponeurotic origin tendons, lateral origin tendon (LOT), medial origin tendon (MOT), and tendinous arch, which were arranged along the soleus margins. The anterior bipennate muscle portion (ABP), surrounded by the three origin structures, terminated as the sagittal insertion tendon (SIT), which continued inferiorly to PIT. The posterior main muscle portion behind LOT and MOT was separated into lateral and medial portions by the SIT. The soleus thus possessed a broad musculotendinous junction. Furthermore, ABP exhibited wide structural diversity in shape and size: in extreme cases, it was duplicated or absent. CONCLUSION: Systematic anatomical descriptions of the soleus will be useful for accurate regional diagnosis of its strains with magnetic resonance imaging and ultrasonography.

Nephrocytes are part of the spectrum of filtration epithelial diversity.

The excretory system produces urine by ultrafiltration via a filtration epithelium. Podocytes are widely found as filtration epithelial cells in eucoelomates. In some animal taxa, including insects and crustaceans, nephrocytes serve to separate toxic substances from the body fluid, in addition to podocytes. Drosophila nephrocytes have been recently utilized as a model system to study podocyte function and disease. However, functionality and cellular architecture are strikingly different between Drosophila nephrocytes and eucoelomate podocytes, and the phylogenetic relationship between these cells remains enigmatic. In this study, using focused-ion beam-scanning electron microscopy (FIB-SEM) tomography, we revealed three-dimensional architecture of decapod nephrocytes with unprecedented accuracy-they filled an enormous gap, which can be called "missing link," in the evolutionary diversity of podocytes and nephrocytes. Thus, we concluded that nephrocytes are part of the spectrum of filtration epithelial diversity in animal phylogeny.

Anatomic characterization of the femoral nutrient artery: Application to fracture and surgery of the femur.

The localization of nutrient foramina and direction of nutrient canals have been studied. However, information about the origin and extraosseous course of nutrient arteries is lacking in some types of long tubular and irregular bones. Thus, we aimed to reexamine the origin and course of the femoral nutrient artery (FNA) through cadaveric dissection to clarify its anatomic characteristics. Sixty thighs were collected from 57 cadavers. To fix the cadavers and visualize the small arterial branches, 10% formalin was injected from the femoral artery, followed by an injection of acrylic ink. The arterial tree in the posterior part of the thigh was recorded by line drawings. The femur received single or double FNAs via the femoral nutrient foramina, which were on and along the linea aspera. In cases with single FNA (41 of the 60 thighs), it typically arose from the four parts of the profunda femoris system: profunda femoris artery between the origins of the third and fourth perforating arteries; second perforating artery; third perforating artery; and terminal branch. In cases with double FNAs (remaining 19 thighs), the superior FNA typically arose from the second perforating artery, and the inferior FNA arose from the terminal branch of the profunda femoris artery and popliteal system. FNAs are described as branches of the perforating arteries in Terminologia Anatomica and anatomy textbooks. However, we found that FNAs also frequently arose from the profunda femoris artery and popliteal system, in addition to the perforating arteries. Clin. Anat. 33:479-487, 2020. © 2019 Wiley Periodicals, Inc.

Three-dimensional imaging of podocyte ultrastructure using FE-SEM and FIB-SEM tomography.

Podocytes are specialized epithelial cells used for glomerular filtration in the kidney. They can be divided into the cell body, primary process and foot process. Here, we describe two useful methods for the three-dimensional(3D) visualization of these subcellular compartments in rodent podocytes. The first method, field-emission scanning electron microscopy (FE-SEM) with conductive staining, is used to visualize the luminal surface of numerous podocytes simultaneously. The second method, focused-ion beam SEM (FIB-SEM) tomography, allows the user to obtain serial images from different depths of field, or Z-stacks, of the glomerulus. This allows for the 3D reconstruction of podocyte ultrastructure, which can be viewed from all angles, from a single image set. This is not possible with conventional FE-SEM. The different advantages and disadvantages of FE-SEM and FIB-SEM tomography compensate for the weaknesses of the other. The combination renders a powerful approach for the 3D analysis of podocyte ultrastructure. As a result, we were able to identify a new subcellular compartment of podocytes, "ridge-like prominences" (RLPs).

Three-dimensional architecture of pericardial nephrocytes in Drosophila melanogaster revealed by FIB/SEM tomography.

Nephrocytes are similar in structure to podocytes and play a role in the isolation of toxic substances from hemolymph in insects. Drosophila melanogaster nephrocytes have recently been used to study podocyte function and disease. However, the three-dimensional ultrastructure of nephrocytes is not clearly understood because their surrounding basement membrane makes it difficult to observe using conventional scanning electron microscopy. We reconstructed the three-dimensional ultrastructure of Drosophila pericardial nephrocytes using serial focused-ion beam/scanning electron microscopy (FIB/SEM) images. The basal surfaces were occupied by foot processes and slit-like spaces between them. The slit-like spaces corresponded to the podocyte filtration slits and were formed by longitudinal infolding/invagination of the basal plasma membrane. The basal surface between the slit-like spaces became the foot processes, which ran almost linearly, and had a "washboard-like" appearance. Both ends of the foot processes were usually anastomosed to neighboring foot processes and thus free ends were rarely observed. We demonstrated that FIB/SEM is a powerful tool to better understand the three-dimensional architecture of nephrocytes.

Morphological Processes of Foot Process Effacement in Puromycin Aminonucleoside Nephrosis Revealed by FIB/SEM Tomography.

BACKGROUND: Foot process effacement is one of the pathologic indicators of podocyte injury. However, the morphologic changes associated with it remain unclear. METHODS: To clarify the developmental process, we analyzed puromycin nephrotic podocytes reconstructed from serial focused-ion beam/scanning electron microscopy (FIB/SEM) images. RESULTS: Intact podocytes consisted of four subcellular compartments: cell body, primary process, ridge-like prominence (RLP), and foot process. The RLP, a longitudinal protrusion from the basal surface of the cell body and primary process, served as an adhesive apparatus for the cell body and primary process to attach to the glomerular basement membrane. Foot processes protruded from both sides of the RLP. In puromycin nephrotic podocytes, foot process effacement occurred in two ways: by type-1 retraction, where the foot processes retracted while maintaining their rounded tips; or type-2 retraction, where they narrowed across their entire lengths, tapering toward the tips. Puromycin nephrotic podocytes also exhibited several alterations associated with foot process effacement, such as deformation of the cell body, retraction of RLPs, and cytoplasmic fragmentation. Finally, podocytes were reorganized into a broad, flattened shape. CONCLUSIONS: The three-dimensional reconstruction of podocytes by serial FIB/SEM images revealed the morphologic changes involved in foot process effacement in greater detail than previously described.

Reevaluation of the superior radial collateral artery in the human upper arm.

The superior radial collateral artery (SRCA) was described in well-established anatomy textbooks published in the 1800s. According to those textbooks, the SRCA originates from the brachial artery, passes transversely between the coracobrachialis and the humerus, and distributes to the most distal portion of the deltoid. The SRCA is not listed in the international standard on anatomical terminology, Terminologia Anatomica, or in modern anatomy textbooks. In the present study, we reevaluated the anatomical features of the SRCA by cadaveric dissection. We found that two kinds of SRCAs were consistently present in the upper arm. One was similar to the previous descriptions of the SRCA in terms of origin and course, but the distribution was somewhat different. The other was similar to the previous descriptions in terms of the distribution, although it differed in origin and course. The discrepancy between the description of the SRCA in classical textbooks and the actual morphologies of the SRCA presumably prompted previous anatomists to question the existence of the SRCA, resulting in its absence from anatomical textbooks after a particular time point.

Anatomic characterization of the radial and ulnar nutrient arteries in humans.

BACKGROUND: The localization of nutrient foramens has been extensively studied in humans and other vertebrate animals. However, accurate information on the origin and extraosseous course of the nutrient arteries in some types of long tubular bones is lacking. Terminologia Anatomica, the international standard on human anatomic terminology, lists the radial nutrient artery (RNA) and the ulnar nutrient artery (UNA) as branches of the radial and ulnar arteries, respectively. Anatomy textbooks published in both German- and English-speaking countries regard both the RNA and UNA as branches of the anterior interosseous artery. METHODS: To clarify the anatomic characteristics of the RNA and UNA in humans, we reexamined the origin and course of these arteries by cadaveric dissection. RESULTS: Almost all RNAs and UNAs branched from the ulnar artery or its tributaries. In typical cases, the RNA branched from the anterior interosseous artery and the UNA branched from the proximal part of the ulnar artery or the anterior interosseous artery. These findings are reasonable from the perspective of regional anatomy, since the ulnar artery passes more deeply than the radial artery in the proximal forearm and thus the proximal part of the ulnar artery and its major branches are situated more closely to the radial and ulnar nutrient foramens. CONCLUSIONS: Based on our findings, it is necessary to correct the position of the RNA and UNA in the arterial hierarchy of T. Anatomica for accurate morphological description.

Anatomic characterization of the humeral nutrient artery: Application to fracture and surgery of the humerus.

Anatomic characterization of the humeral nutrient artery varies among the several textbooks on human anatomy. To clarify the anatomic characteristics of the humeral nutrient artery, we reexamined its origin and course by cadaveric dissection. In typical cases, one prominent nutrient foramen was situated on the anteromedial surface of the humeral shaft, and the nutrient canal distally penetrated the cortical bone layer. The humeral nutrient artery originated from the brachial artery below the level of the nutrient foramen as a short ascending branch. On reaching near the nutrient foramen, the humeral nutrient artery formed a hairpin loop on the periosteum to enter into the nutrient foramen. In some cases, an accessory nutrient foramen was also found near the groove for the radial nerve on the posterior surface of the humerus. This accessory nutrient foramen received an accessory humeral nutrient artery that originated from the radial collateral artery. The present findings corresponded well with the descriptions in the anatomy textbooks published in English-speaking countries. However, textbooks published in German-speaking countries describe only one type of humeral nutrient artery, the branch of the profunda brachii artery. Terminologia Anatomica, the international standard in human anatomic terminology, most likely adopted the description in the German anatomy textbooks, and thus, it is necessary to correct the position of the humeral nutrient artery in the hierarchy of Terminologia Anatomica for accurate morphological description. Clin. Anat. 30:978-987, 2017. © 2017 Wiley Periodicals, Inc.

Morphological process of podocyte development revealed by block-face scanning electron microscopy.

Podocytes present a unique 3D architecture specialized for glomerular filtration. However, several 3D morphological aspects on podocyte development remain partially understood because they are difficult to reveal using conventional scanning electron microscopy (SEM). Here, we adopted serial block-face SEM imaging, a powerful tool for analyzing the 3D cellular ultrastructure, to precisely reveal the morphological process of podocyte development, such as the formation of foot processes. Development of foot processes gives rise to three morphological states: the primitive, immature and mature foot processes. Immature podocytes were columnar in shape and connected to each other by the junctional complex, which migrated toward the basal side of the cell. When the junctional complex was close to the basement membrane, immature podocytes started to interdigitate with primitive foot processes under the level of junctional complex. As primitive foot processes lengthened, the junctional complex moved between primitive foot processes to form immature foot processes. Finally, the junctional complex was gradually replaced by the slit diaphragm, resulting in the maturation of immature foot processes into mature foot processes. In conclusion, the developmental process of podocytes is now clearly visualized by block-face SEM imaging.