Histological Preparation of Large Vertebral Specimens

A histological method is described for processing human lumbosacral vertebral specimens through fixation, decalcification, dehydration and embedding in a mixture of low viscosity nitrocellulose and celloidin. The large blocks thus obtained were serially sectioned on a Jung Tetrander microtome at a section thickness of 100 microns. Shrinkage of 8.6% was observed for the mounted specimens by comparing the unprocessed specimens (by X-ray measurement) with the mounted sections (measured using a transparent ruler). A horizontally sectioned lumbosacral joint is used to illustrate the excellent histological detail obtained of the anatomy at this level of the spinal column.     

Structural analysis of theridiid spider's testicular cyst using 3D reconstruction rendering

A three dimensional reconstruction technique was used for the analysis of a theridiid spider's (Achaearanea tepidariorum) testicular cyst. Although microscopic techniques have greatly improved, most of the information gathered is still based on two‐dimensional images. Particularly in spiders, it is very difficult to count the exact number of sperm in a single cyst, since their spermatogenetic processes takes place within the spherical cysts through the flagellar coiling process. Since morphological features of spider sperm provide detailed information on the whole spermatogenetic processes, we analyzed the exact number of germ cells per cyst in A. tepidariorum through a three‐dimensional image reconstruction technique. For image processing, serially sectioned histological images were scanned using a light microscope and 3D rendering images were reconstructed from these sections. Based on the three dimensional image analysis of the testicular cyst, the number of secondary spermatocytes per cyst was calculated to be 32 (2⁵). Therefore the total number of sperm produced from a single cyst can be calculated as 64 (2⁶), which indicates that a single spermatogonium undergoes four mitotic divisions and an additional two meiotic divisions to produce mature spermatozoa.     

<Emphasis Type="Italic">In situ</Emphasis> hybridization in the pathology laboratory: General principles,automation, and emerging research applications for tissue-based studies of gene expression

Diagnostic anatomic pathologists play an important role in the care of patients through their careful evaluation of morphological features in routinely prepared histological sections stained with Hematoxylin and Eosin. Morphological assessment of tissue sections, backed by over one hundred years of experience is powerful and allows for the accurate classification and diagnosis of the majority of disease states within pathologically altered tissues. However, the appearance of cells and their architectural arrangement within a morphologically complex tissue represents only a fraction of the information, which is contained within a histological section. These tissues also contain all of the cellular proteins and expressed genes, which help to ultimately determine the biological behavior of cells, as well as provide clues to the origins and pathogenesis of disease states. Technical and theoretical advances in our understanding of cellular biology have provided pathologists with powerful tools to probe beyond pure morphology into the abnormalities in both protein and gene expression that underlie human disease. These tools, which include immunohistochemistry and in situ hybridization, are playing an increasingly important role in diagnostic pathology, as well as in translational research. This review will focus on the emerging role of in situ hybridization within clinical and research laboratories, and will highlight a number of technical advances that have expanded the application of this technology.     

Phenotypic variability in human embryonic holoprosencephaly in the Kyoto Collection

BACKGROUND: Holoprosencephaly (HPE) is one of the most common developmental disorders of the brain associated with specific craniofacial dysmorphogenesis. Although numerous postnatal cases have been reported, early phases of its pathogenesis are not well understood. We examined over 200 cases of HPE human embryos both grossly and histologically, and studied their phenotypic variability and stage-specific characteristics. METHODS: Among over 44,000 human embryos in the Kyoto Collection of Human Embryos, 221 embryos have been diagnosed as HPE. Their developmental stages ranged from Carnegie stage (CS) 13 to CS 23. They were examined grossly and were also serially sectioned for detailed histological analysis. RESULTS: HPE embryos after CS 18 were classified into complete (true) cyclopia, synophthalmia (partially fused eyes in a single eye fissure), closely apposed separate eyes (possible forerunners of ethmocephaly and cebocephaly), and milder HPE with median cleft lip (premaxillary agenesis). At CS 13-17, when facial morphogenesis is not completed, HPE embryos had some facial characteristics that are specific to these stages and different from those in older HPE embryos. The midline structures of the brain, including the pituitary gland, were lacking or seriously hypoplastic in HPE embryos. Complete cyclopia was found in two cases after CS 18 but none at earlier stages. CONCLUSIONS: The early development of HPE in human embryos was systematically studied for the first time. The pathogenesis of craniofacial abnormalities, especially eye anomalies, in HPE was discussed in the light of recent studies with mutant laboratory animals.     

Serially etched shark enameloid observed by incident light microscopy

Longitudinal and transverse tooth sections of Isurus oxyrinchus were serially etched in 2.6% nitric acid. The changing optical properties of the etched surfaces were observed during the serial etchings, and the descent of the enameloid surfaces was measured. Shark enameloid seems to be less effectively etched by acid than human enamel; this difference may be due to differences in solubility between fluorapatite and hydroxyapatite. Most of the information regarding the structure of the enameloid was gained during the first five of ten etchings. The reflection of light from the surface was influenced by the orientation of the crystallites, longitudinally sectioned crystallites reflecting the light better than transversely sectioned crystallites. The dentinal extensions were continuous with and of the same structure as the underlying dentine. The radial fibers originated from the dentinal extensions, and they both contained organic material and were accompanied by crystallites. When the specimens were imbibed with water the distinctness of the dentinal extensions and radial fibers was improved.     

Morphological observations in normal primary palate and cleft lip embryos in the Kyoto collection

Normal developmental events during human primary palate formation and alterations associated with cleft lip remain poorly defined. The purpose of this study was to analyze serially sectioned human embryos to identify morphological changes during normal palatal closure and alterations associated with failure of palatal formation. Normal and cleft embryos from the histological collection at the Congenital Anomaly Research Center at the University of Kyoto were studied and photographed for detailed evaluation. Seven serially sectioned cleft lip embryos of stages shortly after primary palate formation (Streeter-O'Rahilly stages 19, 20, and 22) with unilateral or bilateral clefts with varying degrees of clefting were studied. In the normal Kyoto embryos, initial nasal fin (epithelial seam) formation was observed between the medial nasal process and the lateral nasal and maxillary processes at stage 17. During stages 18 and 19, the nasal fin epithelium was replaced by an enlarging mesenchymal bridge, as the maxillary processes united with the medial nasal processes to form the primary palate. The most prominent features observed in the cleft embryos were a reduced thickness of mesenchymal bridging between the medial nasal and maxillary processes, with an excessive amount of epithelium at the junctions between these processes. With ingrowth of the maxillary processes, greater cell dispersion and apparent extracellular matrix accumulation were observed in the medial nasal region. During closure of the primary palate, terminal branches of the maxillary nerve crossed the mesenchymal bridge to the medial nasal region. The partial clefts had reduced maxillary ingrowth and smaller union areas with the medial nasal process. Detailed studies of experimental animal models are required to identify regional growth required for contact between the facial prominences, to clarify the mechanisms of mesenchymal ingrowth and epithelial displacement during palatal formation, and to identify local and/or general factors causing alterations that lead to primary palatal clefting.     

Detection and Phenotypic Characterization of Adult Neurogenesis

Studies of adult neurogenesis have greatly expanded in the last decade, largely as a result of improved tools for detecting and quantifying neurogenesis. In this review, we summarize and critically evaluate detection methods for neurogenesis in mammalian and human brain tissue. Besides thymidine analog labeling, cell-cycle markers are discussed, as well as cell stage and lineage commitment markers. Use of these histological tools is critically evaluated in terms of their strengths and limitations, as well as possible artifacts. Finally, we discuss the method of radiocarbon dating for determining cell and tissue turnover in humans.Detection of neurogenesis in vivo requires the ability to image at a cellular resolution, which currently precludes noninvasive imaging approaches, such as magnetic resonance imaging (MRI). In vivo microscopy, using deeply penetrating UV illumination with multiphoton microscopy, or by the recently available endoscopic confocal microscopy, may provide new opportunities for longitudinal studies of neurogenesis in the living animal with single-cell resolution. These newer microscopy approaches are particularly compelling when coupled with transgenic mice expressing phenotype-specific fluorescent reporter genes. Additionally, an advanced method using ¹⁴C carbon dating of postmortem DNA from specific cell populations of the brain revealed insights into adult human neurogenesis. Nevertheless, at present, the predominant approach for studying neurogenesis relies on traditional histological methods of fixation, production of tissue sections, staining, and microscopic analysis.This review discusses methodological considerations for detection of neurogenesis in the adult brain according to our current state of knowledge. This will include the use of exogenous or endogenous markers of cell cycle, as well as phenotype markers that contribute to resolving stages of neuronal lineage commitment. The accurate analysis of cell phenotype will be discussed, including suggestions for accurate detection and reliable quantification of cell numbers. Finally, we will present the newly developed ¹⁴C carbon dating of nuclear DNA for quantitative analysis of neurogenesis in human tissue.     

Structural strength of the macaque femur

New techniques in bone mechanics, and the demonstration that locomotor function can be interpreted based on patterns of structural strength delineated by these new techniques, lay the foundation for analyses of structural strength in nonhuman primate long bones. The present paper details topographic variability in structural strength of the femoral diaphysis of Macaca as a basis for further quantifying form-function interactions in pronograde primates. The femoral diaphyses of 42 macaques were serially sectioned. These sections were digitized, and coordinate points were submitted to the SCADS computerized stress analysis program. This analysis indicated that the femoral diaphysis of Macaca is better adapted proximally than distally to resist axial loads. The proximal third of the femur is better able to resist bending loads in the posterolateral/anteromedial direction than in the standard planes. The distal femur is geometrically well suited to resist high bending loads, particularly in the mediolateral plane. The elliptical construction of the distal femur is designed to resist high torsional loads as well. When compared with density data on the macaque femoral diaphysis, these data indicate extremely high rigidity in the mediolateral plane. The inverse relationship between density and structural rigidity distally indicates the presence of compensatory mechanisms between structural strength, geometry, and density. Similarities in femoral mechanics in macaques and humans suggest uniformity of stress patterns of the lower extremity in terrestrial quadrupedal and bipedal locomotion.     

Apoptosis of odontoclasts under physiological root resorption of human deciduous teeth

This study was designed to establish the apoptosis of odontoclasts during physiological root resorption of human deciduous teeth. Deciduous teeth were fixed, decalcified, and embedded in paraffin for immunohistochemical (IHC) observations and in Epon for transmission electron microscopy (TEM). Apoptotic cells were identified by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL), and then tartrate-resistant acid phosphatase (TRAP) activity was determined on the same sections. Epon-embedded specimens were sectioned serially into 0.5-μm semithin sections; some of these sections were re-embedded in Epon, sectioned into 0.1-μm ultrathin sections, and observed by TEM. IHC revealed that the nuclei of TRAP-positive odontoclasts on the dentine were generally TUNEL-negative. Around these odontoclasts, a few TRAP-positive structures were present together with TUNEL-positive structures, e.g., a TRAP-positive structure with one TUNEL-positive nucleus, a TRAP-positive structure with one TUNEL-positive nucleus plus one or two TUNEL-negative nuclei, or a TRAP-positive structure with no nucleus. By TEM, some odontoclasts showed nuclear fragments including compacted chromatin. The results suggest that, during apoptosis, odontoclasts fragment into variously sized cellular parts including three or fewer nuclei. This study was supported by a grant from the Japanese Ministry of Education, Science, Sports, Culture, and Technology (grant no. 16591819) and by a grant from the Ministry of Education, Science, Sports, Culture, and Technology to promote multidisciplinary research projects (2003).     

Apparatus for Bone Sectioning

A new type of apparatus for cutting 100-200µ sections of bone is described. This apparatus consists of an especially designed bone vise and a mounted circular saw with three directional movement controls. The saw is driven by one D.C. motor and its vertical movement assembly is driven by another. Their speeds can be regulated individually. The bone sections may be made serially at intervals of 0.3 to 0.5 mm. and bones as large as 2 cm. in diameter or longitudinal slabs of larger bones may be sectioned.     

Pathophysiology and functional consequences of human partial epilepsy: lessons from positron emission tomography studies

Positron emission tomography (PET) is a powerful clinical and research tool that, in the past two decades, has provided a great amount of novel data on the pathophysiology and functional consequences of human epilepsy. PET studies revealed cortical and subcortical brain dysfunction of a widespread brain circuitry, providing an unprecedented insight in the complex functional abnormalities of the epileptic brain. Correlation of metabolic and neuroreceptor PET abnormalities with electroclinical variables helped identify parts of this circuitry, some of which are directly related to primary epileptogenesis, while others, adjacent to or remote from the primary epileptic focus, may be secondary to longstanding epilepsy. PET studies have also provided detailed data on the functional anatomy of cognitive and behavioral abnormalities associated with epilepsy. PET, along with other neuroimaging modalities, can measure longitudinal changes in brain function attributed to chronic seizures as well as therapeutic interventions. This review demonstrates how development of more specific PET tracers and application of multimodality imaging by combining structural and functional neuroimaging with electrophysiological data can further improve our understanding of human partial epilepsy, and helps more effective application of PET in presurgical evaluation of patients with intractable seizures.     

The importance of scientific collecting and natural history museums for comparative neuroanatomy

The comparative study of vertebrate brains is inherently dependent upon access to a sufficient number of species and specimens to perform meaningful comparisons. Although many studies rely on compiling published information, continued specimen collection, in addition to more extensive use of existing brain collections and natural history museums, are crucial for detailed neuroanatomical comparisons across species. This review highlights the importance of collecting species through a variety of means, details a marsupial brain collection, and stresses the potential of natural history museums as a resource for comparative neuroanatomy. By taking advantage of as many of these resources as possible, researchers can rapidly increase species coverage and generate a better understanding of how the brain evolves.     

A Procedure for Preparing Undecalcified and Unembedded Bone Sections for Light Microscopy

We have developed a procedure for light microscopic investigation of undecalcified and unembeddedbone sections. Biopsy samples of human metatarsus and femur and rat femur were fixed in aldehydes and sectioned with a cutting machine equipped with a diamond saw blade. Free sections 100-150 μm thick, stained with toluidine blue and von Kossa, did not show artifacts following the cutting, and the spatial relations of mineralized and nonmineralized components remained intact. Compact and trabecular bone, bone marrow and all cell types appeared well preserved and easily recognizable. Our procedure provides a simple and rapid method for preparing bone sections which undergo no chemical treatment other than fixation. This method is a useful alternative to standard histological protocols for studying bone specimens.     

Diagnostic value of micro-CT in comparison with histology in the qualitative assessment of historical human skull bone pathologies

Cases of pathologically changed bone might constitute a diagnostic pitfall and frequently need histological methods to be etiologically properly evaluated. With micro‐computed tomography (μCT), a new epoch of 2D and 3D imaging has been launched. We evaluated the diagnostic investigation of this analytical method versus well established histological investigations of historical human bone. Pathological changes due to various etiologies (infectious, traumatic, endocrinological, neoplasia) observed in autopsy‐based macerated human skulls (Galler Collection, Natural History Museum Basel, Switzerland) were investigated by μCT and compared with histological thin ground sections using polarized light. Micro‐CT images visualize the architecture of the bone with high spatial resolution without preparation or destruction of the sample in the area to be sectioned. Changes in the bone surfaces as well as alterations of the diploë can be assessed. However, morphological patterns caused by reactive response, such as typical arrangements of collagen fibers, can only be visualized by the microscopic investigation of thin ground sections using polarized light. A great advantage of μCT is the high number of slices obtained so that spatial differences within the areas of the specimen become visible. Micro‐CT is a valuable tool for the diagnosis of vestiges of skull bone diseases. Its advantages over histology are the fast, automated image acquisition and the fact that the specimen is not completely destroyed. Only excision of the area to be scanned is necessary, if the specimen is too large to be scanned as a whole. Further, the 3D visualization of the micro‐architecture allows an easy orientation within the sample, for example, for the choice of the location of the histological slices. However, the need to differentiate woven from lamellar bone still makes histology an indispensable method. Am J Phys Anthropol, 2007. © 2007 Wiley‐Liss, Inc.     

Concentric Layers in the Granules of Human Nervous Lipofuscin Demonstrated by Silver Impregnation

Representative pieces of human brain were fixed in 10% formalin, embedded in paraffin and sectioned at 5 μ. Paired sections were used, one of which was oxidized in equal parts of 0.5% potassium permanganate and 0.5% sulfuric acid for 1-2 min, while the other was left unoxidized. Both the oxidized and unoxidized sections were impregnated with silver diamine. The lipofuscin granules in the nerve cells appeared as small intensely stained black dots, surrounded by a clear unstained zone, in the unoxidized sections, while in the oxidized sections there was an outer ring of intensely blackened material surrounding a central unstained dot.     

Locating tissue collections in tissue economies—deriving value from biomedical research

Abstract

This paper examines diverging notions of value in the use of tissue sample collections and other information resources using a case study of hereditary colorectal cancer research in Finland. Recent science and technology policies that emphasize the production of commercial value derived from tissue sample collections are challenged by varying conceptions of value, as well as structural factors that relate to the combination of different public population information systems in the Finnish research system. Such challenges reflect a tension in the economic aspirations of the ideology of the knowledge society in relation to the goals of national health care policies, as well as the role of the state as a mediator of knowledge production and commercial development.     

Securing a future for China's plant biodiversity through an integrated conservation approach

The severely threatened Chinese flora urgently needs a new, well adapted to China and properly formulated conservation strategy. The present review provides a detailed conservation methodology that complements previously described guidelines for preservation of plant species with extremely small populations (PSESP) in China. This review adds to the above concept in several aspects, making it relevant to all threatened Chinese plant species. The proposed integral conservation strategy has the following crucial components: -ecoregional basis for conservation planning and implementation; -a unified scoring system that is used in regional systematic planning for reserve design, monitoring and assessment of efficiency of a reserve network, and creation of seed banks and living collections; -a focus on population demography and the presence of naturally occurring regeneration as the key criteria for defining the conservation status of a species and the appropriate major focus of the species recovery plan; -creation of multi-species living collections that preserve species genetic variation and provide material for in situ actions; -experimental translocation of threatened species into multiple locations within and outside their known range. Adopting and implementing these strategies successfully and more fully in China requires that the country changes PA legislation and improves PA management, the National Science Foundation of China (NSFC) re-prioritizes the type of research that receives research funds, and local scientists improve their approach toward information sharing.     

SPECT neuroimaging in translational research of CNS disorders

High resolution SPECT imaging is an emerging field and there are only limited studies as yet available in this direction. Still there is continuous effort to achieve better spatial and temporal resolution in order to obtain detailed structural and functional information of different brain regions in small experimental animals. Recently, SPECT imaging system has been used to perform in vivo imaging using specific radioligands to further elucidate the role of dopaminergic, serotonergic, and cholinergic neurotransmission in relation to regional cerebral blood flow in various human CNS disorders and in gene-manipulated mouse models of neurodegeneration. Although in vivo and non-invasive translational research can be performed by high-resolution microPET imaging system, its limited spatial resolution restricts detailed anatomical and functional information of different brain regions involved in disease process. Recently developed NanoSPECT/CT imaging system has a better spatial resolution hence can be used to correlate and confirm microPET imaging data and determine the precise structural and functional anatomy of CNS disorders and their remission. Moreover SPECT imaging system reduces the cost and number of animals and provides detailed information of CNS disorders at the cellular, molecular and genetic level. Furthermore, SPECT system is economical, provides less radiation burden, and can be used to study bio-distribution of newly synthesized radioligands with increased target to non-target ratios, quality control, and clinical applications. It is envisaged that high-resolution SPECT imaging system will further improve in vivo non-invasive translational research on CNS disorders of unknown etiopathogenesis and their treatment in future.