A simple and rapid section embedding technique for sequential light and electron microscopic examination of individually stained central neurons.

A method for section embedding of central nervous tissue, and its application to light and electron microscopic examination of neurons stained by intracellular injection of horseradish peroxidase, is described. After primary fixation in aldehydes, thick (10--80 micrometer) sections are cut on a Vibratome. They are then treated for the histochemical demonstration of peroxidase, postfixed in osmium tetroxide, dehydrated and infiltrated with Spurr's low viscosity resin. Infiltrated sections are embedded between glass slides and coverslips coated with a transparent layer of teflon and polymerized. The resulting mount allows protracted storage and high resolution light microscopic examination of sections. When desired, the glass components can be removed and the specimen cemented to a blank block for thin sectioning and electron microscopic examination.     

Novel applications of common stereology software to represent the complete distribution, density and spatial organization of anterogradely labelled fibers in neuroanatomical tract-tracing studies

In a recent study we have adapted for the first time available software used in spatial neuronal organization studies to represent the complete fiber distribution of a brain projection [Leite-Almeida H, Valle-Fernandes A, Almeida A. Brain projections from the medullary dorsal reticular nucleus: an anterograde and retrograde tracing study in the rat. Neuroscience 2006;140:577-95]. Here we describe the technique in detail using the injection of biotinylated-dextran amine (BDA) in the rat cuneate nucleus and its projection through the pyramidal decussation as an example. Camera-lucida-like computer drawings were produced with StereoInvestigator software. Using high magnification lens, rat brain sections were screened and lines superimposed with virtually all fibers and terminal boutons present in each brain section. Additionally, in each screening field of the section, all focal planes were inspected and lines depicted only on focused fibers and/or fiber segments. Later, NeuroExplorer software was used to interpret fiber depth information and obtain the three-dimensional pathway of each fiber bundle along the rostrocaudal extension of each section. This computer methodology presents several advantages over classical camera-lucida hand-made drawings: (i) the software is prepared to work interchangeably in different magnifications without misplacements, which allows the representation of virtually all labelled fibers and, therefore, results in a much more accurate fiber location in each brain section; (ii) three-dimensional information of all individual fibers and fiber bundles is recorded; (iii) the method is more flexible allowing, for example, to colour differently labelled fibers according to their profiles (e.g. terminal or ramified fiber arborizations and terminal boutons).     

How children show positive and negative relationships on their drawings

This study analyses, whether pictures of children showing a positive relationship are significantly different from those showing a negative one with respect to several criteria. The study involved a random selection of 45 children aged 4;6 to 11;6 years. The children painted a picture with themselves and a person they liked and a picture of themselves with someone they disliked. For the most part, the children drew pictures of themselves with peers both with respect to positive as well as negative images. In an interview afterwards, the children specified the criteria in their drawings by which the quality of the particular relationship can be identified. Positive and negative relationship paintings differ in the character of activity described. The sun as an element in children's paintings is painted not more frequent on positive compared to negative pictures. The colour black is used more often in the drawings signifying negative relationships. While girls used more colour in negative relationship drawings, boys used more colour in the positive ones. There was no significant difference in the use of favourite colours and decorative elements between the two groups. Only in negative relationship drawings people were looking away from each other. Smiling individuals were more common in the positive relationship pictures and in pictures painted by the 6 to 8 year olds. A greater distance between the individuals emerged on negative relationship drawings of the girls.     

An atlas of the rat subpostremal nucleus tractus solitarius.

The nucleus tractus solitarius (NTS) in the dorsal medulla is the principal visceral sensory relay nucleus in the brain. In the rat, numerous lines of evidence indicate that the caudal NTS at the level of the area postrema serves as a major integrating site for coordinating cardiorespiratory reflexes and viscerobehavioral responses. This region of the caudal NTS not only exhibits high densities of binding sites for an impressive array of transmitters and modulators but microinjections of many of these same neuroactive substances into the rat subpostremal NTS elicit pronounced cardiorespiratory and visceral response patterns. This report provides an abbreviated atlas of the rat subpostremal NTS consisting of a series of transverse, sagittal, and horizontal plates. Photomicrographs, together with their corresponding schematic drawings, are provided for the serial sections generated from each reference plane.     

An atlas of the prenatal mouse brain: gestational day 14.

A prenatal atlas of the mouse brain is presently unavailable and is needed for studies of normal and abnormal development, using techniques including immunocytochemistry and in situ hybridization. This atlas will be especially useful for researchers studying transgenic and mutant mice. This collection of photomicrographs and corresponding drawings of Gestational Day (GD) 14 mouse brain sections is an excerpt from a larger atlas encompassing GD 12-18. In composing this atlas, available published studies on the developing rodent brain were consulted to aid in the detailed labeling of embryonic brain structures. C57Bl/6J mice were mated for 1 h, and the presence of a copulation plug was designated as GD 0. GD 14 embryos were perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer and embedded in paraffin. Serial sections (10 microns thickness) were cut through whole heads in sagittal and horizontal planes. They were stained with hematoxylin and eosin and photographed. Magnifications were 43X and 31X for the horizontal and sagittal sections, respectively. Photographs were traced and line drawings prepared using an Adobe Illustrator on a Macintosh computer.     

A simple microcomputer-based three-dimensional serial section reconstruction system (MICROS)

We have developed a computer system which enters and aligns serial sections and displays the completed reconstructions at different rotations in space. The system uses commercially available hardware, including a Hewlett-Packard 9845T microcomputer and an H-P 9874A digitizer. The software for the system is written in the BASIC language.The system consists of two programs, one for section digitization, the other for rotation and display of the reconstructions. Sections are digitized directly from micrographs or back-projected slides. The outlines of cells or other structures are traced from these media using a hand-held cursor on the digitizer. The positions of elements (inputs) which contact the structure and fiducials are also digitized. The sections are aligned by simultaneously displaying two consecutive sections on the graphics CRT screen. The sections are coarsely superimposed by centering around screen center using a centering algorithm. They are precisely aligned by rotating and translating the images with a reference cursor. Special functions for inserting and deleting sections and rapid section scanning are available for editing. The aligned sections are stored using a linked-list file structure on either floppy disks or tape cartridges. The rotation program replots the completed reconstructions on the graphics CRT or digital plotter. The program will reproduce the reconstructions at any scale and at any rotation in the x-, y- or z-planes. A hidden line algorithm removes hidden lines to give a 3-dimensional (3-D) perspective to the reconstructions. The positions of inputs and fiducials are represented by symbols. We use the system to reconstruct cells and neural processes. The 3-D reconstructions allow us to: (a) examine the spatial distribution and density of synaptic contacts on neurons; (b) study complex neuronal shapes; (c) examine the vectors of neural processes. The computer reconstruction system, which is moderately priced, should also prove useful for reconstructing many other types of biological profile.     

Location of single neurons containing HRP for electron microscopy

A technique is described for finding single neurons containing transported horseradish peroxidase (HRP) for study in the electron microscope. Tissue chopper sections which have been reacted to demonstrate HRP are cleared in glycerol and examined with darkfield light microscopy. The granular reaction product is clearly seen, enabling accurate drawings to be made for later dissection for ultramicrotomy. The neuron sought can then be found within a few microns of the cutting face of the block. While some distortion does occur, most of the cellular morphology remains intact, enabling recognition of specific organelles such as neurosecretory granules in cells which have transported HRP from the neurohypophysis.     

CT-assisted stereotactic brain biopsy: value of intraoperative frozen section diagnosis.

In 100 recent CT-guided brain biopsies, the value of intraoperative histologic examination using frozen section technique was evaluated. In 87 of these cases, the biopsy was performed stereotactically. In the remaining 13 cases, a CT-guided free hand technique was used. Of the 100 biopsies performed, adequate tissue for histopathologic diagnosis was obtained in 97, and in three the biopsy was nondiagnostic. In 61 procedures the initial biopsy specimen was adequate for diagnosis. Two specimens were required in 25 and in the remaining cases it was necessary to obtain three to four biopsy specimens before a definitive diagnosis could be made. Ultimately, the histologic diagnosis was made on frozen section examination in 93 of the cases. The lesions identified were neoplastic disease in 83 cases, vascular disease in seven, infectious disease in five, demyelinating disease in one, and radiation necrosis in one. Comparison between the frozen section diagnosis and the final diagnosis based on the permanent sections revealed that they matched in 89 cases (92%). Of the 83 cases of neoplasms the exact grade of malignancy was determined by frozen section to make a final diagnosis revealed that even if the specimen volume was less than 2 mm3, the biopsy was generally successful. The disadvantages of the small sample size obtained through needle biopsy are best overcome by careful targeting and assessment of sample quality by intraoperative frozen section examinations, which will give the definitive diagnosis in most of the cases without paraffin-embedded sections.     

Simple microcomputer system for mapping tissue sections with the light microscope

We describe a method for two-dimensional mapping of tissue sections that makes use of a drawing tube, microscope stage encoders and a microcomputer. The drawing tube views the graphics monitor and superimposes the image of the screen cursor and on-screen menus on the specimen image. Thus, the position of every landmark in each microscopic field can be mapped without stage movement while directly viewing the specimen through the microscope. A mouse is used for data entry and program control. Fields mapped in this way are then assembled into a complete map, which can include line drawings as well as up to 20 landmark types. The coordinate values of all landmarks mapped are stored and remain accessible for editing and analysis. High resolution plots are produced. Specialized functions include grain counting, area and perimeter calculations as well as a perimeter limiter that predefines the area to be mapped. The system uses general purpose hardware that is widely available. Many hitherto time-consuming tasks, such as detailed mapping of cell positions, regions of immunocytochemical staining, degenerating fibers, neuronal connections or any other anatomical feature, can be done in a fraction of the time and effort previously involved. These labor savings can be realized while maintaining the highest resolution and enabling statistical analysis since the data are already in digital form.     

Three-dimensional reconstruction of tissue using computer-generated images

In the study of brain ventricles for both teaching and research, it is often of considerable advantage to graphically display the reconstructed shape of the specimen. This paper describes the making of two-dimensional serial cross-sections and their storage for subsequent manipulation and display on a personal computer; the three-dimensional (3D) reconstructions may have hidden lines removed and various parts coloured for definition of areas of interest, for example the density and position of pyknotic (dead) nuclei. Current research involved the investigation of the effects of maternal hyperthermia on early embryonic brains. The 3D reconstructions were found ideal for understanding the temporal changes occurring in embryonic brains subjected to defined maternal heat stresses. The method involved 4 X 4 matrices using homogenous coordinate theory, being the most ideal and allowing a constant mechanism for all transformations. Total time from examination of sections to obtaining an accurate printed 3D reconstruction is approximately 1 h if 22 sections are used.     

A novel frozen brain tissue array technique: immunohistochemical detection of neuronal paraneoplastic autoantibodies

We introduce a modification of the tissue microarray technique in which several frozen brain tissue specimens are collected to a single frozen brain array block. In the present application, we use it for the detection of neuronal paraneoplastic anti-Hu autoantibodies. Representative samples from 15 different brain regions were collected according to a standard neuropathological autopsy protocol. Cryostat sections from each block were cut and conventionally stained. From representative areas, cylinder tissue samples from each specimen were punched and then arrayed into a recipient array block. Using the cryostat sections of this brain array, autoantibodies from seven anti-Hu-positive patient sera (confirmed by immunoblotting) were screened by immunohistochemistry. Neuronal architecture was well preserved and immunohistochemical staining was comparable to that of conventional cryostat sections. Because of the variable staining pattern in different brain areas, two anti-Hu-positive sera could be detected immunohistochemically by the one brain array. With the present array technique, it is possible to characterize the variable staining patterns of neuronal paraneoplastic autoantibodies in different locations of the human brain. The frozen brain array also allows the detection of RNA and DNA targets involved in neurological diseases.     

Evaluating factors that can influence spirography ratings in patients with essential tremor

Spirograph drawings are used in most comprehensive assessments of essential tremor (ET). Nevertheless, several different paradigms are used and no effort has been made to compare these. We used two different cohorts to assess different aspects of spiral rating. In the first, we had subjects simulate different levels of effort by writing (1) 'normally', (2) 'slowly and carefully', (3) 'softly', and (4) 'rapidly' using both their dominant and non-dominant hands. In the second, subjects (1) drew in between the lines of two drawn spirals, (2) traced a previously drawn spirograph, and (3) drew freehand. Subjects drew each with both 'supported' (regular writing) and 'unsupported' writing. The spirals were coded, randomized and blindly rated on a 0-9 scale. Unsupported drawings were consistently rated as worse than supported spirals, and the dominant hand was generally better than the non-dominant hand. Spiral drawn freehand spirals were consistently rated with the lowest scores. Inter-rater and intra-rater reliability were also best for unsupported drawings and tended to be best for 'freehand'. 'Effort' had little effect on ratings. Based on our results, we recommend that assessment of ET include unsupported, freehand spirals.     

Three-dimensional counting: an accurate and direct method to estimate numbers of cells in sectioned material

We introduce a way to count and measure cells in an optically defined volume of tissue called a counting box. This method--direct three-dimensional counting (3DC)--eliminates the need for correction factors, such as that introduced by Abercrombie (Anat. Rec. 94:239-247,'46), to determine the number of cells per unit volume (NV). Problems caused by irregular cell shape and cell size, nonrandom orientation, and splitting of cells by the knife during sectioning are overcome. Furthermore, 3DC is insensitive to large variations in section thickness. The innovative feature of 3DC is the definition of a counting box with top and bottom sides located inside the section a precise distance away from the cut surfaces of the tissue. The positions of the top and bottom sides of the counting box are delimited by using a digital length gauge and a Z-axis control unit. Sections of tissue between 8 and 100 micron thick are examined with a high numerical aperture objective in combination with video-enhanced differential interference contrast optics (DIC). Cells are marked on a television screen while the microscopist scans systematically from the top to the bottom of the counting box. Cells that are located completely inside the box and cells that only cross through its top, right, or back sides are counted. All cells that cross the planes that define the bottom, left, and front sides of the counting box are not counted. Direct 3DC provides an accurate, simple, and reliable way to count cells, nuclei, nucleoli, or other objects in sectioned material.(ABSTRACT TRUNCATED AT 250 WORDS)     

The tunica muscularis of human brain arteries: three-dimensional measurements of alignment of the smooth muscle mechanical axis,by polarized light and the universal stage

The four-axis universal stage, with the polarizing microscope, was assessed for making precise three-dimensional measurements of alignment on muscle cells. The physiological dimensions of the arteries (obtained at autopsy) and the co-alignment of the myofilaments with the lonaxis of the muscle cells was assured by the fixation of the arteries at normal distending pressure. They were embedded in paraffin wax, eitherperpendicular to the cutting plane which permitted controlled realignment of the block later, or along with a rectangular block of previously fixed liver parenchyma (providing a cartesian reference). The test for accuracy showed that smooth muscle, when unstained, was measured to within ±5°. The same tissue measured after staining by Gomori's silver impregnation, could be repeatedly measured to within 2.4°. The optical axis, parallel to the morphological axis, was not altered by staining. Data from 11 tissue sections revealed that the tunica muscularis is a precisely organized structure, not only with an average alignment which is almost perfectly circumferential, but with a higher degree of alignment among the muscle fibres than has been previously reported. Smooth muscle tissue is abundant and perhaps highly ordered in many structures. The universal stage could be employed to reveal that order.     

The tunica muscularis of human brain arteries: three-dimensional measurements of alignment of the smooth muscle mechanical axis, by polarized light and the universal stage

The four-axis universal stage, with the polarizing microscope, was assessed for making precise three-dimensional measurements of alignment on muscle cells. The physiological dimensions of the arteries (obtained at autopsy) and the co-alignment of the myofilaments with the long axis of the muscle cells was assured by the fixation of the arteries at normal distending pressure. They were embedded in paraffin wax, either perpendicular to the cutting plane which permitted controlled realignment of the block later, or along with a rectangular block of previously fixed liver parenchyma (providing a cartesian reference). The test for accuracy showed that smooth muscle, when unstained, was measured to within +/- 5 degrees. The same tissue measured after staining by Gomori's silver impregnation, could be repeatedly measured to within 2.4 degrees. The optical axis, parallel to the morphological axis, was not altered by staining. Data from 11 tissue sections revealed that the tunica muscularis is a precisely organized structure, not only with an average alignment which is almost perfectly circumferential, but with a higher degree of alignment among the muscle fibres than has been previously reported. Smooth muscle tissue is abundant and perhaps highly ordered in many structures. The universal stage could be employed to reveal that order.     

A computer-assisted video technique for preparing high resolution pictures and stereograms from thick specimens

A computer-assisted video technique is presented for rapidly and accurately gathering, storing and depicting three-dimensional anatomical structures in thick specimens. Several optical sections through the specimen are combined to produce high-resolution photographs with essentially infinite depth-of-field. Further, the depth information implicit in the series of optical sections makes the creation of stereoscopic pairs relatively simple. The technique employs a real-time digitizing frame store and a computer. A video camera is attached to a microscope and successive optical sections are stored digitally as the plane of focus is systematically changed. After storage, the image of each optical section is enhanced to emphasize elements that are sharply focussed. The final two-dimensional image is generated by selecting for each point in the final picture the darkest grey value occurring at the corresponding point in any of the pictures in the through-focus series. A picture with essentially infinite depth-of-field is produced when points of correspondence in the series are determined by a ray passing normal to the plane of optical section. Right and left pictures for a stereoscopic pair are produced when points of correspondence are determined by a ray slanting either left or right of normal. This technique is illustrated with cobalt chloride-filled neurons from whole-mounted cricket ganglia, with HRP-filled axons from whole-mounted goldfish tectum, with Golgi-Kopsch-impregnated neurons from cat visual cortex, and with sections of cobalt chloride-filled antennal afferents in cricket.     

Montage: a system for three-dimensional reconstruction by personal computer

This paper describes a simplified system for serial section three-dimensional (3-D) reconstruction. A set of 9 software programs runs on a standard personal computer and produces camera-ready illustrations suitable for publication. The user enters trace points on a digitizing tablet from sections that have been already aligned. A 3-D view of the reconstructed object is generated which can be displayed with hidden lines removed. Analysis of volume, surface area and autoradiographic grain density are performed automatically. A relational database query language allows display and analysis of a selected subset of the data. The system runs under the UNIX operating system which allows the programs to be easily transported to new hardware or modified for other purposes.     

A computer-aided method for the quantitative analysis of dendritic arborizations reconstructed from serial sections

We present a methodology for measuring precisely defined morphological parameters on complete dendritic arborizations. Brains are sectioned through anatomical planes which are defined with reference to ventricular landmarks. For each neuron, drawn through the camera lucida, dendritic points are defined and identified by means of a numerical topological codification. The 3-dimensional coordinates of each point are measured on a video computer microscope with reference to a cartesian system of axes which are oriented with reference to the anatomical planes of the brain. The data points from several serial sections are stored section by section and re-ordered by a computer program. Quantitative data concerning the diameters of the dendrites and the number and the dimensions of their spines are also stored. From these data, various quantitative morphological parameters may be computed. The accuracy of the video computer microscope is measured. The different existing computerized systems and the contribution of computerized techniques are discussed.     

Features of ligand binding in homogenate and section preparations

The binding of [3H]N-methyl scopolamine (NMS) to muscarinic binding sites in rat forebrain was compared in two types of preparations: homogenates and slide-mounted sections. Under standard assay conditions, [3H]NMS bound to the muscarinic binding site with an apparent Kd that was almost one order of magnitude lower in homogenates (Kd = 0.15 nM) than sections (Kd = 1.25 nM). In addition, the muscarinic agonist carbachol inhibited [3H]NMS binding more effectively in homogenates (Ki = 5 microM) than sections (Ki = 100 microM). The higher Kd observed in sections was dependent upon the thickness of the section since the apparent Kd decreased as the thickness of the section was reduced. A slower equilibration rate may account, in part, for the higher apparent Kd seen in thicker sections. The results support previous evidence that certain ligands exhibit different binding profiles in homogenate and section preparations.     

Cocaine induces cell death within the primate fetal cerebral wall

Transferase dUTP nick-end labelling (TUNEL) analysis was used to compare the occurrence of cell death in the cerebral wall of cocaine-exposed and drug-naïve monkey fetuses. The rhesus monkeys providing the drug-exposed fetuses received 10 mg/kg of cocaine orally (in fruit treats) in the morning and in the evening between pregnancy days 50 and 65. The control pregnant animals received fruit treats only. The fetuses were removed for analysis by Caesarean section 10 h after the last cocaine treatment. The sections of the cerebral wall from the cocaine-exposed fetuses contained significantly higher numbers of TUNEL-positive nuclei (counted either per section area or per 1000 unlabeled nuclei) than the matching sections from the drug-naïve fetuses. This elevation in the number of TUNEL-positive cells was observed through the entire depth of the fetal cerebral wall including its proliferative and intermediate zones, cortical plate and the marginal zone. The present study demonstrates that consumption of cocaine during pregnancy can result in increased occurrence of cell death in the developing cerebrum.