Stereology, morphometry, and mapping: the whole is greater than the sum of its parts.

The latest developments in computer-based stereology build upon the similarities of classical stereology and computer microscopy to provide refined and effective spatial analyses that also permit mapping of anatomical regions. Classical stereology and computer microscopy have developed along independent pathways as methodologies to provide a quantitative understanding of the structure of the brain. They approach brain morphology and brain morphometry from different points of view. On one hand, stereology has concentrated upon the unbiased numerical estimation of parameters, such as length, area, volume, and population size that characterize entire regions of the brain, e.g. hippocampus, as well as individual elements within them, e.g. cell volume. On the other hand, computer microscopy has concentrated upon providing accurate three-dimensional maps of the morphology of entire regions of the brain as well as of individual elements within them, e.g. neuronal dendrite and axon systems. The differences in point of view are not so extensive as to keep the two methodologies separate. They share, after all, a similar manner of controlling microscope data input and analyzing the images the microscope provides. The incorporation of data archiving permits easier access to previous studies, as well as the sharing of stereological findings and their related maps throughout the scientific community. Some of the stereological systems now integrate spatial mapping with stereological analyses to provide more comprehensive methods to analyze brain tissue.

Inherited predisposition to colon cancer.

Colorectal cancer is one of the most common malignancies in the United States. Although both genetic and environmental factors play a role in colorectal tumorigenesis, recent advances in genetics have more clearly defined the impact of inheritance in the multistep process of the disease. Researchers have identified single genes that confer a susceptibility to familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC). Because these genes are inherited in an autosomal dominant fashion, offspring of carriers have a 50% chance of inheriting the gene mutation and its associated risk. The FAP gene, when mutated, initiates the neoplastic process. HNPCC gene mutations disrupt mismatch repair, thus inducing progression of tumor formation. Discovery of these genes has helped our understanding of sporadic colon cancer as well. Genetic testing for the FAP and HNPCC genes is now available, and results of this testing have implications for surveillance and management. In addition, testing raises complex psychosocial and ethical issues. At present, genetic testing is primarily conducted in the research setting, but it will soon be available in the clinical arena. To prepare for the challenges that these new advances will present, nurses must begin now to enhance their knowledge of genetics and its application to oncology.

The coefficient of error of optical fractionator population size estimates: a computer simulation comparing three estimators.

The optical fractionator is a design-based two-stage systematic sampling method that is used to estimate the number of cells in a specified region of an organ when the population is too large to count exhaustively. The fractionator counts the cells found in optical disectors that have been systematically sampled in serial sections. Heretofore, evaluations of optical fractionator performance have been made by performing tests on actual tissue sections, but it is difficult to evaluate the coefficient of error (CE), i.e. the precision of a population size estimate, by using biological tissue samples because they do not permit a comparison of an estimated CE with the true CE. However, computer simulation does permit making such comparisons while avoiding the observational biases inherent in working with biological tissue. This study is the first instance in which computer simulation has been applied to population size estimation by the optical fractionator. We used computer simulation to evaluate the performance of three CE estimators. The estimated CEs were evaluated in tests of three types of non-random cell population distribution and one random cell population distribution. The non-random population distributions varied by differences in 'intensity', i.e. the expected cell counts per disector, according to both section and disector location within the section. Two distributions were sinusoidal and one was linearly increasing; in all three there was a six-fold difference between the high and low intensities. The sinusoidal distributions produced either a peak or a depression of cell intensity at the centre of the simulated region. The linear cell intensity gradually increased from the beginning to the end of the region that contained the cells. The random population distribution had a constant intensity over the region. A 'test condition' was defined by its population distribution, the period between consecutive sampled sections and the spacing between consecutive sampled disectors. There were 1000 independently simulated cell populations for each test condition, and a 'trial' was conducted for each of these cell populations. In each trial we calculated the (unique) true CE of the population size estimate and the three CE estimates obtained by applying the Scheaffer-Mendenhall-Ott (SMO) and both Gundersen-Jensen (GJ) estimators. We compared the estimated CEs with the true CEs for each population distribution. We found that the CE estimates obtained by the SMO estimator were closer to the true CEs and had less scatter than those of the nugget-modified GJ estimator. Both had small positive bias. The CE estimates obtained by the unmodified GJ estimator exhibited widely varying bias and large scatter. In all the population distributions we tested, the average true CE was very nearly proportional to 1/square root of QT, where QT is the average number of cells counted in the two-stage systematic sample.

The changing architecture of the neonatal rat ovary during histogenesis.

The purpose of this study was to describe the changing histological organization of the rat ovary during postpartum days one through three (p1-p3). A PC-based image-combining microscope system was used to reconstruct the ovary in three dimensions. On p1, cyclindrical pocket-like structures radiated from the core of the ovary that were open toward the surface epithelium. The walls of the pockets contained connective tissue cells and capillaries (stroma). By p2, these pockets had completely closed; each pocket enclosed a small nest of oocytes and a few presumptive granulosa cells. By p3, the pocket-like organization had disappeared. On p1, only one or two primordial follicle-like structures were observed in the core and toward the periphery of the ovary; most oocytes were not enclosed in follicles. By p3, very few naked oocytes remained; primordial follicles predominated in all the regions of the ovary and some of the follicles had multiple layers of granulosa cells. There were changes in location, area, and volume of the rete tubules during these postnatal days. The extraovarian rete was visible on all 3 days but changed its orientation relative to the ovary. The connecting rete was found beneath the epithelial layer of the ovary on all 3 days and showed dramatic increase in area on p2. The wide lumen of the intraovarian rete was in direct contact with some of the oocytes near by on all 3 days, but these "communication points" were most abundant on p2. Based on our observations of different cell-cell associations during this time period, we hypothesize (1) that the mesenchymal-presumptive granulosa cell association is essential for the completion of folliculogenesis, and (2) the rete ovarii may have an inductive role in follicle assembly. These observations suggest that the first 3 days postpartum are critically important for studying the heterogeneous cell interactions that lead to the assembly of primordial follicles. The regional differences in tissue organization during this formative period may have significant implications on later aspects of follicular development.

Neuron imaging with Neurolucida--a PC-based system for image combining microscopy.

Neurolucida is a new software package for performing 3-D neuron mapping and tracing to 0.5 micron precision through the oculars of a microscope. It incorporates computer controlled stage movement in three dimensions. Sections are limited in size only by the stage dimensions. An on-line, direct view image editor is provided as are dynamic image rotations in color and morphometric analyses. Neurolucida is written in 'C' and runs on PC ATs, XTs, and PS/2s using DOS and VGA, EGA, or NTSC graphics. Neurolucida has a video mode compatible with frame grabbers, thereby permitting its application to video microscopy.

Postnatal development of lamina III/IV nonpyramidal neurons in rabbit auditory cortex: quantitative and spatial analyses of Golgi-impregnated material.

We have studied the postnatal development of lamina III/IV spine-free nonpyramidal neurons in the auditory cortex of the New Zealand white rabbit. The morphology and dendritic branching pattern of single cells impregnated with a Golgi-Cox variant were analyzed with the aid of camera lucida drawings and three-dimensional reconstructions obtained with a computer microscope. Sample sizes of 20 neurons were obtained at birth (day 0), postnatal day (PD) 3, 6, 9, 12, 15, 21, and 30 days of age. Normative data were also available from PD-60 and young adult rabbits studied previously. At birth, lamina II-IV have not yet emerged from the cortical plate; immature nonpyramidal neurons at the bottom of the cortical plate (presumptive layer IV) are characterized by short, vertically oriented dendrites. Growth-cone-like structures are present along the shafts and at the tips of the dendrites. At birth, soma area and total dendritic length are, respectively, 34 and 10% of adult values. The cortical plate acquires a trilaminar appearance at PD-3. The six-layered cortex is present by PD-6. During the first postnatal week dendritic length increases fourfold and is accompanied by a significant increase in both terminal and preterminal dendritic growth cones. At the onset of hearing at PD-6, there is a significant proliferation of dendrites and branches to 144 and 200% of adult levels, respectively. These supernumerary dendrites are rapidly lost during the second postnatal week, at which time the somata and dendrites become covered with spines. The loss of higher-order dendrites occurs more gradually; the number of dendritic branches is still 116% of adult values at PD-30. Spine density peaks between days PD-12 and PD-15, and then gradually diminishes until the cells are sparsely spined or spine free by PD-30. Total dendritic length increases in a linear fashion up to PD-15, at which time it is 80% of adult values. An analysis of terminal and intermediate branches demonstrated that the increase in total dendritic length after PD-6 is due entirely to the growth of terminal dendrites. Total dendritic length attains adult levels by PD-30. Spatial analyses revealed that a vertical orientation of dendrites is present at birth. Associated with the onset of hearing at PD-6, there is an explosive elaboration of dendrites toward the pial surface.(ABSTRACT TRUNCATED AT 400 WORDS)

Neonatal deafening alters nonpyramidal dendrite orientation in auditory cortex: a computer microscope study in the rabbit.

In order to examine the influence of afferent input on nonpyramidal dendrite development in the auditory cortex, unilateral deafening was carried out in neonatal rabbits at birth, approximately 6 days prior to the onset of hearing. Deafening was produced by surgical removal of the incus and stapes ossicles, aspiration of the cochlear perilymph, and kanamycin injection into the oval window. At 60 days of age, acoustic stimulation of the deafened ear was unable to evoke auditory brainstem responses. The brains of experimental and littermate control rabbits were processed according to the Golgi-Cox Nissl method. The dendritic systems of lamina III/IV spine-free nonpyramidal cells in the auditory cortex contralateral to the deafened ear were digitized from 340-micron-thick coronal sections with the aid of a computer microscope. Three-dimensional spatial and statistical analyses revealed that nonpyramidal dendrite length in neonatally deafened rabbits increased 27% relative to littermate controls. A fan-in projection analysis revealed that the increased dendrite length in the deafened animals was maximum in the tangential direction and toward the white matter. Computer rotation of digitized neurons from neonatally deafened rabbits also revealed evidence of abnormal dendritic growth in the form of recurved dendrites. We interpret our results to indicate that unilateral cochlear destruction early in development causes a reorganization of the ascending auditory pathway which extends to the contralateral cerebral cortex. Because the auditory cortex contralateral to the deafened ear still receives acoustic input from the undamaged ipsilateral ear, normal nonpyramidal dendritic growth in the auditory cortex is, in part, dependent upon afferent activity arising from both ears.

Auditory cortical responses to neonatal deafening: pyramidal neuron spine loss without changes in growth or orientation.

Neonatal rabbits were unilaterally deafened at birth by surgical removal of the stapes, aspiration of the cochlear lymph, and kanamycin injection into the oval window. At 60 days of age, all rabbits were screened with brain stem evoked response tests in order to establish the efficacy of the deafening procedure. The auditory cortex contralateral to the destroyed cochlea was processed according to Golgi-Cox/Nissl procedures. Temporal bone histology revealed nearly complete outer hair cell loss in the damaged cochlea. The dendritic system of lamina III/IV pyramidal neurons contralateral to the deafened ear was digitized from frontal sections using a computer microscope system. Spine counts were also made along the basal dendrites. Spine counts revealed that neonatally deafened rabbits and 38.7% fewer spines along their basal dendrites. No differences between experimental and control rabbits were found in terms of soma cross-sectional area, total number of basal dendrites, total number of dendritic branches and total basal dendritic length. A fan-in projection of the dendritic system revealed no changes in the radial growth of basal dendrites resulting from the early acoustic trauma. In a prior study, spine-free nonpyramidal neurons in the same sections revealed altered dendritic growth and abnormally recurved dendrites. The separate response of pyramidal and nonpyramidal cell types to early cochlear damage is evidence for the different role of epigenetic determinants of dendritic form and orientation in sensory neocortical neurons.

Semiautomatic microscopy as a tool for cytologists.

Despite impressive advances in the application of computer image analysis to cytology, many of the identification tasks that cytologists are called on to perform remain refractory to automated image analysis. The major reason is that a large fraction of these images, though simple for a human to deal with, are too complex to yield to current image analysis methodologies. It may be years before automated computer image analysis is reduced to clinical practicality. Even then, it is not clear that all cytologic image analyses will prove amenable to automation. In the meantime, semiautomatic image analysis (computer-aided microscopy) can provide a viable alternative, especially to persistently difficult image analysis problems. In semiautomatic image analysis, the onerous tasks of data acquisition--e.g., stage movement, data entry and storage--are left to the computer, while the decision-making tasks-e.g., identifying a cell's morphologic class--are left to the observer. Such a system proves to be easy and flexible to use as well as economical to build. It can also provide a reliable data base for the later evaluation of fully automated systems as they are developed. One such semiautomatic system, the Image Combining Computer Microscope (ICCM), is described, and the range of its application is illustrated. Some of the examples of ICCM applications discussed are: neuronal cell plots, three-dimensional dendrite tracking, serial section reconstruction of axons and mapping of plaques and tangles in Alzheimer's disease. They illustrate how powerful a semiautomated system can be in handling complex image analysis problems. It is suggested that semiautomated image analysis provides a viable long-range alternative to many cytologic image analysis problems.

The fan-in projection method for analyzing dendrite and axon systems.

The fan-in projection is a computer graphical method of projecting onto a half plane the branching patterns of dendrites and axons that have already been 3-dimensionally digitized. It is suited for neurons possessing an axis of orientation (or elongation), such as cortical neurons. This axis is taken as the polar axis of a spherical coordinate system whose center is the soma. For cortical neurons, the equatorial plane corresponds to the tangential plane. Co-latitude is measured with respect to the positive polar axis. Longitude is discarded. What results is a projection in which dendrites and axons appear to be growing in a half plane whose boundary is the polar axis. The projection eliminates many of the distorting effects of depth foreshortening seen in conventional projections. In so doing it helps one to visualize branching properties that would otherwise be obscure.

Morphometry of spine-free nonpyramidal neurons in rabbit auditory cortex.

A study of the morphometry and laminar distribution of spine-free nonpyramidal neurons in electrophysiologically verified primary auditory cortex was carried out in adult rabbits. By using image-combining computer microscopy, the locations of all impregnated neurons in 300-micrometers Golgi-Cox Nissl sections through the auditory cortex were determined. Spine-free non-pyramidal neurons constitute nearly 72% of the nonpyramidal neurons present. They are distributed in a band extending from 450 to 750 micrometers beneath the pial surface corresponding to laminae III and IV. A combination of dendritic stick, Fourier, and statistical analyses revealed a highly significant spatial orientation of their dendrite systems along a vertical axis parallel to the apical dendrites of pyramidal neurons. A significant tangential orientation of dendrites along a dorsal-ventral axis was also found. A radial analysis of the dendrite systems revealed that the pronounced vertical orientation of spine-free nonpyramidal neurons is due to (1) directed dendritic growth along the vertical axis, (2) decreased branching and rapid termination of tangentially oriented dendrites, and (3) increased branching of vertically growing dendrites. The radial analysis also revealed that the longest branches are those directed toward the white matter.

The image-combining computer microscope--an interactive instrument for morphometry of the nervous system.

The image-combining computer microscope is a new configuration of light microscope and computer graphics instrumentation which provides remarkable morphometric capabilities over a wide range of applications. The key component is the well known camera lucida, an image-combining device that is used to superimpose the computer graphics image upon the microscope's image of the object under study. Illumination need not be limited to the bright-field variety. The image superposition permits the investigator to study the object and acquire data from it while always looking through the microscope oculars. A computer controlled stepping motor driven stage is integrated with translations of the graphics image to permit image superposition and data acquisition at maximum visual magnification over the full expanse of a 25 X 25 mm tissue section. The investigator controls the system by means of a graphics tablet and a "Menu" area visible to him in the microscope's field of view. Spatial data in all 3 dimensions can be acquired easily since the z-axis is also stepping motor controlled. EM micrograph transparencies can be studied similarly. Stereological applications are possible by superimposing test grids upon the image. The system is self-contained and carries out data analysis programs written in C or Basic. Simplicity of operation has been emphasized throughout.

Morphology and laminar distribution of nonpyramidal neurons in the auditory cortex of the rabbit.

A study of the morphology and laminar distribution of nonpyramidal neurons in Golgi-Nissl preparations of electrophysiologically verified auditory cortex was carried out in the adult rabbit. Nonpyramidal neurons were located primarily within laminae I-IV and were only infrequently seen in lamina V and VI. In lamina I, four nonpyramidal cell types were observed: (1) small, spine-free horizontal neurons, (2) small, sparsely spined multipolar neurons with radiate dendrites, (3) large, multipolar neurons with fusiform somata and vertically aligned, sparsely spined dendrites, and (4) small, spine-free neurogliform neurons. The horizontal and small multipolar neurons had tangentially running axons confined to lamina I. The large, fusiform cells had descending axons which arborized in lamina II and occasionally reached lamina III. In lamina II and the upper part of lamina III, seven nonpyramidal cell types were observed: (1) spine-free bipolar neurons with vertically aligned dendrites and axonal arbors; (2) large, (3) medium, and (4) small, spine-free and sparsely spined multipolar neurons, all with locally ramifying axons; (5) pear-shaped cells with highly oriented dendrites which branched toward the pial surface and vertically arborizing axons; (6) multipolar cells with tangentially and vertically oriented dendrites and ascending axons which entered lamina I, and (7) tufted cells with local axons. Three types of nonpyramidal cells were observed in lamina IV and the lower part of lamina III: (1) large, multipolar cells with radiate, spine-free dendrites and stout axons which arborized locally, (2) spiny multipolar cells with vertically aligned dendrites and ascending axons which arborized in lamina II and III via long horizontal collaterals, and (3) spine-free bipolar cells with vertical dendrites and axons which arborized in a narrow vertical column adjacent to the dendrites. Nonpyramidal neurons in lamina V and VI were primarily multipolar cells with sparsely spined and spine-free dendrites. A comparison of these data with those of other species indicates that the neuronal organization of the rabbit auditory cortex is similar to that of the sensory cortex of the rodent but is strikingly different from that of carnivores and primates.

Analysis of thick brain sections by obverse-reverse computer microscopy: application of a new, high clarity Golgi-Nissl stain.

Exceptionally clear Golgi-Nissl sections of 300 micron thickness have been morphometrically studied by light microscopy using oil immersion objectives. The clarity results from a new variation of a staining procedure that combines Golgi and Nissl images in one section. A viewing technique has been developed that permits a histologic preparation to be examined from its obverse (or normally viewed) side and its reverse (or under) side. The technique was designed for use with a computer microscope but can be employed with any light microscope whose stage position can be measured within 100 micron. Sections thicker than 300 micron can be studied dependent on the working distance of the objective lens, provided that the clarity of the material permits it.