Nebojša T. Milošević

Application of modified Sholl analysis to neuronal dendritic arborization of the cat spinal cord

The drawings of Golgi-impregnated neurons from laminae I to VI in dorsal horn of the cat spinal cord were analysed morphometrically with a modified Sholl method of concentric circles. In order to advance the Sholl analysis of neuronal dendritic arborization patterns, we developed a new method of data presentation using polynomial regression and defining three parameters: the critical value of the circle radius (which defines the place of a possible circle intersecting maximum number of dendrites), the maximum number of dendritic intersections with the circles (counted for consecutive circles placed starting at the cell body to the border of the dendritic tree), and the mean value of the fitted polynomial function (which describes an average property concerning numbers of branches of dendritic tree over the whole region occupied by the dendritic arbor). For that purpose we also used the Sholl regression coefficient as well as the Schoenen ramification index. As an illustration of our model, we demonstrate that proposed modification of the Sholl method can successfully discriminate neuronal populations among different laminae of the cat spinal cord.

Morphology and classification of large neurons in the adult human dentate nucleus: A qualitative and quantitative analysis of 2D images

The dentate nucleus represents the most lateral of the four cerebellar nuclei that serve as major relay centres for fibres coming from the cerebellar cortex. Although many relevant findings regarding to the structure, neuronal morphology and cytoarchitectural development of the dentate nucleus have been presented so far, very little quantitative information has been collected on the types of large neurons in the human dentate nucleus. In the present study we qualitatively analyze our sample of large neurons according to their morphology and topology, and classify these cells into four types. Then, we quantify the morphology of such cell types taking into account seven morphometric parameters which describe the main properties of the cell soma, dendritic field and dendrite arborization. By performing appropriate statistics we prove out our classification of the large dentate neurons in the adult human. To the best of our knowledge, this study represents the first attempt of quantitative analysis of morphology and classification of the large neurons in the adult human dentate nucleus.

Changes in fractal dimension and lacunarity as early markers of UV-induced apoptosis

The aim of our study was to employ fractal analysis for evaluation of ultrastructural changes during early stages of apoptosis. Apoptosis was induced in U251 human glioma cell line by exposure to UVB light. The cells were visualized by optical phase-contrast microscopy and photographed before the UV treatment, immediately after the treatment, as well as at 30 min intervals during 5h observation period. For each of the 32 cells analyzed, cellular and nuclear fractal dimension, as well as nuclear lacunarity, were determined at each time point. Our data demonstrate that cellular ultrastructural complexity determined by fractal dimension and lacunarity significantly decreases after the UV irradiation, with the nuclear lacunarity being a particularly sensitive parameter in detecting early apoptosis. Importantly, fractal analysis was able to detect cellular apoptotic changes earlier than conventional flow cytometric analysis of phosphatidylserine exposure, DNA fragmentation and cell membrane permeabilization. These results indicate that fractal analysis might be a powerful and affordable method for non-invasive early identification of apoptosis in cell cultures.

Fractal analysis of the laminar organization of spinal cord neurons.

Images of Golgi impregnated neurons from different laminae of the human and rat dorsal horns were subjected to a quantitative analysis to support the Rexeds laminar scheme in mammals. Four methods of fractal analysis were performed in the proceedings: box-counting, mass-radius, cumulative intersection, and vectorized intersection. The results show that the box-counting method is more precise than the other fractal methods performed, and offers support for the conclusion that fractal analysis can successfully discriminate the neuron populations among different laminae. The analysis supports the concept of Rexeds cytoarchitectonic lamination of the dorsal horn.

Application of fractal analysis to neuronal dendritic arborisation patterns of the monkey dentate nucleus.

The deep nuclei of the cerebellar cortex have not yet received adequate exploratory attention. An exception is represented by the pioneering work of Chan-Palay, published in 1977, on the dentate nucleus morphology. She has classified each individual cell in the dentatus of the monkey into one of six types. Although fractal analysis is presently the most prominent quantitative method for morphometric neuronal studies, no article referring to applications of this method to the analysis of cell types of the dentate nucleus has so far been published. In the present study we apply fractal analysis to this unsolved problem and calculate the fractal dimension for each dendritic arbour of a neuron. We will hereby prove that by application of fractal analysis to the dendritic arbours of these cells whilst ignoring other neuronal attributes allows for clear discrimination of only three cell types.

Quantitative analysis of dendritic morphology of the alpha and delta retinal ganglion cells in the rat: A cell classification study

Type I retinal ganglion cells in the rat have been classified into several groups based on the cell body size and dendritic morphology. Considerable overlap and heterogeneity within groups have been reported, which is especially obvious for the morphology of the dendritic tree. For that purpose, we analysed quantitatively the dendritic morphology of the alpha and delta rat retinal ganglion cells, using parameters which provide information on the dendritic field size, shape of the dendritic tree and dendritic branching complexity. We show that the alpha and delta cells have significantly different dendritic field sizes. Taking into account the level of stratification of the dendritic tree, we found a difference in the properties of the dendritic morphology between alpha inner and alpha outer cells, while the opposite result was obtained for the delta inner and delta outer delta cells. In this study we also call attention to the relationship between morphological parameters and retinal eccentricity. The significance of our quantitative results in terms of present alpha and delta rat retinal ganglion cell classification is discussed.

FRACTAL ANALYSIS OF DENDRITIC ARBORISATION PATTERNS OF STALKED AND ISLET NEURONS IN SUBSTANTIA GELATINOSA OF DIFFERENT SPECIES

Through analysis of the morphology of dendritic arborisation of neurons from the substantia gelatinosa of dorsal horns from four different species, we have established that two types of cells (stalked and islet) are always present. The aim of the study was to perform the intra- and/or inter-species comparison of these two neuronal populations by fractal analysis, as well as to clarify the importance of the fractal dimension as an objective and usable morphological parameter. Fractal analysis was carried out adopting the box-counting method. We have shown that the mean fractal dimensions for the stalked cells are significantly different between species. The same is true for the mean fractal dimensions of the islet cells. Still, no significant differences were found for the fractal dimensions of the stalked and islet cells within a particular species. The human species has shown as the only exception where fractal dimensions of these two types of cells differ significantly. This study shows once more that the fractal dimension is a useful and sensitive morphological descriptor of neuronal structures and differences between them.

Morphology and cell classification of large neurons in the adult human dentate nucleus: a quantitative study.

The dentate nucleus represents the most lateral of the four cerebellar nuclei that serve as a major relay centres for fibres coming from the cerebellar cortex. Although many relevant findings regarding to the three-dimensional structure, the neuronal morphology and the cytoarchitectural development of the dentate nucleus have been presented so far, very little quantitative information has been collected to further explain several types of large neurons in the dentate nucleus. In this study we quantified the morphology of the large dentate neurons in the adult human taking, into account seven morphometric parameters that describe the main properties of the cell soma, the dendritic field and the dendritic branching pattern. Since the lateral cerebellar nucleus in the cat and other lower mammals is homologous to the dentate nucleus in primates and man, we have classified our sample of large neurons in accordance with the shape of the cell body, the dendritic arborization and their location within the dentate nucleus. By performing the appropriate statistical analysis, we have proved that our sample of human dentate neurons can be classified into four distinct types. In that sense, our quantitative analysis verifies the validity of previous qualitative conclusions concerning the large neurons in the developing human dentate nucleus. Furthermore, the present study represents the first attempt to perform a quantitative analysis and cell classification of the large projection neurons in the adult human dentate nucleus.

The morphology and classification of alpha ganglion cells in the rat retinae: A fractal analysis study

Rat retinal ganglion cells have been proposed to consist of a varying number of subtypes. Dendritic morphology is an essential aspect of classification and a necessary step toward understanding structure-function relationships of retinal ganglion cells. This study aimed at using a heuristic classification procedure in combination with the box-counting analysis to classify the alpha ganglion cells in the rat retinae based on the dendritic branching pattern and to investigate morphological changes with retinal eccentricity. The cells could be divided into two groups: cells with simple dendritic pattern (box dimension lower than 1.390) and cells with complex dendritic pattern (box dimension higher than 1.390) according to their dendritic branching pattern complexity. Both were further divided into two subtypes due to the stratification within the inner plexiform layer. In the present study we have shown that the alpha rat RCGs can be classified further by their dendritic branching complexity and thus extend those of previous reports that fractal analysis can be successfully used in neuronal classification, particularly that the fractal dimension represents a robust and sensitive tool for the classification of retinal ganglion cells. A hypothesis of possible functional significance of our classification scheme is also discussed.

Age-related reduction of structural complexity in spleen hematopoietic tissue architecture in mice.

The effects of aging on structural complexity in hematopoietic tissue are unknown. In this work, in a mouse experimental model, we report the age-related reduction of spleen hematopoietic tissue (SHT) complexity. Spleen tissue was obtained from the total of 64 male Swiss albino mice divided into 8 age groups: newborns (0 days old), 10 days, 20 days, 30 days, 120 days, 210 days, 300 and 390 days old. SHT was stained using conventional hematoxylin/eosin, and DNA-binding toluidine blue dyes. Fractal dimension as an indicator of cellular complexity, and lacunarity as indicator of tissue heterogeneity were determined based on the binarized SHT micrographs. Results indicate that fractal dimension of mice spleen hematopoietic tissue decreases with age, while lacunarity increases. These changes/trends have been detected in SHT stained both with toluidine blue and conventional hematoxylin/eosin. Fractal dimension was negatively correlated with lacunarity. The detected reduction in complexity suggests that age-related structural changes are present in mouse SHT both in general tissue architecture and progenitor cell DNA.