Method For Morphometric Analysis Research Articles

Time-dependent urinary bladder remodeling in the streptozotocin-induces diabetic rat model

Urinary bladder dysfunction and remodeling are well-recognized phenomena in diabetes but detailed assessments of tissue morphological changes have not been conducted. We studied time-dependent morphological changes in bladders from diabetic rats (streptozotocin model) and evaluated the usefulness of automated digital imaging technology as an unbiased, reproducible, and convenient method for the bladder morphometric analysis. Urinary bladders were isolated from diabetic (3 days, 2 weeks or 5 weeks after single injection of streptozotocin, 65 mg/kg) or control rats (0 or 5 weeks) and were processed for histochemical evaluations (hematoxylin/eosin and Mason's trichrome staining). Digital image analysis was used to quantify equatorial cross-sectional areas of bladder tissue and lumen, as well as relative prevalence of the three primary tissue components viz. smooth muscle, urothelium, and extracellular matrix. Digital imaging and color segmentation provided reliable and unbiased evaluations of the bladder tissue sections. Progressive increases in total bladder tissue and lumen area were observed in the diabetic animals relative to controls (p<0.05), demonstrating classic hypertrophy and dilation. Prevalence of smooth muscle and urothelium (% of total tissue) both increased significantly, but collagen content decreased. Average bladder wall thickness and urothelium thickness were unchanged. Bladder remodeling during experimental diabetes is associated with time-dependent chamber dilation and increased tissue mass. Changes in bladder wall composition also occurred in a time-dependent manner, most notably increased smooth muscle and urothelium and decreased collagen prevalence. Furthermore, automated digital imaging technologies provide an unbiased, reproducible, and convenient method for detailed morphometric analysis of bladder tissues.

Long-acting calcium channel blocker benidipine suppresses expression of angiogenic growth factors and prevents cardiac remodelling in a Type II diabetic rat model.

Calcium channel blockers, widely used for the treatment of hypertension and angina, could prevent cardiovascular complications in patients with diabetes. They can improve cardiac remodelling in animal models of a variety of cardiovascular diseases. Here, we examined the therapeutic effect of benidipine, a long-acting calcium channel blocker, on cardiac remodelling in Otsuka-Long-Evans-Tokushima-Fatty (OLETF) rats, a Type II (non-insulin-dependent) diabetes mellitus model. The methods for morphometric analysis included double staining for coronary capillaries, dye-binding staining for collagen content and Masson's trichrome staining for perivascular fibrosis. Immunohistochemical and in situ hybridization techniques were used for detecting protein and mRNA expressions for vascular endothelial growth factors (VEGF), basic fibroblast growth factors (bFGF) and TGF-beta(1), endothelial nitric oxide synthase (eNOS), and anti- and pro-apoptotic markers. OLETF rats showed an increased coronary capillary density, a reduced venular capillary proportion, an increased cardiac collagen content and prominent cardiac perivascular fibrosis. In OLETF rat hearts, significant increases in vascular expressions for VEGF, bFGF and TGF- beta(1) were found. Furthermore, the apoptosis signalling pathways, involving eNOS and apoptotic markers, were markedly altered, and coronary endothelial cell apoptosis was lower. These alterations with the exception of eNOS expression were significantly blocked by benidipine treatment. The suppressive effect of benidipine on overproduction of angiogenic growth factors could prevent cardiac angiogenesis and fibrosis, resulting in an improvement of cardiac remodelling in diabetes. As VEGF and bFGF potently block endothelial cell apoptosis execution, physiological apoptosis revived by benidipine treatment could also contribute to coronary vessel regression.

Análise morfométrica da bacia do Rio do Tanque, MG - Brasil

Os indicadores físicos específicos da bacia do Rio do Tanque, afluente do rio Santo Antônio, localizada no médio vale do Rio Doce, borda sudeste do Cráton São Francisco, foram determinados quantitativamente, através da análise de seus parâmetros morfométricos. Entre os diversos parâmetros, alguns se mostraram de fundamental importância no estudo dessa bacia, tais como: relação de relevo, densidade hidrográfica, densidade de drenagem, coeficiente de manutenção, gradiente de canais, índice de circulação e índice de sinuosidade. Dos dados obtidos, pôde-se inferir algumas peculiaridades da bacia, como, por exemplo, o fato de os seus cursos d'água possuírem baixo nível de escoamento, sendo que alguns encontram-se bastante encaixados, enquanto outros apresentam caráter tipicamente meandrante, além de áreas de drenagens homogêneas, levando-se em consideração o padrão de drenagem e a tipologia dos canais. Essas características se confirmam pela presença de dois níveis de base locais que se configuram em áreas de retenção de sedimentos e se localizam sempre a montante das porções dos canais controladas por falhas, fraturas e contatos geológicos.

Phylogenetic analysis of skull shape evolution in marmotine squirrels using landmarks and thin-plate splines

Abstract Several studies have shown that the recently developed techniques of geometric morphometrics are extremely powerful descriptive tools. And yet, one potential use of the resulting descriptions, phylogenetic analysis, has generally been neglected. This neglect is understandable because prominent systematists as well as prominent morphometricians have objected to the use of morphometric data in phylogenetic systematics. We agree that some methods of morphometric analysis produce results that cannot be used in phylogenetic systematics, and that some methods of incorporating morphometric results into statements about character transformation are not appropriate. However, we do not agree that these objections to specific techniques support a blanket rejection of the use of morphometric data in systematic studies. In this paper, we review the principles of phylogenetic systematics and show that they are equally applicable to qualitative descriptions of triangles and to quantitative descriptions (shape coordinates of the apex) of those same shapes. Then we show how these principles would he applied to complex shapes like skulls of marmotine squirrels, and that the resulting analysis leads to legitimate hypotheses about marmotine phylogeny and the evolution of skull shape in these animals.

Novel image analysis of corpus cavernous tissue in impotent men

Objectives. To objectively evaluate the contents of corpus cavernous tissue in impotent men using an automated novel image analysis system. Methods. Thirty-three impotent men and 2 normal potent men (controls) underwent corpus cavernous biopsies. The procedures were performed using a Biopty gun under local anesthesia. The obtained specimens were stained with Masson’s trichome technique, and the collagen fiber contents were evaluated by a computerized morphometric analysis method. In addition, we estimated the intraobserver and interobserver reliability of this automated image analysis system. Results. No major complication was noted during or after the biopsies. Of the 33 impotent patients, 3 were diagnosed as having psychogenic, 11 as having arteriogenic, 13 as having venogenic, 1 as having neurogenic, and 5 as having idiopathic impotence. The collagen fiber percentages in the 35 men were as follows: normal potent: 48.2% ± 1.4%, psychogenic 55.2% ± 11.6%, arteriogenic 73.2% ± 4.4%, venogenic 66.5% ± 4.2%, neurogenic 76.9%, and idiopathic 77.4% ± 4.2%. Significant differences were found between the normal potent and arteriogenic groups ( P <0.05) and between the normal potent and idiopathic groups ( P <0.05). Patients older than 60 years had a higher collagen fiber content (70.4% ± 3.7%) than those younger than 50 years old (58.6% ± 5.2%). The interobserver and intraobserver variances were both negligible for this automated image analysis system. This method significantly reduced the amount of variation introduced by the intra-rater reliability of a technician compared with the manual method. Conclusions. The present automated image analysis system is believed to be a reliable, accurate quantitative measurement tool for studies of penile tissue. Cavernous biopsy is a rapid, safe, and representative modality to study penile disease. An increase in cavernous collagen fibers (or corporal fibrosis) is considered an important factor in impaired erectile function.

Method for morphometric analysis of axons in experimental peripheral nerve reconstruction

A new method for morphometric analysis of axons in experimental peripheral nerve reconstruction is presented. Twelve adult female rabbits were used. In nine animals the saphenous nerve was transected and stitched epineurially. Three animals functioned as control. After 3, 6, and 12 months, the nerves were harvested, fixed in Kryofix and embedded in Histowax. Transverse sections of 6 microm were cut, immunohistochemically stained for NF 90, and counterstained by Sirius Red. Quantification of nerve fibers in cross sections was performed by using a confocal laser scanning microscope (CLSM), and the images were stored digitally. Data analyzing was performed by the Optimas program (5.2). Calculations were done with Microsoft Excel. The total number of axons, the mean axon diameter and the percentage axon area/fascicle area were evaluated statistically. This method for morphologic analysis provides automatically complete registration of axons and so different methods of experimental nerve reconstruction can be compared in a fast and reliable way.

Generalizing and extending the eigenshape method of shape space visualization and analysis

Outline-based morphometric methods have been more or less restricted to the consideration of closed curves and plagued by problems related to the maintenance of close biological correspondence across all forms within a sample. Methods developed herein generalize and extend the eigenshape method of outline analysis along the following lines: (1) consideration of open curves, (2) improvement of interobject correspondence via incorporation of information provided by landmarks, and (3) extension to the analysis of three-dimensional (open and closed) curves. In addition, techniques for using eigenshape results to create models of shape variation and for more consistently assessing the digital resolution necessary to represent an object are discussed and illustrated. These improvements are then placed in context via discussions of previous attempts to extend morphometric outline analysis methods, the relation between landmark and outline-based morphometric methods, the use of morphometric analyses to test biological hypotheses, and the nature of morphometric shape spaces (with special reference to studies of morphological disparity).

Nitric oxide suppresses bFGF- and IL-1-alpha-mediated but not VEGF165-mediated angiogenesis in natively vascularized mammalian tissue.

Using the rat mesenteric window angiogenesis assay, we studied the systemic effect of the nitric oxide synthase inhibitor Nw-nitro-L-arginine methyl ester, L-NAME, on angiogenesis induced by basic fibroblast growth factor (bFGF), interleukin-1-alpha (IL-1) or vascular endothelial growth factor (VEGF165). Using technically independent morphometric and image analysis methods, the angiogenic response was quantified in variables related to (i) microvascular spatial extension and (ii) microvascular density. The test tissue, which is natively vascularized and lacks significant angiogenesis physiologically, was unaffected by surgical intervention. Two daily intraperitoneal injections of bFGF (2.20 nmole), IL-1 (1.18 nmole) and VEGF165 (480 pmole) for 5 days elicited significant angiogenesis in the mesenteric windows. L-NAME (0.5 g/L in drinking water) caused further enhancement of the angiogenic response produced by human recombinant bFGF (p<0.001), bovine purified bFGF (p<0.05) or murine recombinant IL-1 (p<0.05). In contrast, the L-NAME treatment did not affect the angiogenic response produced by human and murine recombinant VEGF165. These data suggest that nitric oxide can act as an endogenous suppressor of mammalian de novo angiogenesis, which is a new finding, and, moreover, that angiogenesis induced by VEGF165 on the one hand and by bFGF and IL-1 on the other in the rat mesenteric window depends on different pathways.

Iron overload in bone marrow transplant recipients.

We examined the degree of hepatic iron overload in patients receiving marrow transplant for hematologic malignancy and evaluated a new method of morphometric analysis of marrow iron content as a means of estimating hepatic iron stores in these patients. The iron content of marrow and liver specimens from 10 consecutive patients who died between 50 and 100 days after transplant was determined by spectrophotometry. Their mean age was 34.9 years (range 10-59). The mean time to death from disease onset was 2.2 years (0.5-8.7). Patients had received 30.2 +/- 17.4 units of red cells during the transplant period and 47.6 +/- 25.9 red cell units from diagnosis to death. The median hepatic iron concentration (HIC) was 4307 microg/g dry weight (range 1832-13120; normal 530-900) and the median hepatic iron index (HIC (micromol/g dry weight/age (years)) was 3.85 (0.76-8.14). The median biochemical marrow iron content was 1999 microg/g dry weight (range 932-3942). Morphometric analysis of the marrow iron content was performed on digital photomicrographs of a single Prussian blue-stained section of marrow. Strong correlations were demonstrated between morphometric marrow iron content and (1) biochemical marrow iron content (r = 0.8, P= 0.006) and (2) biochemical hepatic iron index (r = 0.82, P = 0.004). We conclude that marrow transplant recipients have a high liver iron content at 50-100 days post transplant with the hepatic iron index in the hereditary hemochromatosis range. Computerized morphometric marrow iron determination is a readily available means of estimating hepatic iron stores in these patients.

Landmark Based Thin Plate Spline Relative Warp Analysis of Gastropod Shells

In the analysis of organismal form, landmark-based geometrical morphometrics now are preferred to traditional morphometrics by many systematists. However, certain forms have eluded geometrical morphometric analysis because landmarks on them are difficult to de® ne ; this situation is a problem of logistics rather than an inadequacy of the landmark- based approach. In this article, a landmark-based geometrical morphometric analysis is per- formed on one such form (the gastropod shell), and the results obtained are compared to those obtained using more traditional methods of morphometric analysis. (Gastropods ; geo- metric morphometrics ; landmarks ; principal components ; thin-plate spline. )

A COMPARISON OF COMPUTERIZED IMAGE ANALYSIS AND STEREOLOGY AS TOOLS FOR MORPHOLOGICAL STUDY OF ALGAL CELLS

A computerized image analysis package (ImagePro+) was evaluated as an alternative method for morphometric analysis of electron micrographs of microalgal cells. The morphometric analysis was demonstrated with micrographs of the marine alga Nannochloropsis sp. grown under high and low light intensity. We applied the ImagePro+ package to estimate the relative volume of an organelle based on the ratio of perimeters of the organelle and the cell. The measurements included the volumes of chloroplasts, mitochondria, nuclei, vacuoles, and accumulation bodies, all relative to cell volume. The length of thylakoids was measured using the same package. The results obtained by ImagePro+ were compared to those of the traditional manual and laborious method involving the superimposition of an array of short lines on the micrograph. A high correlation between the methods was found. The following correlations were found for chloroplast, nucleus, and accumulation bodies: 0.96, 0.92, and 0.75, respectively. The correlation between length of thylakoids (ImagePro+) and surface area of thylakoids (superimposition) was 0.82.

Comparing Wing Shape of Bats: The Merits of Principal-Components Analysis and Relative-Warp Analysis

Size and shape are fundamental features of organisms. Ideally, the methods used to describe size and shape must be sensitive enough to detect small differences, and at the same time provide interpretation that is visually satisfying. I compared shape of wings among Eptesicus fuscus, Myotis lucifugus, Pipistrellus hesperas , and Tadarida brasiliensis using three methods of morphometric analysis; principal-components analysis on traditional measures of lengths and areas, principal-components analysis of interlandmark distances, and relative-warp analysis. These species were chosen to test the efficacy of each morphometric analysis at discovering and describing differences in both size and shape. Although principal-components analyses provided similar aggregations of species based on variable loadings, only relative-warps analysis provided clear pictures as to how species differ. The molossid bat, T. brasiliensis , had narrower wings than the vespertilionid bats; this shape was not due to compression of the entire wing, but to a complicated rotation around the fifth digit. The vespertilionid bats were difficult to discern based on wing shape alone, but the warp analysis showed that P. Hesperus and M. lucifugus are more similar in shape of wing than either is to E. fuscus . Each method of analysis of shape has strengths, but relative-warp analysis provided both the power to discriminate among minor differences in shape of species within families, and the graphical capability to display those differences in a visually pleasing way.

Quantitative analysis of the lamina cribrosa in vivo using a scanning laser opthalmoscope

PURPOSE. Structural changes in the lamina cribrosa have been implicated in the pathogenesis of glaucomatous optic nerve atrophy. The purpose of this study was to develop a method for morphometric analysis of the lamina cribrosa pores in vivo, using a scanning laser ophthalmoscope. METHODS. A prototype Zeiss confocal laser scanning ophthalmoscope was used to acquire images of the lamina cribrosa. The images were digitised and aligned to compensate for eye movements. Thirty-two consecutive images were averaged to reduce noise. The images were processed to adjust for luminance gradients prior to segmentation and analysis. Details of the image processing are described. RESULTS. The end result of processing the images was a binary (black and white) image that can be used for automated computer assisted measurements. The pores of the lamina cribrosa are well represented and retain their overall shape in the binary image, as judged by superimposing the binary image on the unprocessed image. We also established the repeatability, reproducibility and intercession variability of this technique. Repeated images of the internal lamina cribrosa of 10 patients were acquired, by two observers in two separate visits, and the images were processed before automated computer measurements. The parameters evaluated were number of pores, area covered by the pores and area covered by the visible lamina cribrosa. The coefficient of variation for number of pores, pore area and lamina area was 6.9%, 2.1% and 4.3% for observer A and 5.5%, 2.1% and 5.8% for observer B. Pearson product moment correlation coefficient between the two observers was 0.94, 0.99 and 0.97 for the above parameters respectively. There was no significant difference between the measurements on visit 1 and 2 for both observers. CONCLUSIONS. The technique described allows, for the first time, in vivo morphometry of the internal lamina cribrosa surface. This method has good reproducibility, suggesting future clinical applications.

Detrended correspondence analysis in the ordination of data for phenetics and cladistics

SummaryParnell, J. &amp; Waldren, S.: Detrended correspondence analysis in the ordination of data for phenetics and cladistics. – Taxon 45: 71‐84. 1996. – ISSN 0040‐0262.Most methods of morphometric and phylogenetic analysis do not permit the incorporation of both categorical and quantitative data. Detrended correspondence analysis (DCA) was used to produce ordination diagrams of strictly categorical data from a cladistic analysis, and mixed data from a morphometric study of hybridization. In both cases it proved preferable to standardize the data prior to DCA, because increased separation on ordination was found when larger scale ranges were used. A standardization is suggested, such that characters are scaled on a range from 1 to the total number of characters scored; this will produce more informative ordinations if large numbers of characters are scored. When used in conjunction with appropriate cladograms this phenetic technique may help reveal features such as homoplasy which are not readily apparent in cladograms, while the capability of DCA to combine both categorical and quantitative characters is especially useful in morphometric studies and in areas of evolutionary biology, e.g. chronistic studies of extinction events in morphospace.

A simple, automatic method for morphometric analysis of the left ventricle in rats with myocardial infarction

Induction of acute myocardial infarction in the rat is an established model for studying effects of therapeutic interventions. Images of sections of the rat left ventricle, stained with nitroblue tetrazolium, were digitized and several parameters estimated by dedicated software on an image analyzer (IBAS 2.0). The method was tested on 7 rats with 48-hr-old myocardial infarction and 4 sham-operated controls. Infarct size can be evaluated by two largely used methods, based on area or on angular extension of the lesion. Results of the two methods are linearly correlated, but area calculations give values half of those obtained from angular extension. Five minutes were needed for a complete evaluation of a section of the left ventricle. Estimates of the parameters showed a relatively low between- and within-operator variability and a good correlation with a classic, but time-consuming, planimetric method. The method simultaneously measures infarct size and left ventricular geometry in the rat. The advantages over previous nonautomatic methods are simplicity, good reproducibility, and speed of execution, which make it particularly useful in the evaluation of drug effects.

A quantitative method for morphometric analysis in neuronal cell culture: unbiased estimation of neuron area and number of branch points

The morphology and fine structure of neurons in vivo as well as in vitro are influenced by a variety of cell-adhesion and extracellular matrix molecules and soluble growth factors. To examine the effects of such molecules, we have developed a new method for the quantitation of several parameters associated with the morphology of neurons in culture. Whereas methods which have been traditionally used to perform quantitative morphometric analysis of neurons in vitro are often time-consuming and subjective, the methods we describe provide a rapid, efficient, and unbiased approach to morphometric analysis of cultured neurons.

Morphometry of sinusoids and portal hypertension in non-alcoholic cirrhosis

To examine whether structural changes in hepatocytes and/or sinusoidal areas contribute to the portal hypertensive state in non-alcoholic cirrhosis, a new method of morphometric analysis using a computer-aided color image analyzer was performed in 16 patients with non-alcoholic cirrhosis, which allowed quantitative evaluation of various morphometric parameters of sinusoids and hepatocytes. The sinusoidal pressure gradient was estimated theoretically with these and clearance parameters using Poiseulle's equation and compared with the hepatic venous pressure gradient measure by hepatic vein cannulation. A significant relationship was found between the hepatic venous pressure gradient and sinusoidal volumetric ratio (r = -0.598, p < 0.05), but not between mean hepatocyte volume and sinusoidal volumetric ratio (r = 0.416, NS), or the hepatic venous pressure gradient (r = 0.371, NS). The estimated sinusoidal pressure gradient showed a significant relationship with the hepatic venous pressure gradient (r = 0.637, p < 0.01). However, the absolute values of the former were much lower than those of the latter. Therefore, in non-alcoholic cirrhosis, although sinusoidal stenosis not caused by hepatocyte swelling may lead to increased vascular resistance, other factors must also play a significant role.

Comparison between wedge and needle biopsies for evaluating the degree of cirrhosis

To examine whether the biopsy method could affect histological evaluation, the volumetric ratio of human liver parenchyma was estimated in specimens from subcapsular and intralobar areas, and also in specimens obtained by needle biopsy (Tru-Cut needle). A new method of morphometric analysis was performed using a computer-aided color image analyzer. Eighteen cirrhotic, 7 fibrotic, and 4 normal liver biopsies were taken during hepatic resection and analyzed. The parenchymal cell volume ratio in the intralobar area was significantly correlated with that in the subcapsular area, and less significantly with needle biopsy samples (r = 0.844, p < 0.001; r = 0.577, p < 0.01, respectively). Both showed one-to-one correspondence. These results suggest that both wedged and needle biopsy samples are appropriate for assessing the degree of fibrosis or cirrhosis, although the sampling variability of the latter is greater than the former.

Effect of titanium implants on primary mineralization following 6 and 14 days of rat tibial healing

The effect of pure commercial titanium implants on the process of primary mineralization was studied. This was examined by insertion of titanium implants into rat tibial bone after ablation. The effects of the titanium were studied through the behaviour of extracellular matrix vesicles (MV). Methods of morphometric analysis at the TEM level were applied. The insertion of titanium implants was followed by an increase in the number of MV as well as vesicular diameter and by a decrease in vesicular distance from the calcified front when compared to normal healing. These results suggest that the process of MV maturation around titanium implants was delayed when compared to normal primary bone formation during bone healing. The delay in mineralization was compensated by an increase in vesicle production, resulting in an enhancement of primary mineralization by the titanium.

Quantitative analysis of age-related dendritic changes in medium spiny I (MSI) striatal neurons of C57BL/6N mice

Our study used quantitative morphometric analysis and Golgi staining methods to evaluate postnatal changes in the dendritic architecture of MSI neurons of the striatum between 1 and 30 months of age. Morphological changes and chronological age were also correlated with functional testing in order to identify subpopulations of aged mice with dendritic alterations that may be more characteristic of a motor deficit rather than the normal aging process. We found that the overall size of the dendritic arbor of MSI neurons in the rostral striatum remained stable with age, while caudal MSI neurons exhibited a significant elongation of terminal dendritic segments between 25 and 30 months of age. In addition, our correlation analysis of motor performance and chronological age found that neither striatal-motor deficits nor their associated anatomical correlates were inevitable consequences of senescence but were characteristic for a select subpopulation of aged mice with striatal-motor deficits. We found that mice that tested poorly on the balance rod had a significant increase in the number of MSI neurons with small dendritic arbors in various stages of atrophic degeneration. Conversely, 30-month-old mice that had no functional impairment showed no significant change in the number of neurons with atrophic dendrites. These data reinforce the premise that the correlation of structure and function plays an important role in the analysis of an aging population since data may vary based on the number of functionally impaired or unimpaired mice that make up an experimental group.