Use Of Computer-assisted Image Analysis Research Articles

Use of computer-assisted image analysis for semi-quantitative histology of the hepatopancreas in whiteleg shrimp Penaeus vannamei (Boone).

Despite the increasing use of novel molecular techniques in pathology, histology remains the standard method for monitoring tissue alterations and for assessing pathology. Histopathological evaluation is generally laborious and subjective with risk of discrepancies in semi-quantitative scoring between pathologists. In contrast, computer-assisted image analysis (CAIA) is potentially faster, more objective and thus suitable for routine screening. Limited research has been carried out on CAIA in crustacean histopathology, and the methods described were not fully automated. Therefore, the aim of this study was to develop CAIA in whiteleg shrimp (Penaeus vannamei) for the study of the hepatopancreas. Paraffin sections were immunohistochemically stained with monoclonal antibodies WSH8 against haemocytes and counterstained with Mayer's haematoxylin for detection of haemocytes and B-cell vacuoles, and modified toluidine blue protocol was used for detection of F-cells; frozen sections were stained with Oil Red O for detection of lipid droplets within R-cells. Visiopharm® software was used to develop and validated protocols for the quantification of morphological parameters (areas of haemocyte infiltration, F-cells, B-cell vacuoles, lipid droplets and their ratios to total tissue area and total lumen area). These protocols enable the future use of CAIA for determination of the nutritional and pathological condition of this organ.

The use of computer-assisted image analysis in the evaluation of the effect of management systems on changes in the color, chemical composition and texture of m. longissimus dorsi in pigs

The effect of management systems on selected physical properties and chemical composition of m. longissimus dorsi was studied in pigs. Muscle texture parameters were determined by computer-assisted image analysis, and the color of muscle samples was evaluated using a spectrophotometer. Highly significant correlations were observed between chemical composition and selected texture variables in the analyzed images. Chemical composition was not correlated with color or spectral distribution. Subject to the applied classification methods and groups of variables included in the classification model, the experimental groups were identified correctly in 35–95%. No significant differences in the chemical composition of m. longissimus dorsi were observed between experimental groups. Significant differences were noted in color lightness (L*) and redness (a*).

Radiographic analysis in castor bean seeds (Ricinus communis L.)

Castor bean cropping has great social and economic value, but its production has been affected by factors such as low quality seeds used for sowing. The quick and precise evaluation of seed quality by x-ray test is known as an effective method to evaluate seed lots, but little is known about the interpretation between of the type of radiographic image and the seed quality correlation. The potential of x-ray analysis as a marker of seed physiological quality and as an initial process for the implementation of the use of computer-assisted image analysis was investigated using castor bean seeds of the different cultivars. The seeds were classified according to internal morphology visualized in the radiography and subjected to the germination test, emergency and seedling growth rate. It was possible to identify the different types of internal tissues, morphological and physical damage in castor bean seeds using the x-ray test. Tissues generating translucent images, embryo deformation, or tissues with less than 50% of endosperm reserves or spotted, negatively affected the physiological potential of the seed lots. Radiographic analysis is effective as an instrument to improve castor bean seed lot quality. This non destructive analysis allows the prediction of seedling performance and enabled the selection of high-quality seeds under the standards of a sustainable and precision agriculture

Immunohistochemical evaluation of cyclooxygenase expression in corneal squamous cell carcinoma in horses

To evaluate expression of cyclooxygenase (COX)-1 and COX-2 in the cornea, eyelid, and third eyelid of healthy horses and those affected with squamous cell carcinoma (SCC) by use of immunohistochemical techniques. 15 horses with SCC involving ocular tissues and 5 unaffected control horses. SCC-affected tissues were obtained from the cornea (n = 5 horses), eyelid (5), and third eyelid (5). Site-matched control tissues were obtained from 5 horses unaffected with SCC. Tissue sections of affected and control cornea, eyelid, and third eyelid were stained immunohistochemically for COX-1 and COX-2 via standard techniques. Stain uptake was quantified by use of computer-assisted image analysis of digital photomicrographs. Immunoreactivity for both COX-1 and COX-2 was significantly greater in equine corneas with SCC than in control corneas. No significant differences in COX-1 or COX-2 immunoreactivity were detected in eyelid and third-eyelid SCC, compared with site-matched control tissues. Immunoreactivity for COX-1 and COX-2 is high in equine corneal SCC, possibly indicating that COX plays a role in oncogenesis or progression of this tumor type at this site. Pharmacologic inhibition of COX may represent a useful adjunctive treatment for corneal SCC in horses.

Digital subtraction angiography and computer assisted image analysis for the evaluation of the antiangiogenetic effect of ionizing radiation on tumor angiogenesis

The aim of the present study was to evaluate and quantify the antiangiogenetic effect of ionizing radiation on tumor angiogenesis using digital subtraction angiography (DSA) in conjunction with computer assisted image analysis (CAIA). Walker 256 carcinosarcoma was inoculated in both glutei of 12 Wistar rats. When the tumors reached a diameter of 1.5 cm, local irradiation of the right gluteus was performed. The left gluteus of each animal served as a control. After 24 hours of irradiation, angiography was performed, and images where digitized and subsequently processed. The effect of irradiation was observed both in big and small vessels (smaller or greater than 200 microm). Irradiated areas of both small and big vessels showed a statistically significant reduction in both total vessel area and length. Small vessels showed a greater trend toward suppression by irradiation (not statistically significant). Irradiation had a deleterious effect in both macro- and micro-blood supply of a tumor. The use of CAIA enhanced the efficacy of DSA and enabled the in vivo identification of the effect of irradiation on various caliber vessels as well as the ratios of total length and total area of small and big vessels.

Effect of aging on the substance P receptor, NK–1, in the spinal cord of rats with peripheral nerve injury

Substance P (SP) levels in the spinal cords of very old rats are less than the levels in younger rats (Bergman et al., 1996). After injury to a peripheral nerve in young rats, immunoreactivity (ir) to the SP receptor, NK–1 (neurokinin-1), increases in the spinal cord ipsilateral to the injury and the increases are correlated with the development of thermal hyperalgesia (Goff et al., 1998). Thus we postulated that aged rats might display an increased sensitivity to thermal stimulation before peripheral nerve injury and that they might respond differently to injury than do younger rats. To test this hypothesis, we used the Bennett and Xie model (1988) of chronic constriction injury (CCI) to the sciatic nerve to induce a neuropathic pain condition. We investigated the effect of age on changes in NK-1 ir in superficial layers of the dorsal horn and on numbers of NK ir cells in deeper laminae at the L4-L5 levels of the spinal cord after CCI. NK-1 receptors were tagged immunohistochemically and their distribution quantified by use of computer-assisted image analysis. NK-1 ir changes were related to alterations in thermal and tactile sensitivity that developed after CCI in young, mature and aged (4-6, 14-16, and 24-26 months) Fischer F344 BNF1 hybrid rats. No differences in thermal or tactile sensitivity of young and aged rats were seen in the absence of nerve injury. After injury, aged rats developed thermal hyperalgesia and tactile allodynia more slowly than did the younger rats. NK-1 receptor ir and numbers of NK-1 ir cells in the dorsal horn increased with time post-injury in all three groups. NK-1 ir increases were correlated with the development of thermal hyperalgesia in those rats that displayed hyperalgesia. However, some rats developed an increased threshold to thermal stimuli (analgesia) and that also was correlated with increases in NK-1 ir. Thus NK-1 ir extent, while correlated with thermal sensitivity in the absence of injury, is not a specific marker for disturbances in one particular sensory modality; rather it increases with peripheral nerve injury per se.

Histamine-induced biphasic macromolecular leakage in the microcirculation of the conscious hamster: evidence for a delayed nitric oxide-dependent leakage.

1. Late effects (up to 3 h) of intravenously-injected histamine on FITC-dextran extravasation were investigated in the conscious hamster, by use of computer-assisted image analysis of fluorescence distribution in a microscopic window of dorsal skin fold preparations. This analysis allowed measurement of local (skin) and general (all organs) extravasations caused by a bolus injection of histamine (1 mg kg(-1), i.v.) 2. Histamine doses higher than 0.01 mg kg(-1) caused biphasic local and general extravasations. Initial phases developed fully within 15 min (for local) and 60 min (for general) and were followed by late phases beginning 90 min after histamine injection. Although the initial and late phases of histamine-induced extravasations had differential apparent reactivities to the autacoid, all the effects of histamine on the microcirculation (1 mg kg[-1]) were inhibited by pyrilamine (1 mg kg(-1), i.v.) but not by cimetidine (1 mg kg(-1), i.v.). 3. Pretreatment with N(G)-monomethyl-L-arginine (L-NMMA, 30 mg kg(-1), i.v.) or N(G)-nitro-L-arginine methyl ester (L-NAME, 100 mg kg(-1), i.v.) did not affect the initial phases but did prevent the late phases of local and general extravasations triggered by 1 mg kg(-1) histamine. The inhibitory effects of L-NAME were reversed by L-arginine (30 mg kg[-1]) but not by D-arginine (30 mg kg[-1]) according to the enantioselectivity of nitric oxide synthase (NOS). A late NO-mediated venular dilatation occurred in response to plasma histamine. 4. A low dose of aminoguanidine (1 mg kg(-1), i.v.), a selective inhibitor of the inducible isoform of NOS (iNOS), mimicked the inhibitory effects of L-NAME on the late phases of histamine-induced macromolecular extravasations and venular dilatation. 5. Pretreatment with dexamethasone (1 mg kg(-1), i.v.) prevented both the initial and late phases of histamine-induced extravasations. Fucoidan (1 or 25 mg kg(-1), i.v.) prevented the late phases without affecting initial phases, consistent with a role for leukocytes adhesion in the development of the late NO-mediated effects of histamine. 6. We conclude that intravenous injection of histamine triggers a biphasic inflammatory cascade via initial activation of H1 receptors which induces a late NO-mediated PMN-dependent extravasation process.

Use of computer-assisted image analysis for noninvasive evaluation of oral lichenoid reactions and oral leukoplakia

The objective of the present study was to elaborate and to evaluate a method in which the clinical appearance of a mucosal lesion could be expressed by means of color and morphologic features extracted from digitized images. The ability to express mucosal changes with image features was evaluated, as was the accuracy of a classification of the lesions on the basis of image feature analysis. Computer-assisted image analysis was performed on digitized color slides from 76 patients with lichenoid reactions that included both oral lichen planus and contact lesions and 20 patients with homogenous oral leukoplakia. Color features were measured according to the intensity-hue-saturation color system, and morphologic features were estimated by tracing the hyperkeratotic area. Hyperkeratinizing and inflammatory tissue reactions could be demonstrated and defined by image analysis. Image features indicating inflammatory tissue reactions were more pronounced in cases of oral lichenoid reactions. Morphologic parameters could express the anatomic appearance of the lesions. The comparison between histopathologic diagnosis and a classification through computerized analysis demonstrated that 90.5% of oral lichenoid reactions could be separated from homogenous oral leukoplakia by linear discriminant analysis.

Observer variation in quantification of immunocytochemistry by image analysis

This paper reports the findings of a study designed to examine observer variation as a source of inaccuracy inherent in the use of computer-assisted image analysis to measure areas of stained tissue. The rat pituitary immunostained for prolactin and galanin was used as an example to estimate patterns of immunoreactivity exhibited by different cell types. Six observers, with differing experience, selected grey level threshold values on 40 fields of images of stained tissue making three repeats of each field. The 40 fields consisted of 20 serial pairs of colocalized fields, one immunostained for prolactin, the other for galanin. The 20 pairs consisted of four pairs from each of five animals. Analysis of observer variation in the selection of threshold values showed large differences in the within- and between-observer variation. Analysis of the components of variance in the estimation of the ratios of stained tissues showed that the major source of variation was the within-observer component. An additional experiment using two observers, where half of the images were compared to the original microscope images before setting threshold levels, showed that the opportunity to make a comparison did not reduce observer variation. It is suggested that any study which uses semi-automatic methods to segment regions of a digital image can benefit from an analysis of this kind so that the sources of variation can be determined to enable maximum discriminating power in future studies.

Quantification of vascular graft seeding by use of computer-assisted image analysis and genetically modified endothelial cells

Current methods for the evaluation of retention of endothelial cells seeded on vascular grafts are limited by the inability to specifically identify and quantitate seeded cells on a long-term basis. To address this problem we developed a method of quantification of graft surface coverage using genetic labeling of endothelial cells combined with computer-assisted image analysis. Rabbit aortic endothelial cells were transduced with a marker gene (lac-Z) and seeded on polytetrafluoroethylene grafts. After histochemical staining in which the genetically labeled cells turn blue, computer-assisted image analysis was used to measure the percentage of graft surface covered by the seeded cells. The utility of the method was evaluated by using it to assess the effect on graft coverage of seeded cell density and by precoating with fibronectin. Quantification of surface area coverage was automated and reproducible both between scans and between observers. Use of this method allowed the determination of a linear correlation between cell density in the seeding suspension and graft coverage (r2 = 0.93, p less than 0.0001). The method also permitted confirmation of the positive contribution of fibronectin coating to graft coverage by seeded cells: 73% coverage coated versus 8% coverage uncoated (p less than 0.0001). The ability of this method to specifically identify genetically marked endothelial cells and their progeny makes it attractive for use in studies targeted at optimization of graft coverage in vivo.

Quantitation of oncogene products by computer-assisted image analysis and flow cytometry.

The use of antibodies permits the study of oncogene product expression in cells and tissues. However, quantitation of the levels of expression in immunohistochemical preparations is beset by difficulties, and the available scoring system provide semiquantitative data at best. Here we describe the use of computer-assisted image analysis for determination of oncoprotein levels in a model system and compare the results with those generated by flow cytometric analysis. The oncogene products measured are located in the nucleus (c-myc p62 and c-fos p55), the inner surface of the membrane (c-ras p21), and both sides of the membrane (c-erbB-2 p185). In each instance, both analytic modalities yielded concordant results. Our data indicate that computer-assisted image analysis is a useful tool for quantitating cell components in immunohistochemical preparations.

Cryosurgery of rhesus monkey skin--evaluation of epidermal thickening and effect on elastic component.

Summary The skin of the rhesus monkey was subjected to one and two-cycle freezing with liquid nitrogen spray. Temperatures of –40 to –60°C were used and the sites were evaluated from 7 to 182 days. Overall healing was excellent. Long-term specimens showed a consistent increase in epidermal thickness at the site of the freeze. This was quantified by use of computer-assisted image analysis. In addition, the elastic component of the skin, seldom studied in repair following skin surgery, did not regenerate.