Semi-automatic Image Analysis System Research Articles

Development and Validation of an Image Analysis System for the Measurement of Cell Proliferation in Mammary Glands of Rats.

Reliable detection and measurement of cell proliferation are essential in the preclinical assessment of carcinogenic risk of therapeutics. In this context, the assessment of mitogenic potential on mammary glands is crucial in the preclinical safety evaluation of novel insulins. The existing manual counting is time-consuming and subject to operator bias. To standardize the processes, make it faster, and resistant to errors, we developed a semiautomated image analysis system (CEPA software, which is open-source) for counting of proliferating cells in photomicrographs of mammary gland sections of rats labeled with Ki-67. We validated the software and met the predefined targets for specificity, accuracy, and reproducibility. In comparison to manual counting, the respective mean differences in absolute labeling indices (LIs) for CEPA software were 3.12% for user 1 and 3.05% for user 2. The respective regression analysis revealed a good correlation between the CEPA software user and manual counting. Moreover, the CEPA software showed enhanced reproducibility between independent users. The interuser variability is centered around 0 and the absolute difference was about 0.53% LI. Based on validation data, our software has superiority to the manual counting and is a valid and reliable tool for the routine analysis of cell proliferation in mammary glands from rats exposed to insulin analogs.

Impact of HIV/simian immunodeficiency virus infection and viral proteins on adipose tissue fibrosis and adipogenesis

HIV-infected patients receiving antiretroviral treatment (ART) often present adipose tissue accumulation and/or redistribution. adipose tissue has been shown to be an HIV/SIV reservoir and viral proteins as Tat or Nef can be released by infected immune cells and exert a bystander effect on adipocytes or precursors. Our aim was to demonstrate that SIV/HIV infection per se could alter adipose tissue structure and/or function. Morphological and functional alterations of subcutaneous (SCAT) and visceral adipose tissue (VAT) were studied in SIV-infected macaques and HIV-infected ART-controlled patients. To analyze the effect of Tat or Nef, we used human adipose stem cells (ASCs) issued from healthy donors, and analyzed adipogenesis and extracellular matrix component production using two dimensional (2D) and three-dimensional (3D) culture models. Adipocyte size and index of fibrosis were determined on Sirius red-stained adipose tissue samples. Proliferating and adipocyte 2D-differentiating or 3D-differentiating ASCs were treated chronically with Tat or Nef. mRNA, protein expression and secretion were examined by RT-PCR, western-blot and ELISA. SCAT and VAT from SIV-infected macaques displayed small adipocytes, decreased adipogenesis and severe fibrosis with collagen deposition. SCAT and VAT from HIV-infected ART-controlled patients presented similar alterations. In vitro, Tat and/or Nef induced a profibrotic phenotype in undifferentiated ASCs and altered adipogenesis and collagen production in adipocyte-differentiating ASCs. We demonstrate here a specific role for HIV/SIV infection per se on adipose tissue fibrosis and adipogenesis, probably through the release of viral proteins, which could be involved in adipose tissue dysfunction contributing to cardiometabolic alterations of HIV-infected individuals.

Characterization of cracking in pavement distress using image processing techniques and k-Nearest neighbour

<p class="Abstract">This study presents characterization of cracking in pavement distress using image processing techniques and <em>k</em>-nearest neighbour (kNN) classifier. The proposed semi-automated detection system for characterization on pavement distress anticipated to minimize the human supervision from traditional surveys and reduces cost of maintenance of pavement distress. The system consists of 4 stages which are image acquisition, image processing, feature extraction and classification. Firstly, a tool for image acquisition, consisting of digital camera, camera holder and tripod, is developed to capture images of pavement distress. Secondly, image processing techniques such as image thresholding, median filter, image erosion and image filling are applied. Thirdly, two features that represent the length of pavement cracking in <em>x</em> and <em>y</em> coordinate system namely <em>delta_x</em> and <em>delta_y</em> are computed. Finally, the computed features is fed to a kNN classifier to build its committee and further used to classify the pavement cracking into two types; transverse and longitudinal cracking. The performance of kNN classifier in classifying the type of pavement cracking is also compared with a modified version of kNN called fuzzy kNN classifier. Based on the results from images analysis, the semi-automated image processing system is able to consistently characterize the crack pattern with accuracy up to 90%. The comparison of analysed data with field data shows good agreement in the pavement distress characterization. Thus the encouraging results of semi-automated image analysis system will be useful for developing a more efficient road maintenance process.</p>

High-Content and Semi-Automated Quantification of Responses to Estrogenic Chemicals Using a Novel Translucent Transgenic Zebrafish.

Rapid embryogenesis, together with genetic similarities with mammals, and the desire to reduce mammalian testing, are major incentives for using the zebrafish model in chemical screening and testing. Transgenic zebrafish, engineered for identifying target gene expression through expression of fluorophores, have considerable potential for both high-content and high-throughput testing of chemicals for endocrine activity. Here we generated an estrogen responsive transgenic zebrafish model in a pigment-free "Casper" phenotype, facilitating identification of target tissues and quantification of these responses in whole intact fish. Using the ERE-GFP-Casper model we show chemical type and concentration dependence for green fluorescent protein (GFP) induction and both spatial and temporal responses for different environmental estrogens tested. We also developed a semiautomated (ArrayScan) imaging and image analysis system that we applied to quantify whole body fluorescence responses for a range of different estrogenic chemicals in the new transgenic zebrafish model. The zebrafish model developed provides a sensitive and highly integrative system for identifying estrogenic chemicals, their target tissues and effect concentrations for exposures in real time and across different life stages. It thus has application for chemical screening to better direct health effects analysis of environmental estrogens and for investigating the functional roles of estrogens in vertebrates.

O.4 - A comparative study of the cytogenetic responses for homo- and heterogeneous exposures of human T-lymphocytes

Introduction: Biological dosimetry is central in the assessment and mitigation of large scale radiation accidents and occupational over-exposures. Several biological endpoints have been successfully employed to estimate doses for both in vitro and in vivo exposures. Comprehensive statistical analysis of the dose–response parameters can yield more information than mere quantitative dose estimates. Qualitative aspects of the radiation type and dose distribution can be predicted, which is important for real world biodosimetry applications. Materials and Methods: Equivalent total body dose estimates were prepared for blood samples exposed to 60Co gamma-rays at doses ranging from 1 to 6Gy. The samples were subsequently dilutedwith sham irradiated blood to yield a range of total body dose equivalents between 0 and 4 Gy. Dicentric formation frequency in lymphocyte metaphase spreads was quantified using a semi-automated image analysis system. Results: Cytogenetic aberration frequencies for each irradiation and dilution condition were translated to whole body dose values using dose– response curves established in the laboratory. The measured values were in agreement with the calculated estimates (P-value > 0.05). The overdistribution of chromosomal aberrations correlated with the non-uniformity of the radiation applied; which is reflected by the indices of dispersion (σ2/ μ > 1.2) for heterogeneously exposed samples. Conclusion: The degree of non-uniformity of exposures can be predicted on the basis of over-dispersion parameters calculated using advanced statistical analysis of dicentric frequency distributions.

The effect of dietary estimates calculated using food frequency questionnaires on micronuclei formation in European pregnant women: a NewGeneris study.

The use of biomarkers of early genetic effects, predictive for cancer, such as micronuclei (MN) in lymphocytes, may help to investigate the association between diet and cancer. We hypothesised that the presence of mutagens in the diet may increase MN formation. A 'pooled' standardised analysis was performed by applying the same experimental protocol for the cytokinesis block micronucleus assay in 625 young healthy women after delivery from five European study populations (Greece, Denmark, UK, Spain and Norway). We assessed MN frequencies in mono- and binucleated T-lymphocytes (MNMONO and MNBN) and the cytokinesis blocked proliferation index using a semi-automated image analysis system. Food frequency questionnaires (FFQs) were used to estimate intake of fatty acids and a broad range of immunotoxic and genotoxic/carcinogenic compounds through the diet. Pooled difference based on delivery type revealed higher MNMONO frequencies in caesarean than in vaginal delivery (P = 0.002). Statistical analysis showed a decrease in MNMONO frequencies with increasing calculated omega-6 PUFA concentrations and a decrease in MNBN frequencies with increasing calculated omega-3 PUFA concentrations. The expected toxic compounds estimated by FFQs were not associated with MN formation in mothers after delivery. In pregnant women, an omega-3 and -6 rich diet estimated by FFQ is associated with lower MN formation during pregnancy and delivery.

Correlation between 3D microstructural and 2D histomorphometric properties of subchondral bone with healthy and degenerative cartilage of the knee joint.

Cartilage degeneration of the knee joint is considered to be a largely mechanically driven process. We conducted a microstructural and histomorphometric analysis of subchondral bone samples of intact cartilage and in samples with early and higher- grade arthritic degeneration to compare the different states and correlate the findings with the condition of hyaline cartilage. These findings will enable us to evaluate changes in biomechanical properties of subchondral bone during the evolution of arthritic degeneration, for which bone density alone is an insufficient parameter. From a continuous series of 80 patients undergoing implantation of total knee endoprosthesis 30 osteochondral samples with lesions macroscopically classified as ICRS grade 1b (group A) and 30 samples with ICRS grade 3a or 3b lesions (group B) were taken. The bone samples were assessed by 2D histomorphometry (semiautomatic image analysis system) and 3D microstructural analysis (high-resolution micro-CT system). The cartilage was examined using the semiquantitative real-time PCR gene expression of collagen type I and II and aggrecan. Both histomorphometry and microstructural and biomechanical analysis of subchondral bone in groups A and B consistently revealed progressive changes of both bone and cartilage compared with healthy controls. The severity of cartilage degeneration as assessed by RT PCR was significantly correlated with BV/TV (Bone Volume Fraction), Tb.Th (Trabecular Thickness) showed a slight increase. Tb.N (Trabecular Number), Tb.Sp (Trabecular separation) SMI (Structure Model Index), Conn.D (Connectivity Density) and DA (Degree of Anisotropy) were inversely correlated. We saw sclerotic transformation and phagocytic reticulum cells. Bone volume fraction decreased with an increasing distance from the cartilage with the differences compared with healthy controls becoming greater in more advanced cartilage damage. The density of subchondral bone alone is considered an unreliable parameter for classifying changes evolving over time. The progressive damage of subchondral bone seen in the present study correlates well with cartilage changes. Trabecular orientation is also impaired, which explains the changes in biomechanical parameters and the inadequate load transfer and excessive loading of cartilage. Besides subchondral bone density, which in turn correlates with cartilage thickness, other parameters such as structure model index and grade of anisotropy best reflect mechanical properties such as Young modulus, compressive strength, tensile stress, and failure energy. However, it remains unclear whether the mechanical interaction of the mineralized subchondral tissues with articular cartilage works vice versa. The possibility of a biochemical signalling from the degenerating cartilage via the synovial fluid and bone- cartilage crosstalks via subchondral pores may indeed explain a certain depth-dependency of subchondral bone changes.

Semi-Automatic Segmentation and Classification of Pap Smear Cells

Cytologic screening has been widely used for detecting the cervical cancers. In this study, a semiautomatic PC-based cellular image analysis system was developed for segmenting nuclear and cytoplasmic contours and for computing morphometric and textual features to train support vector machine (SVM) classifiers to classify four different types of cells and to discriminate dysplastic from normal cells. A software program incorporating function, including image reviewing and standardized denomination of file names, was also designed to facilitate and standardize the workflow of cell analyses. Two experiments were conducted to verify the classification performance. The cross-validation results of the first experiment showed that average accuracies of 97.16% and 98.83%, respectively, for differentiating four different types of cells and in discriminating dysplastic from normal cells have been achieved using salient features (8 for four-cluster and 7 for two-cluster classifiers) selected with SVM recursive feature addition. In the second experiment, 70% (837) of the cell images were used for training and 30% (361) for testing, achieving an accuracy of 96.12% and 98.61% for four-cluster and two-cluster classifiers, respectively. The proposed system provides a feasible and effective tool in evaluating cytologic specimens.

Abstract 1924: Thrombospondin-2 expression is down-regulated in prostate cancer and is associated with inhibition of prostate cancer progression.

Abstract Introduction: Thrombospondin 2 (THBS2 or TSP2) is a secreted glycoprotein associated with tumor growth and angiogenesis inhibition. TSP2 expression levels are altered in many tumors, but, to date, there is no detailed analysis of TSP2 expression and function in prostate cancer (PCa). Objectives: The aim of this work was to characterize TSP2 expression levels in PCa and its potential usefulness for the differential diagnosis between PCa and benign prostatic hyperplasia (BPH), besides characterizing TSP2 functions in PCa. Methods: TSP2 transcript expression levels were analyzed by quantitative real time PCR, in PCa and BPH tissue samples and tumor and non-tumor cell lines. TSP2 protein expression levels were assessed by immunohistochemistry in a tissue microarray platform, containing 204 PCa and 66 BPH tissue samples, using a semi-automated image analysis system. To investigate TSP2 functions in PCa cells, PC3 and DU145 cell lines, were treated with recombinant TSP2 protein (rTSP2) (50 ug/ml) and were subjected to proliferation end point assays and cell cycle analysis. PC3 cells were also subjected to phosphoproteomic analysis using a phospho-antibody array. Results: Our data showed that TSP2 transcript expression levels are down-regulated in PCa tissue samples and cell lines, compared to BPH and non-tumoral samples (p<0.01). Receiving Operating Curve (ROC) analysis demonstrated that TSP2 transcript levels could better distinguish PCa from BPH tissue samples (p<0.01) than serum PSA levels (p=0.299). TSP2 protein expression was observed in the cytoplasm of both PCa and BPH epithelial and stromal cell compartments. TSP2 stromal staining scores were significantly lower in PCa than in BPH tissues (p<0.01), while similar TSP2 epithelial staining patterns were observed. Notably, TSP2 epithelial staining score was positively correlated to vascular invasion and biochemical recurrence in PCa (p<0.05). Treatment of PC3 and DU145 cells with rTSP2 significantly decreased the proliferation rate of these cells, while increased the percentage of cells in sub-G1 phase of the cell cycle. The phosphoproteomic analysis of PC3 cells treated with rTSP2 showed significantly lower phosphorylation levels of ERK and JNK proteins, in contrast to the higher phosphorylation status of p53, p27, paxillin and RSK. Conclusions: Our data indicate that lower TSP2 expression levels in PCa has potential application for the differential diagnosis of PCa and BPH, and suggests possible stromal and epithelial cross talks modulating PCa progression. Additionally, our results point to a putative role of TSP2 on negatively modulating PCa cell proliferation and survival, which could be partially associated with modulation of phosphorylation levels of proteins involved in the MAPK pathway and controlling the cell cycle. Citation Format: Aline R. Matos, Claudia M. Coutinho-Camillo, Eloisio A. Silva, Francisco P. Fonseca, Luiz C S Thuler, Fernando A. Soares, Etel R P Gimba. Thrombospondin-2 expression is down-regulated in prostate cancer and is associated with inhibition of prostate cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1924. doi:10.1158/1538-7445.AM2013-1924

Significance of AgNORs and Ki-67 Proliferative Markers in Differential Diagnosis of Thyroid Lesions

We aimed to assess the utility of quantitative analysis of AgNORs and Ki67 labeling index (LI) in the differential diagnosis of different thyroid lesions. This study included: 25 papillary carcinomas, 7 follicular carcinomas, 21 follicular adenomas and 27 nodular goiters. Using a semiautomatic image analysis system, Ag NORs parameters were measured and calculated including: total area of AgNORs, mean Ag NOR number in nuclei, nuclear area, mean area of AgNOR dots per each nucleus, number of central and marginal AgNOR dots, and the relative ratio of total area of AgNOR dots/total area of nucleus. Ki67 immunostaining was performed and the LI was determined. There was a significant difference between groups of thyroid lesions regarding total area of AgNORs, Ag NOR number and number of marginal Ag NOR dots. According to receiver operating characteristic curve, Ag NORs number =2.91 and marginal Ag NORs = 2.67 were useful cut off values above which follicular carcinoma can be diagnosed with 100% sensitivity, 79% specificity, 76% PPV, 100% NPV and 85% diagnostic accuracy for both parameters. Mean Ki67 LI in our study was 14.12 ± 2.29, 61.42 ± 3.77, 34.90 ± 3.49 and 18.60 ± 1.96 for papillary carcinoma, follicular carcinoma, follicular adenoma and nodular goiter respectively. Ki67 LI showed statistically significant difference between follicular carcinoma and follicular adenoma (p = 0.026) and between papillary carcinoma and follicular adenoma (p = 0.007). Quantification of Ag NORs and Ki67 LI could be used as helpful ancillary methods in the differentiation between different thyroid lesions.

Abstract C37: Thrombospondin 2 is differentially expressed between prostate cancer and benign prostatic hyperplasia tissues

Abstract Introduction: Thrombospondin 2 (TSP2) is a glycoprotein secreted to the extracellular matrix and behaves as an angiogenic and cellular adhesion modulator. Due to its classical role on inhibiting angiogenesis, TSP2 became an interesting target to be studied in prostate cancer (PCa) tumorigenic process. Objectives: The aim of this study was to investigate TSP2 expression profiling in PCa and benign prostate hyperplasia (BPH) in order to characterize its putative application on the differential diagnosis of these prostatic diseases and trying to understand TSP2 roles on PCa progression steps. Methods: TSP2 transcript expression was analyzed by quantitative real time PCR in PCa, BPH and prostate nontumoral samples. The ability of TSP2 transcript expression level to distinguish between PCa and BPH samples was evaluated by receiver operating characteristic (ROC) curve analysis. TSP2 protein expression was assessed by immunohistochemistry analysis on a tissue microarray containing 204 PCa and 66 BPH tumor specimens, using a semi-automated cellular image analysis system. Tissue areas corresponding to tumor epithelia and stromal compartments were manually selected and analyzed independently for TSP2 protein expression by evaluating the percentage of positively stained area and TSP2 staining intensity. Results: TSP2 transcript expression presented a significant downregulated expression pattern in PCa tissues, as compared to BPH samples and prostate non-tumoral samples (p < 0.01). ROC curve analysis of TSP2 transcript expression level can efficiently distinguish PCa from BPH samples and outperformed serum PSA levels to differentiate these two groups of patients (p < 0.01). By using the TMA approach, our data demonstrated that TSP2 is expressed both at the stromal and epithelial compartments of BPH and PCa tissue samples. There were no significant differences in TSP2 mean expression levels between PCa and BPH epithelial compartments. However, we observed a significant downregulation of TSP2 expression at PCa stromal cell compartment, as compared to the corresponding BPH niches (p < 0.05). There were no significant correlation between TSP2 protein expression and several clinical-pathological data from patients included in this cohort. Conclusions: In summary, our data suggest that TSP2 transcript differential expression levels between PCa and BPH samples seems to be an auxiliary tool for PCa diagnosis. Although TSP2 presents a down regulated expression pattern, both at transcript and protein expression levels on PCa samples, it seems that the expression of TSP2 is differentially modulated at the prostate epithelial and stromal cell compartments. These data indicate that the down regulation of TSP2 at the PCa stromal cell compartment could be a positive modulator of PCa progression, as evidenced by the classical roles of TSP2 on inhibiting angiogenesis and activating cell adhesion. Citation Format: Aline Rocha Matos, Claudia M. Coutinho-Camillo, Aline Silva Damascena, Eloisio Alexsandro Silva, Helena Paula Brentani, Francisco P. Fonseca, Fernando Augusto Soares, Etel Rodrigues Pereira Gimba. Thrombospondin 2 is differentially expressed between prostate cancer and benign prostatic hyperplasia tissues [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C37.

Karyomorphometrical analysis of Spilanthes Jacq. (Asteraceae) using image analysis system

This study was aimed to bring out the Karyotype characteristics of four species of Spilanthes to check for marked symmetry or asymmetry in the chromosomal complements with the aid of semi-automatic image analysis system. The chromosome number of Spilanthes calva and S. radicans was 2n = 78, and in S. ciliata and S. uliginosa it was 2n = 52. The four investigated taxa of Spilanthes are monobasic with the secondary number X 2 = 13. The size of chromosome ranged from 0.50–1.62 μm. The chromosome pairs with secondary constriction are varying from one to three in various taxa. The karyotypes show that considerable variation exists in chromosome morphology and structure among the species studied. In Spilanthes the lesser values for all the parameters of karyomorphology show its rather evolved nature. Such type of variation is important from the evolutionary view point.

Evaluation of estimates of crown condition in forest monitoring: comparison between visual estimation and automated crown image analysis

In long-term forest monitoring, tree crown condition has been visually rated to diagnose tree vigor and forest condition. However, visual estimates are subjective. A semiautomatic image analysis system, called CROCO, was developed to estimate crown condition quantitatively. CROCO calculates a DSO value which decrease with increasing crown transparency. This study aims to verify visual estimates objectively using CROCO and to assess characteristics of visual estimates and DSO values by comparing the effectiveness as indicators of radial growth. Crown condition of Abies mariesii was visually rated using a vitality index, and DSO values of the same trees were calculated. When the top of the tree was intact, the trees with a higher vitality index showed a significantly higher DSO. Vitality index showed the strongest relationship with DBH increment for 8 years. DSO had a significant relationship with DBH increment by adding information of the crown top condition. Analysis of tree crown images is effective to verify visual estimates. Vitality index is a synthetic index involving factors affecting radial growth. DSO could be utilized similarly to vitality index, as an indicator of radial growth, by addition of information on crown size and/or shape.

A relation between the AgNORs motifs and T lymphocyte subgroups in phytohemagglutinin-stimulated human T lymphocytes

The relevance of silver-stained NORs for classifications and ,prognosis was investigated inbreast tissue. Paraffin sections from 137 cases of invasive ductal breast carcinomas and 12 cases with non-tumorous ductus epithelium as controls were stained according to a modified technique and analysed. From the cancer cases follow-up data up to 10 years (45 to 165 months) and in addition clinical, histological and several DNA distribution parameters were available. The nuclei and the silver grains were measured by means of a semiautomatic image analysis system. Significant differences in AgNOR features were found between controls and diploid tumors (p ≤ 0.001), diploid and aneuploid tumors (p ≤ 0.001), Bloom-Richardson-gradings I, II, and III (p ≤ 0.001), and between the tumor cells from patients developing metastases within 5 years and those without (p ≤ 0.002). The prognostic significance of AgNORs was estimated using Cox regression analysis. Four AgNOR features were correlated significantly with survival time. In a multivariate approach offering all parameters available an AgNOR parameter (CV of relative area AgNORs) ranked at the third position beyond the SD of DNA distribution and pTNM-staging. Considering the metastases-free interval of patients the same AgNOR feature showed an independent prognostic validity.

Immunohistochemical Expression of Notch Signaling in the Lining Epithelium of Periapical Cysts

Introduction In this study we evaluated the immunohistochemical expression of the receptors Notch 1 and Notch 2, the ligand Delta 1, and the transcription factors HES 1 and HES 5 in the epithelium of well-defined periapical cysts. Methods Immunohistochemistry was carried out on 55 formalin-fixed and paraffin-embedded, well-defined periapical cysts with minimum inflammation, obtained from the archival tissue database of the Department of Oral Pathology and Surgery. Western blotting was performed to evaluate the specificity of the anti-Notch antibody and the expression of Notch signaling in 5 fresh-frozen periapical cysts. The levels of staining intensity were estimated by the performance of a semiautomated image analysis system. Descriptive statistic of mean values obtained by computerized image analysis method was performed. Results Immunostaining reaction of all Notch signaling components was observed in the cytoplasm and/or the cytoplasmic membrane in the majority of epithelial cells of periapical cysts. Nuclear staining was observed occasionally in all cases. Notch 2 showed strong staining in 52.83% of the cases, followed by Notch 1 (35.85%), HES 1 and HES 5 moderate staining in 72.73% and 57.69% of the cases, respectively, and Delta 1 weak staining in 58.33% of the cases. No statistical correlation was found between the antibodies and the sex or the age of the study group. Conclusions Notch is an evolutionarily conserved signaling mechanism that regulates cell fate decisions during development and postnatal life in organisms as diverse as worms, flies, and humans. The present observations indicate that Notch pathway is active downstream in the lining epithelium of periapical cysts, suggesting an involvement of this pathway in periapical cyst growth and expansion.

Optimization of a 96-Well Electroporation Assay for Postnatal Rat CNS Neurons Suitable for Cost–Effective Medium-Throughput Screening of Genes that Promote Neurite Outgrowth

Following an injury, central nervous system (CNS) neurons show a very limited regenerative response which results in their failure to successfully form functional connections with their original target. This is due in part to the reduced intrinsic growth state of CNS neurons, which is characterized by their failure to express key regeneration-associated genes (RAGs) and by the presence of growth inhibitory molecules in CNS environment that form a molecular and physical barrier to regeneration. Here we have optimized a 96-well electroporation and neurite outgrowth assay for postnatal rat cerebellar granule neurons (CGNs) cultured upon an inhibitory cellular substrate expressing myelin-associated glycoprotein or a mixture of growth inhibitory chondroitin sulfate proteoglycans. Optimal electroporation parameters resulted in 28% transfection efficiency and 51% viability for postnatal rat CGNs. The neurite outgrowth of transduced neurons was quantitatively measured using a semi-automated image capture and analysis system. The neurite outgrowth was significantly reduced by the inhibitory substrates which we demonstrated could be partially reversed using a Rho Kinase inhibitor. We are now using this assay to screen large sets of RAGs for their ability to increase neurite outgrowth on a variety of growth inhibitory and permissive substrates.

Effects of Increased Renal Tubular Vascular Endothelial Growth Factor (VEGF) on Fibrosis, Cyst Formation, and Glomerular Disease

The role of vascular endothelial growth factor (VEGF) in renal fibrosis, tubular cyst formation, and glomerular diseases is incompletely understood. We studied a new conditional transgenic mouse system [Pax8-rtTA/(tetO)(7)VEGF], which allows increased tubular VEGF production in adult mice. The following pathology was observed. The interstitial changes consisted of a ubiquitous proliferation of peritubular capillaries and fibroblasts, followed by deposition of matrix leading to a unique kind of fibrosis, ie, healthy tubules amid a capillary-rich dense fibrotic tissue. In tubular segments with high expression of VEGF, cysts developed that were surrounded by a dense network of peritubular capillaries. The glomerular effects consisted of a proliferative enlargement of glomerular capillaries, followed by mesangial proliferation. This resulted in enlarged glomeruli with loss of the characteristic lobular structure. Capillaries became randomly embedded into mesangial nodules, losing their filtration surface. Serum VEGF levels were increased, whereas endogenous VEGF production by podocytes was down-regulated. Taken together, this study shows that systemic VEGF interferes with the intraglomerular cross-talk between podocytes and the endocapillary compartment. It suppresses VEGF secretion by podocytes but cannot compensate for the deficit. VEGF from podocytes induces a directional effect, attracting the capillaries to the lobular surface, a relevant mechanism to optimize filtration surface. Systemic VEGF lacks this effect, leading to severe deterioration in glomerular architecture, similar to that seen in diabetic nephropathy.

Deoxyribonucleic acid (DNA) content of morphologically different sperm types from normal and subfertile human males.

20 male patients (G2), affected by idiopathic oligozoospermia, and 10 normal fertile males (G1) were investigated. The DNA content of 50 Feulgen stained sperm heads per subject was determined, using single cell photometry. In a second step area, circumference, length, and width of the same heads were measured, using a semi-automatic image analysis system. Considering only morphologically normal heads, the mean DNA content is only slightly smaller in G2 than in G1. On the contrary, the DNA variation is strongly higher in G2 than in G1. Unfortunately, morphologically defect heads show an increased DNA variation, too. Thus, determining the DNA variation of a semen, containing many morphologically defect heads, a combined determination of the DNA content and head morphology is necessary. This can only be achieved by single cell cytophotometry, which is therefore superior to flow cytophotometry as regards this problem. Many heads with a strongly abnormal DNA content do not show any morphological abnormalities. These subcellular changes can only be detected by the determination of the DNA content.

Améliorations en vue d’un suivi standardisé de l’état sanitaire des houppiers en plantations monoclonales de peuplier

• This work aims at developing new tools for rapid assessment of forest health indicators in poplar plantations. • Crown transparency and discoloration were visually evaluated in all trees of four 15 m-radius subplots in 32 poplar clonal plantations, which were chosen according to a factorial scheme with three factors: tree age, site quality and understorey vegetation management. A subset of trees was assessed using digital photos processed with a semi-automatic image analysis system (the CROCO software) in order to compare visual and digital crown transparency estimates. • Poplar crown conditions were better in young stands and rich sites. Harrowing understorey vegetation improved tree health in poor sites. Samples of 20 trees per stand provided the same information about crown transparency and discoloration as 60 trees. Calibration curves of digital crown transparency estimates were successfully fitted against visual crown transparency estimates. The same effects of stand age and site quality could be detected with digital crown transparency as response

A comparison of two methods for estimating polymegethism in cell areas of the human corneal endothelium

To compare the outcome measures from two different methods for estimating the coefficient of variation (COV) in cell area for human corneal endothelia. A single non-contact specular micrograph was obtained from the central region of the corneal endothelium of 100 healthy non-contact lens-wearing white European subjects, aged from 32 to 62 years. The captured image file was either assessed using a machine-based algorithm in which 21 cells were marked and their areas reported (designated as the TOPCON 'centre-dot' method), or by an overlay and semi-automated computer-based image analysis system of the entire image of around 200 cells (designated as the AUTO method). The average cell area values were used to calculate the endothelial cell density (ECD), while the COV was calculated from the standard deviation of the cell area measures. The TOPCON and AUTO methods yielded comparable data for average cell area (395 vs 391 microm(2)) and estimated ECD (2566 vs 2575 cells mm(-2)) that were not statistically different (p > or = 0.351), although there was a slight bias between the two methods. However, the mean COV values were very different at 43.5 and 31.4% (p < 0.001). The overestimation of the COV was related to the difference in the largest cell area domain identified by the two methods (p < 0.001, r = > or =0.748). A centre-dot method using a small number of cells generates useful data on cell area and ECD, but it should be used cautiously for estimates of COV.