Computed tomography image of the mediastinal and axillary lymph nodes in clinically sound Rottweilers

Trough computed tomography (CT), it is possible to evaluate lymph nodes in detail and to detect changes in these structures earlier than with radiographs and ultrasound. Lack of information in the veterinary literature directed the focus of this report to normal aspects of the axillary and mediastinal lymph nodes of adult dogs on CT imaging. A CT scan of 15 normal adult male and female Rottweilers was done. To define them as clinically sound, anamnesis, physical examination, complete blood count, renal and hepatic biochemistry, ECG, and thoracic radiographs were performed. After the intravenous injection of hydrosoluble ionic iodine contrast medium contiguous 10mm in thickness thoracic transverse images were obtained with an axial scanner. In the obtained images mediastinal and axillary lymph nodes were sought and when found measured in their smallest diameter and their attenuation was compared to musculature. Mean and standard deviation of: age, weight, body length and the smallest diameter of the axillary and mediastinal lymph nodes were determined. Mean and standard deviation of parameters: age 3.87±2.03 years, weight 41.13±5.12, and body length 89.61±2.63cm. Axillary lymph nodes were seen in 60% of the animals, mean of the smallest diameter was 3.58mm with a standard deviation of 2.02 and a minimum value of 1mm and a maximum value of 7mm. From 13 observed lymph nodes 61.53% were hypopodense when compared with musculature, and 30.77% were isodense. Mediastinal lymph nodes were identified in 73.33% of the dogs; mean measure of the smallest diameter was 4.71mm with a standard deviation of 2.61mm and a minimum value of 1mm, and a maximum value of 8mm. From 14 observed lymph nodes 85.71% were isodense when compared with musculature and 14.28% were hypodense. The results show that it is possible to visualize axillary and mediastinal lymph nodes in adult clinically sound Rottweilers with CT using a slice thickness and interval of 10mm. The smallest diameter of the axillary and mediastinal lymph nodes not surpassed 7mm and 8mm respectively. Their attenuations were equal or smaller than that of musculature in the post contrast scan.

Morphoquantitative description of bovine digital cushion

Abstract The digital cushion is characterized as a modified subcutaneous tissue that absorbs the shock during gait, assists venous return of the hoof and supports a considerable part of body weight. Digital cushions have particular importance in the pathogenesis of the hoof, since they need to properly work in order to prevent compression and traumas in soft tissues. This study aimed to measure and determine how is the arrangement of these structures, and for this it was established the proportions of connective, adipose, vascular tissues and collagen fibers and collagen types found in palmar and plantar digital cushion of bovine using fore and hindlimbs of twelve adult zebu cattle of both sexes, 11 male and one female, with 269kg average carcass weight and without limb disorders. Fragments of cushions were subjected to conventional histology, cut to a thickness of 4µm and stained with Red Picrosirius. With digital optical microscope, the quantification of the connective tissue and differentiation of types of collagen used the Image Pro Plus® software, and of adipose and vascular tissue, the test point system. The mean and standard error were estimated with the GraphPad Prism 5.0 software, and then data were subjected to Kolmogorov-Smirnov normality test and Student's t-test with significance level set at 5% for determining the amount of different tissues between fore and hindlimbs of studied animals. In forelimbs the mean and standard error of the connective tissue proportion was 50.10%+1.54, of the adipose tissue was 21.34%+1.44, and of vascular tissue was 3.43%+0.28. Hindlimbs presented a proportion of connective tissue of 61.61%+1.47, 20.66%+1.53 of adipose tissue, and 3.06%+0.20 of vascular tissue. A significant difference (p<0.001) was detected in the connective tissue proportion between fore and hindlimbs. Types I and II collagen fibers have presented, respectively, a proportion of 31.89% and 3.9% in forelimbs and 34.05% and 1.78% in hindlimbs. According to the used methodology, digital cushions had a clear differentiation relative to adipose tissue between fore and hindlimbs.

The Shadow Moiré method using the phase shifting technique and digital image processing: computational implementation and application to the 3D-reconstruction of a buckled plate

The Shadow Moiré fringe patterns are level lines of equal depth generated by interference between a master grid and its shadow projected on the surface. In simplistic approach, the minimum error is about the order of the master grid pitch, that is, always larger than 0,1 mm, resulting in an experimental technique of low precision. The use of a phase shift increases the accuracy of the Shadow Moiré technique. The current work uses the phase shifting method to determine the surfaces three-dimensional shape using isothamic fringe patterns and digital image processing. The current study presents the method and applies it to images obtained by simulation for error evaluation, as well as to a buckled plate, obtaining excellent results. The method hands itself particularly useful to decrease the errors in the interpretation of the Moiré fringes that can adversely affect the calculations of displacements in pieces containing many concave and convex regions in relatively small areas.

Phytochemistry and quantification of polyphenols in extracts of the Asteraceae weeds from Diamantina, Minas Gerais state, Brazil

Asteraceae weeds are rich in chemicals that have biological and pharmaceutical activities. The aims of this work were to describe the phytochemistry and quantify the polyphenols in ethanol extracts from leaves of 12 species of Asteraceae weeds collected in Diamantina, Minas Gerais State, Brazil. The screening of Asteraceae extracts revealed the presence of tannins, steroids, triterpenes, anthocyanins, and flavonoids. The total phenolic content was high in extracts of Lychnophora ericoides (147.97 ± 2.66), Lepidaploa lilacina (141.11 ± 1.99), and Eremanthus elaeagnus (134.61 ± 7.81) and low in extracts of Lychnophora ramosissima (32.65 ± 0.70), and Lychnophora sp. (54.03 ± 0.73). Extracts of Asteraceae weeds from Diamantina could have potential for biological studies that are searching for new pesticides and drugs.

Image analysis and definition of nuclear phenotypes

Some basic topics concerned with the extraction of textural and geometric information from cell nucleus images as well as description and characterization of chromatin supraorganization and consequent classification of nuclear phenotypes are presented.

Comparison of the quantification of caffeine in human plasma by gas chromatography and ELISA

In the present study we evaluated the precision of the ELISA method to quantify caffeine in human plasma and compared the results with those obtained by gas chromatography. A total of 58 samples were analyzed by gas chromatography using a nitrogen-phosphorus detector and routine techniques. For the ELISA test, the samples were diluted to obtain a concentration corresponding to 50% of the absorbance of the standard curve. To determine whether the proximity between the I50 of the standard curve and that of the sample would bring about a more precise result, the samples were divided into three blocks according to the criterion of difference, in modulus, of the I50 of the standard curve and of the I50 of the sample. The samples were classified into three groups. The first was composed of 20 samples with I50 up to 1.5 ng/ml, the second consisted of 21 samples with I50 ranging from 1.51 to 3 ng/ml, and the third of 17 samples with I50 ranging from 3.01 to 13 ng/ml. The determination coefficient (R² = 0.999) showed that the data obtained by gas chromatography represented a reliable basis. The results obtained by ELISA were also reliable, with an estimated Pearson correlation coefficient of 0.82 between the two methods. This coefficient for the different groups (0.88, 0.79 and 0.49 for groups 1, 2 and 3, respectively) showed greater reliability for the test with dilutions closer to I50.

Standard-curve competitive RT-PCR quantification of myogenic regulatory factors in chicken embryos

The reverse transcription-polymerase chain reaction (RT-PCR) is the most sensitive method used to evaluate gene expression. Although many advances have been made since quantitative RT-PCR was first described, few reports deal with the mathematical bases of this technique. The aim of the present study was to develop and standardize a competitive PCR method using standard-curves to quantify transcripts of the myogenic regulatory factors MyoD, Myf-5, Myogenin and MRF4 in chicken embryos. Competitor cDNA molecules were constructed for each gene under study using deletion primers, which were designed to maintain the anchorage sites for the primers used to amplify target cDNAs. Standard-curves were prepared by co-amplification of different amounts of target cDNA with a constant amount of competitor. The content of specific mRNAs in embryo cDNAs was determined after PCR with a known amount of competitor and comparison to standard-curves. Transcripts of the housekeeping ß-actin gene were measured to normalize the results. As predicted by the model, most of the standard-curves showed a slope close to 1, while intercepts varied depending on the relative efficiency of competitor amplification. The sensitivity of the RT-PCR method permitted the detection of as few as 60 MyoD/Myf-5 molecules per reaction but approximately 600 molecules of MRF4/Myogenin mRNAS were necessary to produce a measurable signal. A coefficient of variation of 6 to 19% was estimated for the different genes analyzed (6 to 9 repetitions). The competitive RT-PCR assay described here is sensitive, precise and allows quantification of up to 9 transcripts from a single cDNA sample.

Measurement of epidermal thickness in a patient with psoriasis by computer-supported image analysis

The aim of the present study was to measure full epidermal thickness, stratum corneum thickness, rete length, dermal papilla widening and suprapapillary epidermal thickness in psoriasis patients using a light microscope and computer-supported image analysis. The data obtained were analyzed in terms of patient age, type of psoriasis, total body surface area involvement, scalp and nail involvement, duration of psoriasis, and family history of the disease. The study was conducted on 64 patients and 57 controls whose skin biopsies were examined by light microscopy. The acquired microscopic images were transferred to a computer and measurements were made using image analysis. The skin biopsies, taken from different body areas, were examined for different parameters such as epidermal, corneal and suprapapillary epidermal thickness. The most prominent increase in thickness was detected in the palmar region. Corneal thickness was more pronounced in patients with scalp involvement than in patients without scalp involvement (t = -2.651, P = 0.008). The most prominent increase in rete length was observed in the knees (median: 491 µm, t = 10.117, P = 0.000). The difference in rete length between patients with a positive and a negative family history was significant (t = -3.334, P = 0.03), being 27% greater in psoriasis patients without a family history. The differences in dermal papilla distances among patients were very small. We conclude that microscope-supported thickness measurements provide objective results.

DNA extraction and quantification from touch and scrape preparations obtained from autopsy liver cells

The objective of the present study was to develop a simplified low cost method for the collection and fixation of pediatric autopsy cells and to determine the quantitative and qualitative adequacy of extracted DNA. Touch and scrape preparations of pediatric liver cells were obtained from 15 cadavers at autopsy and fixed in 95% ethanol or 3:1 methanol:acetic acid. Material prepared by each fixation procedure was submitted to DNA extraction with the Wizard® genomic DNA purification kit for DNA quantification and five of the preparations were amplified by multiplex PCR (azoospermia factor genes). The amount of DNA extracted varied from 20 to 8,640 µg, with significant differences between fixation methods. Scrape preparation fixed in 95% ethanol provided larger amount of extracted DNA. However, the mean for all groups was higher than the quantity needed for PCR (50 ng) or Southern blot (500 ng). There were no qualitative differences among the different material and fixatives. The same results were also obtained for glass slides stored at room temperature for 6, 12, 18 and 24 months. We conclude that touch and scrape preparations fixed in 95% ethanol are a good source of DNA and present fewer limitations than cell culture, tissue paraffin embedding or freezing that require sterile material, culture medium, laboratory equipment and trained technicians. In addition, they are more practical and less labor intensive and can be obtained and stored for a long time at low cost.

Abnormalities of hippocampal signal intensity in patients with familial mesial temporal lobe epilepsy

Mesial temporal lobe epilepsy (MTLE) is associated with hippocampal atrophy and hippocampal signal abnormalities. In our series of familial MTLE (FMTLE), we found a high proportion of hippocampal abnormalities. To quantify signal abnormalities in patients with FMTLE we studied 152 individuals (46 of them asymptomatic) with FMTLE. We used NIH-Image® for volumetry and signal quantification in coronal T1 inversion recovery and T2 for all cross-sections of the hippocampus. Values diverging by 2 or more SD from the control mean were considered abnormal. T2 hippocampal signal abnormalities were found in 52% of all individuals: 54% of affected subjects and 48% of asymptomatic subjects. T1 hippocampal signal changes were found in 34% of all individuals: 42.5% of affected subjects and 15% of asymptomatic subjects. Analysis of the hippocampal head (first three slices) revealed T2 abnormalities in 73% of all individuals (74% of affected subjects and 72% of asymptomatic subjects) and T1 abnormalities in 59% (67% of affected subjects and 41% of asymptomatic subjects). Affected individuals had smaller volumes than controls (P < 0.0001). There was no difference in hippocampal volumes between asymptomatic subjects and controls, although 39% of asymptomatic patients had hippocampal atrophy. Patients with an abnormal hippocampal signal (133 individuals) had smaller ipsilateral volume, but no linear correlation could be determined. Hippocampal signal abnormalities in FMTLE were more frequently found in the hippocampal head in both affected and asymptomatic family members, including those with normal volumes. These results indicate that subtle abnormalities leading to an abnormal hippocampal signal in FMTLE are not necessarily related to seizures and may be determined by genetic factors.

Quantification of fecal estradiol and progesterone metabolites in Syrian hamsters (Mesocricetus auratus)

Alternative methods to the utilization of laboratory animal blood and its by-products are particularly attractive, especially regarding hamsters due to their small size and difficulties in obtaining serial blood samples. Steroid hormone metabolite quantification in feces, widely used in studies of free-ranging or intractable animals, is a non-invasive, non-stressor, economical, and animal saving technique which allows longitudinal studies by permitting frequent sampling of the same individual. The present study was undertaken to determine the suitability of this method for laboratory animals. Estradiol and progesterone metabolites were quantified by radioimmunoassay in feces of intact, sexually mature female Syrian hamsters during the estrous cycle (control) and in feces of superovulated females. Metabolites were extracted by fecal dilution in ethanol and quantified by solid phase radioimmunoassay. Median estrogen and progesterone concentrations were 9.703 and 180.74 ng/g feces in the control group, respectively. Peaks of estrogen (22.44 ± 4.54 ng/g feces) and progesterone (655.95 ± 129.93 ng/g feces) mean fecal concentrations respectively occurred 12 h before and immediately after ovulation, which is easily detected in this species by observation of a characteristic vaginal postovulatory discharge. Median estrogen and progesterone concentrations (28.159 and 586.57 ng/g feces, respectively) were significantly higher in superovulated animal feces (P < 0.0001). The present study demonstrated that it is possible to monitor ovarian activity in Syrian hamsters non-invasively by measuring fecal estradiol and progesterone metabolites. This technique appears to be a quite encouraging method for the development of new endocrinologic studies on laboratory animals.

Comparative quantification of umbilical cord blood CD34+ and CD34+ bright cells using the ProCount™-BD and ISHAGE protocols

The total number of CD34+ cells is the most relevant clinical parameter when selecting human umbilical cord blood (HUCB) for transplantation. The objective of the present study was to compare the two most commonly used CD34+ cell quantification methods (ISHAGE protocol and ProCount™ - BD) and analyze the CD34+ bright cells whose 7-amino actinomycin D (7AAD) analysis suggests are apoptotic or dead cells. Twenty-six HUCB samples obtained at the Placental Blood Program of New York Blood Center were evaluated. The absolute numbers of CD34+ cells evaluated by the ISHAGE (with exclusion of 7AAD+ cells) and ProCount™ (with exclusion of CD34+ bright cells) were determined. Using the ISHAGE protocol we found 35.6 ± 19.4 CD34+ cells/µL and with the ProCount™ method we found 36.6 ± 23.2 CD34+ cells/µL. With the ProCount™ method, CD34+ bright cell counts were 9.3 ± 8.2 cells/µL. CD34+ bright and regular cells were individually analyzed by the ISHAGE protocol. Only about 1.8% of the bright CD34+ cells are alive, whereas a small part (19.0%) is undergoing apoptosis and most of them (79.2%) are dead cells. Our study showed that the two methods produced similar results and that 7AAD is important to exclude CD34 bright cells. These results will be of value to assist in the correct counting of CD34+ cells and to choose the best HUCB unit for transplantation, i.e., the unit with the greatest number of potentially viable stem cells for the reconstitution of bone marrow. This increases the likelihood of success of the transplant and, therefore, the survival of the patient.

Measurement of annexin V uptake and lactadherin labeling for the quantification of apoptosis in adherent Tca8113 and ACC-2 cells

Phosphatidylserine (PS) exposure occurs during the cell death program and fluorescein-labeled lactadherin permits the detection of PS exposure earlier than annexin V in suspended cell lines. Adherent cell lines were studied for this apoptosis-associated phenomenon to determine if PS probing methods are reliable because specific membrane damage may occur during harvesting. Apoptosis was induced in the human tongue squamous carcinoma cell line (Tca8113) and the adenoid cystic carcinoma cell line (ACC-2) by arsenic trioxide. Cells were harvested with a modified procedure and labeled with lactadherin and/or annexin V. PS exposure was localized by confocal microscopy and apoptosis was quantified by flow cytometry. The detachment procedure without trypsinization did not induce cell damage. In competition binding experiments, phospholipid vesicles competed for more than 95 and 90% of lactadherin but only about 75 and 70% of annexin V binding to Tca8113 and ACC-2 cells. These data indicate that PS exposure occurs in three stages during the cell death program and that fluorescein-labeled lactadherin permitted the detection of early PS exposure. A similar pattern of PS exposure has been observed in two malignant cell lines with different adherence, suggesting that this pattern of PS exposure is common in adherent cells. Both lactadherin and annexin V could be used in adherent Tca8113 and ACC-2 cell lines when an appropriate harvesting procedure was used. Lactadherin is more sensitive than annexin V for the detection of PS exposure as the physical structure of PS in these blebs and condensed apoptotic cell surface may be more conducive to binding lactadherin than annexin V.

Manual and semi-automatic quantification of in vivo ¹H-MRS data for the classification of human primary brain tumors

In vivo proton magnetic resonance spectroscopy (¹H-MRS) is a technique capable of assessing biochemical content and pathways in normal and pathological tissue. In the brain, ¹H-MRS complements the information given by magnetic resonance images. The main goal of the present study was to assess the accuracy of ¹H-MRS for the classification of brain tumors in a pilot study comparing results obtained by manual and semi-automatic quantification of metabolites. In vivo single-voxel ¹H-MRS was performed in 24 control subjects and 26 patients with brain neoplasms that included meningiomas, high-grade neuroglial tumors and pilocytic astrocytomas. Seven metabolite groups (lactate, lipids, N-acetyl-aspartate, glutamate and glutamine group, total creatine, total choline, myo-inositol) were evaluated in all spectra by two methods: a manual one consisting of integration of manually defined peak areas, and the advanced method for accurate, robust and efficient spectral fitting (AMARES), a semi-automatic quantification method implemented in the jMRUI software. Statistical methods included discriminant analysis and the leave-one-out cross-validation method. Both manual and semi-automatic analyses detected differences in metabolite content between tumor groups and controls (P < 0.005). The classification accuracy obtained with the manual method was 75% for high-grade neuroglial tumors, 55% for meningiomas and 56% for pilocytic astrocytomas, while for the semi-automatic method it was 78, 70, and 98%, respectively. Both methods classified all control subjects correctly. The study demonstrated that ¹H-MRS accurately differentiated normal from tumoral brain tissue and confirmed the superiority of the semi-automatic quantification method.

Effect of image resolution manipulation in rearfoot angle measurements obtained with photogrammetry

The aim of this study was to investigate the influence of image resolution manipulation on the photogrammetric measurement of the rearfoot static angle. The study design was that of a reliability study. We evaluated 19 healthy young adults (11 females and 8 males). The photographs were taken at 1536 pixels in the greatest dimension, resized into four different resolutions (1200, 768, 600, 384 pixels) and analyzed by three equally trained examiners on a 96-pixels per inch (ppi) screen. An experienced physiotherapist marked the anatomic landmarks of rearfoot static angles on two occasions within a 1-week interval. Three different examiners had marked angles on digital pictures. The systematic error and the smallest detectable difference were calculated from the angle values between the image resolutions and times of evaluation. Different resolutions were compared by analysis of variance. Inter- and intra-examiner reliability was calculated by intra-class correlation coefficients (ICC). The rearfoot static angles obtained by the examiners in each resolution were not different (P > 0.05); however, the higher the image resolution the better the inter-examiner reliability. The intra-examiner reliability (within a 1-week interval) was considered to be unacceptable for all image resolutions (ICC range: 0.08-0.52). The whole body image of an adult with a minimum size of 768 pixels analyzed on a 96-ppi screen can provide very good inter-examiner reliability for photogrammetric measurements of rearfoot static angles (ICC range: 0.85-0.92), although the intra-examiner reliability within each resolution was not acceptable. Therefore, this method is not a proper tool for follow-up evaluations of patients within a therapeutic protocol.