Sural nerve involvement in experimental hypertension: morphology and morphometry in male and female normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR)

Background: The sural nerve has been widely investigated in experimental models of neuropathies but information about its involvement in hypertension was not yet explored. The aim of the present study was to compare the morphological and morphometric aspects of different segments of the sural nerve in male and female spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. Rats aged 20 weeks (N = 6 in each group) were investigated. After arterial pressure and heart rate recordings in anesthetized animals, right and left sural nerves were removed and prepared for epoxy resin embedding and light microscopy. Morphometric analysis was performed with the aid of computer software, and took into consideration the fascicle area and diameter, as well as myelinated fiber number, density, area and diameter. Results: Significant differences were observed for the myelinated fiber number and density, comparing different genders of WKY and SHR. Also, significant differences for the morphological (thickening of the endoneural blood vessel walls and lumen reduction) and morphometric (myelinated fibers diameter and G ratio) parameters of myelinated fibers were identified. Morphological exam of the myelinated fibers suggested the presence of a neuropathy due to hypertension in both SHR genders. Conclusions: These results indicate that hypertension altered important morphometric parameters related to nerve conduction of sural nerve in hypertensive animals. Moreover the comparison between males and females of WKY and SHR allows the conclusion that the morphological and morphometric parameters of sural nerve are not gender related. The morphometric approach confirmed the presence of neuropathy, mainly associated to the small myelinated fibers. In conclusion, the present study collected evidences that the high blood pressure in SHR is affecting the sural nerve myelinated fibers.

Rheological Measurements and Thermal Characterization of Lamellar Gel Phase Emulsions Developed with Cetearyl Alcohol/Nonionic Ethoxylated Surfactants

The aim of this research was to characterize emulsion systems (ES) containing nonionic ethoxylated surfactants by using rheological, microscopic, and thermogravimetric assays. Three formulations were developed: ES-1: 8.0% (w/w) oleth-20; ES-2: 4.0% (w/w) oleth-20/4.0% (w/w) steareth-21; and ES-3: 8.0% (w/w) steareth-21. The systems showed typical non-Newtonian pseudo-plastic behavior. The presence of a lamellar gel phase was observed for all systems, with ES-2 being more pronounced. Through thermogravimetry, the profiles of the three systems were found to be similar, consisting of two main events, the first one being characterized by loss of water and, beyond 110 degrees C, by loss of the oil phase.

Evaluation of epoxy resin sealer after three root canal filling techniques by confocal laser scanning microscopy

The aim of this study was to evaluate the penetration of endodontic sealer into the dentin tubules, the integrity of the sealer layer perimeter, and the sealer area at the apical third after different filling techniques by confocal laser scanning microscopy (CLSM). Forty-five mandibular premolars were mechanically prepared with ProTaper files, until F5 file. Thereafter, they were filled with an epoxy-resin sealer (AH Plus) mixed with Rhodamine B dye (0.1% proportion) and allocated in three groups: Group 1, single master cone; Group 2, cold lateral compaction; and Group 3, Thermafil. For confocal laser scanning microscopy analysis, the specimens were transversely sectioned at 4 mm from the apex. The images at x10 and x40 were analyzed by Imagetool 3.0 software. Significant differences were not found among the three experimental groups according the dentin-impregnate area by the sealer (P = 0.68) and between the sealer and root canal perimeter (P = 0.18). However, root canal filling techniques were significantly different when apical sealer areas were compared (P = 0.001). Thermafil group showed smaller sealer areas (8.09%) while cold lateral compaction and gutta-percha master cone showed similar areas (17.37 and 21.18%, respectively). The dentin-impregnated area was not dependent on the root canal filling technique. Single master cone, cold lateral condensation and Thermafil techniques presented integrity of the sealer perimeter close to 100% and Thermafil resulted in a significantly thinner sealer layer. Microsc. Res. Tech. 75:12771280, 2012. (C) 2012 Wiley Periodicals, Inc.

Towards a precise test for malaria diagnosis in the Brazilian Amazon: comparison among field microscopy, a rapid diagnostic test, nested PCR, and a computational expert system based on artificial neural networks

Abstract Background Accurate malaria diagnosis is mandatory for the treatment and management of severe cases. Moreover, individuals with asymptomatic malaria are not usually screened by health care facilities, which further complicates disease control efforts. The present study compared the performances of a malaria rapid diagnosis test (RDT), the thick blood smear method and nested PCR for the diagnosis of symptomatic malaria in the Brazilian Amazon. In addition, an innovative computational approach was tested for the diagnosis of asymptomatic malaria. Methods The study was divided in two parts. For the first part, passive case detection was performed in 311 individuals with malaria-related symptoms from a recently urbanized community in the Brazilian Amazon. A cross-sectional investigation compared the diagnostic performance of the RDT Optimal-IT, nested PCR and light microscopy. The second part of the study involved active case detection of asymptomatic malaria in 380 individuals from riverine communities in Rondônia, Brazil. The performances of microscopy, nested PCR and an expert computational system based on artificial neural networks (MalDANN) using epidemiological data were compared. Results Nested PCR was shown to be the gold standard for diagnosis of both symptomatic and asymptomatic malaria because it detected the major number of cases and presented the maximum specificity. Surprisingly, the RDT was superior to microscopy in the diagnosis of cases with low parasitaemia. Nevertheless, RDT could not discriminate the Plasmodium species in 12 cases of mixed infections (Plasmodium vivax + Plasmodium falciparum). Moreover, the microscopy presented low performance in the detection of asymptomatic cases (61.25% of correct diagnoses). The MalDANN system using epidemiological data was worse that the light microscopy (56% of correct diagnoses). However, when information regarding plasma levels of interleukin-10 and interferon-gamma were inputted, the MalDANN performance sensibly increased (80% correct diagnoses). Conclusions An RDT for malaria diagnosis may find a promising use in the Brazilian Amazon integrating a rational diagnostic approach. Despite the low performance of the MalDANN test using solely epidemiological data, an approach based on neural networks may be feasible in cases where simpler methods for discriminating individuals below and above threshold cytokine levels are available.

Physiological and structural aspects of fruit and vegetable mild processing

Over the past years fruit and vegetable industry has become interested in the application of both osmotic dehydration and vacuum impregnation as mild technologies because of their low temperature and energy requirements. Osmotic dehydration is a partial dewatering process by immersion of cellular tissue in hypertonic solution. The diffusion of water from the vegetable tissue to the solution is usually accompanied by the simultaneous solutes counter-diffusion into the tissue. Vacuum impregnation is a unit operation in which porous products are immersed in a solution and subjected to a two-steps pressure change. The first step (vacuum increase) consists of the reduction of the pressure in a solid-liquid system and the gas in the product pores is expanded, partially flowing out. When the atmospheric pressure is restored (second step), the residual gas in the pores compresses and the external liquid flows into the pores. This unit operation allows introducing specific solutes in the tissue, e.g. antioxidants, pH regulators, preservatives, cryoprotectancts. Fruit and vegetable interact dynamically with the environment and the present study attempts to enhance our understanding on the structural, physico-chemical and metabolic changes of plant tissues upon the application of technological processes (osmotic dehydration and vacuum impregnation), by following a multianalytical approach. Macro (low-frequency nuclear magnetic resonance), micro (light microscopy) and ultrastructural (transmission electron microscopy) measurements combined with textural and differential scanning calorimetry analysis allowed evaluating the effects of individual osmotic dehydration or vacuum impregnation processes on (i) the interaction between air and liquid in real plant tissues, (ii) the plant tissue water state and (iii) the cell compartments. Isothermal calorimetry, respiration and photosynthesis determinations led to investigate the metabolic changes upon the application of osmotic dehydration or vacuum impregnation. The proposed multianalytical approach should enable both better designs of processing technologies and estimations of their effects on tissue.

Aktivität der humanen Meprin-Metalloproteasen unter Berücksichtigung potentieller Aktivatoren

Die Metalloproteasen Meprin α und Meprin β sind an essentiellen (patho)physiologischen Prozessen beteiligt. Um die Funktion dieser Proteasen zu verstehen, ist es von Bedeutung, sie nicht isoliert, sondern im gesamten proteolytischen Netzwerk zu betrachten.rnDie Meprine werden in einer Vielzahl von Geweben, in Leukozyten, aber auch in Krebszellen exprimiert. In der Haut konnten die beiden Enzyme in unterschiedlichen dermalen Schichten detektiert werden, wo sie u.a. an der Kollagenassemblierung durch Abspaltung der Propeptide beteiligt sind. rnIm Zuge von Proteomics Analysen konnten mehr als 3000 proteolytische Schnittstellen von fünf Astacin-Metalloproteasen (Meprin α, Meprin β, Astacin, LAST und LAST_MAM) in Peptiden und nativen Substraten identifiziert werden und somit eine Aussage über die Spaltspezifität getroffen werden. In der vorliegenden Arbeit konnten diese Spaltspezifitäten mit Hilfe von fluorogenen Substraten in vitro verifiziert werden. Bemerkenswert hierbei ist die starke Präferenz der beiden Meprine und LAST_MAM für die Aminosäuren Aspartat und Glutamat in der P1‘ Position. rnMeprine werden als Zymogene exprimiert und müssen durch proteolytische Prozessierung einer tryptischen Protease aktiviert werden. Ein Schwerpunkt der vorliegenden Arbeit waren Aktivitätsbestimmungen beider Meprine unter Berücksichtigung potentieller Aktivatoren und Substrate. Es konnten die kallikrein-related peptidases (KLK) 4, 5 und 8 als spezifische Aktivatoren identifiziert werden, wobei nur KLK5 beide Proteasen aktiviert. Sowohl KLK4 als auch KLK8 sind lediglich in der Lage, das Propeptid von Meprin β abzuspalten. Außerdem konnte biochemisch und mittels Proteomics gezeigt werden, dass proKLK7 von Meprin β prozessiert wird. Durch N-terminale Sequenzierung wurde eine Schnittstelle zwei Aminosäuren N-terminal der eigentlichen Aktivierungsstelle identifiziert. Dieser Schritt beschleunigt die Aktivierung von KLK7, wenn durch Trypsin noch das verbliebene Dipeptid abgespalten wird. rnDa einige Vertreter der humanen kallikrein-related peptidases (KLK) als Meprin-Aktivatoren identifiziert werden konnten, sollten diese im Zuge dieser Arbeit im Modellorganismus Danio rerio untersucht werden. Durch in silico und RT-PCR Analysen konnte gezeigt werden, dass keine funktionellen KLK-Homologe im Zebrafisch codiert sind. Da somit andere tryptische Proteasen an der Aktivierung der Meprine beteiligt sein müssen, wurde die Transmembran-Serinprotease TMPRSS4 analysiert. In der Tat zeigte die Reduktion des Expressionslevels von TMPRSS4 durch Morpholino-Injektion drastische Störungen in der embryonalen Entwicklung von Zebrabärblingen. Mittels Licht- und Rasterelektronenmikroskopie ließ sich eine Fehlbildung der epidermalen Haut bis zu einem Ablösen der Keratinozyten von dem darunter liegenden Gewebe feststellen. rn

Intracellular imaging of nanoparticles: is it an elemental mistake to believe what you see?

In order to understand how nanoparticles (NPs <100 nm) interact with cellular systems, potentially causing adverse effects, it is important to be able to detect and localize them within cells. Due to the small size of NPs, transmission electron microscopy (TEM) is an appropriate technique to use for visualizing NPs inside cells, since light microscopy fails to resolve them at a single particle level. However, the presence of other cellular and non-cellular nano-sized structures in TEM cell samples, which may resemble NPs in size, morphology and electron density, can obstruct the precise intracellular identification of NPs. Therefore, elemental analysis is recommended to confirm the presence of NPs inside the cell. The present study highlights the necessity to perform elemental analysis, specifically energy filtering TEM, to confirm intracellular NP localization using the example of quantum dots (QDs). Recently, QDs have gained increased attention due to their fluorescent characteristics, and possible applications for biomedical imaging have been suggested. Nevertheless, potential adverse effects cannot be excluded and some studies point to a correlation between intracellular particle localization and toxic effects. J774.A1 murine macrophage-like cells were exposed to NH2 polyethylene (PEG) QDs and elemental co-localization analysis of two elements present in the QDs (sulfur and cadmium) was performed on putative intracellular QDs with electron spectroscopic imaging (ESI). Both elements were shown on a single particle level and QDs were confirmed to be located inside intracellular vesicles. Nevertheless, ESI analysis showed that not all nano-sized structures, initially identified as QDs, were confirmed. This observation emphasizes the necessity to perform elemental analysis when investigating intracellular NP localization using TEM.

Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis

Bacteria are generally difficult specimens to prepare for conventional resin section electron microscopy and mycobacteria, with their thick and complex cell envelope layers being especially prone to artefacts. Here we made a systematic comparison of different methods for preparing Mycobacterium smegmatis for thin section electron microscopy analysis. These methods were: (1) conventional preparation by fixatives and epoxy resins at ambient temperature. (2) Tokuyasu cryo-section of chemically fixed bacteria. (3) rapid freezing followed by freeze substitution and embedding in epoxy resin at room temperature or (4) combined with Lowicryl HM20 embedding and ultraviolet (UV) polymerization at low temperature and (5) CEMOVIS, or cryo electron microscopy of vitreous sections. The best preservation of bacteria was obtained with the cryo electron microscopy of vitreous sections method, as expected, especially with respect to the preservation of the cell envelope and lipid bodies. By comparison with cryo electron microscopy of vitreous sections both the conventional and Tokuyasu methods produced different, undesirable artefacts. The two different types of freeze-substitution protocols showed variable preservation of the cell envelope but gave acceptable preservation of the cytoplasm, but not lipid bodies, and bacterial DNA. In conclusion although cryo electron microscopy of vitreous sections must be considered the 'gold standard' among sectioning methods for electron microscopy, because it avoids solvents and stains, the use of optimally prepared freeze substitution also offers some advantages for ultrastructural analysis of bacteria.

Pattern of mineralization after regenerative periodontal therapy with enamel matrix proteins

A derivative (EMD) of enamel matrix proteins (EMPs) is used for periodontal regeneration because EMPs are believed to induce the formation of acellular extrinsic fiber cementum (AEFC). Other reports, however, indicate that EMPs have osteogenic potential. The aim of this study was to characterize the nature of the tissue that forms on the root surface following application of EMD. Ten human teeth affected by periodontitis and scheduled for extraction were treated with EMD. Four to six weeks later, they were extracted and processed for analysis by light microscopy and transmission electron microscopy. Immunocytochemistry with antibodies against bone sialoprotein (BSP) and osteopontin (OPN) was performed to determine the mineralization pattern. The newly formed tissues on the root were thick and contained embedded cells. Small mineralization foci were regularly seen, and large organic matrix patches were occasionally seen, but a distinct mineralization front was lacking. While labeling for BSP was always associated with small mineralization foci and large matrix patches, OPN labeling was seen inconsistently. It is concluded that tissues resembling either cellular intrinsic fiber cementum or a type of bone were observed. The mineralization pattern mostly resembled that found in bone, except for a few areas that exhibited a hitherto undescribed mineralization pattern.

Regional structural characteristics of bovine periodontal ligament samples and their suitability for biomechanical tests

Mechanical testing of the periodontal ligament requires a practical experimental model. Bovine teeth are advantageous in terms of size and availability, but information is lacking as to the anatomy and histology of their periodontium. The aim of this study, therefore, was to characterize the anatomy and histology of the attachment apparatus in fully erupted bovine mandibular first molars. A total of 13 teeth were processed for the production of undecalcified ground sections and decalcified semi-thin sections, for NaOH maceration, and for polarized light microscopy. Histomorphometric measurements relevant to the mechanical behavior of the periodontal ligament included width, number, size and area fraction of blood vessels and fractal analysis of the two hard-soft tissue interfaces. The histological and histomorphometric analyses were performed at four different root depths and at six circumferential locations around the distal and mesial roots. The variety of techniques applied provided a comprehensive view of the tissue architecture of the bovine periodontal ligament. Marked regional variations were observed in width, surface geometry of the two bordering hard tissues (cementum and alveolar bone), structural organization of the principal periodontal ligament connective tissue fibers, size, number and numerical density of blood vessels in the periodontal ligament. No predictable pattern was observed, except for a statistically significant increase in the area fraction of blood vessels from apical to coronal. The periodontal ligament width was up to three times wider in bovine teeth than in human teeth. The fractal analyses were in agreement with the histological observations showing frequent signs of remodeling activity in the alveolar bone - a finding which may be related to the magnitude and direction of occlusal forces in ruminants. Although samples from the apical root portion are not suitable for biomechanical testing, all other levels in the buccal and lingual aspects of the mesial and distal roots may be considered. The bucco-mesial aspect of the distal root appears to be the most suitable location.

Do mature pulmonary lobes grow after transplantation into an immature recipient?

BACKGROUND The use of reduced-size adult lung transplants could help solve the profound pediatric donor lung shortage. However, adequate long-term function of the mature grafts requires growth in proportion to the recipient's development. METHODS Mature left lower lobes from adult mini-pigs (age: 7 months; mean body weight: 30 kg) were transplanted into 14-week-old piglets (mean body weight: 15 kg). By the end of the 14-week holding period, lungs of the recipients (n = 4) were harvested. After volumetric measurements, the lung morphology was studied using light microscopy, scanning, and transmission electron microscopy. Changes of alveolar airspace volume were determined using a computer aided image analysis system. Comparisons were made to age- and weight-matched controls. RESULTS Volumetric studies showed no significant differences (p = 0.49) between the specific volume (mL/kg body weight) of lobar grafts and left lower lobes of adult controls. Morphologic studies showed marked structural differences between the grafts and the right native lungs of the recipients, with increased average alveolar diameter of the grafts. On light microscopy and scanning electron microscopy, alveoli appeared dilated and rounded compared to the normal polygonal shape in the controls. The computer generated semi-quantitative data of relative alveolar airspace volume tended to be higher in transplanted lobes. CONCLUSIONS The mature pulmonary lobar grafts have filled the growing left hemithorax of the developing recipient. Emphysema-like alterations of the grafts were observed without evidence of alveolar growth in the mature lobar transplants. Thus, it can be questioned whether mature pulmonary grafts can guarantee sufficient long-term gas exchange in growing recipients.

Vaginal cytokines do not differ between postmenopausal women with and without symptoms of vulvovaginal irritation.

OBJECTIVE The aim of this exploratory pilot study was to determine if there are differences in vaginal cytokine levels between postmenopausal women with and without vulvovaginal irritative symptoms (itching, burning, or pain). METHODS Postmenopausal women (n = 34) not using hormone therapy and presenting with or without symptoms of vulvovaginal irritation were asked to volunteer for this study. Each participant underwent a vaginal examination and screening for vaginitis using Amsel criteria, pH, and light microscopy. A vaginal lavage with 5.0 mL of sterile saline was carried out, and a peripheral blood sample was obtained. The vaginal lavage and serum samples were assayed for interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α by specific enzyme-linked immunosorbent assays. Results were adjusted for total protein concentration and presented as the amount of cytokines per protein (pg/μg protein). Statistical analysis was performed using SAS version 9.3 (SAS Institute, Cary, NC). The means and SDs of all variables among women with and without vulvovaginal irritation were compared using independent-samples Student's t test. RESULTS A total of 26 postmenopausal women were enrolled into the study (symptomatic, n = 15; asymptomatic, n = 11). The mean (SD) vaginal pH for all participants was 5.9 (1.2). There were no significant differences (P > 0.05) in age, age at menopause, vaginal pH, and vaginal and serum cytokines and chemokines (IL-1β, IL-6, IL-8, and tumor necrosis factor-α) among symptomatic versus asymptomatic women. IL-8 was the most abundant vaginal cytokine, with mean (SD) vaginal IL-8 levels being 4.1 (3.4) and 3.1 (3.9) pg/μg protein in the symptomatic versus asymptomatic groups, respectively (P = 0.55). There were no significant linear correlations (P > 0.05) between serum and vaginal cytokine levels for all endpoints. CONCLUSIONS The presence or absence of postmenopausal vulvovaginal symptoms does not significantly differentiate vaginal inflammatory markers. Serum and vaginal cytokines are not significantly linearly correlated among postmenopausal women with and without symptoms commonly associated with vaginal atrophy, implying that this is a local reaction.

Temporal evolution of strut light intensity after implantation of bioresorbable polymeric intracoronary scaffolds in the ABSORB cohort B trial-an application of a new quantitative method based on optical coherence tomography.

BACKGROUND Quantitative light intensity analysis of the strut core by optical coherence tomography (OCT) may enable assessment of changes in the light reflectivity of the bioresorbable polymeric scaffold from polymer to provisional matrix and connective tissues, with full disappearance and integration of the scaffold into the vessel wall. The aim of this report was to describe the methodology and to apply it to serial human OCT images post procedure and at 6, 12, 24 and 36 months in the ABSORB cohort B trial. METHODS AND RESULTS In serial frequency-domain OCT pullbacks, corresponding struts at different time points were identified by 3-dimensional foldout view. The peak and median values of light intensity were measured in the strut core by dedicated software. A total of 303 corresponding struts were serially analyzed at 3 time points. In the sequential analysis, peak light intensity increased gradually in the first 24 months after implantation and reached a plateau (relative difference with respect to baseline [%Dif]: 61.4% at 12 months, 115.0% at 24 months, 110.7% at 36 months), while the median intensity kept increasing at 36 months (%Dif: 14.3% at 12 months, 75.0% at 24 months, 93.1% at 36 months). CONCLUSIONS Quantitative light intensity analysis by OCT was capable of detecting subtle changes in the bioresorbable strut appearance over time, and could be used to monitor the bioresorption and integration process of polylactide struts.

Osteoclast-like cells on deproteinized bovine bone mineral and biphasic calcium phosphate: light and transmission electron microscopical observations.

OBJECTIVES The occurrence of multinucleated giant cells (MNGCs) on bone substitute materials has been recognized for a long time. However, there have been no studies linking material characteristics with morphology of the MNGCs. The aim was to analyze the qualitative differences of MNGCs on two commercially available calcium phosphate bone substitute materials retrieved from bone defects. MATERIAL AND METHODS Six defects were prepared bilaterally in the mandibular body of three mini pigs. The defects were randomly grafted with either deproteinized bovine bone mineral (DBBM) or biphasic calcium phosphate (BCP). After a healing period of four weeks, bone blocks were embedded in LR White resin. Three consecutive sections per defect were analyzed as follows: two with light microscopy using toluidine blue and tartrate-resistant acid phosphatase (TRAP) staining and one with transmission electron microscopy. RESULTS Multinucleated giant cells appeared on both biomaterials. On BCP, MNGCs had a flat morphology and were not observed in resorption lacunae. On DBBM, the MNGCs appeared more round and were often found in shallow concavities. MNGCs on both biomaterials demonstrated a varying degree of TRAP staining, with a tendency toward higher staining intensity of MNGCs on BCP. At the ultrastructural level, signs of superficial dissolution of BCP together with phagocytosis of minor fragments were observed. MNGCs on the surface of DBBM demonstrated sealing zones and ruffled borders, both features of mature osteoclasts. CONCLUSION MNGCs demonstrated distinctly different histological features depending on the bone substitute material used. Further research is warranted to understand the clinical implications of these morphological observations.

Different bacterial models for in vitro induction of non-cavitated enamel caries-like lesions: Microhardness and polarized light miscroscopy analyses

The aim of this study was to compare different bacterial models for in vitro induction of non-cavitated enamel caries-like lesions by microhardness and polarized light microscopy analyses. One hundred blocks of bovine enamel were randomly divided into four groups (n = 25) according to the bacterial model for caries induction: (A) Streptococcus mutans, (B) S. mutans and Lactobacillus acidophilus, (C) S. mutans and L. casei, and (D) S. mutans, L. acidophilus, and L. casei. Within each group, the blocks were randomly divided into five subgroups according to the duration of the period of caries induction (4-20 days). The enamel blocks were immersed in cariogenic solution containing the microorganisms, which was changed every 48 h. Groups C and D presented lower surface hardness values (SMH) and higher area of hardness loss (ΔS) after the cariogenic challenge than groups A and B (P < 0.05). As regards lesion depth, under polarized light microscopy, group A presented significantly lower values, and groups C and D the highest values. Group B showed a higher value than group A (P < 0.05). Groups A and B exhibited subsurface caries lesions after all treatment durations, while groups C and D presented erosion-type lesions with surface softening. The model using S. mutans, whether or not it was associated with L. acidophilus, was less aggressive and may be used for the induction of non-cavitated enamel caries-like lesions. The optimal period for inducing caries-like lesions was 8 days.