The effects of moderate and low levels of acoustic overstimulation on stereocilia and their tip links in the guinea pig

Guinea pigs were exposed to pure tones of 10 kHz at intensities between 98 and 115 dB SPL for 5-30 min, to produce varying degrees of acoustic trauma. Changes in auditory thresholds were measured electrophysiologically, and the animals were immediately fixed for scanning electron microscopy. Correlation between morphological changes to the hair bundle and losses in threshold, showed that with the smallest degrees of trauma (98 dB SPL for 15 min, mean maximum threshold loss of 22 dB), damage was confined to a small stretch of inner hair cells (IHC), with only subtle changes to the stereocilia of the outer hair cells (OHC). At exposure intensities greater than 102 dB SPL (duration: 15 min) the IHC stereocilia in the centre of the lesion were always substantially disarrayed. Substantial damage to the OHC bundles was seen only with exposures above 110 dB SPL(duration: greater than or equal to 5 min), producing threshold losses of 50 dB or more. Tip links were lost only where the stereocilia were disarrayed. It is concluded that the tip links are not the most vulnerable components of the cochlear hair cell, but that relatively low levels of acoustic stimulation can cause significant damage to the stereociliary bundle of the IHCs.

Improved method for counting virus and virus like particles

An improved method for counting virus and virus like particles by electron microscopy (EM) was developed. The procedure involves the determination of the absolute concentration of pure or semi-pure particles once deposited evenly on EM grids using either centrifugation or antibody capture techniques. The counting of particles was done with a Microfiche unit which enlarged approximately 50 x the image of particles on a developed negative film which had been taken at a relatively low magnification (2500 x) by EM. Initially, latex particles of a known concentration were counted using this approach, to prove the accuracy of the technique. The latex particles were deposited evenly on an EM grid using centrifugation (Modified Beckmen EM-90 Airfuge technique). Subsequently, recombinant Bluetongue virus (BTV) core-like particles (CLPs) captured by a Monoclonal antibody using a hovel sample loading method were counted by the Microfiche unit method and by a direct EM method. Comparison of the simplified counting method developed with a conventional method, showed good agreement. The method is simple, accurate, rapid, and reproducible when used with either pure particles or with particles from crude cell culture extracts.

Effects of Time on Ultrastructural Integrity of Parathyroid Tissue Before Cryopreservation

Cryopreservation of parathyroid tissue is used in the surgical treatment of secondary hyperparathyroidism. After surgical resection, the tissue is temporarily maintained in a cell culture solution until it arrives at the specialized laboratory where the cryopreservation process will take place. The present study evaluates the time that the human hyperplastic parathyroid gland tissue can wait before cryopreservation, based on parathyroid cell ultrastructural integrity. This prospective study included 11 patients who underwent total parathyroidectomy with heterotopic autotransplantation and cryopreservation of parathyroid tissue fragments. Part of the tissue was kept in cell culture solution at 4A degrees C. Five time periods between 2 and 24 h were defined, and parathyroid fragments were kept in the solution for that length of time. At the end of each period, the fragments were removed from the transport solution, fixed, and prepared for ultrathin sections. Of the 11 cases studied, 10 showed ultrastructural findings consistent with cellular viability in tissue fragments that remained in the transport solution up to 12 h. Electron microscopy revealed that cell adhesion and the integrity of plasma membranes, nuclei, and mitochondria were preserved in one case for up to 24 h. Changes in mitochondrial structure represented the most constant ultrastructural damage seen in the cases studied, in addition to the presence of edema and cell vacuoles. Analysis of the ultrastructure of hyperplastic parathyroid gland tissue showed that ultrastructural integrity was in most cases properly maintained in fragments stored up to 12 h in a cell culture solution at 4A degrees C.

Annexin 1 mimetic peptide protects against renal ischemia/reperfusion injury in rats

Inflammation is currently recognized as a key mechanism in the pathogenesis of renal ischemia-reperfusion (I/R) injury. The importance of infiltrating neutrophil, lymphocytes, and macrophage in this kind of injury has been assessed with conflicting results. Annexin 1 is a protein with potent neutrophil anti-migratory activity. In order to evaluate the effects of annexin A1 on renal I/R injury, uninephrectomized rats received annexin A1 mimetic peptide Ac2-26 (100 mu g) or vehicle before 30 min of renal artery clamping and were compared to sham surgery animals. Annexin A1 mimetic peptide granted a remarkable protection against I/R injury, preventing glomerular filtration rate and urinary osmolality decreases and acute tubular necrosis development. Annexin A1 infusion aborted neutrophil extravasation and attenuated macrophage infiltration but did not prevent tissue lymphocyte traffic. I/R increased annexin A1 expression (assessed by transmission electron microscopy) in renal epithelial cells, which was attenuated by exogenous annexin A1 infusion. Additionally, annexin A1 reduced I/R injury in isolated proximal tubules suspension. Annexin A1 protein afforded striking functional and structural protection against renal I/R. These results point to an important role of annexin A1 in the epithelial cells defense against I/R injury and indicate that neutrophils are key mediators for the development of tissue injury after renal I/R. If these results were confirmed in clinical studies, annexin A1 might emerge as an important tool to protect against I/R injury in renal transplantation and in vascular surgery.

Sheina orri (Myodocopa: Cypridinidae), an ostracod parasitic on the gills of the epaulette shark, Hemiscyllium ocellatum (Elasmobranchii: Hemiscyllidae)

The cypridinid ostracod, Sheina orri, was found on the gills of healthy epaulette sharks, Hemiscyllium, ocellatum, collected from the Great Barrier Reef, Australia, Seventeen of the 28 fish examined had ostracods attached to their gills. Detailed investigation of the gills and ostracods using light microscopy and scanning electron microscopy revealed that ostracods anchor themselves to the gill tissues using their mandibular and maxillular claws. These claws appear to be adapted for this purpose and the process of attachment causes some damage to the host tissues. The observation that ostracods were often located in distinct pockets, formed by local distortion of shark respiratory lamellae, strongly suggests that they had been attached to the gills for considerable time. (C) 1997 Australian Society for Parasitology.

Abnormal expression of telomerase/apoptosis limits type II alveolar epithelial cell replication in the early remodeling of usual interstitial pneumonia/idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis is a distinctive, usually fatal, type of chronic fibrosing interstitial pneumonia of unknown cause that increases in prevalence with advanced age, characterized by failure of alveolar re-epithelization and progressive scar formation. Recently, limitation of the replicative capacity of tissues determined by telomerase/apoptosis balance has been implicated in pathogenesis of age-related diseases. In this study, we validated the importance of the expression of type 2 alveolar epithelial cells telomerase protein and studied the relationships between telomerase and apoptosis in early remodeling of usual interstitial pneumonia. We determined type 2 alveolar epithelial cells density, telomerase expression, and apoptosis in surgical lung biopsies from 24 patients with usual interstitial pneumonia, and in normal lung tissues from 18 subjects. We used immunohistochemistry, deoxynucleotidyl transferase method of end labeling, electron microscopy, and histomorphometry to evaluate the amount of type 2 alveolar epithelial cells staining for surfactant-A, telomerase, and in situ detection of apoptotic cells. Unaffected areas of usual interstitial pneumonia and normal lung tissue had similar densities of type 2 alveolar epithelial cells, but a significant minor subpopulation of type 2 alveolar epithelial cells was telomerase positive and a large population was telomerase negative. A significant inverse association was found between low type 2, alveolar. epithelial cell telomerase expression and high apoptosis in unaffected areas of usual interstitial pneumonia. Although type 2 alveolar epithelial cell telomerase expression was higher than apoptosis in NLT group, no significant association was found between them. Electron microscopy confirmed epithelial apoptosis, alveolar collapse, and initial fibroplasia. We conclude that abnormal type 2 alveolar epithelial cells telomerase/apoptosis balance may reduce alveolar epithelial regenerative capacity, thus contributing to the early remodeling response in usual interstitial pneumonia. (C) 2010 Elsevier Inc. All rights reserved.

A systemic parvo-like virus in the freshwater crayfish Cherax destructor

Systemic Cowdry Type A inclusions (CAs) were observed in a moribund Cherax destructor collected at an aquaculture farm in South Australia. Inclusions were most common in the gills and were associated with multifocal necrosis of the main gill axis and lamellae. The hepatopancreas was necrotic; however, only one CA was observed in the interstitial tissues. CAs were associated with necrosis in the abdominal and gut musculature. CAs were also observed in the spongy connective tissues and the epicardium. Empty capsids (17.5 +/- 0.5 nm) and microfilaments were most commonly observed within these inclusions by transmission electron microscopy. Complete icosahedral viral particles (20.8 +/- 1.2 nm) were difficult to distinguish within the viroplasm, but were visualised better in aggregates between the viroplasm and the inner nuclear membrane. The nucleolus was closely associated with the developing viroplasm, and was hypertrophied and segregated into its fibrillar and granular components. The virus was named Cherax destructor systemic parvo-like virus (CdSPV) on the basis of its histopathology, cytopathology and morphology. CdSPV is the first systemic virus described in a freshwater crayfish.

Characterization of Superparamagnetic Iron Oxide Coated with Silicone Used as Contrast Agent for Magnetic Resonance Image for the Gastrointestinal Tract

The present work is a report of the characterization of superparamagnetic iron oxide nanoparticles coated with silicone used as a contrast agent in magnetic resonance imaging of the gastrointestinal tract. The hydrodynamic size of the contrast agent is 281.2 rim, where it was determined by transmission electron microscopy and a Fe(3)O(4) crystalline structure was identified by X-ray diffraction, also confirmed by Mossbauer Spectroscopy. The blocking temperature of 190 K was determined from magnetic measurements based on the Zero Field Cooled and Field Cooled methods. The hysteresis loops were measured at different temperatures below and above the blocking temperature. Ferromagnetic resonance analysis indicated the superparamagnetic nature of the nanoparticles and a strong temperature dependence of the peak-to-peak linewidth Delta H(pp), giromagnetic factor g, number of spins N(S) and relaxation time T(2) were observed. This behavior can be attributed to an increase in the superexchange interaction.

Characterization of Adherent Umbilical Cord Blood Stromal Cells Regarding Passage, Cell Number, and Nano-biomarking Utilization

Adherent umbilical cord blood stromal cells (AUCBSCs) are multipotent cells with differentiation capacities. Therefore, these cells have been investigated for their potential in cell-based therapies. Quantum Dots (QDs) are an alternative to organic dyes and fluorescent proteins because of their long-term photostability. In this study we determined the effects of the cell passage on AUCBSCs morphology, phenotype, and differentiation potential. QDs labeled AUCBSCs in the fourth cell passage were differentiated in the three mesodermal lineages and were evaluated using cytochemical methods and transmission electron microscopy (TEM). Gene and protein expression of the AUCBSCs immunophenotypic markers were also evaluated in the labeled cells by real-time quantitative PCR and flow cytometry. In this study we were able to define the best cellular passage to work with AUCBSCs and we also demonstrated that the use of fluorescent QDs can be an efficient nano-biotechnological tool in differentiation studies because labeled cells do not have their characteristics compromised.

In vitro study of CD133 human stem cells labeled with superparamagnetic iron oxide nanoparticles

Superparamagnetic iron oxide nanoparticles (SPIONs) are applied in stem cell labeling because of their high magnetic susceptibility as compared with ordinary paramagnetic species, their low toxicity, and their ease of magnetic manipulation. The present work is the study of CD133(+) stem cell labeling by SPIONs coupled to a specific antibody (AC133), resulting in the antigenic labeling of the CD133+ stem cell, and a method was developed for the quantification of the SPION content per cell, necessary for molecular imaging optimization. Flow cytometry analysis established the efficiency of the selection process and helped determine that the CD133 cells selected by chromatographic affinity express the transmembrane glycoprotein CD133. The presence of antibodies coupled to the SPION, expressed in the cell membrane, was observed by transmission electron microscopy. Quantification of the SPION concentration in the marked cells using the ferromagnetic resonance technique resulted in a value of 1.70 x 10 (13) mol iron (9.5 pg) or 7.0 x 10 (6) nanoparticles per cell ( the measurement was carried out in a volume of 2 mu L containing about 6.16 x 10 5 pg iron, equivalent to 4.5 x 10 (11) SPIONs). (c) 2008 Elsevier Inc. All rights reserved.

Trypan blue staining for capsulorhexis: Ultrastructural effect on lens epithelial cells and capsules

PURPOSE: To evaluate the ultrastructural effect of trypan blue 0.1% staining for capsulorhexis on lens epithelial cells (LECs) and capsules SETTING: Division of Ophthalmology. University of Sao Paulo, Sao Paulo, Brazil METHODS: Before capsulorhexis, patients were randomly assigned to 1 of 2 groups Trypan blue 0 1% staining was performed in the treatment group No trypan blue was used in the control group Samples of capsules with LECs were fixed and analyzed with routine optical microscopy techniques. immunohistochemistry for beclin-1 expression (a marker of autophagy), terminal deoxynucleotidyl transf erase-mediated dUTP-biotin nick-end labeling to detect apoptosis, and transmission electron microscopy (TEM) Morphometric analyses were performed. and the 2 sets of data were compared. RESULTS: Each group comprised 15 patients Cell death by autophagy and apoptosis was observed in the treatment group but not in the control group The TEM images of subcapsular epithelium cells showed mitochondria` rupture, dilation of the cisterns of the endoplasmic reticulum, increased cytoplasmic and nuclear electron density, and abnormalities in the nuclear profile of trypan blue-stained cells. Morphometric analysis showed statistically significant differences between the 2 groups in the longest nuclear axes and the ratio between the total nuclear perimeter and the cell area (P = .03) The difference in capsule thickness between groups was not significant. CONCLUSION: Trypan blue caused LEC death, which supports the hypothesis that staining with trypan blue 0 1% can help reduce the incidence of posterior capsule pacification after cataract surgery

Early keratocyte apoptosis after epithelial scrape injury in the human cornea

Animal studies in mice, rats, rabbits, pigs and hens demonstrated that anterior keratocytes undergo programmed cell death or apoptosis after corneal epithelial injury. Many other wound healing changes subsequently follow the keratocyte apoptosis response. This study evaluated early keratocyte apoptosis after corneal epithelial scrape injury in human eyes scheduled for enucleation for malignancy. Two eyes had corneal epithelial scrape 1 h prior to the enucleation and another eye served as a control and had no corneal scrape prior to enucleation. One additional eye was enucleated, washed with balanced salt solution, and then had the corneal epithelium scraped 1 h prior to processing for analysis. Apoptosis was identified by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and confirmed by transmission electron microscopy (TEM). Anterior keratocyte apoptosis was detected in the three corneas that had epithelial scrape injury, but not in the control unwounded cornea. This study confirmed that keratocyte apoptosis is also an early response to corneal epithelial injury in humans and showed that tears are not essential for keratocyte apoptosis to occur in response to epithelial injury. (C) 2009 Elsevier Ltd. All rights reserved.

Osteoblastlike Cell Adhesion on Titanium Surfaces Modified by Plasma Nitriding

Purpose: The aim of this study was to evaluate the characteristics of various titanium surfaces modified by cold plasma nitriding in terms of adhesion and proliferation of rat osteoblastlike cells. Materials and Methods: Samples of grade 2 titanium were subjected to three different surface modification processes: polishing, nit riding by plasma direct current, and nitriding by cathodic cage discharge. To evaluate the effect of the surface treatment on the cellular response, the adhesion and proliferation of osteoblastlike cells (MC3T3) were quantified and the results were analyzed by Kruskal-Wallis and Friedman statistical tests. Cellular morphology was observed by scanning electron microscopy. Results: There was more MC3T3 cell attachment on the rougher surfaces produced by cathodic cage discharge compared with polished samples (P < .05). Conclusions: Plasma nitriding improves titanium surface roughness and wettability, leading to osteoblastlike cell adhesion. INT J ORAL MAXILLOFAC IMPLANTS 2011;26:237-244

Collagen V and vascular injury promote lung architectural changes in systemic sclerosis

Background: Systemic sclerosis (SSc) is a multisystem disorder characterized by inflammation, fibrosis and vascular damage. The aim of this study was to evaluate the interactions between basement membrane disruption, endothelial injury and collagen V deposition on the vascular wall, as well as their association with pulmonary function tests in patients with SSc. Method: The endothelial apoptosis was assessed by TUNEL and electron microscopy, and quantified through the point-counting technique. To evaluate basement membrane integrity, laminin immunostaining and electron microscopy were used. Immunofluorescence and morphometric analysis were used to determine the amount of collagen V in the vascular walls in 23 open lung biopsies of patients with SSc without pulmonary hypertension. Normal lung tissue was obtained from five individuals who had died of traumatic injuries. Results: The apoptosis index in SSc was higher in the endothelial cells (13.83 +/- 6.83) when compared with the control (2.51 +/- 2.06) group (P < 0.001) and confirmed by electron microscopy. We observed an important disruption of the basement membrane on the vascular wall shown by discontinuous laminin immunostaining and electron microscopy. An increase in collagen V on the vascular wall of the SSc group was observed (45.28 +/- 13.21), when compared with control group (22.90 +/- 4.13, P < 0.001), and this difference was statistically significant. An inverse correlation was found between vital capacity, forced vital capacity, forced expiratory volume in 1 s, vascular collagen V and endothelial apoptosis (P < 0.05). Conclusions: We conclude that the endothelial apoptosis and vascular collagen V interaction reinforce the vascular pathway in the SSc pathogenesis. Further studies are needed to determine whether this relationship is causal or consequential. Please cite this paper as: Parra ER, Aguiar AC Jr, Teodoro WR, de Souza R, Yoshinari NH and Capelozzi VL. Collagen V and vascular injury promote lung architectural changes in systemic sclerosis. The Clinical Respiratory Journal 2009; 3: 135-142.

Pulmonary lesion induced by low and high positive end-expiratory pressure levels during protective ventilation in experimental acute lung injury

Objective: To investigate the effects of low and high levels of positive end-expiratory pressure (PEEP), without recruitment maneuvers, during lung protective ventilation in an experimental model of acute lung injury (ALI). Design: Prospective, randomized, and controlled experimental study. Setting: University research laboratory. Subjects: Wistar rats were randomly assigned to control (C) [saline (0.1 ml), intraperitoneally] and ALI [paraquat (15 mg/kg), intra peritoneally] groups. Measurements and Main Results: After 24 hours, each group was further randomized into four groups (six rats each) at different PEEP levels = 1.5, 3, 4.5, or 6 cm H(2)O and ventilated with a constant tidal volume (6 mL/kg) and open thorax. Lung mechanics [static elastance (Est, L) and viscoelastic pressure (Delta P2, L)] and arterial blood gases were measured before (Pre) and at the end of 1-hour mechanical ventilation (Post). Pulmonary histology (light and electron microscopy) and type III procollagen (PCIII) messenger RNA (mRNA) expression were measured after 1 hour of mechanical ventilation. In ALI group, low and high PEEP levels induced a greater percentage of increase in Est, L (44% and 50%) and Delta P2, L (56% and 36%) in Post values related to Pre. Low PEEP yielded alveolar collapse whereas high PEEP caused overdistension and atelectasis, with both levels worsening oxygenation and increasing PCIII mRNA expression. Conclusions: In the present nonrecruited ALI model, protective mechanical ventilation with lower and higher PEEP levels than required for better oxygenation increased Est, L and Delta P2, L, the amount of atelectasis, and PCIII mRNA expression. PEEP selection titrated for a minimum elastance and maximum oxygenation may prevent lung injury while deviation from these settings may be harmful. (Crit Care Med 2009; 37:1011-1017)