Study of the Effect of Hyperbaric Oxygen Therapy on the Viability of Dorsal Cutaneous Flaps in Tobacco-Exposed Rats.

BACKGROUND: Smoking causes a threefold increase in the risk of surgical complications in flaps. Hyperbaric oxygen therapy (HBOT) increases the viability of chronic wounds. However, there are few studies concerning the effects of HBOT on surgical flaps in patients who smoke. This study aimed to analyze the effect of HBOT on the viability of cutaneous flaps in tobacco-exposed rats. METHODS: Twenty Wistar rats were exposed to tobacco smoke for two months. Following this period, all animals underwent a dorsal cutaneous flap (3 × 10 cm) surgery and were divided into two groups: control (n = 10) and HBOT (n = 10). HBOT was performed in seven daily sessions (2 ATA, 90 min). After seven days, the animals were euthanized. The outcomes were total area, viable area, viable area/total area rate, analysis of dermal appendages and angiogenesis (hematoxylin-eosin), and gene expression analysis of iNOS and VEGF-a biomarkers. RESULTS: The HBOT group showed an increase in viable area compared with the control group (84% versus 47%, p = 0.009, respectively). The HBOT group also showed an increase in appendage units (1.69 ± 0.54 versus 1.87 ± 0.58, p = 0.04) and angiogenesis density (1.29 ± 0.45 versus 1.82 ± 0.64, p < 0.001) compared to the control group. There was a difference between the control and HBOT groups in iNOS levels (0.926 ± 1.4 versus 0.04 ± 0.1 p = 0.002, respectively). However, this study did not show a difference between the groups concerning the gene expression of VEGF-a. CONCLUSION: The use of hyperbaric oxygen therapy increased the viability of cutaneous flaps in tobacco-exposed rats and decreased iNOS mRNA levels; however, it did not change VEGF-a levels. Level of Evidence IV This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Effects of an individually ventilated cage system on the airway integrity of rats (Rattus norvegicus) in a laboratory in Brazil.

The ventilation method used in the management of laboratory rats is important in maintaining their health. Rats kept under general diluting ventilation (GDV) are exposed to high levels of pollutants present in the environment (dust, airborne bacteria, etc.) or those pollutants produced by animal metabolism and excretion inside the boxes (e.g. ammonia and carbon dioxide). These pollutants may contribute to respiratory pathologies. An alternative experimental ventilation system for laboratory animal housing using intracage ventilation technology (individually ventilated cage system, IVC) was developed. In this system, ammonia levels decreased and rats exhibited better reproductive performance and a lower incidence of pneumonia than rats maintained under GDV. Using two different levels of air speed (0.03-0.26 m/s: IVC(1); 0.27-0.80 m/s: IVC(2)), the effects of IVC were compared with GDV (control) in Wistar rats in terms of respiratory mucus properties, on the nasal epithelium (as measured by quantitative morphometry) and on the lungs (as determined by the cellular composition obtained by bronchoalveolar lavage). Mucus of the respiratory system was evaluated using the following techniques: rheology (viscoelasticity) by microrheometer, in vitro mucociliary transportability (frog palate) and contact angle (an indicator of adhesivity). Also, membrane transepithelial potential difference was measured as a biomarker of airway integrity. After bedding was changed, ammonia concentrations inside the cages on day 3 were significantly higher for GDV than for IVC(1) and IVC(2). The potential-difference values for IVC(1), IVC(2) and GDV in the epiglottis and in the trachea also showed differences. Although some significant differences were observed across the three groups in counts of some cell types, the intragroup results were highly variable among individuals and inconsistent between sexes. No significant differences in the other parameters were found across groups. These results establish that rats maintained under GDV in relatively unregulated conditions are exposed to factors that can lead to deleterious effects on the ciliated epithelium of the airways, and that these effects can be prevented by the use of IVC.

Pollen mother cells of Tradescantia clone 4430 and Tradescantia pallida var. purpurea are equally sensitive to the clastogenic effects of X-rays

The Tradescantia micronucleus test is a sensitive bioassay for mutagenesis that may be employed both under field and laboratory conditions. This test has been standardized mostly on the basis of the results obtained with clone 4430. However, this clone is not well adapted to tropical weather, frequently showing problems with growth and flowering. In addition, it is attacked by parasites and insects, a fact that limits its use in field studies aiming at the biomonitoring of air pollution. In the city of Säo Paulo, Tradescantia pallida (Rose) Hunt. var. purpurea Boom is widely distributed as an ornamental plant in gardens and along roadsides and streets, mostly because of its natural resistance and its easy propagation. In this report, we present dose-response curves indicating that the sensitivity of T. pallida and clone 4430 to X-radiation (1, 10, 25 and 50 cGy) is similar. The results confirm our previous suggestion that T. pallida represents a good alternative for in situ mutagenesis testing in tropical regions, especially biomonitoring studies in which the exposure conditions may not be fully controllable

Pollen mother cells of Tradescantia clone 4430 and Tradescantia pallida var. purpurea are equally sensitive to the clastogenic effects of X-rays.

The Tradescantia micronucleus test is a sensitive bioassay for mutagenesis that may be employed both under field and laboratory conditions. This test has been standardized mostly on the basis of the results obtained with clone 4430. However, this clone is not well adapted to tropical weather, frequently showing problems with growth and flowering. In addition, it is attacked by parasites and insects, a fact that limits its use in field studies aiming at the biomonitoring of air pollution. In the city of São Paulo, Tradescantia pallida (Rose) Hunt. var. purpurea Boom is widely distributed as an ornamental plant in gardens and along roadsides and streets, mostly because of its natural resistance and its easy propagation. In this report, we present dose-response curves indicating that the sensitivity of T. pallida and clone 4430 to X-radiation (1, 10, 25 and 50 cGy) is similar. The results confirm our previous suggestion that T. pallida represents a good alternative for in situ mutagenesis testing in tropical regions, especially biomonitoring studies in which the exposure conditions may not be fully controllable.

Effects of a heat and moisture exchanger and a heated humidifier on respiratory mucus in patients undergoing mechanical ventilation.

OBJECTIVE: To evaluate the effects of a heat and moisture exchanger and a heated humidifier on respiratory mucus and transportability by cilia and cough in patients undergoing invasive mechanical ventilation (up to 72 hrs). DESIGN: Prospective, randomized, clinical study. SETTING: General intensive care unit and university research laboratory. PATIENTS: A total of 32 consecutive patients with acute respiratory failure, who were intubated and mechanically ventilated in the intensive care unit setting, were enrolled in the study. INTERVENTIONS: Patients were randomly assigned to receive as a humidifying system a heat and moisture exchanger (HME) or heated humidified water (HHW) at the onset of mechanical ventilation (time 0). Respiratory mucus samples were collected by suction using a sterile technique at time 0, 24, 48, and 72 hrs of mechanical ventilation. MEASUREMENTS AND MAIN RESULTS: Eleven patients were excluded from this study because of either extubation or death before 72 hrs of mechanical ventilation, leaving 12 patients in the HME group and nine patients in the HHW group. Ventilatory variables including minute volume, mean airway pressure, positive end-expiratory pressure, Fio2, as well as Pao2/Fio2 ratio, fluid balance (last 6 hrs), furosemide, and inotrope administration (last 4 hrs) were recorded. In vitro mucus transportability by cilia was evaluated on the mucus-depleted frog palate model, and the results were expressed as the mucus transport rate. Cough clearance (an estimation of the interaction between the flow of air and the mucus lining the bronchial walls) was measured using a simulated cough machine, the results being expressed in millimeters. Mucus wettability was measured by the contact angle between a mucus sample drop and a flat glass surface. Mucus rheologic properties (mechanical impedance [log G*] and the ratio between viscosity and elasticity [tan delta]) were measured using a magnetic microrheometer at 1 and 100 cGy/sec deformation frequency. The two humidification groups were comparable in terms of the Acute Physiology and Chronic Health Evaluation II score, age, gender, ventilatory variables, fluid balance, use of inotropes, and furosemide. CONCLUSION: Ours results indicate that air humidification with either HME or HHW at 32 degrees C (89.6 degrees F) has similar effects on mucus rheologic properties, contact angle, and transportability by cilia in patients undergoing mechanical ventilation, except for transportability by cough, which diminished after 72 hrs of mechanical ventilation in the HME group (p = .0441).

Acute effects of inhalable particles on the frog palate mucociliary epithelium.

This work was designed to evaluate the toxicity of inhalable particles [less than/equal to] 10 microm in aerodynamic diameter (PM(10)) collected from the urban air in São Paulo, Brazil, to the mucociliary apparatus using the frog palate preparation. Seven groups of frog palates were immersed in different concentrations of PM(10) diluted in Ringer's solution during 120 min: 0 (control, n = 31); 50 (n = 10); 100 (n = 9); 500 (n = 28); 1,000 (n = 10); 5,000 (n = 11); and 10,000 microg/m(3) (n = 10). Mucociliary transport and transepithelial potential difference were determined at 0, 30, 60, and 120 min exposure. Additional groups (control and 500 microg/m(3)) were studied by means of morphometric analyses (quantification of the amount of intraepithelial and surface mucins), measurement of cilia beat frequency, and quantification of total glutathione. Mucociliary transport and transepithelial potential difference were significantly decreased at higher concentrations of PM(10) (p = 0.03 and p = 0.02, respectively). Exposure to PM(10) also elicited a significant decrease of total glutathione (p = 0. 003) and depletion of neutral intraepithelial mucins (p = 0.0461). These results show that PM(10) can promote significant alterations in ciliated epithelium in vitro.

Exploring the clastogenic effects of air pollutants in São Paulo (Brazil) using the Tradescantia micronuclei assay.

This study was designed to determine the clastogenicity of particulate matter (aerodynamic diameter smaller than 10 microm) in the urban polluted air in the city of São Paulo. The Tradescantia-micronucleus (Trad-MCN) assay was used throughout this study to evaluate the clastogenicity of the extracts of the particulate matter. Tradescantia pallida (Rose) Hunt. cv. purpurea, an indigenous cultivar, was used in the Trad-MCN assay. The efficacy of this plant material for the Trad-MCN assay was validated with dose-response studies using formaldehyde and beta radiation. Dose-response curves were established with these known mutagens. The extracts of the PM10 particles at concentrations between 5 and 50 ppm induced a dose-related increase in MCN frequencies. The results indicate that T. pallida is equally sensitive to mutagens as the standard Tradescantia clone 4430 or 03 and the particulate matter in the urban air are clastogenic to the chromosomes of this plant. Inhalation of these particles by urban dwellers may affect their health by inducing similar genetic damage.

The use of the frog palate preparation to assess the effects of oxidants on ciliated epithelium.

This work was designed to develop a simple method based on the frog palate preparation to study the effects of hydrogen peroxide (H2O2) on ciliated epithelium. For this purpose, five sets (n = 10 per set) of frog palate preparations (Rana catesbeiana) were studied during 35 min after immersion in increasing concentrations of H2O2: 1, 8, 16, 32, and 64 microM. The effects of H2O2 on ciliated epithelium were assessed by measuring transepithelial potential difference (PD) and mucociliary transport (MT). Measurements were performed at 5-min intervals. In addition, the palates submitted to the 64 microM dose were immersed in Ringer's solution and followed by another 30 min to assess the possible recovery after maximal injury. Transepithelial potential difference (PD) was measured by means of agar-filled microelectrodes connected to the high input of a grounded electrometer. Mucociliary transport (MT) was determined by directly monitoring the movement of autologous mucus along the palate surface. Significant decrease in MT was observed in 16 microM and beyond and significant change in PD was observed in 32 microM and 64 microM. Palates submitted to 64 microM of H2O2 returned to their baseline levels of PD and MT within 30 min of recovery in Ringer's solution. In conclusion, the frog palate preparation was shown to be an efficient experimental tool to assess the deleterious effects of H2O2 on the ciliated epithelium.

Effects of water pollution on the gill apparatus of fish.

The study was designed to investigate the influence of water pollution on gill apparatus. Specimens of Nile tilapia were collected from a polluted site in the São Paulo metropolitan area (Billings reservoir) and from a "clean" area. Fish from the polluted site showed a chronic inflammatory process in the distal region of the gill filaments, with epithelial hyperplasia. The raker length was increased and hypersecretion occurred, with a considerable volume of alcian blue-positive mucin on the epithelium of the rakers and a smaller volume of periodic acid-Schiff-positive mucin on the epithelial surface of the filaments. The rigidity of mucus in fish from the polluted site was increased, but no abnormalities in the viscosity to elasticity ratio or in mucus "wettability" were observed.

Methods for studying respiratory mucus and mucus clearance

We describe some basic procedures for studying the properties of mucus. These techniques can be applied in both clinical and physiological studies to improve the understanding of the mechanisms related to epithelial defense in health and disease. Mucus collection - A major difficulty is the lack of simple and noninvasive methods for collecting normal mucus in sufficient quantity for later analysis. Physical properties of mucus - A. Rheology: Mucus exhibits both solid and liquid properties and the important factor governing the actual behavior is time. The magnetic microrheometer provides an elegant method for measuring rheological properties of microsamples. B. Adhesivity: It characterizes the forces of attraction between an adherent surface and an adhesive system and can be calculated by measuring the contact angle between a mucus drop and a surface. Mucus Transport - A. Mucus transport by cilia: Mucus is primarily cleared by the continuous ciliary beating, which can be studied using techniques such as the frog palate preparation as well as direct measurement. i.e., in situ mucus clearance. B. Cough clearance: It is essential for elimination of secretions in diseases leading to hypersecretory states. The cough machine simulates the flow-time profile of human coughing. Transepithelial potential difference - A potential difference exists between the epithelial surface and the submucosa and is the net result of the activity of the ion-transport system of the pulmonary epithelium. The potential can be measured using appropriate microelectrodes. Quantitative morphology - Methods may be used to characterize the epithelial surface condition that continuously changes during aggressive conditions.

Methods for studying respiratory mucus and mucus clearance.

We describe some basic procedures for studying the properties of mucus. These techniques can be applied in both clinical and physiological studies to improve the understanding of the mechanisms related to epithelial defense in health and disease. Mucus collection--A major difficulty is the lack of simple and noninvasive methods for collecting normal mucus in sufficient quantity for later analysis. Physical properties of mucus--A. Rheology: Mucus exhibits both solid and liquid properties and the important factor governing the actual behavior is time. The magnetic microrheometer provides an elegant method for measuring rheological properties of microsamples. B. Adhesivity: It characterizes the forces of attraction between an adherent surface and an adhesive system and can be calculated by measuring the contact angle between a mucus drop and a surface. Mucus Transport--A. Mucus transport by cilia: Mucus is primarily cleared by the continuous ciliary beating, which can be studied using techniques such as the frog palate preparation as well as direct measurement, i.e., in situ mucus clearance. B. Cough clearance: It is essential for elimination of secretions in diseases leading to hypersecretory states. The cough machine simulates the flow-time profile of human coughing. Transepithelial potential difference--A potential difference exists between the epithelial surface and the submucosa and is the net result of the activity of the ion-transport system of the pulmonary epithelium. The potential can be measured using appropriate microelectrodes. Quantitative morphology--Methods may be used to characterize the epithelial surface condition that continuously changes during aggressive conditions.

Nasal mucus clearance in rats: differences with sex and phase of the oestrous cycle.

We studied the changes in airway mucus rheology and clearability, as well as in morphometric indices, between male rats and females in either the oestrous or dioestrous phases of the oestrous cycle. Three-month-old Wistar rats were studied and the phases of the oestrous cycle were determined based on analysis of vaginal smears stained by a modified Shorr's procedure. Nasal mucus samples were analysed by means of magnetic rheometry and determination of in vitro transport rate in the frog palate preparation. In situ clearance on the exposed nasal septum was also determined. The mucociliary velocity in situ was significantly affected by both sex and the oestrous cycle phase. In female rats, dioestrous phase clearance was significantly slower than the oestrous phase one. Clearance in male rats was faster than that of both the phases studied in females. Mucus rigidity of females in the oestrous phase was more rigid than that of females in the dioestrous phase and that of males. Mucus viscosity/elasticity ratio for deformations performed at high frequencies was greater for females in comparison with males. Cough clearability computed on the basis of rheological parameters was predicted to be more favourable in females. There were no significant differences between the three groups in mucociliary clearance in vitro. Morphometric studies of the nasal epithelium showed that epithelial and glandular volumes did not exhibit sex- or oestrous-phase-related differences, but the amount of epithelial acidic glycoproteins was lower in females in the oestrous phase in comparison with males and those in the dioestrous phase, the same trend being observed in the glands of the lamina propria.(ABSTRACT TRUNCATED AT 250 WORDS)

Rheological determinants of mucociliary transport in the nose of the rat.

The present work was designed to investigate whether the rheological determinants for nasal mucociliary transport are the same in the intact preparation (in situ), as they are when the mucus is added exogenously to the isolated, mucus-depleted frog palate (in vitro). We evaluated the association between estimators of mucociliary transport in both conditions and rheological parameters using multiple regression techniques. Two kinds of rats were used: (a) specific pathogen free (SPF) rats, representing the normal condition of respiratory epithelium; (b) non-SPF rats (NSPF), which have a chronic inflammatory process in the airways. In situ mucociliary clearance (MCC) was determined by measuring the displacement of charcoal particles placed in the nasal septum. In vitro mucociliary transport (MCT) of rat nasal mucus was measured using the isolated frog palate preparation. Mucus rheologic properties were determined by magnetic microrheometry, in oscillatory deformations performed at 1, 10 and 100 radians/sec. No differences were detected between SPF and NSPF rats in terms of rheological parameters. A decreased MCC was found in NSPF in comparison with the SPF group, but no differences were observed between groups in terms of MCT, as could be predicted by rheological data. When all animals were pooled, in situ transport was significantly associated with the viscosity/elasticity ratio, whereas in vitro transport was dependent on the total mechanical impedance of the mucus sample. In conclusion, in situ mucus transport is influenced by other rheological parameters than those associated with in vitro transportability.

Computer-assisted rheological evaluation of microsamples of mucus.

Dynamic viscoelasticity measurements are required in many studies on biological fluids and they can be performed by determining the corresponding strain when a sinusoidal shear stress is applied to a sample. In several circumstances the amount of fluid that can be obtained for analysis in physiological conditions does not exceed microliters. In this context, the microrheometer technique is a useful approach to determine the dynamic rheological profile of the samples. However, the manual calculation of the desired parameters is tedious and time-consuming. This paper describes a menu-oriented program in order to facilitate its use by non-experts. The comparison between manual and computer-aided calculations demonstrated that the program reduced the time of measurement, and reduced intra- and interobserver variations. The program was developed on an IBM compatible PC in Microsoft C 5.1, and tested in a blind study to check the advantages in terms of time and reproducibility of the system verified by the concordance of two independent observers (interobserver influence) in two different occasions (intraobserver influence).

Correlation between rheologic properties and in vitro ciliary transport of rat nasal mucus.

The relationship between mucus rheologic variables and in vitro ciliary transport was investigated in mucus samples collected from the upper airways of 30 Wistar rats. In vitro mucus transportability was determined by means of the frog palate preparation. Rheologic evaluation was done by measuring the rigidity modulus (log G*, representing the vectorial sum of viscosity and elasticity) and the loss tangent (tan delta, i.e. the ratio between viscosity and elasticity) at 1 and 100 radian/s using a magnetic microrheometer. The correlation between the rheologic variables and in vitro mucus transportability was made by stepwise multiple linear regression analysis, with frog palate transport rate considered as the dependent variable. A significant relationship was obtained between the rheologic parameters (log G* and tan delta) measured at 1 radian/s and the frog palate transport ratio. The relative speed of mucus samples was related to rheology according to the following relationship: rat/frog speed ratio = 1.666-0.434 log G*-0.331 tan delta, for G* and delta determined at 1 radian/s (multiple r = 0.666, p < 0.001). Transport rates predicted from the above formula gave a satisfactory fit to those observed in a second set of 30 rats. The present results indicate that the overall mucus impedance, as well as the ratio between viscosity and elasticity, are important in determining the efficiency of clearance. In addition, it was shown that measurements performed by applying relatively low frequency deformations are preferable for predicting ciliary transport.