The levels of pre-P2 may provide clues into the pathogenic mechanisms of infertility. The increased proportion of pre-P2 in some patients with increased P1/P2 ratio suggests an involvement of pre-P2 processing. The positive correlation between TUNEL-positive sperm and pre-P2 at low pre-P2/P2 ratios also suggests a link between deficient protamine processing and decreased DNA integrity.
The sGC-cGMP axis is perturbed by chronic exposure to CS. Treatment of COPD animal models with sGC stimulators can prevent CS-induced PH and emphysema.
An association between DNA fragmentation in sperm determined by the terminal deoxynucleotidyl transferase [TdT]-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay and the incidence of reproductive failure has been reported, either using flow cytometry or optical microscopy. However, the results obtained using each of these two approaches are different. Since there is a relative lack of studies standardizing these two approaches, the direct comparison of the results described in the different articles is difficult at present. To allow the comparison of the TUNEL results obtained using flow cytometry and optical microscopy, we applied these two approaches in a total of 66 human sperm samples. A positive correlation is detected in the TUNEL results as measured by flow cytometry and optical microscopy (Spearman; r = 0.720, P 5 0.001). The percentage of TUNEL-positive spermatozoa assessed by flow cytometry is 2.6 times higher than that detected in optical microscopy (39.7% + 23.1% versus 15.3% + 10.3%). Although there is a good correlation of the TUNEL results obtained by flow cytometry and optical microscopy, the percentages obtained with either technique are different. Therefore, the TUNEL results described in the present work should be valuable to compare the results described in many independent articles, using either optical microscopy or flow cytometry. ' InternationalSociety for Analytical Cytology Key terms DNA fragmentation; TUNEL; flow cytometry; microscopy; infertility; sperm IT is known that an increased DNA fragmentation is present in the sperm cells of infertile patients (1-4). In addition, an altered DNA integrity in spermatozoa leads to poorly assisted reproduction results (2,5-17). The selection of spermatozoa to perform ICSI is presently based on its morphology and motility, but these parameters do not give information about DNA integrity. Spermatozoa with damaged DNA may also result in increased risk of anomalies in newborns and increased risk of childhood cancer (18-21). Therefore, laboratory techniques to evaluate DNA integrity are important toward a better management of the infertile patients. The etiology of these strand breaks may involve aberrant recombination, defective chromatin packaging, abortive apoptosis, and oxidative stress (22).There are several approaches available to evaluate DNA integrity in human spermatozoa (23)(24)(25). One of the most widely used techniques is the terminal deoxynucleotidyl transferase [TdT]-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay. Using this approach, the DNA fragmentation is assessed through the labeling with dUTP of the double-and single-stranded DNA breaks present in spermatozoa (26-28; and also references cited in Table 2).The TUNEL assay has been considered as a measure of the DNA strand breaks and as a biological assay for sperm quality at the assisted reproduction laboratories (5,6,9,10,(28)(29)(30)(31). However, there are some factors that may result in variation in the
BackgroundExposure to cigarette smoke has been shown to lead to vascular remodelling. Computed tomography (CT) imaging measures of vascular pruning have been associated with pulmonary vascular disease, an important morbidity associated with smoking. In this study we compare CT-based measures of distal vessel loss to histological vascular and parenchymal changes.MethodsA retrospective review of 80 patients who had undergone lung resection identified patients with imaging appropriate for three-dimensional (3D) vascular reconstruction (n=18) and a second group for two-dimensional (2D) analysis (n=19). Measurements of the volume of the small vessels (3D) and the cross-sectional area of the small vessels (<5 mm2 cross-section) were computed. Histological measures of cross-sectional area of the vasculature and loss of alveoli septa were obtained for all subjects.ResultsThe 2D cross-sectional area of the vasculature on CT imaging was associated with the histological vascular cross-sectional area (r=0.69; p=0.001). The arterial small vessel volume assessed by CT correlated with the histological vascular cross-sectional area (r=0.50; p=0.04), a relationship that persisted even when adjusted for CT-derived measures of emphysema in a regression model.ConclusionsLoss of small vessel volume in CT imaging of smokers is associated with histological loss of vascular cross-sectional area. Imaging-based quantification of pulmonary vasculature provides a noninvasive method to study the multiscale effects of smoking on the pulmonary circulation.
Cigarette smoke (CS) and chronic hypoxia (CH) can produce pulmonary hypertension. Similarities and differences between both exposures and their interaction have not been explored. The aim of the present study was to investigate the effects of CS and CH, as single factors or in combination, on the pulmonary circulation in the guinea pig.51 guinea pigs were exposed to CS for 12 weeks and 32 were sham-exposed. 50% of the animals in each group were additionally exposed to CH for the final 2 weeks. We measured pulmonary artery pressure (Ppa), and the weight ratio between the right ventricle (RV) and left ventricle plus the septum. Pulmonary artery contractility in response to noradrenaline (NA), endothelium-dependent vasodilatation and distensibility were evaluated in organ bath chambers. The number of small intrapulmonary vessels showing immunoreactivity to smooth muscle (SM) a-actin and double elastic laminas was assessed microscopically.CS and CH induced similar increases of Ppa and RV hypertrophy (p,0.05 for both), effects that were further enhanced when both factors were combined. CH increased the contractility to NA (p,0.01) and reduced the distensibility (p,0.05) of pulmonary arteries. Animals exposed to CS showed an increased number of small vessels with positive immunoreactivity to SM a-actin (p,0.01) and those exposed to CH a greater proportion of vessels with double elastic laminas (p,0.05).We conclude that CH amplifies the detrimental effects of CS on the pulmonary circulation by altering the mechanical properties of pulmonary arteries and enhancing the remodelling of pulmonary arterioles.KEYWORDS: Chronic obstructive pulmonary disease, cigarette smoke, hypoxia, pulmonary hypertension, vascular remodelling P ulmonary hypertension is a common and serious complication of chronic obstructive pulmonary disease (COPD). It is considered to result from the effects of chronic hypoxaemia on pulmonary vessels. Indeed, acute and chronic exposure to hypoxia results in smooth muscle cell (SMC) and adventitial fibroblast proliferation [1]. Nevertheless, structural changes in the pulmonary arteries of COPD patients differ from those observed in subjects exposed to a hypoxic environment. Whereas subjects living at high altitudes show medial hypertrophy, COPD patients show prominent changes in the intima [2]. Intimal hypertrophy is present in nonhypoxaemic COPD patients and in smokers with normal lung function, suggesting that vascular changes may be triggered by cigarette smoke (CS) before hypoxaemia develops.Cigarette smoking is associated with endothelial dysfunction [3], increased expression of growth factors [4] and inflammatory cell infiltrate in pulmonary arteries [5]. These factors may induce SMC proliferation and increase pulmonary vascular resistance. The molecular mechanisms by which CS induces vascular changes remain unknown, but they might be related to oxidative damage [6].The effects of hypoxia on human lungs are difficult to characterise because it is usually related to the presence of primary lu...
Sildenafil, a phosphodiesterase-5 inhibitor used to treat pulmonary hypertension, may have effects on pulmonary vessel structure and function. We evaluated the effects of sildenafil in a cigarette smoke (CS)-exposed model of chronic obstructive pulmonary disease (COPD).42 guinea-pigs were exposed to cigarette smoke or sham-exposed and treated with sildenafil or vehicle for 12 weeks, divided into four groups. Assessments included respiratory resistance, pulmonary artery pressure (PAP), right ventricle (RV) hypertrophy, endothelial function of the pulmonary artery and lung vessel and parenchymal morphometry. CS-exposed animals showed increased PAP, RV hypertrophy, raised respiratory resistance, airspace enlargement and intrapulmonary vessel remodelling. CS exposure also produced wall thickening, increased contractility and endothelial dysfunction in the main pulmonary artery. CS-exposed animals treated with sildenafil showed lower PAP and a trend to less RV hypertrophy than CS-exposed only animals. Furthermore, sildenafil preserved the intrapulmonary vessel density and attenuated the airspace enlargement induced by CS. No differences in gas exchange, respiratory resistance, endothelial function and vessel remodelling were observed.We conclude that in this experimental model of COPD, sildenafil prevents the development of pulmonary hypertension and contributes to preserve the parenchymal and vascular integrity, reinforcing the notion that the nitric oxide-cyclic guanosine monophosphate axis is perturbed by CS exposure.
Cigarette smoke (CS) induces an inflammatory process in the lung that may underlie the development of chronic obstructive pulmonary disease (COPD). The nature and characteristics of this process have not been fully established in animal models. We aimed to evaluate the pulmonary inflammatory reaction and its involvement in structural changes in guinea pigs chronically exposed to CS. 19 Hartley guinea pigs were exposed to 7 cigarettes/day, during 3 or 6 months. 18 control guinea pigs were sham-exposed. Numbers of neutrophils, macrophages and eosinophils and lymphoid follicles were assessed in different lung structures. Airway and vessel morphometry, alveolar space size and collagen deposition were also quantified. After 6 months of exposure, CS-exposed guinea pigs showed increased numbers of neutrophils, macrophages and eosinophils in the airways, intrapulmonary vessels and alveolar septa, as well as lymphoid follicles. Increased numbers of muscularized intrapulmonary vessels were apparent at 3 months. After 6 months of exposure, the airway wall thickened and the alveolar space size increased. Collagen deposition was also apparent in airway walls and alveolar septa after 6 months' exposure. The magnitude of airway wall-thickening correlated with the number of infiltrating inflammatory cells, and the extension of collagen deposition correlated with alveolar space size. We conclude that in the guinea pig, 6 months of CS exposure induces inflammatory cell infiltrate in lung structures, at an intensity that correlates with airway remodelling. These changes resemble those observed in COPD, thus endorsing the pathogenic role of CS and the usefulness of this animal model for its study.
Long-acting muscarinic antagonists are widely used to treat chronic obstructive pulmonary disease (COPD). In addition to bronchodilation, muscarinic antagonism may affect pulmonary histopathological changes. The effects of long-acting muscarinic antagonists have not been thoroughly evaluated in experimental models of COPD induced by chronic exposure to cigarette smoke (CS). We investigated the effects of aclidinium bromide on pulmonary function, airway remodeling, and lung inflammation in a CS-exposed model of COPD. A total of 36 guinea pigs were exposed to CS and 22 were sham exposed for 24 weeks. Animals were nebulized daily with vehicle, 10 μg/ml, or 30 μg/ml aclidinium, resulting in six experimental groups. Pulmonary function was assessed weekly by whole-body plethysmography, determining the enhanced pause (Penh) at baseline, after treatment, and after CS/sham exposure. Lung changes were evaluated by morphometry and immunohistochemistry. CS exposure increased Penh in all conditions. CS-exposed animals treated with aclidinium showed lower baseline Penh than untreated animals (P = 0.02). CS induced thickening of all bronchial wall layers, airspace enlargement, and inflammatory cell infiltrate in airways and septa. Treatment with aclidinium abrogated the CS-induced smooth muscle enlargement in small airways (P = 0.001), and tended to reduce airspace enlargement (P = 0.054). Aclidinium also attenuated CS-induced neutrophilia in alveolar septa (P = 0.04). We conclude that, in guinea pigs chronically exposed to CS, aclidinium has an antiremodeling effect on small airways, which is associated with improved respiratory function, and attenuates neutrophilic infiltration in alveolar septa. These results indicate that, in COPD, aclidinium may exert beneficial effects on lung structure in addition to its bronchodilator action.
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