The Substitution of Fifty Percent of Combustible Tobacco Smoke Exposure With Either Electronic Cigarettes or Heated tobacco Products Did Not Attenuate Acute Lung Injury in an Animal Model.

BACKGROUND: To reduce the harmful health effects of combustible cigarette smoke (CS), some (CS) users attempt to substitute CS with electronic cigarettes (ECIG) and/or heated tobacco products (HTP). In this animal study, we evaluated the acute effects of substituting CS consumption with ECIG or HTP thus mimicking the dual users' approach, on the lungs of a mouse model. METHODS: C57BL/6 mice were divided into Control, ECIG, HTP, CS, ECIG + CS, HTP + CS, and HTP + ECIG groups. Animals were exposed for 3 hours in AM and PM sessions to either air, CS, ECIG, or HTP for seven days. Lung injury was assessed by: wet to dry (W/D) ratio, albumin concentration in bronchoalveolar lavage fluid, expression of IL-1ß, IL-6, and TNF-α, histopathology examination, reactive oxygen species (ROS) production, and assessment of cellular apoptosis. RESULTS: W/D ratio was significantly increased in mice exposed to CS only. Albumin leak and expression of IL-1ß, IL-6, and TNF-a were elevated in CS, ECIG + CS, and HTP + CS. Histological examination revealed significant inflammatory cells infiltration, as well as collagen deposit in CS, ECIG + CS, HTP + CS. ROS production was significantly increased in CS, ECIG + CS, HTP + CS. Finally, cell death was also significantly increased in CS, ECIG + CS, and HTP + CS. CONCLUSION: In this animal model, substituting 50% of daily CS exposure by either ECIG or HTP exposure did not result in significant attenuation of acute lung injury.

Role of diabetes in lung injury from acute exposure to electronic cigarette, heated tobacco product, and combustible cigarette aerosols in an animal model.

BACKGROUND: Patients with diabetes are more vulnerable to the detrimental respiratory effects of combustible cigarette smoke (CS) when compared to the general population. Electronic cigarettes (ECIG) and heated tobacco products (HTP) are marketed as less harmful alternatives to CS. In this study, we compared the effects of acute ECIG, HTP and CS exposure on the lungs of type II diabetes versus non-diabetic mice in an animal model. METHODS: Type II Diabetic (Diab) and Non-Diabetic (Non-Diab) mice were divided into Control, ECIG, HTP and CS groups. Animals were exposed for 6 hrs./day to either air, ECIG, HTP or CS for seven days. Lung injury was determined by a) histopathology, b) wet to dry ratio, c) albumin concentration in bronchoalveolar lavage fluid, d) expression of TNF-α, IL-6, and IL-1 ß, e) reactive oxygen species production (ROS), and f) assessment of cellular apoptosis. RESULTS: Lung histology revealed increased edema and inflammatory cells in diabetic mice exposed to ECIG, HTP and CS. The expression of Inflammatory mediators was, in general, more significant in the Diabetic groups as well. TNF-α expression, for example, was upregulated in Diab + ECIG but not in Non-Diab + ECIG. ROS was significantly increased in Diab + CS, less in Non-Diab + CS and weakly noted in ECIG + Diab. Significant albumin leak was observed in Diab and Non-Diab HTP-exposed animals. CS exposure worsened lung injury in Diab when compared to Non-Diab mice. CONCLUSION: Comorbid medical conditions like diabetes may amplify ill effects of CS, ECIG or HTP exposure.

Antioxidant activity of pomegranate juice reduces emphysematous changes and injury secondary to cigarette smoke in an animal model and human alveolar cells.

BACKGROUND: Cigarette smoke (CS) increases oxidative stress (OS) in the lungs. Pomegranate juice (PJ) possesses potent antioxidant activities, attributed to its polyphenols. This study investigates the effects of PJ on the damaging effects of CS in an animal model and on cultured human alveolar cells (A549). METHODS: Male C57BL/6J mice were divided into the following groups: Control, CS, CS + PJ, and PJ. Acute CS exposure was for 3 days, while chronic exposure was for 1 and 3 months (5 days of exposure/week). PJ groups received daily 80 µmol/kg via bottle, while other groups received distilled water. At the end of the experiments, different parameters were studied: 1) expression levels of inflammatory markers, 2) apoptosis, 3) OS, and 4) histopathological changes. In vitro, A549 cells were pretreated for 48 hours with either PJ (0.5 µM) or vehicle. Cells were then exposed to increasing concentrations of CS extracted from collected filters. Cell viability was assessed by counting of live and dead cells with trypan blue staining. RESULTS: Acutely, a significant increase in interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α expression, apoptosis, and OS was noted in CS when compared to Control. PJ significantly attenuated the expression of inflammatory mediators, apoptosis, and OS. Chronically (at 1 and 3 months), increased expression of TNF-α was observed, and lung sections demonstrated emphysematous changes when compared to Control. PJ supplementation to CS animals attenuated the increased expression of TNF-α and normalized lung cytoarchitecture. At the cellular level, CS extract reduced cellular proliferation and triggered cellular death. Pretreatment with PJ attenuated the damaging effects of CS extract on cultured human alveolar cells. CONCLUSION: The expression of inflammatory mediators associated with CS exposure and the emphysematous changes noted with chronic CS exposure were reduced with PJ supplementation. In vitro, PJ attenuated the damaging effects of CS extract on cultured human alveolar cells.

The SF-36 and 6-Minute Walk Test are Significant Predictors of Complications After Major Surgery.

BACKGROUND: Major surgeries are associated with postoperative morbidity and mortality. Current preoperative evaluation fails to identify patients at increased risk of postoperative complications. This study is aimed to determine whether the Short Form-36 health survey (SF-36) and the 6-minute walk test (6-MWT) are useful predictors of postoperative complications after major surgery. METHODS: All patients scheduled to undergo major surgery were eligible for the study. Major surgeries include patients undergoing thoracotomy, sternotomy, or upper abdominal laparotomy. The SF-36 health survey and 6-MWT were administered prior to surgery. Spirometry and other preoperative testing, ordered by the surgeon, like echocardiography were included in the study. Patients were then followed-up for postoperative complications for 30 days. RESULTS: One-hundred and seventeen subjects undergoing major surgery were recruited to the study. The mean age was 58 years and 66 (56.4%) were male. Physical Functioning as a component of the SF-36 positively correlated with decreased length of hospital stay (LOS). The 6-MWT had a negative correlation with LOS (p < 0.0001) and with severity of postoperative complications (p < 0.0001). Spirometry and echocardiography did not correlate with LOS or grade of complications. CONCLUSIONS: SF-36 (Physical Functioning) and 6-MWT are useful indicators for predicting postoperative complications and LOS. Patients undergoing major surgery answered SF-36 and performed 6-MWT. Physical Functioning as a component of the SF-36 correlated with LOS. The 6-MWT had a negative correlation with LOS and with complication grade. SF-36 and 6-MWT are useful predictors of postoperative complications.

Antioxidant activity of pomegranate juice reduces acute lung injury secondary to hyperoxia in an animal model.

BACKGROUND: Hyperoxia triggers the release of toxic reactive oxygen species (ROS). Pomegranate Juice (PJ) is a rich source of potent antioxidants. We assessed the effects of PJ supplementation on Acute Lung Injury (ALI) in adult rats exposed to hyperoxia for 5 days. METHODS: Adult rats were divided into four different groups: control, hyperoxia, hyperoxia + PJ and PJ. Animals were placed in chambers containing either room air or oxygen above 95% for a total of 5 days. Two different PJ concentrations were utilized and the control group received placebo water. Animals were euthanized and their lungs were excised. Assessment of lung injury was accomplished by: a) wet to dry ratio (W/D) method, b) measurement of albumin concentration in the bronchoalveolar lavage fluid (BALF), c) oxidative stress, d) histological evaluation of the lung e) apoptosis and f) transcriptional expression levels of the inflammatory mediators IL-1ß, IL-6 and TNF-alpha. RESULTS: An increase in the W/D and albumin leak was noted in Hyperoxia (p < 0.05). Those findings were attenuated by the higher dose of PJ supplementation. Hyperoxia increased ROS production. Again PJ significantly reduced oxidative stress. Lung sections showed significant reduction in inflammation, edema, and infiltrating neutrophils in Hyperoxia + 80 µmol/kg when compared with Hyperoxia. TUNEL demonstrated significant apoptosis in the Hyperoxia, which was diminished in the Hyperoxia + 80 µmol/kg. Furthermore, increase in IL-1ß and IL-6 was noted in Hyperoxia. Again, 80 µmol/kg of PJ significantly reduced the expression of inflammatory mediators. CONCLUSION: In this animal model, PJ supplementation attenuated ALI associated with hyperoxia.

Apoptosis and the activity of ceramide, Bax and Bcl-2 in the lungs of neonatal rats exposed to limited and prolonged hyperoxia.

BACKGROUND: The aim of the study is to examine the effect of limited and prolonged hyperoxia on neonatal rat lung. This is done by examining the morphologic changes of apoptosis, the expression of ceramide, an important mediator of apoptosis, the expression of inflammatory mediators represented by IL-1beta and the expression of 2 proto-oncogenes that appear to modulate apoptosis (Bax and Bcl-2). METHODS: Newborn rats were placed in chambers containing room air or oxygen above 90% for 7 days. The rats were sacrificed at 3, 7 or 14 days and their lungs removed. Sections were fixed, subjected to TUNEL, Hoechst, and E-Cadherin Staining. Sections were also incubated with anti-Bcl-2 and anti-Bax antisera. Bcl-2 and Bax were quantitated by immunohistochemistry. Lipids were extracted, and ceramide measured through a modified diacylglycerol kinase assay. RT-PCR was utilized to assess IL-1beta expression. RESULTS: TUNEL staining showed significant apoptosis in the hyperoxia-exposed lungs at 3 days only. Co-staining of the apoptotic cells with Hoechst, and E-Cadherin indicated that apoptotic cells were mainly epithelial cells. The expression of Bax and ceramide was significantly higher in the hyperoxia-exposed lungs at 3 and 14 days of age, but not at 7 days. Bcl-2 was significantly elevated in the hyperoxia-exposed lungs at 3 and 14 days. IL-1beta expression was significantly increased at 14 days. CONCLUSION: Exposure of neonatal rat lung to hyperoxia results in early apoptosis documented by TUNEL assay. The early rise in Bax and ceramide appears to overcome the anti-apoptotic activity of Bcl-2. Further exposure did not result in late apoptotic changes. This suggests that apoptotic response to hyperoxia is time sensitive. Prolonged hyperoxia results in acute lung injury and the shifting balance of ceramide, Bax and Bcl-2 may be related to the evolution of the inflammatory process.

Regulation of the sphingolipid signaling pathways in the growing and hypoxic rat heart.

Sphingolipids (SLs) have a biomodulatory role in physiological as well as pathological cardiovascular conditions. This study aims to assess the variation of SL mediators and metabolizing enzymes in the growing and hypoxic rat heart. Sprague-Dawley rats were placed in a hypoxic environment at birth. Control animals remained in room air. In control animals, activities of acidic-sphingomyelinase (A-SMase), sphingomyelin synthase (SMS), glucosylceramide synthase (GCS), and ceramidase decreased with age in both ventricles whereas activity of neutral-sphingomyelinase (N-SMase) increased with age. Hypoxic RV mass was 171 and 229% that of controls, at 4 and 8 weeks, respectively. This was accompanied by an increase in RV myocardial ceramide synthesis, consumption and breakdown, with a net effect of suppression of ceramide accumulation and increase in diacylglycerol (DAG) concentration. In addition, significant increase in activities of: A-SMase by 26 and 29%, SMS by 108 and 40%, and ceramidase by 66 and 35%, in the hypoxic RV rats as compared to controls, was noted at 4 and 8 weeks of age, respectively. Sphingolipids and their regulating enzymes appear to play a role in adaptive responses to chronic hypoxia in the neonatal rat heart.

Tissue-specific ceramide response in the chronically hypoxic rat model mimicking cyanotic heart disease.

BACKGROUND AND OBJECTIVE: Acute hypoxia is associated with apoptosis and increase in ceramide levels in various organs. To assess the effect of chronic hypoxia on ceramide accumulation in the lungs and kidneys, we utilized an animal model mimicking cyanotic heart disease. METHODS: Rats were placed in a hypoxic environment at birth and oxygen levels were maintained at 10% in an air-tight Plexiglas chamber. Controls remained in room air. Animals were sacrificed and the lung and kidneys were harvested and weighed at 1 and 4 weeks, respectively. Ceramide levels were measured using a modified diacylglycerol kinase assay. RESULTS: Significant polycythemia developed in the hypoxic rats at 1 and 4 weeks. Indexed lung and kidney masses were significantly increased in the hypoxic animals as compared to controls at 1 and 4 weeks, respectively. The ceramide levels in the hypoxic lungs and kidneys were not significantly different from control groups at 1 and 4 weeks. [Ceramide/phosphate ratio in the kidneys was 1.28 +/- 0.17 (C) versus 1.18 +/- 0.12 (H) at 1 week; P = 0.39, and 1.46 +/- 0.08 (C) versus 1.33 +/- 0.15 (H) at 4 weeks (P = 0.44)] and [ceramide/phosphate ratio (pmol/nmol) in the lungs was 2.29 +/- 0.14 (C) versus 1.98 +/- 0.12 (H) at 1 week (P = 0.17), and 2.42 +/- 0.16 (C) versus 2.30 +/- 0.05 (H) at 4 weeks, P = 0.34]. CONCLUSION: The response of lungs and kidneys to chronic hypoxia includes increase in indexed mass and lack of ceramide accumulation. This is similar to the response previously reported in the chronically hypoxic brain and heart. Thus, various organs appear to have similar ceramide response pattern to chronic hypoxia.

Cardiac growth patterns in response to chronic hypoxia in a neonatal rat model mimicking cyanotic heart disease.

OBJECTIVE: Myocardial growth during fetal life is accomplished by the proliferation of myocytes. Shortly after birth, normal myocytes largely lose their capability to replicate. The present study aims to assess the effect of persistent postnatal hypoxia on myocardial growth patterns in an animal model mimicking cyanotic heart disease. METHODS: Sprague-Dawley rats were placed in a normobaric hypoxic environment at birth and oxygen levels were maintained at 10% (group H). Controls (group C) remained in room air. The animals were sacrificed and the hearts were harvested at one, four and eight weeks. RESULTS: Significant polycythemia developed in the hypoxic rats. There was a significant increase in indexed right ventricle (RV) and left ventricle (LV) masses compared with controls. Myocardial DNA concentrations were significantly increased in both ventricles of the hypoxic rats. For the RV, the increase in DNA content for group H was 135%, 132% and 112% that of group C values at one, four and eight weeks, respectively. RV and LV myocardial protein:DNA ratios were lower in the one-week- and four-week-old hypoxic rats, and significantly higher in the hypoxic eight-week-old rats. Polyploidy, hydroxyproline concentrations and dry:wet weight ratios were not significantly different in the LV and RV between both group H and group C animals. CONCLUSION: Cardiac mass increases in response to chronic hypoxia were greater in the RV than the LV. This increase was mainly due to myocardial proliferation in the first four weeks of life. Although the three groups of hypoxic rats had significant elevations in DNA concentration compared with controls, there was a shift from proliferation to hypertrophy after week 4 of life. The age of the myocyte appears to be the most important factor in triggering proliferation in this hypoxic animal model.

Lack of apoptosis in the hypoxic brain of a rat model mimicking cyanotic heart disease.

OBJECTIVE: To assess the effect of chronic hypoxia on brain neuronal apoptosis, an animal model mimicking cyanotic heart disease was utilized. METHODS: Rats were placed in an hypoxic environment at birth and oxygen levels were maintained at 10% in an air-tight Plexiglass chamber. Controls remained in room air. Animals were sacrificed and the brains were harvested at 1 and 4 weeks, respectively. RESULTS: Significant polycythemia developed in the hypoxic rats at 1 and 4 weeks. Indexed brain mass to body weight was significantly increased in the hypoxic groups by 18% (p < 0.01) and 38% (p < 0.01) as compared to controls at 1 and 4 weeks, respectively. There was no difference in the number of apoptotic neurons between the chronically hypoxic rats and controls, as assayed by TUNEL labelling and Hoechst staining. The role of the sphingolipid ceramide was then examined because of its reported role in stress response, growth suppression and apoptosis. It was found that the brain ceramide accumulation was not significantly different in the hypoxic and control groups at 1 and 4 weeks. CONCLUSION: A protective adaptive response to chronic hypoxia in the neonatal brain may exist.

Modulation of ceramide content and lack of apoptosis in the chronically hypoxic neonatal rat heart.

To assess the effect of chronic hypoxia on cardiomyocyte apoptosis, we used an animal model that mimics cyanotic heart disease. Rats were placed in a hypoxic environment at birth, and oxygen levels were maintained at 10% in an air-tight Plexiglas chamber. Controls remained in room air. Animals were killed, and the hearts were harvested at 1 and 4 wk. Significant polycythemia developed in the hypoxic rats at 1 and 4 wk. Right ventricular mass in the hypoxic rats was 192% and 278% that of controls, and hypoxic left ventricular mass was 140% and 178% that of the controls at 1 and 4 wk, respectively. The increase in cardiac mass was paralleled by only mild hypertrophy (10 to 20%). Contrary to previous reports showing increased apoptosis in response to hypoxia in cultured cardiomyocytes, there was no difference in the number of apoptotic cardiomyocytes between the chronically hypoxic rats and controls, as assayed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and Hoechst staining. We then examined the role of the sphingolipid ceramide because of its reported role in the stress response, growth suppression, and apoptosis. We found that the right ventricular ceramide content was significantly decreased in the hypoxic rats to 73% of control levels at the age of 4 wk. We suggest that the decrease in the ceramide content in the hypoxic right ventricular rat heart may be an adaptive response to chronic hypoxia and pulmonary hypertension. Lower ceramide levels may help suppress apoptosis and allow compensatory right ventricular cardiomyocyte proliferation.