Elastase-induced Emphysema In Rats Research Articles

Nebulization of Risedronate Sodium Microspheres for Potential Attenuation of Pulmonary Emphysema: a Promising New Insight of Alveolar Macrophage Apoptosis.

Risedronate sodium (RS) is a potent nitrogen-containing bisphosphonate which is known to induce osteoclast apoptosis. As a drug repurposing approach, the current work explored the potential of nebulizable RS-chitosan (CS) microspheres to induce alveolar macrophage apoptosis. RS-CS microspheres were assessed for lung deposition, cytotoxicity, and cellular uptake percentage in Calu-3 cells. The potential of nebulizable microspheres for treating elastase-induced emphysema in rats was investigated, compared to RS marketed oral tablets®, with respect to histopathological, immunohistochemical, and flow cytometric studies. The in vitro lung deposition pattern suggested deep alveolar deposition of RS microspheres, with respect to high FPF% and suitable MMAD (66% and 1.506 μm, respectively, at a flow rate of 28.3 L min-1). No apparent cytotoxicity was observed, with a cell viability > 90%. The inhalation of RS-CS microspheres was suggested to inhibit airspace enlargement and lung rarefaction after elastase instillation and reduce the macrophage accumulation in alveolar parenchyma. Immunohistochemical and cytometric analyses revealed significant low expression levels of CD68 and CD11b surface markers, respectively, with significantly (P < 0.05) lower detected numbers of intact alveolar macrophages following inhalation of RS-CS microspheres. The nebulization of RS-CS microspheres could induce apoptosis in alveolar macrophages and be promisingly adopted for attenuation of pulmonary emphysema.

Gallic acid suppresses inflammation and oxidative stress through modulating Nrf2-HO-1-NF-κB signaling pathways in elastase-induced emphysema in rats.

Emphysema is associated with an abnormal airspace enlargement distal to the terminal bronchioles accompanied by destructive changes in the alveolar walls and chronic inflammation. Air pollution can cause respiratory diseases such as chronic obstructive pulmonary disease (COPD) and emphysema in urban areas. As a natural antioxidant compound, gallic acid may be effective in controlling inflammation and preventing disease progression. In this research, we investigated the protective role of gallic acid in the inflammatory process and the possible signaling pathway in the elastase-induced emphysema. Forty-eight rats were divided into six different groups including the following: control, gallic acid (7.5, 15, and 30 mg/kg), porcine pancreatic elastase (PPE), and PPE+gallic acid 30 mg/kg. Oxidative stress indexes such as malondialdehyde and antioxidant enzyme activity were measured in all groups. The gene expression levels of heme oxygenase-1 (HO-1), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) were determined as key regulators of antioxidant and inflammation system. The PPE group showed pulmonary edema and a significant change in arterial blood gas values, which was associated with decreased antioxidant activity of enzymes and changes in NF-κB, HO-1, and Nrf2 gene expression in comparison to the control group. Co-treatment with gallic acid preserved all these changes approximately to the normal levels. The results confirmed that elastase-induced emphysema leads to lung injuries, which are associated with oxidative stress and inflammation. Also, the results suggested that gallic acid as a natural antioxidant agent can modulate the Nrf2 signaling pathway to protect the lung against elastase-induced emphysema. Therefore, we documented the evidence for the importance of NF-κB inhibitors and Nrf2 activators as a target for new treatments in respiratory dysfunction caused by oxidative agents.

All Trans Retinoic Acid (ATRA) progresses alveolar epithelium regeneration by involving diverse signalling pathways in emphysematous rat

IntroductionPulmonary emphysema is characterized by destruction of alveoli leading to inadequate oxygenation, disability and frequently death. This destruction was understood so far as irreversible. Published data has shown that ATRA (All Trans Retinoic Acid) reverses elastase-induced emphysema in rats. However, the molecular mechanisms governing regeneration process are so far unknown. ObjectiveTo examine the therapeutic potential of ATRA on various molecular pathways and their coordination towards governance of alveolar epithelial regeneration in emphysematous rats. MethodsEmphysema was induced by elastase versus saline in Sprague-Dawley rats. On days 26–37, rats received daily intraperitoneal injections with ATRA (500 μg/kg b.w.) versus olive-oil. Lungs were removed at day 38 for histopathology and investigation of relative mRNA and protein expressions. ResultsHistopathological analysis has shown that losses of alveoli were recovered in therapy (EA) group. Moreover, expressions of markers genes for alveolar cell proliferation, differentiation and EMT events at mRNA and protein levels were significantly increased in EA group than emphysema group (ES). Upon validation at genomics level, expressions of components of Notch, Hedgehog, Wnt, BMP and TGFβ pathways were significantly attenuated in EA group when compared with ES and were well comparable with the healthy group. ConclusionTherapeutic supplementation of ATRA rectifies the deregulated Notch, Hedgehog, Wnt, BMP and TGFβ pathways in emphysema condition, resulting in alveolar epithelium regeneration. Hence, ATRA may prove to be a potential drug in the treatment of emphysema. Nevertheless, elaborated studies are to be conducted.

Therapeutic Treatment with Abdominal Adipose Mesenchymal Cells Does Not Prevent Elastase-Induced Emphysema in Rats.

Emphysema and chronic bronchitis have different pathophysiologies but both are significant components of chronic obstructive lung disease (COPD). The levels of Matrix metalloproteinase (MMP)-9 in the bronchoalveloar lavage fluid (BALF) and in serum indicate the presence of emphysema. Intratracheal administration of elastase has been used to create a rat model of emphysema. Adipose tissue-derived mesenchymal stem cells (MSC) have been postulated to prevent or reverse emphysema, however, this has not been examined in the rat model of elastase-induced emphysema. In this study, 31 Wistar albino rats aged 6-8 weeks and weighing 250-300 g were assessed. On day 1, the animals were treated intratracheally with 0.5 mL saline (control group, n=10), i.e., 0.5 mL saline solution containing 0.1 IU porcine pancreatic elastase (PPE) (Elastase group, n=12) or PPE plus MSC (Elastase-MSC group, n=9) was adminstered per animal. MSCs suspended in serum were injected via the caudal vein on day 21. At least 106 cells were injected. All animals were sacrificed on day 42 and the emphysema index (EI) was calculated, along with measuring the BALF and serum MMP-9 concentrations. Porcine pancreatic elastase induced a significant degree of emphysema in the PPE groups as compared to the control group, which was determined by the EI index (p=0.008). This was not reversed by MSC treatment. The EI remained significantly low in comprison with the controls (p=0.001) and measured no different from the Elastase-treated animals. There was no statistically significant difference between the BALF and serum MMP-9 levels between the control and treatment groups. Our findings suggest that therapeutic treatment with adipose tissue-derived MSC in rats has no effect on emphysema or on MMP9 expression, which is a known marker of emphysema.

ATRA reduces inflammation and improves alveolar epithelium regeneration in emphysematous rat lung

Pulmonary emphysema characterized by alveolar wall destruction is resultant of persistent chronic inflammation. All-trans retinoic acid (ATRA) has been reported to reverse elastase-induced emphysema in rats. However, the underlying molecular mechanisms are so far unknown. To investigate the therapeutic potential effect of ATRA via the amelioration of the ERK/JAK-STAT pathways in the lungs of emphysematous rats. In silico analysis was done to find the binding efficiency of ATRA with receptor and ligands of ERK & JAK-STAT pathway. Emphysema was induced by porcine pancreatic elastase in Sprague-Dawley rats and ATRA was supplemented as therapy. Lungs were harvested for histopathological, genomics and proteomics analysis. In silico docking, analysis confirms that ATRA interferes with the normal binding of ligands (TNF-α, IL6ST) and receptors (TNFR1, IL6) of ERK/JAK-STAT pathways respectively. ATRA restored the histology, proteases/antiproteases balance, levels of inflammatory markers, antioxidants, expression of candidate genes of ERK and JAK-STAT pathways in the therapy group. ATRA ameliorates ERK/JAK-STAT pathway in emphysema condition, resulting in alveolar epithelium regeneration. Hence, ATRA may prove to be a potential drug in the treatment of emphysema.

Evaluation of the Anti-inflammatory Activity of Atorvastatin and its Effect on Alveolar Diameter in a Model of Elastase-induced Emphysema in Rats.

Statins are cholesterol-lowering agents and some of them, like simvastatin, have anti-inflammatory effects. In this study, we evaluated the effect of atorvastatin on nitric oxide (NO) release, leukocytes levels and alveolar diameter related to the inflammatory process associated with elastase-induced emphysema in rats. 32 rats were divided into 4 groups, n=8: control (C), atorvastatin (A), emphysema (E), and emphysema+atorvastatin (EA). On day 0 (zero), groups C and A received intratracheal instillation of saline (0.2 ml), and groups E and EA received elastase (0.2 ml). Groups A and EA received atorvastatin (20 mg/kg) and C and E received vehicle, by gavage, for 25 days. Animals were euthanized, slices of lung stained and the alveolar diameters measured. Data obtained show that the treatment with atorvastatin (EA group) did not reduce the alveolar diameter (35.3 vs. 32.3), NO (2.7 vs. 3.0 μM) or the leukocyte count (111 vs. 136) compared with the E group, indicating that different statins, like simvastatin or atorvastatin, have different behavior in inflammatory processes like in elastase-induced development of emphysema in rats.

Functional and morphological assessment of early impairment of airway function in a rat model of emphysema

The aim of this study was to evaluate airway structure-function relations in elastase-induced emphysema in rats. Sprague-Dawley rats were treated intratracheally with 50 IU porcine pancreatic elastase (PPE, n = 8) or saline (controls, n = 6). Six weeks later, lung volumes [functional residual capacity (FRC), residual volume (RV), and total lung capacity (TLC)] and low-frequency impedance parameters (Newtonian resistance, R(N); tissue damping; tissue elastance, H) were measured, and tracheal sounds were recorded during slow inflation to TLC following in vivo degassing. The lungs were fixed and stained for standard morphometry, elastin, and collagen. In the PPE group, FRC and RV were higher [4.53 ± 0.7 (SD) vs. 3.28 ± 0.45 ml; P = 0.003 and 1.06 ± 0.35 vs. 0.69 ± 0.18 ml; P = 0.036, respectively], and H was smaller in the PPE-treated rats than in the controls (1,344 ± 216 vs. 2,178 ± 305 cmH(2)O/l; P < 0.001), whereas there was no difference in R(N). The average number of crackles per inflation was similar in the two groups; however, the crackle size distributions were different and the lower knee of the pressure-volume curves was higher in the PPE group. Microscopic images revealed different alveolar size distributions but similar bronchial diameters in the two groups. The treatment caused a slight but significant decrease in the numbers of alveolar attachments, no difference in elastin and slightly increased mean level and heterogeneity of collagen in the bronchial walls. These results suggest that tissue destruction did not affect the conventionally assessed airway resistance in this emphysema model, whereas the alterations in the recruitment dynamics can be an early manifestation of impaired airway function.

Effect of Nartogra stim, a Recombinant Human Granulocyte Colony-stimulating Factor on Elastase-induced Emphysema in Rats

Granulocyte colony-stimulating factor (G-CSF) is known to mobilize stem cells to various organs and that it participates in tissue regeneration. Effect of the recombinant human G-CSF nartograstim (CAS 134088-74-7) was tested on elastase-induced emphysema. Porcine pancreas elastase (PPE) was administered intratracheally to male Sprague-Dawley rats to induce parenchymal destruction which was assessed by measuring the mean linear intercept (Lm) in tissue sections as an indicator of air space size. Lung alveoli were destructed and Lm value was significantly increased 2 weeks after PPE instillation. Increase in Lm was sustained for 8 weeks after PPE instillation. Two weeks after PPE instillation, 100 and 200 microg/kg of G-CSF injected for 5 d, followed by once and 3 injections a week for 5 weeks had reversed the increase in Lm by 28.7% (P = 0.02) and 35.2% (P = 0.004), respectively. Coadministration of 100 microg/kg x 5 injection of G-CSF with all-trans-retinoic acid (ATRA; 3 mg/ kg/d) for 3 weeks from 2 weeks after PPE instillation significantly inhibited the increase in Lm by 36% (p < 0.01), whereas administration of G-CSF or ATRA alone did not produce significant improvement. Preventive administration of G-CSF, which was treated for 4 weeks from 4 days after PPE instillation, did not improve enlargement of Lm. These data indicate that the administration of G-CSF is beneficial for the recovery of destructed alveoli.

Retinoic acid as a therapy for emphysema?

In concert with its action as a morphogen during embryonal development, retinoic acid appears to be able to regenerate lung alveoli in an experimental model of elastase-induced emphysema in rats, thereby inhibiting manifestation of the disease. The application to humans is now an interesting possibility.

Oral N-Acetylcysteine Attenuates Elastase-Induced Pulmonary Emphysema in Rats

To study the effect of the antioxidant N-acetylcysteine (NAC) in the development of elastase-induced emphysema in rats. Wistar rats (n = 72) were orotracheally instilled with 75 IU elastase or saline solution. Eighteen rats from each group received the antioxidant NAC from 2 days before induction of the lesion until they were killed 2, 8, and 28 days after instillation. The effects of treatment were assessed by measuring collagen content for the left lung, a histopathology evaluation (ie, mean alveolar internal surface area (AIA) and mean linear intercept measurement), and lung function. Twenty-eight days after elastase instillation, rats treated with NAC showed significant attenuation of the lesion in comparison with rats treated only with elastase, including the following: normalization of mean (+/- SEM) collagen content (1.23 +/- 0.09 vs 1.51 +/- 0.10 mg per left lung, respectively; p < 0.05); partial inhibition of mean AIA (14,860 +/- 1,135 vs 19,622 +/- 1,294 micro m(2), respectively; p < 0.05) and mean linear intercept (108.8 +/- 3.7 vs 123.0 +/- 4.2 micro m, respectively; p < 0.05); and increases and improvement in expiratory flows (27.8 +/- 1.2 vs 23.4 +/- 1.3 mL/s, respectively; p < 0.05). NAC was not able to avoid the compliance increase in the elastase-plus-NAC group. Consistent with the results of anatomic, pathologic, and functional studies, NAC is able to attenuate the lesions induced by elastase in rats, which is in accordance with previous data supporting the idea that oxidant injury could contribute to the development of elastase-induced emphysema.

A novel oral neutrophil elastase inhibitor (ONO-6818) inhibits human neutrophil elastase-induced emphysema in rats.

We investigated the effects of a novel oral neutrophil elastase inhibitor (ONO-6818) on acute lung injury and pulmonary emphysema induced by human neutrophil elastase (HNE). Young male Wistar rats were divided into four treatment groups: (1) control group (saline); (2) HNE group (HNE 200 U + 0.5% carboxymethyl-cellulose [solution for ONO-6818]); (3) low-dose ONO-6818 group (HNE 200 U + ONO-6818 10 mg/kg); and (4) high-dose ONO-6818 group (HNE 200 U + ONO-6818 100 mg/kg). Saline and HNE were applied via the trachea using a microsprayer. ONO-6818 was administered orally 1 hour before HNE application. Six hours after HNE application, neutrophil counts and hemoglobin concentration in bronchoalveolar lavage fluid and lung tissue myeloperoxidase activity were determined. Eight weeks after the application, FRC, TLC, lung compliance, and mean linear intercept were estimated. ONO-6818 attenuated dose-dependently HNE-induced increases in lung myeloperoxidase activity, hemoglobin, and neutrophil count in bronchoalveolar lavage fluid. Furthermore, it significantly attenuated HNE-induced increases in FRC, TLC, lung compliance, and mean linear intercept. ONO-6818 inhibited acute lung injury induced by HNE by minimizing lung hemorrhage and accumulation of neutrophils in the lung. ONO-6818 also inhibited the development of HNE-induced emphysematous changes including lung hyperinflation, degradation of elastic recoil, and airspace enlargement.

Sarcomeres Are Added in Series to Emphysematous Rat Diaphragm After Lung Volume Reduction Surgery

The diaphragm adapts to its shortened state in experimental emphysema primarily by losing sarcomeres in series, thus reducing its optimal operating length. One would expect improved diaphragmatic function after lung volume reduction surgery (LVRS) only if the muscle can readapt to its elevated, lengthened postoperative position by either adding back sarcomeres or lengthening sarcomeres. We used a model of elastase-induced emphysema in rats to test the hypothesis that sarcomere addition occurs following LVRS. A cohort of emphysematous rats was created by the intratracheal instillation of elastase. Five months after the instillation, one group of rats underwent measurement of in situ costal diaphragm length via laparotomy, the determination of optimal muscle fiber operating length (Lo) on stimulated diaphragm strips in vitro, and the measurement of sarcomere length by electron microscopy on strips fixed at Lo. Another group of rats underwent LVRS or sham sternotomy 5 months after the instillation, and 5 months following the operation these animals underwent the same series of diaphragmatic studies. Lo was significantly greater in rats that underwent LVRS than those that underwent sternotomy (mean [+/- SE] Lo after LVRS, 2.50 +/- 0.08 cm; mean Lo after sternotomy, 2.27 +/- 0.06 cm; p = 0.013). There was no significant difference in sarcomere lengths between the two groups (2.95 +/- 0.04 vs 3.04 +/- 0.04 microm, respectively; p = 0.10). Using Lo as the length basis, the mean sarcomere number was calculated to be 8,712 +/- 192 in animals that had undergone LVRS and 7,144 +/- 249 in animals that had undergone sternotomy (p < 0.001). Sarcomere length is not significantly altered but sarcomeres are added in series following LVRS in this experimental model of emphysema/LVRS. It is likely that this sarcomere addition is a prerequisite to the improvement in inspiratory muscle function that has been observed following LVRS in humans.

Pulmonary Vagal Reflexes and Breathing Pattern are not Altered in Elastase-Induced Emphysema in Rats

The role of nonmyelinated and myelinated vagal afferents in pulmonary reflexes and breathing pattern was examined in elastase-treated emphysemic rats. Fourteen to 17 days after intratracheal instillation of 1 IU/gm of porcine pancreatic elastase or 0.5 mL of saline, elastase-treated rats had a decreased alveolar surface area to volume of parenchyma (Sv) (42.44 ± 1.7 vs. 31.51 ± 1.1 mm2/mm3), increased quasistatic compliance (QSC) (1.05 ± 0.06 vs. 1.25 ± 0.09 mL/cm H2O), functional residual capacity (FRC) (4.31 ± 0.10 vs. 5.88 ± 0.37 mL), residual volume (RV) (3.02 ± 0.14 vs. 4.27 ± 0.31 mL), and total lung capacity (TLC) (14.04 ± 0.28 vs. 15.58 ± 0.54 mL). There were no changes in the strength of the pulmonary chemoreflex, the strength of the Hering-Breuer inflation reflex, or breathing pattern before or after vagal perineural capsaicin treatment (VPCT) or vagotomy. There were, however, significant negative correlations between Sv and TLC, FRC and RV, and a near significant (p < . 09) negative correlation between Sv and QSC, but no significant correlations between Sv and indices of either the pulmonary chemoreflex or Hering-Breuer inflation reflex. The results indicate that pulmonary vagal nonmyelinated and myelinated reflex activity and breathing pattern are not affected by elastase-induced emphysema in rats.

Digestion of proteoglycans in porcine pancreatic elastase-induced emphysema in rats

Pulmonary emphysema was induced in rats by a single intratracheal instillation of pancreatic elastase. The short-term effects of elastase instillation on basement membrane components were evaluated using immunohistochemical and biochemical methods. Lung alveoli showed a decrease in heparan sulphate proteoglycan content (especially of its heparan sulphate chains) 3 h to 7 days after induction. Type IV collagen, laminin and fibronectin were not affected. The glycosaminoglycan content of the lung was decreased during the first 3 days after induction, while the glycosaminoglycan concentration in urine was increased during the first 4 days by an increase of heparan sulphate and dermatan sulphate. The increase in urinary glycosaminoglycan content was positively correlated with the extent of emphysema developed after 40 days. We conclude that proteoglycans are target molecules for elastase, and may be involved in the pathogenesis of emphysema.

Behavior of morphometric indices in pancreatic elastase-induced emphysema in rats

Two morphometric indices, the destructive index (DI), a measure of alveolar wall destruction, and the proportion of destroyed alveolar attachments to the airways (AA), have been proposed as measures of early lung destruction in human smokers. The aim of this study was to compare DI and AA to the usual measure of airspace enlargement--the mean linear intercept (Lm)--in experimental emphysema. Porcine pancreatic elastase was administered intratracheally to 2 groups of Brown Norway rats (high-dose, n = 8, 1 IU/g body weight; low-dose, n = 4, 0.7 IU/g; control, n = 7). Total lung capacity (TLC), functional residual capacity (FRC) and pressure-volume curves were measured 3 weeks after administration of elastase. Lung elasticity was assessed by chord compliance (Cst). Administration of high-dose, but not low-dose, elastase led to significant increases in FRC and TLC. Cst significantly increased after high-dose elastase compared to controls (p less than 0.01). Lm increased after both low-dose and high-dose elastase compared to controls (p less than 0.01); DI and AA were increased only after high-dose elastase. Significant correlations were found between each morphometric index and Cst; the highest correlation was with AA. Behavior of the morphometric indices in this model differed from that reported in human smokers: Lm was a more sensitive measure of destruction than DI, reflecting a process marked by predominance of airspace enlargement over alveolar septal breaks. These differences from human smokers may result from a differing underlying pathogenesis of lung destruction.

Augmentation of elastase-induced emphysema by cigarette smoke. Description of a model and a review of possible mechanisms.

Rats were treated with a single endotracheal dose of purified porcine pancreatic elastase (400 IU/kg), exposed to undiluted cigarette smoke from Kentucky 2RI reference cigarettes (one 35-ml puff/min for 10 min daily, 5 days per week, for 12 wk) or with a combination of elastase followed by smoke exposure. A number of significant functional abnormalities were observed in the lungs of rats receiving elastase; these included reduced spontaneous ventilation, enlarged subdivisions of lung volume, loss of elastic recoil, and diminished gas exchange capacity. Rats receiving both elastase and cigarette smoke had significantly more severe pulmonary dysfunction than did rats treated with elastase alone. Morphometric measurements of mean linear intercept demonstrated a loss of alveolar fine structure, with enlargement of distal air spaces in elastase-treated rats. These changes were significantly more severe in rats treated with both elastase and cigarette smoke. Pulmonary function tests and morphometric measurements in sham-treated rats and in rats exposed to cigarette smoke only were not significantly different from those in untreated control animals. It is concluded that elastase-induced emphysema in rats is enhanced by exposure to whole cigarette smoke.