Alexander Zagariya

Cell death and lung cell histology in meconium aspirated newborn rabbit lung.

Abstract Meconium aspiration syndrome (MAS) is a major cause of newborn mortality and morbidity. In this study we investigated the inflammatory responses and morphological changes in the newborn lung to debris-free meconium instillation. We developed a model for studies of MAS using 2-week-old rabbit pups. Cell death was assessed by DNA staining and detection of DNA fragmentation by in situ end labeling. Cell death was seen in association with an increase of inflammatory cytokines levels, studied by ELISA. Necrotic cells were detected by staining of lavage cells with ethidium bromide and 4′,6′-diamino-2′-phenylidon. Meconium instillation resulted selectively in loss of airway and alveolar epithelial cells followed by cell death, which increased with time. Necrotic cells looked smaller and damaged with maximal counts at 24 h after instillation. Conclusion Meconium instillation into lungs caused massive cell death, possibly by apoptosis, and necrosis that may have been activated by the inflammatory cytokine production.

Angiotensin II receptor blockade inhibits pneumocyte apoptosis in experimental meconium aspiration.

Lung tissue inflammation and apoptosis are implicated in the pathogenesis of meconium aspiration–induced lung injury in the newborn, but the mechanisms of these reactions are still poorly known. We investigated the time-dependent leukocyte influx and appearance of apoptosis, as well as the contribution of angiotensin (ANG) II receptor action on these processes in the meconium-induced lung injury. Experimental meconium aspiration was induced by intratracheal instillation of human meconium in 18 rats, and eight rats were further pretreated with an unspecific ANG II receptor inhibitor saralasin. Rats were ventilated with 60% oxygen for 1, 3, or 5 h, and the lungs were then studied histologically for tissue injury and with DNA nick-end labeling and electron microscopy for apoptotic cell death. Lung tissue myeloperoxidase activity and expression of angiotensinogen mRNA and endothelial monocyte–activating polypeptide (EMAP) II protein were also analyzed. The meconium-instilled lungs showed increasing neutrophil migration and histologic injury after the first hour, whereas the number of epithelial apoptotic cells was elevated from the control level throughout the study. Myeloperoxidase activity was high, and the angiotensinogen mRNA and EMAP II protein was up-regulated at 5 h after the meconium insult. Pretreatment with saralasin significantly prevented the increase in lung tissue myeloperoxidase activity, EMAP II, and lung epithelial apoptosis. The results suggest that pulmonary meconium insult rapidly results in epithelial apoptosis, before significant neutrophil sequestration into the lungs. Apoptotic cell death is further connected with ANG II receptor action in the meconium-contaminated lung tissue.

Cytokine expression in meconium-induced lungs.

Objective : In this article the authors present relationship between meconium exposure and inflammatory cytokine release in newborn lungs.Methods : The authors used forty 2-week-old rabbit pups for the study. One-half of the group were instilled with meconium and the other half with saline. Rabbits were sacrificed at 0, 2, 4, 8, and 24 hrs after installation and lung lavage was obtained and was examined for cytokine mRNA expression using RT-PCR and for cytokine proteins using ELISA technique. The data were collected in each of the study group.Results : Meconium instillation caused significant expression of inflammatory cytokines TNFα, IL-6, and IL-8 (p<0.05) with a peak at 8 hrs after meconium instillation. Levels of IL-10 were insignificant (p> 0.05). Also, we found significant increase in necrotic cells and neutrophils (p<0.05), compared to the control, saline instilled rabbit lungs.Conclusion: The present studies demonstrates that meconium induces inflammatory response and cytokines gene and protein expression in the lungs.

Meconium Increases Type 1 Angiotensin II Receptor Expression and Alveolar Cell Death

The pulmonary renin-angiotensin system (RAS) contributes to inflammation and epithelial apoptosis in meconium aspiration. It is unclear if both angiotensin II receptors (ATR) contribute, where they are expressed and if meconium modifies subtype expression. We examined ATR subtypes in 2 wk rabbit pup lungs before and after meconium exposure and with and without captopril pretreatment or type 1 receptor (AT1R) inhibition with losartan, determining expression and cellular localization with immunoblots, RT-PCR and immunohistochemistry, respectively. Responses of cultured rat alveolar type II pneumocytes were also examined. Type 2 ATR were undetected in newborn lung before and after meconium instillation. AT1R were expressed in pulmonary vascular and bronchial smooth muscle and alveolar and bronchial epithelium. Meconium increased total lung AT1R protein approximately 3-fold (p = 0.006), mRNA 29% (p = 0.006) and immunostaining in bronchial and alveolar epithelium and smooth muscle, which were unaffected by captopril and losartan. Meconium also increased AT1R expression >3-fold in cultured type II pneumocytes and caused concentration-dependent cell death inhibited by losartan. Meconium increases AT1R expression in newborn rabbit lung and cultured type II pneumocytes and induces AT1R-mediated cell death. The pulmonary RAS contributes to the pathogenesis of meconium aspiration through increased receptor expression.

Elevated immunoreactive endothelin-1 levels in newborn rabbit lungs after meconium aspiration

Objective Our prime objective was to study the production of big endothelin-1 (Big ET-1) and its conversion to ET-1 in the lungs of newborn rabbits exposed to meconium. Our second objective was to study the effect of captopril on endothelin expression. Design Prospective, comparative study. Setting Research laboratory of the Michael Reese Hospital and the University of Illinois, Chicago. Subjects Two-wk-old rabbit pups. Interventions Rabbit pups were instilled with meconium or saline into the lungs. Another group, pretreated with captopril, was also instilled with either meconium or saline. Measurements and Main Results After meconium or saline instillation, lung lavage was performed. Big ET-1 and ET-1 were measured in lung lavage fluid by using a commercially available enzyme-linked immunosorbent assay kits in all groups. Also, lungs were studied by histochemistry analysis for a morphologic evaluation of meconium-induced damage. In the lavage fluid of saline-instilled pups, ET-1 remained low and no increase in Big ET-1 levels was observed. In meconium-instilled animals, bioactive ET-1 levels were significantly higher, with a peak at 8 hrs after instillation. The conversion ratio of Big ET-1 to ET-1 in the meconium group increased from 2.19 at the initial period to 7.19 at 8 hrs after meconium instillation. Conclusions Our conclusion is that aspiration of meconium causes lung injury in the newborn and that this injury is associated with a significant increase in ET peptide production in the lungs. We also showed that ET production is inhibited by pretreatment of rabbits with captopril before meconium-induced injury. ET-1 and its conversion from ET-1 in response to meconium may play important roles in increasing pulmonary vascular resistance and lung cell death, even in the absence of hypoxia. In general, we conclude, that ET-1 levels are significantly elevated in meconium-instilled rabbits compared with saline-instilled ones, and both can be significantly inhibited by pretreatment with captopril. Whether ET-1 contributes directly to the pathophysiology of or is simply a marker of meconium aspiration syndrome remains speculative.

Characterization of serine/cysteine protease inhibitor α1‐antitripsin from meconium‐instilled rabbit lungs

We have recently purified from meconium‐instilled rabbit lungs a novel serine proteinase inhibitor, with an apparent molecular mass of 50 kDa, which we assign to be α1‐antitripsin. We hypothesize that serpin may attenuate pulmonary inflammation and improve surfactant function after meconium aspiration. α1‐antitripsin is a member of the proteinase inhibitor (serpin) superfamily and inhibitor of neutrophil elastase, and it can be identified as a member of the family by its amino acid sequence due to the high degree of conserved residues. α1‐antitripsin is synthesized by epithelial cells, macrophages, monocytes, and neutrophils. Deficiency in α1‐antitripsin leads to exposure of lungs to uncontrolled proteolytic attack from neutrophil elastase or other damaging factors culminating in lung destruction and cell apoptosis. We hypothesize that accumulation of α1‐antitripsin in the lungs serves as a predisposed protection against meconium‐induced lung injury. In this paper, we show how this knowledge can lead to the development of novel therapeutic approaches for treatment of MAS.

A novel method for detection of apoptosis

There are two different Angiotensin II (ANG II) peptides in nature: Human type (ANG II) and Bovine type (ANG II). These eight amino acid peptides differ only at position 5 where Valine is replaced by Isoleucine in the Bovine type. They are present in all species studied so far. These amino acids are different by only one atom of carbon. This difference is so small, that it will allow any of ANG II, Bovine or Human antibodies to interact with all species and create a universal method for apoptosis detection. ANG II concentrations are found at substantially higher levels in apoptotic, compared to non-apoptotic, tissues. ANG II accumulation can lead to DNA damage, mutations, carcinogenesis and cell death. We demonstrate that Bovine antiserum can be used for universal detection of apoptosis. In 2010, the worldwide market for apoptosis detection reached the

The TNF-α, IL-1-β, IL-6, IL-8, INF-γ, PGE-2 and IL-10 Expression in Meconium Aspirated Newborn Lungs

20 billion mark and significantly increases each year. Most commercially available methods are related to Annexin V and TUNNEL. Our new method based on ANG II is more widely known to physicians and scientists compared to previously used methods. Our approach offers a novel alternative for assessing apoptosis activity with enhanced sensitivity, at a lower cost and ease of use.

The Cell Death and Dramatic Changes in Lung Cell Morphology in Meconium Aspirated Newborn Lungs

The TNF-α, IL-1-β, IL-6, IL-8, INF-γ, PGE-2 and IL-10 Expression in Meconium Aspirated Newborn Lungs

Apoptosis of airway epithelial cells in response to meconium

The Cell Death and Dramatic Changes in Lung Cell Morphology in Meconium Aspirated Newborn Lungs