Shay Giles

Enhanced expression of inducible nitric oxide synthase without vasodilator effect in chronically infected lungs

We hypothesized that abnormal ventilation-perfusion matching in chronically infected lungs was in part due to excess nitric oxide (NO) production after upregulation of inducible NO synthase (iNOS) expression. Rats were anesthetized and inoculated intratracheally with Pseudomonas aeruginosa incorporated into agar beads (chronically infected) or with sterile agar beads (placebo inoculated) and killed 10-15 days later. Immunohistochemistry demonstrated increased expression of iNOS and reduced expression of endothelial NOS (eNOS) in chronically infected compared with placebo-inoculated or noninoculated lungs. In isolated lungs from chronically infected rats, NOS inhibition with N ω-nitro-l-arginine methyl ester increased the mean perfusion pressure (14.4 ± 2.7 mmHg) significantly more than in the placebo-inoculated (4.8 ± 1.0 mmHg) or noninoculated (5.3 ± 0.8 mmHg) lungs ( P < 0.01). Although the chronically infected lungs were more sensitive to NOS inhibition, further evidence suggested that the increased iNOS expression was not associated with enhanced iNOS activity. Selective inhibitors of iNOS did not produce an increase in vascular resistance similar to that produced by nonselective inhibitors. Accumulation of nitrate/nitrite in the perfusate of isolated lungs was unchanged by chronic infection. Thus although iNOS expression was increased in chronic pulmonary infection, iNOS activity in the intact lung was not. Nonetheless, endogenous NO production was essential to maintain normal vascular resistance in these lungs.We hypothesized that abnormal ventilation-perfusion matching in chronically infected lungs was in part due to excess nitric oxide (NO) production after upregulation of inducible NO synthase (iNOS) expression. Rats were anesthetized and inoculated intratracheally with Pseudomonas aeruginosa incorporated into agar beads (chronically infected) or with sterile agar beads (placebo inoculated) and killed 10-15 days later. Immunohistochemistry demonstrated increased expression of iNOS and reduced expression of endothelial NOS (eNOS) in chronically infected compared with placebo-inoculated or noninoculated lungs. In isolated lungs from chronically infected rats, NOS inhibition with N(omega)-nitro-L-arginine methyl ester increased the mean perfusion pressure (14.4 +/- 2.7 mmHg) significantly more than in the placebo-inoculated (4.8 +/- 1.0 mmHg) or noninoculated (5.3 +/- 0.8 mmHg) lungs (P < 0.01). Although the chronically infected lungs were more sensitive to NOS inhibition, further evidence suggested that the increased iNOS expression was not associated with enhanced iNOS activity. Selective inhibitors of iNOS did not produce an increase in vascular resistance similar to that produced by nonselective inhibitors. Accumulation of nitrate/nitrite in the perfusate of isolated lungs was unchanged by chronic infection. Thus although iNOS expression was increased in chronic pulmonary infection, iNOS activity in the intact lung was not. Nonetheless, endogenous NO production was essential to maintain normal vascular resistance in these lungs.

Visualizing expression patterns of Shh and Foxf1 genes in the foregut and lung buds by optical projection tomography

Congenital malformations of the foregut are common in humans. The respiratory and digestive tubes are both derived by division of the foregut primordium. Sonic hedgehog (Shh) and Fork head box F1 (Foxf1) genes encode regulatory molecules that play a pivotal role in gut and lung morphogenesis and are therefore important candidate genes to be examined in models of foregut developmental disruption. Optical projection tomography (OPT) is a new, rapid and non-invasive technique for three-dimensional (3D) imaging of small biological tissue specimens that allows visualization of the tissue distribution of RNA in developing organs while also recording morphology. To explore the application of OPT in this context, we visualized Shh and Foxf1 gene expression patterns in the mouse foregut and lung buds at several stages of development. Time-mated CBA/Ca mice were harvested on embryonic days 9–12. The embryos were stained following whole mount in situ hybridization with labelled RNA probes to detect Shh and Foxf1 transcripts at each stage. The embryos were scanned by OPT to obtain 3D representations of gene expression domains in the context of the changing morphology of the embryo. OPT analysis of Shh and Foxf1 expression in the foregut and lung buds revealed extra details of the patterns not previously reported, particularly in the case of Foxf1 where gene expression was revealed in a changing pattern in the mesenchyme around the developing lung. Shh expression was also revealed in the epithelium of the lung bud itself. Both genes were detected in complementary patterns in the developing bronchi as late as E12, showing successful penetration of molecular probes and imaging at later stages. OPT is a valuable tool for revealing gene expression in an anatomical context even in internal tissues like the foregut and lung buds across stages of development, at least until E12. This provides the possibility of visualizing altered gene expression in an in vivo context in genetic or teratogenic models of congenital malformations.

Type 2 nitric oxide synthase and protein nitration in chronic lung infection.

Inflammation in the lung can lead to increased expression of inducible nitric oxide synthase (iNOS) and enhanced NO production. It has been postulated that the resultant highly reactive NO metabolites may have an important role in host defence, although they might also contribute to tissue damage. However, in a number of inflammatory lung diseases, including bronchiectasis, iNOS expression is increased but no elevation of airway NO can be detected. A potential explanation for this finding is that NO is rapidly scavenged by reaction with superoxide radicals, forming peroxynitrite, which is preferentially metabolized via nitration and nitrosation reactions. To test this hypothesis, anaesthetized, specific pathogen‐free rats were inoculated with Pseudomonas aeruginosa incorporated into agar beads (chronically infected group) or sterile agar beads (control group). Ten to 15 days later, the lungs were isolated and fixed. Pseudomonas organisms were isolated from the lungs of the chronically infected group. These lungs showed extensive inflammatory cell infiltration and tissue damage, which were not observed in control lungs. Expression of iNOS was increased in the chronically infected group when compared with the control group. However, the mean number of cells staining for nitrotyrosine in the chronically infected group was not significantly different from that in the controls, nor was there an excess of nitrotyrosine, nitrate, nitrite or nitrosothiol concentrations in the infected lungs. Thus, no evidence was found of increased NO metabolites in chronically infected lungs, including products of the peroxynitrite pathway. These findings suggest that chronic infection does not cause increased iNOS activity in the lung, despite increased expression of iNOS. Copyright

Persistent otitis media with effusion: A new experimental model

Objectives/Hypothesis: Numerous mechanical animal models for the creation of otitis media with effusion (OME) have been described since the 1920s. However, there are many problems associated with these models, including high infection rates, unreliability, and high resolution rates. The aim of the current study was to create a suitable mechanical animal model that would produce a sterile and long‐lasting effusion. Study Design: A new technique using an external surgical approach on specific pathogen–free rats is described. Method: The eustachian tubes of 56 rats were obstructed in the mid portion along the skull base with gutta percha. Results: All animals developed an effusion within 1 week of the procedure. The resolution rate was 8%, with 80% maintaining sterile effusions for up to 1 year. Conclusions: This new procedure for an OME model has proved consistently reliable in creating a persistent and long‐lasting effusion. It has a low infection rate and should benefit future studies on the prolonged effects of OME on the tympanic membrane and middle ear.

The Adriamycin rat/mouse model and its importance to the paediatric surgeon

The teratogenic effect of Adriamycin (doxorubicin) in the rat model, and more recently in the mouse, has provided paediatric surgeons with a reliable, easily reproducible method of studying the embryology and molecular biology for a range of complex congenital anomalies. Concomitantly these animal models have stimulated interest among embryologists for the effect on the notochord, shedding more light on the important organizational role of this structure in the developing embryo. Finally, as more is learnt of the pathogenesis of the various malformations induced by Adriamycin, future therapeutic interventions involving gene therapy, drugs or surgery may arise. This article reviews the establishment of the Adriamycin rat and mouse models, examines their impact on various congenital malformations, and suggests targets for further research.