Gene expression analysis in interstitial lung edema induced by saline infusion.

To investigate the molecular events taking place during the development of hydraulic interstitial edema, we analyzed by microarray and conventional molecular techniques the variation of gene expression in lung from rabbits treated with slow-rate saline infusions. This analysis indicates that even a condition characterized by a small increase in extravascular water can have a significant influence on the inflammatory milieu. In this regard, cytokines, in particular TNFalpha, can be considered early mediators capable of inducing secondary effects on the injured tissue. Moreover, two MT1 genes were strongly up-regulated, data consistent with their role as protective molecules.

Endothelial cells as early sensors of pulmonary interstitial edema.

We studied responses of endothelial and epithelial cells in the thin portion of the air-blood barrier to a rise in interstitial pressure caused by an increase in extravascular water (interstitial edema) obtained in anesthetized rabbits receiving saline infusion (0.5 ml.kg(-1).min(-1) for 3 h). We obtained morphometric analyses of the cells and of their microenvironment (electron microscopy); furthermore, we also studied in lung tissue extracts the biochemical alterations of proteins responsible for signal transduction (PKC, caveolin-1) and cell-cell adhesion (CD31) and of proteins involved in membrane-to-cytoskeleton linkage (alpha-tubulin and beta-tubulin). In endothelial cells, we observed a folding of the plasma membrane with an increase in cell surface area, a doubling of plasmalemma vesicular density, and an increase in cell volume. Minor morphological changes were observed in epithelial cells. Edema did not affect the total plasmalemma amount of PKC, beta-tubulin, and caveolin-1, but alpha-tubulin and CD-31 increased. In edema, the distribution of these proteins changed between the detergent-resistant fraction of the plasma membrane (DRF, lipid microdomains) and the rest of the plasma membrane [high-density fractions (HDFs)]. PKC and tubulin isoforms shifted from the DRF to HDFs in edema, whereas caveolin-1 increased in DRF at the expense of a decrease in phosphorylated caveolin-1. The changes in cellular morphology and in plasma membrane composition suggest an early endothelial response to mechanical stimuli arising at the interstitial level subsequently to a modest (approximately 5%) increase in extravascular water.

Compositional changes in lipid microdomains of air-blood barrier plasma membranes in pulmonary interstitial edema.

We evaluated in anesthetized rabbits the compositional changes of plasmalemmal lipid microdomains from lung tissue samples after inducing pulmonary interstitial edema (0.5 ml/kg for 3 h, leading to approximately 5% increase in extravascular water). Lipid microdomains (lipid rafts and caveolae) were present in the detergent-resistant fraction (DRF) obtained after discontinuous sucrose density gradient. DRF was enriched in caveolin-1, flotillin, aquaporin-1, GM1, cholesterol, sphingomyelin, and phosphatidylserine, and their contents significantly increased in interstitial edema. The higher DRF content in caveolin, flotillin, and aquaporin-1 and of the ganglioside GM1 suggests an increase both in caveolar domains and in lipid rafts, respectively. Compositional changes could be ascribed to endothelial and epithelial cells that provide most of plasma membrane surface area in the air-blood barrier. Alterations in lipid components in the plasma membrane may reflect rearrangement of floating lipid platforms within the membrane and/or lipid translocation from intracellular stores. Lipid traffic could be stimulated by the marked increase in hydraulic interstitial pressure after initial water accumulation, from approximately -10 to 5 cmH2O, due to the low compliance of the pulmonary tissue, in particular in the basement membranes and in the interfibrillar substance. Compositional changes in lipid microdomains represent a sign of cellular activation and suggest the potential role of mechanotransduction in response to developing interstitial edema.

Signal molecules and receptors in the differential development of cerebellum lobules. Acute effects of cisplatin on nitric oxide and glutamate systems in Purkinje cell population.

Three functionally correlated parameters, nitric oxide (NO), glutamate and NMDA receptors were analyzed through enzymehistochemical and immunohistochemical reactions. A single injection of cisplatin (cisPt) was administered to 10-day-old rats in order to study how Purkinje cells differentiation may be early changed by a mild injury due to the drug during postnatal cerebellar histogenesis. In comparison with age-matched control rats, a correlated decreasing expression of nitric oxide synthase (NOS), glutamate and NMDAR1 was observed in the Purkinje cells of lobules VI-VIII 6 h after the treatment. Moreover, at 24 h after cisPt, the expression of glutamate, NMDAR1 and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd) reactivity was further decreased. In the same period, the ionotropic receptor GluR2 evidenced a less developed dendrite of Purkinje neurons in the top of lobules. In addition, the metabotropic receptor mGluR1alpha revealed unstained areas in the molecular layer, which was entirely stained in control rats; on PD11 this altered pattern was observed in all the lobules and in both the outer and the inner parts. Findings show the importance of NO-glutamate interactions via NMDAR1 in the crucial phases of Purkinje cells differentiation and their involvement on Purkinje neurons dendrite branching as demonstrated by the patterns of the other glutamate receptors. Changes were discussed in relation to an important critical event of Purkinje cell differentiation, i.e. regression of perisomatic spines and elimination of climbing fiber synapses on the somata. Finally, lobules VI-VIII appear to be the most vulnerable ones when cisplatin treatment is administered at 10 days of life, which demonstrates that at this stage some critical developmental changes occur in these lobules and that slower/damaged dendritic tree development is different in the outer versus the inner regions of the lobules.

Cell death may regulate visual functionality in the retina of adults of the dipteran Ceratitis capitata.

The white eye mutation in the medfly Ceratitis capitata, like the homologous mutation in Drosophila melanogaster, was shown to impair visual function. Light and electron microscopy, combined with the DNA-end labelling histochemistry (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) technique), were used to investigate whether programmed cell death may contribute to the morpho-functional differences between the retina of adults from the white eye and wild type strains. Several photoreceptor nuclei in mature white eye flies appeared smaller and showed intensely Toluidine Blue-stained chromatin masses. At the ultrastructural level, they showed different stages of degeneration, resembling apoptotic figures. Positive TUNEL labelling in the white eye retina indicates that apoptosis may be a candidate mechanism for retinal cell degeneration in adult flies, where visual functionality is altered, to achieve the proper cell number. Apoptosis also appears to occur in the wild type retina in early adult life during normal tissue development.

Composition, biophysical properties, and morphometry of plasma membranes in pulmonary interstitial edema.

We evaluated the changes in plasma membrane composition, biophysical properties, and morphology of pulmonary endothelial cells in anesthetized rabbits receiving 0.5 ml. kg(-1). min(-1) saline infusion for 180 min, causing mild interstitial edema. Plasma membrane fractions were obtained from lung homogenates with gradient centrifugation, allowing a sixfold enrichment in caveolin-1. In edematous lungs, cholesterol content and phospholipidic phosphorus increased by 15 and 40%, respectively. These data correlated with morphometric analysis of lungs fixed in situ by vascular perfusion with 2.5% glutaraldehyde, suggesting a relative increase in surface of luminal to interstitial front of the capillary endothelial cells, due to a convoluted luminal profile. In edematous lungs, the fraction of double-bound fatty acids increased in membrane lipids; moreover, the phosphatidylcholine/phosphatidylethanolamine and the cholesterol/phospholipid ratios decreased. These changes were consistent with the increase in fluorescence anisotropy of plasma membrane, indicating an increase in its fluidity. Data suggest that mechanical stimuli elicited by a modest (approximately 4%) increase in extravascular water cause marked changes in plasma membranes that may be of relevance in signal transduction and endothelial cell activation.

Morpho-functional analysis of lung tissue in mild interstitial edema.

Mild pulmonary interstitial edema was shown to cause fragmentation of interstitial matrix proteoglycans. We therefore studied compartmental fluid accumulation by light and electron microscopy on lungs of anesthetized rabbits fixed in situ by vascular perfusion after 0.5 ml.kg(-1).min(-1) iv saline infusion for 180 min causing approximately 6% increase in lung weight. Morphometry showed that a relevant portion (44%) of extravascular fluid is detected early in the alveolar septa, 85% of this fluid accumulating in the thick portion of the air-blood barrier. The arithmetic mean thickness of the barrier increased in interstitial edema from 1.06 +/- 0.05 (SE) to 1.33 +/- 0.06 microm. The harmonic mean thickness increased from 0.6 +/- 0.03 to 0.86 +/- 0.07 microm, mostly due to thickening of the thin portion causing an increase in gas diffusion resistance. Despite some structural damage, the air-blood barrier displays a relatively high structural resistance providing a safety factor against the development of severe edema. It is suggested that the increase in extra-alveolar perivascular space occurs as a consequence of fluid accumulation in the air-blood barrier.

Nitric oxide synthase in the frog cerebellum: response of Purkinje neurons to unilateral eighth nerve transection.

When vestibular damage occurs, nitric oxide synthase (NOS) expression in rat cerebellar flocculus is affected. Since compensation for postural symptoms occurs and Purkinje cells play an important role in movement coordination and motor learning, we analyzed in situ the induction of NOS in the Purkinje cell population of the cerebellum (corpus cerebelli) of frog after unilateral transection of the eighth statoacoustic nerve to gain insight into the role of NO in neural plasticity after injury. Three days after neurectomy, the early effects induced NADPH diaphorase reactivity in most of the Purkinje cells on the ipsilateral side, while on the contralateral side the highest labeling was observed at 15 days. This finding can give information on the dynamics of vestibular compensation, in which NOS involvement was investigated. At 30 days, NADPH diaphorase reactivity was present in a large number of Purkinje cells of the whole cerebellum, while at 60 days a down-regulation for NADPH diaphorase reactivity was evident. A similar trend was observed for NOS-immunoreactivity, which was still present at 60 days in a high percentage of Purkinje cells, mainly on the ipsilateral side. On the basis of cell density evaluations, it was proposed that the early induction of NOS after neurectomy was linked to the degeneration of a part of the Purkinje neurons, while the permanence of NOS labeling might be due to a neuroprotective role of NO in the restoration phase of the vestibular compensation process.