Davide Grisafi

Human amniotic fluid stem cells protect rat lungs exposed to moderate hyperoxia.

Treatment of bronchopulmonary dysplasia (BPD) remains as yet an unmet clinical need and recently stem cells have been proposed as a therapeutic tool in animal models. We investigated the role of amniotic fluid stem cells (AFS) in an adult rat model of hyperoxia lung injury.

Cyclosporine and hyperoxia-induced lung damage in neonatal rats ☆

Cyclosporine effects on hyperoxia-induced histopathological and functional changes in the rat adult lung are controversial and the newborn lung has not been studied. Thus, we evaluated the effects of cyclosporine in young rats after 60% hyperoxia exposure postnatally. Experimental categories included: (1) room air for the first 5 postnatal weeks with daily subcutaneous injections of saline from postnatal day (PN)15 to PN35; (2) room air with daily injections of cyclosporine from PN15 to PN35; (3) 60% oxygen from PN0 to PN14 and then daily saline injections during the following three weeks; (4) 60% oxygen from PN0 to PN14 followed by cyclosporine treatment from PN15 to PN35. Hyperoxia significantly reduced the number of secondary crests and microvessel density, and it increased the mean alveolar size and septa thickness. Cyclosporine treatment did not significantly modify the hyperoxia-induced changes. Conversely, in normoxia, cyclosporine reduced microvessel density and the number of secondary crests. In conclusion, cyclosporine did not modify alveolar and microvascular parameters in hyperoxia exposure, although it caused some changes in normoxia.

Fractal analysis of alveolarization in hyperoxia-induced rat models of bronchopulmonary dysplasia

No papers are available about potentiality of fractal analysis in quantitative assessment of alveolarization in bronchopulmonary dysplasia (BPD). Thus, we here performed a comparative analysis between fractal [fractal dimension (D) and lacunarity] and stereological [mean linear intercept (Lm), total volume of alveolar air spaces, total number of alveoli, mean alveolar volume, total volume and surface area of alveolar septa, and mean alveolar septal thickness] parameters in experimental hyperoxia-induced models of BPD. At birth, rats were distributed between the following groups: 1) rats raised in ambient air for 2 wk; 2) rats exposed to 60% oxygen for 2 wk; 3) rats raised in normoxia for 6 wk; and 4) rats exposed to 60% hyperoxia for 2 wk and to room air for further 4 wk. Normoxic 6-wk rats showed increased D and decreased lacunarity with respect to normoxic 2-wk rats, together with changes in all stereological parameters except for mean alveolar volume. Hyperoxia-exposed 2-wk rats showed significant changes only in total number of alveoli, mean alveolar volume, and lacunarity with respect to equal-in-age normoxic rats. In the comparison between 6-wk rats, the hyperoxia-exposed group showed decreased D and increased lacunarity, together with changes in all stereological parameters except for septal thickness. Analysis of receiver operating characteristic curves showed a comparable discriminatory power of D, lacunarity, and total number of alveoli; Lm and mean alveolar volume were less discriminative. D and lacunarity did not show significant changes when different segmentation thresholds were applied, suggesting that the fractal approach may be fit to automatic image analysis.

Lipopolysaccharide-induced chorioamnionitis and postnatal lung injury: The beneficial effects of L-citrulline in newborn rats

Abstract Aim of the study: The lung architecture of newborns appears to be affected by an inflammatory reaction to maternal choriodecidual layer infection. L-citrulline (L-Cit) was administered to pregnant rats exposed to intra-amniotic lipopolysaccharide (LPS)-induced chorioamnionitis to investigate its effect on neonatal lung injury. Materials and Methods: The pups were assigned to four experimental groups: 1- pups exposed to intra-amniotic NaCl but not to postnatal L-Cit (Controls); 2 - pups exposed to intra-amniotic NaCl as well as to postnatal L-Cit treatment (L-Cit group); 3 - pups exposed to prenatal LPS but not to postnatal (LPS); 4- pups exposed to prenatal LPS as well as to postnatal L-Cit treatment (LPS + L-Cit). Some pups in each group were sacrificed on postnatal (P) day 3 and others on day 7. The pups’ lungs were harvested for morphometric analysis; cytokine, arginase 1, and VEGF values were quantified. Serum arginine, citrulline, asymmetric dimethylarginine (ADMA), symmetric dimethylarginine, NG-monomethyl arginine, and homoarginine levels were determined using UPLC-MS/MS. Results: L-Cit attenuated the disruption of alveolar growth in the LPS + L-Cit group. Arginine, homo-arginine, and ADMA levels fell in the LPS treated groups. Arginine and ADMA rose at P7 in the L-Cit group whose members also showed higher VEGF levels with respect to the Controls. The Controls, instead, showed higher IL-10 and IL-1β values with respect to the L-Cit group at P7. Arginase 1 was higher in the LPS groups with respect to the Controls at P7. Conclusions: L-Cit improved alveolar and vascular growth diminishing the lung inflammatory response in the newborn rats exposed to intra-amniotic LPS. The ADMA/DDAH/NO pathway appeared to counteract proinflammatory cytokine production and to sustain macrophage migration.

L-citrulline is protective in hyperoxic lung damage and improves matrix remodelling and alveolarization

Moderate hyperoxia alters alveolar and vascular lung morphogenesis. Nitric oxide (NO) and matrix metalloproteinases (MMP) have a crucial role in the homeostasis of the matrix and bronchoalveolar structure and may be regulated abnormally by exposure to hyperoxia. Disruption of vascular endothelial growth factor (VEGF)-NO signaling impairs vascular growth and contributes to hyperoxia-induced vascular disease in bronchopulmonary dysplasia (BPD). We hypothesize that L-citrulline, by raising the serum levels of L-arginine and enhancing endogenous NO synthesis, might attenuate hyperoxia-induced lung injury in an experimental model of BPD. Neonatal rats (1 day old) were exposed to 60% oxygen or room air for 14 days and administered L-citrulline or a vehicle (sham). Lung morphometry were performed; Serum was tested for arginine level; Matrix metalloproteinases2 (MMP2) gene expression, VEGF gene and protein expression and endothelial NO synthase (eNOS) protein expression were compared. Mean linear intercept was higher in the hyperoxia and sham groups when compared with the room air (RA) and L-citr+hyperoxia treated group (p<0.02). Secondary crests number was higher in L-citrulline treated and RA when compared to hyperoxia and sham group (p<0.02). L-Arginine level rose in the L-citrulline-treated group (p<0.05). L-citrulline did not affect MMP2 gene expression, but it regulated the MMP2 active protein, which rose in bronchoalveolar lavage fluid (p<0.05), presumably due to a post-transductional effect. Compared with RA controls, hyperoxia significantly decreased VEGF and eNOS protein expression. At the same time, an increased lung VEGF gene and protein expression (p<0.05) were also seen in the rats treated with L-citrulline. We conclude that: (i) hyperoxia decreases growth and disrupts VEGF-NO signaling of lung; (ii) the main effects of L-citrulline are an increased serum level of arginine, as a promoter and a substrate of the nitric oxide synthase; and (ii) a better alveolar growth and matrix control than in hyperoxia-induced lung damage seems promising.

Hyperoxia-induced changes in morphometric parameters of postnatal neurogenic sites in rat

In literature many works address the effects of hypoxia exposure on postnatal neurogenesis but few data are available about hyperoxia effects, although high oxygen concentrations are frequently used for ventilation of premature newborns. Thus, the aim of the present study was to compare with controls the morphometrical parameters of the main neurogenic sites (subventricular zone and dentate gyrus) in newborn Sprague-Dawley rats exposed to 60% or 95% oxygen for the first 14 postnatal days. Six rats were studied for each of the three groups. The unbiased quantitative method of the optical disector was applied to analyze neuronal densities, nuclear volumes, and total neuron numbers of the subventricular zone and hippocampal dentate gyrus. Apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling, TUNEL) and proliferation (Ki67) were also studied. The subventricular zone of newborn rats exposed to 95% hyperoxia showed statistically significant higher volume (mean value ± coefficient of variation: 0.40 ± 0.20 mm3) than subventricular zone of rats raised in normoxia (0.20 ± 0.11 mm3) or 60% hyperoxia (0.26 ± 0.18 mm3). Total neuron number was also significantly higher in 95% hyperoxia while neuronal densities did not reach statistically significant differences. TUNEL showed increased apoptotic indexes in hyperoxic rats. The percentage of proliferating KI67 positive cells was also higher in hyperoxia. The dentate gyrus granular layer of the normoxic rats showed higher volume (0.65 ± 0.11 mm3) than both the hyperoxic groups (60% hyperoxia: 0.39 ± 0.14 mm3; 95% hyperoxia: 0.36 ± 0.16 mm3). Total neuron numbers of hyperoxic dentate gyrus were also significantly reduced; neuronal densities were not modified. Hyperoxia-exposed rats also showed higher apoptotic and proliferating indexes in the dentate gyrus. Hyperoxia exposure in the first postnatal period may affect the main neurogenic areas (subventricular zone and dentate gyrus) increasing apoptosis but also inducing a certain reparative response consisting of increased proliferation. In particular, the increased volume of the subventricular zone may be ascribed to compensatory neurogenic response to the hyperoxic damage. Conversely, the decreased volume of the dentate gyrus granular layer could derive by a non sufficient neurogenic response to counterbalance the hyperoxic neuronal injury.