One was measured in lung tissue homogenates by reaction with DTNB
One was measured in lung tissue homogenates by reaction with DTNB (5, 5’dithiobis-2-nitrobenzoic acid) using the Glutathione Assay Kit (Calbiochem, Gibbsontown, NJ), following the manufacturer’s instructions. C. Histological morphometric evaluation: Harvested lungs were fixed at inflation with 10 phosphate-buffered formalin at 20 cm H2O pressure for 30 min. After overnight immersion in fixative, the lungs were embedded in paraffin wax, cut into 5 m-thick sections, and stained with hematoxylin and eosin. Lungs were examined qualitatively, and a quantitative analysis was performed using light-microscopic morphometry. Five animals from each group were evaluated to PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27527552 determine alveolar surface density and alveolar volume density as estimates of surface area and alveolar number, as described previously [25]. Data from each group are expressed as means ?SE.Statistical analysisbetween room air and oxygen-exposed litters every 24 hours to prevent maternal O2 toxicity. There were no mortalities during the 7 day- hyperoxic exposure. At 8 days, pups were euthanized with 150 mg/kg pentobarbital sodium i.p. Lung tissue and bronchoalveolar lavage (BAL), fluid were obtained. One lung from each mouse was used for Anlotinib biological activity biochemical assessments and the contra-lateral lung was used for histopathology. A. Evaluation of the inflammatory markers:BAL cell countsThe trachea was exposed via a midline incision and the lungs were gently lavaged via a tracheal cannula with three aliquots of 0.3 mL Phosphate Buffered Saline (PBS 0.5 M, pH 7.4). The volume of the recovered lavage fluid was recorded, and cell counts were determined using a hemocytometer. Differential counts were performed on cells stained with Wright-Giemsa stain as previously described [36]. Samples with gross hemolysis and with <70 BAL recovery were not analyzed.Myeloperoxidase (MPO)Groups were compared using analysis of variance, and a post hoc Tukey Kramer test was performed to determine statistical differences. p values less than 0.05 were accepted as significant, assuming an error = 0.05 and error = 0.10.ResultsThe effect of combination of hEC-SOD overexpression and Antileukinate on neutrophil influx into lung after acute hyperoxic exposureMPO activity was measured spectrophotometrically in lung tissue homogenates by reaction with o-dianisidine using a microplate assay [36-38].AlbuminThe albumin concentration, in BAL fluid, was measured using the assayMax Mouse ELISA kit (Assaypro, St. Charles, MO), following the manufacturer's instructions. B. Measurements of oxidative stress:8-isoprostaneThere was marked elevation in both the total number of white blood cells and neutrophils in the BAL fluid following hyperoxic exposure. In addition, there was increased MPO activity in the lung tissues of both Antileukinate-non-treated WT and Tg mice after acute hyperoxic exposure. In WT mice, Antileukinate significantly reduced neutrophil counts and MPO activity (p < 0.05) (Figure 1A B). Of particular importance, there were significantly less neutrophils and MPO in the BAL of Antileukinate-treated Tg compared to Antileukinate treated WT group after acute hyperoxic exposure (p < 0.05).The effect of combination of hEC-SOD overexpression and Antileukinate on lung permeability after acute hyperoxic exposureLung tissue was immediately flash frozen in liquid nitrogen at harvest and stored at -80 until analysis to prevent auto-oxidation. Lipid peroxidation is a well-defined mechanism of cellular damage. 8-isoprostane.