Dose adaptation of bleomycin to OA route and comparison of different bleomycin doses in BALB/c mice by UTE-MRI and post-mortem analyses (n = 8 mice for each group except n = 4 mice for histological analysis).

<p>Data are shown as means±STDEV. One-way ANOVA (Two-Way repeated measures ANOVA for MRI-results) with Bonferroni tests was used for statistical analysis. A) Total volume of MRI signals detected in the lung by UTE-MRI The signal at baseline contains contributions from vessels, whereas signals following bleomycin administration reflect additionally the injury induced by the antibiotic. B) Collagen content in left lung lobes detected by picrosirius staining. The picrosirius factor expresses the amount of collagen relative to the mean collagen content in the lungs of saline-treated mice. B) Post-mortem determination of hydroxyproline in right lung lobes (day 23); Data are shown as Mean ± STDEV of µg hydroxyproline per right lung. D-F) mRNA expression level of Col1α1, F4/80, and MMP12 compared to expression of HPRT. Data shown as means±STDEV of fold induction compared to control group.</p

Histology of picrosirius stained lung slices from C57BL/6 mice.

<p>Collagen (green) visualized by picrosirius staining in three slices of the left lung lobe (upper row). Magnified views (×200) of a portion of the corresponding histological slices are shown in the bottom row. A) Stained collagen content in saline treated mouse lung 37 days after administration. B) Stained collagen content in a mouse lung 37 days after last IN bleomycin administration (0.25 mg/kg on six consecutive days). C) Collagen content in a mouse lung 37 days after last OA of bleomycin (0.25 mg/kg on six consecutive days). Compared to the IN administration, the amount of picrosirius was six times higher when bleomycin was oropharyngeally aspirated and the distribution of picrosirus staining was more homogeneous.</p

Correlations between MRI and all other readouts on day 23 analyzed in the present study for detection of bleomycin-induced lung fibrosis in BALB/c mice (n = 32; except for Picrosirius n = 16): total MRI signal volume, picrosirius staining, hydroxyproline, and gene expression of Col1α1, F4/80 and MMP12.

<p>Saline-treated BALB/c animals (n = 8, n = 4 for picrosirius) were used as controls.</p

Details of segmentation procedure.

<p>For each slice, a border is drawn manually to limit the segmentation of high intensity signals to the lung. Images correspond to a rat at A) baseline and B) day 7 after OA of bleomycin (2 mg/kg).</p

Detection of bleomycin-induced lung injury in C57BL/6 mice by UTE-MRI, picrosirius staining, and hydroxyproline determination.

<p>Animals were treated with 0.1 mg/kg bleomycin (n = 9) or vehicle (n = 5) on six consecutive days via OA. Data are shown as means ± STDEV. A) Relative body weight during the course of the experiment. The statistical significance values ***p<0.001 represent comparisons between saline and bleomycin-treated mice, at each time point. B) Total volume of MRI signals in the lung (µL). The signal at baseline contains contributions from vessels, whereas signals following bleomycin administration reflect additionally the injury induced by the antibiotic. The statistical significance values ***p<0.001 indicate comparison to baseline. C) Post-mortem determination of hydroxyproline in right lung lobes D) Picrosirius factor expressing the amount of collagen relative to the mean collagen content assessed in the lungs of saline-treated mice. The statistical significance value ***p<0.001 means comparison to saline treatment.</p

HPLC/MS spectra of bleomycin (0.075 mg/mL) freshly prepared and after six days at 4°C acquired on an Acquity Ultra performance LC instrument (Waters, Milford, Massachusetts, USA).

<p>An Acquity HSS T3 1.8 µm 2.1×50 mm column was used at 60°C; eluent A (water +0.05% formic acid +3.75 mM ammonium acetate) and eluent B (acetonitrile +0.04% formic acid) were used with a gradient of 5 to 98% B in 1.4 min and 1.0 mL/min flow. Bleomycin A2: C55H84N17O21S3+ with a mass of 1415.56 g/mol; bleomycin B2: C55H84N20O21S2 with a mass of 1425.52 g/mol.</p

Comparison of OA to IN administration of bleomycin (6×0.25 mg/kg) in C57BL/6 mice (n = 8 per group) by <i>in vivo</i> MRI and post-mortem analyses.

<p>All data are shown as means ± STDEV. A) Relative body weight and time point of death during the study. Animals have been sacrificed directly after their last measurements. Five out of eight mice receiving OA of bleomycin had to be sacrificed prematurely due to excessive bodyweight loss (A). B) Total volume of MRI signals in the lung (µL). The signal at baseline contains contributions from vessels, whereas signals following bleomycin reflect additionally the injury inflicted by the antibiotic. C) Post-mortem determination of hydroxyproline in right lung lobes (day 37). D) Amount of collagen detected by histology of picrosirius in left lung lobes (day 37). Results are expressed as collagen content relative to mean collagen content in the lungs of saline-treated mice (picrosirius factor).</p

Picrosirius-stained histological slices of C57BL/6 mice observed at bright-field (left column) and at polarization (right column).

<p>The high intensity areas in the polarized view, corresponding to collagen, coincide well with the red areas in the bright-field view. In this work, collagen has been quantified by examining histological slices at bright-field. A) Saline-challenged mouse (OA administration). B) IN administration of bleomycin (6×0.25 mg/kg). C) OA administration of bleomycin (6×0.25 mg/kg).</p

Ventral NIRF images of the lungs from two mice 60 min after instillation of the dye Cy5.5 (0.1 mg/mL) either via the IN route (A) or via OA (B).Yellow arrows point to the trachea.

<p>Ventral NIRF images of the lungs from two mice 60 min after instillation of the dye Cy5.5 (0.1 mg/mL) either via the IN route (A) or via OA (B).Yellow arrows point to the trachea.</p

Expression of transient receptor potential C6 channels in human lung macrophages

Chronic obstructive pulmonary disease (COPD) is associated with pulmonary inflammation with increased numbers of macrophages located in the parenchyma. These macrophages have the capacity to mediate the underlying pathophysiology of COPD; therefore, a better understanding of their function in chronic inflammation associated with this disease is vital. Ion channels regulate many cellular functions; however, their role in macrophages is unclear. This study examined the expression and function of transient receptor potential (TRP) channels in human macrophages. Human alveolar macrophages and lung tissue macrophages expressed increased mRNA and protein for TRPC6 when compared with monocytes and monocyte-derived macrophages. Moreover, TRPC6 mRNA expression was significantly elevated in alveolar macrophages from patients with COPD compared with control subjects. There were no differences in mRNA for TRPC3 or TRPC7. Although mRNA for TRPM2 and TRPV1 was detected in these cells, protein expression could not be determined. Fractionation of lung-derived macrophages demonstrated that TRPC6 protein was more highly expressed by smaller macrophages compared with larger macrophages. Using whole-cell patch clamp electrophysiology, TRPC6-like currents were measured in both macrophage subpopulations with appropriate biophysical and basic pharmacological profiles. These currents were active under basal conditions in the small macrophages. These data suggest that TRPC6-like channels are functional on human lung macrophages, and may be associated with COPD