210 Fig. 3 Representative prosurfactant protein C-stained lung sections from HPS mice at postnatal ages of a 12 and b 17 months. Note the aggregated and misshapen ATII cells in HPS double mutant mice seen at these older ages compared with WT and CHS. Also, TGF 1 levels in HPS BAL were signiWcantly higher at 17 months than those at 9 months of age. Dense staining using IHC for this cytokine was found in AMs of the HPS double mutants as well as WT control and CHS mouse strain throughout all ages tested. Dark staining was observed in cells of the alveolar walls in HPS mice at 12 and 17 months of age, but not in WT or CHS mice (Fig. 5b–e). Discussion This research was undertaken in order to determine whether the HPS double mutant mice exhibited classical signs of lung Wbrosis with aging beyond 1 year of age. Some 300 or more post-natal animals were examined for this study, including 131 HPS double mutants. Twenty-seven HPS mice 16 WT and 18 CHS were raised to 17 months of age or older. Among these, none of the WT control or CHS 123 Histochem Cell Biol (2010) 134:205–213 animals exhibited classical signs of Wbrosis as evidenced by high levels of hydroxyproline or the presence of Wbrotic patches, while 20 (74%) of the double mutant animals at 17 months and beyond had one or both of these symptoms. Most importantly, perhaps, the lung abnormalities in HPS mice, including lung Wbrosis, are unlike those induced by drugs (Borzone et al. 2001), i.e., they are progressive until the end of the animals’ lives and do not heal with time. Masson trichrome staining and hydroxyproline analysis both detected lung Wbrosis in this double mutant at the age of 17 months. The histological method appeared to be more sensitive in detecting collagen deposition in lungs. That is, at 12 months of age, Masson trichrome staining showed obvious blue staining in the alveolar walls, which was not found in WT and CHS mouse strains or HPS at younger ages. Hydroxyproline levels, however, were not signiW- cantly diVerent among these three strains at that age. This may be because pulmonary Wbrosis starts as deposition in alveolar walls with insuYcient amounts of collagen to aVect the whole lung analysis of hydroxyproline levels. Also, hydroxyproline analysis can be aVected by the increased levels of surfactant proteins in ATII cells (Lyerla et al. 2003), which explains the slightly higher levels seen in HPS and CHS mice at younger ages. Many lung abnormalities including emphysema, dys- morphic and highly activated AMs (Lyerla et al. 2003), and higher levels of TGF 1 (this study), appeared much earlier than the overt Wbrosis in the HPS double mutant animals. All of these abnormalities were more severe at 12 months of age, when the alveolar interstitium was thickened with collagen and lined with ATII cells. Thus, the lung abnor- malities of HPS double mutant can be separated into two stages: the preWbrotic, in which early abnormalities are observed and persist to 9 months of age; and accelerated deterioration, after 9 months of age when these symptoms increase in severity as either the causes or eVects of devel- oping Wbrosis. The preWbrotic stage in the HPS double mutant may be useful for studies of early diagnosis and therapeutic inter- ventions of HPSIP. For example, the changes of ATII cell distributions and decrease in number observed in HPS dou- ble mutant from 2 to 9 months of age were unique. This was not observed in earlier studies (Lyerla et al. 2003) and is presumably not detectable in bleomycin-induced animals due to the rapid nature of developing Wbrosis in this model. The decrease of ATII cells in HPS lungs from 2 to 5 months may be due to the abnormal structure of this cell type with giant lamellar bodies. This might also be the rea- son for the trend of ATII cell decreasing in CHS mice with similar but less enlarged giant lamellar bodies. The increase of ATII cell numbers in HPS lungs at 9 months and beyond may be due to proliferation induced by mechanical stress as lung structure deteriorates. This phenomenon has been