The OX-7 mAb binds to a Thy-1 epitope on the surface of mesangial cells and causes complement-dependent cell lysis followed by exuberant matrix synthesis and deposition.41 Normal control animals were injected with the same volume of phosphate-buffered saline (PBS). also histology-based disease diagnosis, staging, and therapeutic screening. This study lays the foundation for further testing CHP as a targeting moiety for theranostic delivery AZD3759 in various animal models. non-green fluorescence channels (Tukey HSD test, 0.05). (C) Fluorescence scans of a heart harvested 7 days after MI show close spatial similarity between signals from B-CHP and macrophages as detected with an anti-CD68 antibody. (D) A magnified view of the infarcted region within the red box in panel (C) with merged fluorescence signals shows that high level of degraded collagen is in areas crowded with macrophages. Scale bars: 1 mm (A, C), 50 m (D). Glomerulonephritis Glomerulonephritis is a group of inflammatory kidney diseases characterized by glomerular damage.39?41 The destruction of glomerular architecture, in turn, accelerates remodeling of the surrounding extracellular matrix, thereby leading to fibrotic scarring (glomerulosclerosis).39?41 In the glomeruli of anti-Thy-1 nephritic rats, a classic animal model of glomerulonephritis, elevated MMP expression, and increased collagen deposition is well documented in the literature.40?43 As such, we first analyzed kidney cryosections harvested from the anti-Thy-1 nephritic and normal control rats with the conventional periodic acidCSchiff (PAS) stain. As expected, the amount of mesangial matrix occupying each glomerulus was higher in the affected kidneys (Figure ?Figure44A), confirming the fibrotic glomerular expansion. Following F-CHP staining, strong signals were recorded in the glomeruli of the nephritic animals with minimal staining intensity detected in normal kidneys (Figure ?Figure44B). Interestingly, the CHP signal is confined to the diseased glomeruli, whereas type IV collagen (visualized by immunofluorescence staining) is uniformly distributed in the tissue, AZD3759 including the interstitial space (Figure ?Figure44B). Similar results were obtained when AZD3759 the sections were stained with B-CHP and further visualized using horseradish peroxidase (HRP) conjugated neutravidin (Figure S4). Given that F-CHP signals and anti-collagen IV antibody staining were colocalized in the glomeruli (Figure ?Figure44B), the unfolded/degraded type IV collagen localized in the basement membrane region is the most likely target of CHP, though we cannot exclude the possibly of CHP binding to minor basement-membrane-associated collagen types.44 Hence, the PAS and CHP staining results validate that nephritic glomeruli contain elevated amounts of collagen with Gfap a partially degraded and unfolded structure. The CHP stain pinpoints the glomerular lesion; such high specificity is nearly impossible to achieve with conventional staining tools, which are typically insensitive to the structural change of their targets. Open in a separate window Figure 4 Glomerulonephritis. Representative micrographs of kidney cryosections from anti-Thy-1 nephritic and normal control rats stained with PAS (A) or F-CHP and an anti-collagen IV antibody (B). Images are representative of similar results from three animals within each group, three stained sections per animal. Level pub: 100 m. Pulmonary Fibrosis Bleomycin is definitely a chemotherapeutic agent with known pulmonary toxicity that leads to fibrotic changes in animal models that mimic those observed in idiopathic pulmonary fibrosis individuals.45 Aberrant wound healing responses, involving both increased MMP activity46,47 and excess matrix deposition,45,48 have been implicated in the pathogenesis of pulmonary fibrosis with this model. Using MMPsense 680, a probe that fluoresces in the near-infrared region after MMP cleavage, we confirmed elevated MMP activity in the fibrotic lungs of bleomycin-treated mice relative to normal settings (Number S5). To visualize the build up of damaged collagen within the redesigning lung matrix in the cells and cellular levels, F-CHP staining was applied to a set of lung cryosections harvested at varying time points after bleomycin delivery an implanted osmotic pump. F-CHP staining exposed the spotty distribution of damaged collagen, indicated from the highly localized, bright F-CHP deposits appearing in samples beginning 1 week after bleomycin treatment (Number ?Number55A). Representative images of F-CHP staining and quantitative analysis shown that (i) the number of bright spots was significantly higher in the subpleural area of the lungs.
