Three-Dimensional Imaging of Pulmonary Fibrotic Foci at the Alveolar Scale Using Tissue-Clearing Treatment with Staining Techniques of Extracellular Matrix

Idiopathic pulmonary fibrosis is a progressive, chronic lung disease characterized by the accumulation of extracellular matrix proteins, including collagen and elastin. Imaging of extracellular matrix in fibrotic lungs is important for evaluating its pathological condition as well as the distribution of drugs to pulmonary focus sites and their therapeutic effects. In this study, we compared techniques of staining the extracellular matrix with optical tissue-clearing treatment for developing three-dimensional imaging methods for focus sites in pulmonary fibrosis. Mouse models of pulmonary fibrosis were prepared via the intrapulmonary administration of bleomycin. Fluorescent-labeled tomato lectin, collagen I antibody, and Col-F, which is a fluorescent probe for collagen and elastin, were used to compare the imaging of fibrotic foci in intact fibrotic lungs. These lung samples were cleared using the ClearT2 tissue-clearing technique. The cleared lungs were two dimensionally observed using laser-scanning confocal microscopy, and the images were compared with those of the lung tissue sections. Moreover, three-dimensional images were reconstructed from serial two-dimensional images. Fluorescent-labeled tomato lectin did not enable the visualization of fibrotic foci in cleared fibrotic lungs. Although collagen I in fibrotic lungs could be visualized via immunofluorescence staining, collagen I was clearly visible only until 40 μm from the lung surface. Col-F staining facilitated the visualization of collagen and elastin to a depth of 120 μm in cleared lung tissues. Furthermore, we visualized the three-dimensional extracellular matrix in cleared fibrotic lungs using Col-F, and the images provided better visualization than immunofluorescence staining. These results suggest that ClearT2 tissue-clearing treatment combined with Col-F staining represents a simple and rapid technique for imaging fibrotic foci in intact fibrotic lungs. This study provides important information for imaging various organs with extracellular matrix-related diseases.

Steric constraints control processing of glycosylphosphatidylinositol anchors in Trypanosoma brucei

The transferrin receptor (TfR) of the bloodstream form (BSF) of Trypanosoma brucei is a heterodimer comprising glycosylphosphatidylinositol (GPI)-anchored expression site–associated gene 6 (ESAG6 or E6) and soluble ESAG7. Mature E6 has five N-glycans, consisting of three oligomannose and two unprocessed paucimannose structures. Its GPI anchor is modified by the addition of 4–6 α-galactose residues. TfR binds tomato lectin (TL), specific for N-acetyllactosamine (LacNAc) repeats, and previous studies have shown transport-dependent increases in E6 size consistent with post-glycan processing in the endoplasmic reticulum. Using pulse-chase radiolabeling, peptide-N-glycosidase F treatment, lectin pulldowns, and exoglycosidase treatment, we have now investigated TfR N-glycan and GPI processing. E6 increased ∼5 kDa during maturation, becoming reactive with both TL and Erythrina cristagalli lectin (ECL, terminal LacNAc), indicating synthesis of poly-LacNAc on paucimannose N-glycans. This processing was lost after exoglycosidase treatment and after RNAi-based silencing of TbSTT3A, the oligosaccharyltransferase that transfers paucimannose structures to nascent secretory polypeptides. These results contradict previous structural studies. Minor GPI processing was also observed, consistent with α-galactose addition. However, increasing the spacing between E6 protein and the GPI ω-site (aa 4–7) resulted in extensive post-translational processing of the GPI anchor to a form that was TL/ECL-reactive, suggesting the addition of LacNAc structures, confirmed by identical assays with BiPNHP, a non-N-glycosylated GPI-anchored reporter. We conclude that BSF trypanosomes can modify GPIs by generating structures reminiscent of those present in insect-stage trypanosomes and that steric constraints, not stage-specific expression of glycosyltransferases, regulate GPI processing.

Abstract WP426: Early Non-white Matter Changes in Cerebrovascular Integrity in Mouse Brain Following Bilateral Carotid Artery Stenosis

Bilateral carotid artery stenosis (BCAS) is an experimental model of vascular dementia which leads to white matter lesions and cognitive dysfunction in mice. Unfortunately, with time the white matter pathology worsens and spreads to the hippocampus and cortex. While some variability in the temporal and spatial distribution of brain injury may result from inter-mouse strain differences in cerebrovascular anatomy, coil size employed to constrict the carotids and surgical technique, it is generally accepted that hippocampal, striatal and cortical pathology is not significantly present prior to 30 days. However, as changes in cerebrovascular integrity, i.e. blood-brain barrier (BBB) permeability, are known to precede more overt brain pathology in stroke, we hypothesized that BBB changes could occur earlier after BCAS in the hippocampus, striatum and cortex and be a precursor of longer term pathology in these regions. In our study, 3 month old male C57/Bl6 mice underwent BCAS with 0.18 mm coils or sham surgery control and cerebrovascular integrity was analyzed by collagen IV (vascular basement membrane component), tomato-lectin (marker of endothelial cells) and Ki-67 (marker of cell proliferation) immunohistochemistry after 7, 14, or 21 days (n=4 animals per group per day). By day 14 we noted that collagen IV staining density was significantly less in the hippocampus compared to sham controls. Surprisingly, both collagen IV and tomato-lectin staining pattern indicate blood vessel disruption in not only the hippocampus but the striatum as well. Expression of Ki-67 increased in both of these regions, and further co-labeling studies will shed light on cell specificity. Similar differences were noted at all days tested, with few changes observed in the cortex. In conclusion, this study demonstrates for the first time that changes in cerebrovascular integrity occur earlier than expected after BCAS and suggests that such changes might underlie the gradual development of BCAS non-white matter pathology.