Acid phosphatase reaction (A-F) and anti CD68 immunofluorescence staining (G-L).

<p>Resident in untreated (A, G) and migrating macrophages-like cells in treated leeches (B-E) and (H-L), located under the epithelium (e) and among the muscle fibers (m), are positive for acid phosphatase reaction (arrowheads in A-F) and for anti-CD68 (G-L). (F) Quantitative evaluation of cell numbers. Column 1: number of cells in untreated sample, columns 2–10: number of cells in MWCNTs treated sample from 1h up to 5 weeks. *p<0.01. (J) Combined transmission and fluorescence images showing CD68⁺ cells (in red) in spatial association with MWCNTs aggregates (circled). Bars in A-E, G-I, K-L: 100μm; Bar in F, J: 10μm.</p

Morphological analysis (A-L).

<p>Morphological at optical (A-E, C, I) and transmission electron microscopes (F, H, J-L) analyses of sections from <i>H</i>. <i>medicinalis</i> body wall. The body wall of untreated leeches results practically avascular (A) and the botryoidal tissue (b) appears as a solid chord of clustered cells (B). After 1 hour from MWCNTs treatment, numerous neo-vessels (v) are found among muscles (m) and under the epithelium (e) (C). Within the new cavities (c) lined by the botryoidal tissue (b), immunocompetent circulating precursors cells (arrowhead) are clearly distinguishable (D). After 3 hours from treatment, numerous fibroblasts are visible immersed in an abundant extracellular matrix ECM (E, F). After 1 (G, H) up to 5 weeks (I) from MWCNTs treatment, numerous vessels (v) and fibroblasts (arrowheads) are still visible in the body wall. (J-L) Detail of MWCNTs (arrowheads) freely dispersed in the cytoplasm of macrophage-like cells. Bars in A, C, G: 100μm; Bars in B, D-E, I: 10μm; Bar in F: 5μm; Bar in H: 2μm; Bar in J: 500nm; Bars in K-L: 200nm.</p

Determination of MWCNTs presence in tissues (A-D).

<p>(A) MWCNTs extracted by means of KOH digestion from exposed-animals tissues. (B-D) MWCNTs crude powder (used as control) raw (B), after sonication (C) and after KOH treatment (D). Bars in A-D: 500nm.</p

Anti-IL18 (A-F) and Thioflavine-T staining (G-L).

<p>(A-F) Localization of IL-18. Note the population of resident in untreated (A) and migrating (B- F) immune-responsive cells (arrowheads) located under the epithelium (e) and among the muscle (m). Nuclei are counterstained with DAPI (blue). (G-K) Thioflavin-T method. Amyloid material is stained in yellow (arrowheads). (L) Double-staining of Thioflavin-T (yellow) and macrophage markers CD68 (red) in a cryosection of 3 hours MWCNTs treated leech body wall. Bars in A-E, G-K: 100μm; EDS analysis.</p

Characterization of circulating precursors cells (A-D).

<p>Morphological (A) and immunohistochemical (B) analysis of cryosections from <i>H</i>. <i>medicinalis</i> body wall. The circulating precursors (arrowhead) visible in lumen of neo-vessels are highly positive for anti-CD45 antibody. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI; blue). (C-D) Western blot analysis. A protein extracts from the body wall of untreated (n.t) and MWCNTs treated leeches from 1 to 12 hours and from 1 to 5 weeks were probed with the anti-CD45 antibody. The housekeeping protein D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. In all samples, the anti-CD45 detected specific immunoreactive band 24 of about 145 kDa (C), according to the molecular weight ladder (kDa). CD45 protein was quantified by densitometry from three experiments. *P<0.05 compared with untreated leeches (D). Bars in A-B: 10μm.</p