NFP treatment causes collagen deficiency and impairment of caudal fin regeneration. Electron microscopy performed on longitudinal sections revealed severe structural alteration after NFP treatment. 3dpa (a) and 7dpa (b) represented overview of the fish ray containing bones, RBCs deposition and blastema. a’–a’’’ display clusters and single RBCs that are in direct contact with surrounding macrophages MΦ, and areas of atypical, amorphous ECM. The latter contains disintegrated cells and cell debris (asterisk) as well apoptotic cells (a’’’, arrow). In contrast to the controls and BAPN treatment, granulocytes (Gr) and MФ are common features. Classical collagen fibers are not detectible. Amputation plane at 7dpa containing perfused vessels is displayed in b; the endothelial coverage is partially interrupted; the EC are activated containing round nuclei and multiple intravascular protrusions. ECM contains less collagen fibers (CF), those do not build classical bundles. The intercellular space is loose, amorphous with irregular distribution representing electron microscopically “empty areas” (b’’’, asterisks). Immunofluorescence staining on transversal sections at the amputation site in control animal (c) and NFP-treated animal (d) at 3dpa are documented. Blood vessels (ECs) are labeled in green, active collagen 1α2 producing cells in red and collagen I purple (c). c demonstrates intact blood vessels, active collagen 1α2 producing cells, and accumulation of collagen I in the vicinity of the blood vessels. d displays blood vessels, very tiny scattered fragments of collagen I and absence of active collagen 1α2 producing cells.
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