Figure 1

Unstimulated and stimulated H. medicinalis: optical and TEM microscopy. (a–f) Light microscopy, semi-thin sections of healthy (unstimulated) leech. (a) General view of cross sectioned body of leech (Crystal violet staining). Under the epithelium (E), the body wall is mainly composed of tightly packed helical muscle fibers (Mf) embedded in a scarce and loose connective tissue. Under the longitudinal layer of muscle fibers the botryoidal tissue is visible (B). (b) Detail of muscular body wall. Among muscle fibers (Mf), embedded in the loose connective tissue, interstitial cells showing telocyte phenotype are visible (white arrowheads). (c) Specimen stained with methylene blue, which is considered the best dye in identifying telocyte feature. In the connective tissue HmTCs, showing small cell bodies and thin long cellular processes (white arrowheads) are easily recognizable in respect to granulocytes (G), fibroblasts (F) and macrophages (M). (d) Specimen May-Grunwald Giemsa. HmTCs (arrowheads) in close contact by their tubular long cytoplasmic processes are embedded in the loose connective tissue. (e,f) Specimens Toluidine blue stained. HmTCs’ telopodes (arrowhead) surround a small vessel (V) and ganglia (nervous system-NS). (g–i) Semi-thin sections of leeches stimulated by LPS injection (g,h) or subjected to surgical lesion (i). In the specimens stained with crystal violet (g) and with methylene blue (h) telocytes (white arrowheads), wide distributed in the connective tissue, among muscle fibers (Mf) and granulocytes (G), are more numerous than controls. In leech surgically lesioned (specimen stained with May-Grunwald Giemsa) (i), the outlined wedge-shaped regenerating tissue is characterized by a large amount of newly synthesized connective tissue infiltrated with proliferating (expressing stemness factors) telocytes (arrowheads). (Mf: muscle fibers). (l,m) Cryosections of leech body wall. HmTCs (arrowheads) are Nonspecific esterase+ (NSE+) (l) and Succinic dehydrogenase+ (SDH+) (m). Nonspecific esterase reaction is linked to microsomal and lysosomal activity while SDH reaction is attributable to mitochondrial activity. Intense spotted SDH staining evidences mitochondria localized in podoms (arrowheads). (j,k,n) Ultrastructural analysis (TEM) confirm the telocyte phenotype characterized by spindle-shape cell body and long and thin telopods (arrows). Note the localization of mitochondria within podoms (n) (arrowheads) that validates the SDH moniliform positivity (m). Scale bars. j: 2 μm; k: 0.9 μm; n: 2.2 μm.