Nerve growth factor involvement in liver cirrhosis and hepatocellular carcinoma

Figure 1 NGF distribution in tissues from healthy donors. B, C, D, F, G: Confocal microscopy images of immuno-stained sections; in negative hepatocytes, endothelial cells, biliary epithelial cells and Kupffer cells the immunoreaction is the same as the background obtained in control performed by substitution of primary antisera with non-immune rabbit serum; positive immunoreaction (red hue) is only observed on lymphocytes in blood vessels. Green hue represents the auto-fluorescence used to visualize liver tissue morphology; A, E, H: Images of H&E stained sections close to that used for immunohistochemistry. Differently coloured arrows indicate the different cell types (see legend). bec: biliary epithelial cells; hep: hepatocytes.

Figure 2 NGF distribution in HCC tissue. E, F, G, I, J, K: Confocal microscopy images of immuno-stained sections showing NGF immunoreaction (red hue) in the cytoplasm (F) and on nuclei (I) of some hepatocytes, on endothelial cells, on lymphocytes and on Kupffer cells; no immunoreaction was observed in controls performed by substitution of primary antisera with non-immune rabbit serum. A: Images, at two different magnification, of H&E stained sections; B, C, D, H: Images of semithin sections from samples embedded in Spurr epoxy resin. Differently coloured arrows indicate the different cell types (see legend). n: nucleus; hep: hepatocyte; bvl: blood vessel lumen.

Figure 3 NGF and trkA^NGF distribution in cirrhotic tissues by confocal microscopy. A₁: Liver specimens obtained before transplantation from patients with cirrhosis but without HCC (Child-Pugh C); A₂: NGF distribution; A₃: trkA^NGF distribution; B₁: Specimens from patients with cirrhosis, also suffering from HCC, at early staging (Child-Pugh A); B₂: NGF distribution; B₃: trkA^NGF distribution. NGF or trkA^NGF immunoreaction (red hue) is particularly evident on biliary epithelial cells and on spindle-shaped cells, in cirrhotic tissue from patient at early staging (Child-Pugh A), but in presence of HCC, while no immunoreaction is observed in liver specimens resected before transplantation (Child-Pugh C) from patients without HCC. No immunoreaction is observed in controls performed by substitution of primary antisera with non-immune rabbit serum. bec: biliary epithelial cells; ssc: spindle-shaped cells.

Figure 4 NGF distribution in cirrhotic tissue from patient also suffering from HCC. B, C, D, G, H, I, M, J, K: Confocal microscopy images of immuno-stained sections showing NGF immunoreaction (red hue) in the cytoplasm of some hepatocytes, on biliary epithelial cells and on spindle-shaped cells; no immunoreaction is observed in controls performed by substitution of primary antisera with non-immune rabbit serum. J, K: Double staining on spindle-shaped cells using anti-NGF and antibody against the myofibroblast marker smooth muscle actin (SMA); A, L: Images of H&E staining; E, F: Images of semithin sections from samples embedded in Spurr epoxy resin. Differently coloured arrows indicate the different cell types (see legend). bec: biliary epithelial cells; n: nucleus; hep: hepatocyte; ssc: spindle-shaped cells.

Figure 5 NGF distribution in HCC tissue by immunogold labelling. A: Transmission electron micrographs of hepatocyte; B, C: Positive immunogold reaction on cytoplasmic vesicles; D: Positive immunogold reaction on nuclei; E: Positive immunogold reaction on endoplasmic reticulum. ER: endoplasmic reticulum. Scale bars = A: 2 μm; B-E: 100 nm.

Figure 6 NGF distribution in cirrhotic tissue from patient with HCC by immunogold labelling. A, B: Transmission electron micrographs of hepatocytes showing positive immunogold reaction on cytoplasmic vesicles (black arrows), free in the cytoplasm (double pointed arrows) and along endoplasmic reticulum (white arrows); C, C₁, C₂: Endothelial cells; positive immunogold reaction on cytoplasmic vesicles (black arrows) and free in the cytoplasm (double pointed arrows); D, D₁: Spindle-shaped cells; E, D₁: Biliary epithelial cells. In C₁, C₂, D₁ and E₁ details at higher magnification are shown. hep: hepatocytes; cf: collagen fibers; ER: endoplasmic reticulum. Scale bars = A, C, D, E: 2 μm; B, C₁, C₂, D₁, E₁: 100 nm.

Figure 7 Trk^ANGF distribution in HCC and cirrhotic tissues. A: HCC tissue; B: Cirrhotic tissue. A₁, A₂, B₁, B₂, B₃, B₆: Confocal microscopy images of immuno-stained sections. No immunoreaction was observed in controls performed by substitution of primary antisera with non-immune rabbit serum; A₄, B₄, B₅, B₇: Images of HE stained sections; A₃: Images of semithin section from samples embedded in Spurr epoxy resin. Differently coloured arrows indicate the different cell types (see legend). bec: biliary epithelial cells.

Figure 8 TrkA^NGF distribution in cirrhotic tissue from patient with HCC by immunogold labeling. A, A₁: Transmission electron micrographs of biliary epithelial cells showing positive immunogold reaction on cell membrane and cytoplasmic vesicles (black arrows) near the ductal lumen; B: Transmission electron micrographs of endothelial cells and lymphocytes; B₁: Immunoreactivity free in the cytoplasm of endothelial cells beneath the cell membrane (arrowheads); B₂: Gold particles on cell membrane of a lymphocyte. In A₁, B₁ and B₂ details at higher magnification are shown. bvl: blood vessel lumen; cf: collagen fibers. Scale bars = A, B: 2 μm; A₁, B₁, B₂: 200 nm.

Figure 9 Bar diagram illustrating the circulating NGF levels, determined by test ELISA, in patients with documented cirrhosis/HCC. NGF amounts, reported with regard to the etiology, is calculated either as mean ± SD (all patients examined) or as mean values for Child-Pugh class A (score = 5-6), for Child-Pugh class B (score = 7-9) and for Child-Pugh class C (score = 10-15). As a control, mean ± SD of circulating NGF levels from some healthy individuals is also reported.