Growth hormone abolishes the negative effects of everolimus on intestinal wound healing

Figure 1 Bursting pressure of the colonic anastomosis (mmHg). Everolimus treatment decreased the bursting pressure significantly. Additional treatment with human growth hormone (hGH) partial antagonized this effect. V: Vehicle, E: Everolimus 3.0 mg/kg, E + GH: Everolimus 3.0 mg/kg + hGH 2.5 mg/kg; ^aP < 0.05, V vs E, E vs E + GH.

Figure 2 Hematoxylin and eosin staining from the anastomotic region. In the hematoxylin and eosin stained samples there were significant changes in anastomotical architecture in everolimus-treated animals, indicated by decreased cell density and reduced angiogenesis (B) which are indicators for granulation tissue. Simultaneous treatment with human growth hormone (hGH) showed more granulation tissue (C). A: Vehicle; B: Everolimus 3.0 mg/kg; C: Everolimus 3.0 mg/kg + hGH 2.5 mg/kg (magnification × 100).

Figure 3 Azan staining from the anastomotic region. The collagen fibres appear blue. Everolimus treated animals show a disturbed arrangement of collagen fibres, which is antagonized by additional human growth hormone (hGH)-treatment (A-C). A: Vehicle; B: Everolimus 3.0 mg/kg; C: Everolimus 3.0 mg/kg + hGH 2.5 mg/kg. Hydroxyproline concentration in the anastomotic region in mg per mg dry weight (DW). The decrease of hydroxyproline under everolimus-treatment is reduced by additional hGH-treatment (D) (magnification × 400). V: Vehicle, E: Everolimus 3.0 mg/kg, E + GH: Everolimus 3.0 mg/kg + hGH 2.5 mg/kg; ^aP < 0.05, vs E group, V vs E + GH group.

Figure 4 Immunohistochemistry for PCNA- (A) and MPO- (B) positive cells from the anastomotic region. Treatment with everolimus alone reduced significantly the amount of PCNA-positive cells in the anastomotic region while increasing the number of MPO-positive cells, indicating a prolonged inflammation. Simultaneous treatment with hGH led to nearly normal values for PCNA- and MPO-positive cells. V: Vehicle, E: Everolimus 3.0 mg/kg, E + GH: Everolimus 3.0 mg/kg + hGH 2.5 mg/kg; V vs E, E + GH vs E, ^aP < 0.05.

Figure 5 Zymographic activities of the pro-inflammatory matrix-metalloproteinases MMP2 and MMP9. Zymography gel showing increased proteolytic activity under treatment with everolimus which is reduced by simultaneous treatment with human growth hormone (hGH) (A). Numeric calculation of the proteolytic activity of MMP2 (B) and MMP9 (C). V: vehicle, E: everolimus 3.0 mg/kg, E+GH: everolimus 3.0 mg/kg + hGH 2.5 mg/kg; V vs E, E + GH vs E, ^aP < 0.05.