Fig 1: Upregulation of PDGF at the periablational area induced liver regeneration after nsPEF ablation through recruitment of activated HSCs. (a) a-Smooth muscle actin (a-SMA) staining (original magnification, ×40 and ×200) shows the distribution of activated myofibroblasts (activated HSCs) at the border zone at 3 days after nsPEF ablation. (b) Sirius red staining (original magnification, ×200) shows collagen deposition at the border zone at 3 days after nsPEF ablation. (c, d) Representative images of IHC staining for two key molecules for recruitment and activation of HSCs, MCP-1 and PDGF within the periablational area in 3 days after ablation (original magnification, ×40 and ×200). (e–i) A PDGF neutralization IgG antibody (NA) or an isotype IgG antibody (control) were injected into the portal vein of mice in one day pre- and postablation, respectively. Three days after nsPEF ablation, liver tissues from both ablated and unablated lobes were harvested for examining PDGF expression by IHC staining for PDGF, activated HSCs by IHC staining for a-SMA, and liver regeneration by IHC staining for Ki67. Representative images of IHC staining for PDGF, a-SMA, and Ki67 (magnification, ×200) (e) and quantification of a-SMA- (f) or Ki67- (g) positive staining cells within 5 random fields of ×100 microscopy. The amount of HGF was detected within liver tissues (h) and serum (i) by ELISA. Data were presented as mean ± SD (*P < 0.05, **P < 0.01, and ***P < 0.001).
Fig 2: nsPEF induces a more obvious hepatocyte proliferation at the periablational area in comparison to other unablated areas at ablated lobes. (a) Representative image of IHC staining for Ki67 (original magnification, ×200) shows more obvious hepatocyte proliferation at the periablational zone at 3 days after nsPEF ablation compared to other areas at the ablated lobe. (b) Representative image of IHC staining for cox-2 (original magnification, ×40 and ×200) at the periablational zone at 24 hours after nsPEF ablation. (c, d) Mice were administered intraperitoneally with the cox-2 inhibitor celecoxib (50 mg/kg) or vehicle (control) daily for 3 successive days after nsPEF ablation. Then, livers were harvested for the detection of Ki67-positive staining cells. Representative results of IHC staining with magnification of ×200 at the periablational area of the ablated lobe of mice from the celecoxib group and control group (c) and quantification of Ki67-positive cells within 5 random fields of ×100 microscopy (d). (e) Level of HGF within liver tissues in the unablated area and periablated area within the ablated lobe at indicated time points after nsPEF ablation detected by ELISA, respectively. (f, g) Mice were administered intraperitoneally with the c-Met inhibitor PHA (30 mg/kg) (PHA) or vehicle (control) daily for 3 successive days after nsPEF ablation. Then, livers were harvested for the detection of Ki67-positive staining hepatocytes. Representative results of IHC staining for Ki67 with magnification of ×200 at the periablational area of the ablated lobe of mice from the PHA group and control group (f) and quantification of Ki67-positive cells within 5 random fields of ×100 microscopy (g). Data were presented as mean ± SD (*P < 0.05, **P < 0.01, and ***P < 0.001).
Fig 3: The liver regeneration induced by nsPEF is related to the activated HGF/c-Met pathway. (a, b) Level of HGF mRNA (a) and protein (b) within liver tissues at the ablated lobe and unablated lobe at indicated time points after nsPEF ablation detected by qRT-PCR and ELISA, respectively. (c, d) Serum level of HGF (c) and VEGF (d) examined by ELISA at indicated time points after nsPEF ablation. (e–g) Mice were administered intraperitoneally with the c-Met inhibitor PHA (30 mg/kg) (PHA) or vehicle (control) daily for 3 successive days after nsPEF ablation. Then, the liver and serum were harvested for the detection of Ki67-positive hepatocytes by IHC staining and serum VEGF level by ELISA. Representative IHC staining results with magnification of ×200 (e), the quantification of the average number of Ki67-positive cells within 5 random fields of ×100 microscopy (f), and serum VEGF level (g) by ELISA on day 3 after ablation. Data were presented as mean ± SD (*P < 0.05, **P < 0.01, and ***P < 0.001).
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