Tissue shrinkage in microwave ablation of liver: an <i>ex vivo</i> predictive model

<p><b>Purpose:</b> The aim of this study was to develop a predictive model of the shrinkage of liver tissues in microwave ablation.</p> <p><b>Methods:</b> Thirty-seven cuboid specimens of <i>ex vivo</i> bovine liver of size ranging from 2 cm to 8 cm were heated exploiting different techniques: 1) using a microwave oven (2.45 GHz) operated at 420 W, 500 W and 700 W for 8 to 20 min, achieving complete carbonisation of the specimens, 2) using a radiofrequency ablation apparatus (450 kHz) operated at 70 W for a time ranging from 6 to 7.5 min obtaining white coagulation of the specimens, and 3) using a microwave (2.45 GHz) ablation apparatus operated at 60 W for 10 min. Measurements of specimen dimensions, carbonised and coagulated regions were performed using a ruler with an accuracy of 1 mm. Based on the results of the first two experiments a predictive model for the contraction of liver tissue from microwave ablation was constructed and compared to the result of the third experiment.</p> <p><b>Results:</b> For carbonised tissue, a linear contraction of 31 ± 6% was obtained independently of the heating source, power and operation time. Radiofrequency experiments determined that the average percentage linear contraction of white coagulated tissue was 12 ± 5%. The average accuracy of our model was determined to be 3 mm (5%).</p> <p><b>Conclusions:</b> The proposed model allows the prediction of the shrinkage of liver tissues upon microwave ablation given the extension of the carbonised and coagulated zones. This may be useful in helping to predict whether sufficient tissue volume is ablated in clinical practice.</p

Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury <i>In Vitro</i>

<div><p>Several biomedical applications, such as xenotransplantation, require multiple genes simultaneously expressed in eukaryotic cells. Advances in genetic engineering technologies have led to the development of efficient polycistronic vectors based on the use of the 2A self-processing oligopeptide. The aim of this work was to evaluate the protective effects of the simultaneous expression of a novel combination of anti-inflammatory human genes, ENTPD1, E5NT and HO-1, in eukaryotic cells. We produced an F2A system-based multicistronic construct to express three human proteins in NIH3T3 cells exposed to an inflammatory stimulus represented by tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine which plays an important role during inflammation, cell proliferation, differentiation and apoptosis and in the inflammatory response during ischemia/reperfusion injury in several organ transplantation settings. The protective effects against TNF-α-induced cytotoxicity and cell death, mediated by HO-1, ENTPD1 and E5NT genes were better observed in cells expressing the combination of genes as compared to cells expressing each single gene and the effect was further improved by administrating enzymatic substrates of the human genes to the cells. Moreover, a gene expression analyses demonstrated that the expression of the three genes has a role in modulating key regulators of TNF-α signalling pathway, namely <i>Nemo</i> and <i>Tnfaip3</i>, that promoted pro-survival phenotype in TNF-α injured cells. These results could provide new insights in the research of protective mechanisms in transplantation settings.</p></div

Changes in <i>Tnfaip3</i> (<i>A20</i>) mRNA expression in control (Ctrl) and pCX-TRI-2A-transfected cells.

<p>Cells were incubated with 50 ng/ml TNF-α for 16h, alone or in combination with 20 μM hemin and/or 200 μM ATP. Murine <i>Tnfaip3/A20</i> mRNA was quantified by real-time PCR. Data (mean±SD of three independent experiments) are normalized for <i>Gapdh</i> gene and expressed as fold change respect to the untreated cells. [*] indicates a significant difference between pCX-TRI-2A-transfected cells and control cells within the same treatment (t Student, <i>p</i><0.05); [#] indicates a significant difference as compared to untreated cells within the same cell type (ANOVA, <i>p</i><0.05); [a] indicates a significant difference as compared to TNF-α treatment alone within the same cell type (ANOVA, <i>p</i><0.05).</p

Heme oxygenase-1 and ectonucleotidases functional assays.

<p>(<b>A</b>) Heme Oxygenase 1 activity assay on NIH3T3 cells. Lysates from WT, mock- and pCX-TRI-2A-transfected cells were incubated for 2 hours with Hemin, BSA, Biliverdin Reductase A in reaction buffer as described in Materials and Methods. As positive control of the assay, wt or mock-transfected cells were pre-stimulated with 50 μM of HO1 inducer, Cobalt Protoporphyrin for 24 hours (wt CoPP, mock CoPP). Enzymatic activity is reported as nanomoles of bilirubin per hours per milligrams of protein extract. Data are expressed as mean ± SEM of 3–4 independent experiments. *<i>p</i><0.05 versus wt and mock-transfected cells. (B) ENTPD1-mediated AMP production and (C) E5NT-mediated adenosine production by wild type, mock and pCX-TRI-2A transfected-cells. Cells were incubated with 50 μM ATP for 30 min. The nucleotide content of supernatants collected at 0, 5, 15, 30 min time points was measured by reverse phase-HPLC as detailed in Material and Methods. Data shown are mean ± S.D. (n = 3). *<i>p</i><0.05 versus all groups. (D) E5NT-mediated adenosine production by wild type, mock and transfected-cells. Cells were incubated with 50 μM AMP for 30 min. The nucleotide content of supernatants collected at 0, 5, 15, 30 min time points was measured by reverse phase-HPLC ad detailed in Material and Methods. Data shown are mean ± S.D. (n = 3). *<i>p</i><0.05 versus all groups.</p

Changes in <i>Ikgkb</i> (<i>Nemo</i>) mRNA expression in control (Ctrl) and pCX-TRI-2A-transfected cells.

<p>Cells were incubated with 50 ng/ml TNF-α for 16h, alone or in combination with 20 μM hemin and/or 200 μM ATP. Murine <i>Ikgkb</i> mRNA was quantified by real-time PCR. The data (mean±SD of three independent experiments), normalized for <i>Gapdh</i> gene, are expressed as fold change respect to the untreated cells. [*] indicates a significant difference between pCX-TRI-2A-transfected cells and Ctrl cells within the same treatment (t Student, <i>p</i><0.05); [#] indicates a significant difference as compared to untreated cells within the same cell type (ANOVA, <i>p</i><0.05) [a] indicates a significant difference as compared to TNF-α treatment alone within the same cell type (ANOVA, <i>p</i><0.05).</p

pCX-TRI-2A transfected-cells are protected against TNF-α-induced apoptosis.

<p>Caspase 3/7 activities were determined in all cell groups after 16h (<b>A</b>) or 24h (<b>B</b>) of incubation with 50 ng/ml TNF-α alone or in combination with 20 μM hemin and/or 200 μM ATP. Expression of the three human genes in TG cells significantly reduced the activation of effector caspases 3/7. The data are expressed as mean ± SD of three independent experiments. [#] indicates significant difference between pCX-TRI-2A and all the other groups, except for pCX-hHO1, within the same treatment (ANOVA, <i>p</i><0.05). [*] indicates significant difference between pCX-TRI-2A and all the other groups within the same treatment (ANOVA, <i>p</i><0.05); [ϕ] indicates a significant difference between single gene-transfected cells and WT/mock within the same treatment (ANOVA, <i>p</i><0.05); [a] indicates significant difference as compared to TNF-α treatment alone within the same cell type (ANOVA, <i>p</i><0.05); [§] indicates a significant difference as compared to all the other treatments within the same cell type (ANOVA, <i>p</i><0.05).</p

pCX TRI 2A-transfected cells were enriched via FACS for the expression of hE5NT and hENTPD1.

<p>Expression analysis on sorted transfectants showed that E5NT and ENTPD1 were expressed by more than 95% of the pCX-TRI-2A-transfected cells (<b>A</b>) and that more than 93% of those cells expressed both the two ecto-enzyme simultaneously (<b>B</b>). (<b>C</b>) The three human proteins were found strongly expressed into the transfected cells after sorting, and expression levels were unaffected by the number of genes in the construct, as there was no evidence of incomplete separation of individual proteins.</p

Nf-kB1 nuclear translocation in control (Ctrl) and pCX-TRI-2A-transfected cells.

<p>Cells were incubated with 50 ng/ml TNF-α for 16h, alone or in combination with 20 μM hemin and 200 μM ATP. Murine Nf-kB1/p50 protein was detected in nuclear extracts of Ctrl and pCX-TRI-2A-transfected cells by immunoblotting analysis (<b>A</b>). Densitometric analysis for Nf-kB1/p50, normalized for the Lamin B2 signal, showed a 60% or 83.6% increase in Nf-kB1/p50 level in pCX-TRI-2A-transfected cells treated with TNF-α alone or in combination with hemin and ATP, respectively, as compared to control cells (<b>B</b>). Densitometric analysis was performed by using “Gel analyzer” function of ImageJ software. Data are shown as mean ± S.D from at least three independent experiments. [*] indicates a significant difference between pCX-TRI-2A-transfected cells and control cells within the same treatment (t Student, <i>p</i><0.05).</p

Schematic maps of tricistronic pCX-TRI-2A vector and all the constructs used in the work.

<p>The transgenic plasmid is composed of the CAGGS promoter, followed by the coding sequence of human HO-1 gene without the stop codon, fused in frame to the first F2A sequence (F2A), then the coding sequence of human E5NT gene without the stop codon, the second F2A sequence and the coding sequence of human ENTPD1 gene followed by a polyadenilation signal (pA).</p

pCX-TRI-2A transfected cells are protected against TNF-α-induced cytotoxicity.

<p>WT cells and transfected cell lines were incubated with 50 ng/ml TNF-α for 24 h (<b>A</b>) and 48 h (<b>B</b>) alone or in combination with 20 μM hemin and/or 200 μM ATP. Cytotoxicity was assessed by lactate dehydrogenase (LDH) release and expressed as follows: relative cytotoxicity (%)  =  [(<i>A</i> e − <i>A</i> c) / (<i>A</i> b − <i>A</i> c)] × 100 (%), where ‘<i>A</i> e’ is the experimental absorbance, ‘<i>A</i> b’ is the absorbance of lysed controls and ‘<i>A</i> c’ is the absorbance of untreated controls. The data are expressed as mean ± SD of three independent experiments. [#] indicates significant difference between pCX-TRI-2A and all the other groups, except for pCX-hHO1, within the same treatment (ANOVA, <i>p</i><0.05). [*] indicates significant difference between pCX-TRI-2A and all the other groups within the same treatment (ANOVA, <i>p</i><0.05); [ϕ] indicates a significant difference between single gene-transfected cells and WT/mock within the same treatment (ANOVA, <i>p</i><0.05); [a] indicates significant difference as compared to TNF-α treatment alone within the same cell type (ANOVA, <i>p</i><0.05); [b] indicates a significant difference as compared to TNF-α + hemin treatment within the same cell type (ANOVA, <i>p</i><0.05).</p