Effects of ischemic preconditioning in a pig model of
                    large-for-size liver transplantation

Leal, Antonio José Gonçalves; Tannuri, Ana Cristina Aoun; Belon, Alessandro Rodrigo; Guimarães, Raimundo Renato Nunes; Coelho, Maria Cecília Mendonça; Gonçalves, Josiane de Oliveira; Serafini, Suellen; Melo, Evandro Sobroza de; Tannuri, Uenis

doi:10.6061/clinics/2015(02)10

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BASIC RESEARCHS • Clinics 70 (2) • Feb 2015 • https://doi.org/10.6061/clinics/2015(02)10 copy

Effects of ischemic preconditioning in a pig model of large-for-size liver transplantation

Authorship SCIMAGO INSTITUTIONS RANKINGS

OBJECTIVE:

In most cases of pediatric liver transplantation, the clinical scenario of large-for-size transplants can lead to hepatic dysfunction and a decreased blood supply to the liver graft. The objective of the present experimental investigation was to evaluate the effects of ischemic preconditioning on this clinical entity.

METHODS:

Eighteen pigs were divided into three groups and underwent liver transplantation: a control group, in which the weights of the donors were similar to those of the recipients, a large-for-size group, and a large-for-size + ischemic preconditioning group. Blood samples were collected from the recipients to evaluate the pH and the sodium, potassium, aspartate aminotransferase and alanine aminotransferase levels. In addition, hepatic tissue was sampled from the recipients for histological evaluation, immunohistochemical analyses to detect hepatocyte apoptosis and proliferation and molecular analyses to evaluate the gene expression of Bax (pro-apoptotic), Bcl-XL (anti-apoptotic), c-Fos and c-Jun (immediate-early genes), ischemia-reperfusion-related inflammatory cytokines (IL-1, TNF-alpha and IL-6, which is also a stimulator of hepatocyte regeneration), intracellular adhesion molecule, endothelial nitric oxide synthase (a mediator of the protective effect of ischemic preconditioning) and TGF-beta (a pro-fibrogenic cytokine).

RESULTS:

All animals developed acidosis. At 1 hour and 3 hours after reperfusion, the animals in the large-for-size and large-for-size + ischemic preconditioning groups had decreased serum levels of Na and increased serum levels of K and aspartate aminotransferase compared with the control group. The molecular analysis revealed higher expression of the Bax, TNF-alpha, I-CAM and TGF-beta genes in the large-for-size group compared with the control and large-for-size + ischemic preconditioning groups. Ischemic preconditioning was responsible for an increase in c-Fos, IL-1, IL-6 and e-NOS gene expression.

CONCLUSION:

Ischemia-reperfusion injury in this model of large-for-size liver transplantation could be partially attenuated by ischemic preconditioning.

Liver Transplantation; Models; Animal; Reperfusion; Apoptosis; Ischemic Preconditioning

Gene	Primers
β actin	Sense: 5′-tcc aga ggc gct ctt cca-3′
	Antisense: 5′-cgc act tca tga tcg agt tga-3′
Bax	Sense: 5′-ctg gga ctt ctt gct gga ttt gc-3′
	Antisense: 5′-cag ggt gaa gca ggt gaa gat c-3′
Bcl-XL	Sense: 5′-gga gct ggt ggt tga ctt tc-3′
	Antisense: 5′-gtt tcc tct tct gat tca gtc-3′
eNOS	Sense: 5′-aca tct gca acc aca tca agt acg-3′
	Antisense: 5′-cac cag ttg gct gtt cca gat c-3′
IL-6	Sense: 5′-cga gcc cac cag gaa cga aag-3′
	Antisense: 5′-aag cag ccc cag gga gaa ggc-3′
IL-1	Sense: 5′-caa gga cag tgt ggt gat gg-3′
	Antisense: 5′-cat cat tca gga tgc act gg-3′
TGF-beta	Sense: 5′-cct gga tac caa cta ctg ctt c-3′
	Antisense: 5′-gtg tct agg ctc cag atg tag g-3′
TNF-alpha	Sense: 5′-cca cca acg ttt tcc tca ct-3′
	Antisense: 5′-ggc act gag tcg atc atc ct-3′
c-Fos	Sense: 5′-acg gtg act gct atc tcg ac-3′
	Antisense: 5′-tct tct tct ggg gac aac tg-3′
c-Jun	Sense: 5′-acc ttg aaa gcg cag aac tc-3′
	Antisense: 5′-aac agt ctc gcc tca aaa cg-3′
ICAM	Sense: 5′-ttc ctt gga tgg act gtt cc-3′
	Antisense: 5′-cgt ctg cca gca tta tct ca-3′

	Control	LFS	LFS + IPC
Donor weight (kg)	20.3±0.58	44.8±6.21^* *p = 0.005 relative to control.	48.24±2.89^ p = 0.001 relative to control.
Recipient weight (kg)	20.86±1.08	22.2±4.76	20.6±2.79
GBWR	2.9±0.22	6.3±0.73^* *p = 0.005 relative to control.	6.4±0.98^* *p = 0.004 relative to control.
Total ischemia time (min.)	138.8±17.8	147.6±8.3	152.0±14.5
Warm ischemia time (min.)	44.8±3.7	46.8±2.7	47.0±3.4

	Control		LFS^* *p = 0.7 and p = 0.8 relative to the control group at 1 h and 3 h, respectively, for Ki-67.		LFS + IPC^* *p = 0.7 and p = 0.8 relative to the control group at 1 h and 3 h, respectively, for Ki-67.
	1 h	3 h	1 h	3 h	1 h	3 h
Ki-67	10 (5-10)	15 (8-25)	7.5 (3-15)	20 (6-25)	15 (6-25)	10 (3-15)
Cytokeratin	1 (0-3)	2 (0-4)	2 (1-5)	1 (0-3)	1 (0-4)	2 (0-4)

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E-mail: clinics@hc.fm.usp.br

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[1] AUTHOR CONTRIBUTIONS

Leal AJ, Tannuri AC, Belon AR and Guimarães RR performed the experiments. Coelho MC, Gonçalves JO, Serafini S and Melo ES performed the laboratory analyses. Tannuri U performed the final revision of the manuscript.