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Treatment with 17β‐estradiol protects donor heart against brain death effects in female rat
2020; Springer Science+Business Media; Volume: 33; Issue: 10 Linguagem: Inglês
10.1111/tri.13687
ISSN1432-2277
AutoresRoberto Armstrong, Fernanda Yamamoto Ricardo‐da‐Silva, Cristiano de Jesus Correia, Marina Vidal‐dos‐Santos, Lucas Ferreira da Anunciação, Raphael dos Santos Coutinho e Silva, Luíz Felipe Pinho Moreira, Henri G. D. Leuvenink, Ana Cristina Breithaupt‐Faloppa,
Tópico(s)Nitric Oxide and Endothelin Effects
ResumoTransplant InternationalVolume 33, Issue 10 p. 1312-1321 Original ArticleFree Access Treatment with 17β-estradiol protects donor heart against brain death effects in female rat Roberto Armstrong-Jr, orcid.org/0000-0002-5228-3992 Laboratorio de Cirurgia Cardiovascular e Fisiopatologia da Circulação (LIM-11), Instituto do Coração (InCor), Faculdade de Medicina da Universidade de São Paulo, São Paulo, BrazilSearch for more papers by this authorFernanda Yamamoto Ricardo-da-Silva, orcid.org/0000-0003-3495-8551 Laboratorio de Cirurgia Cardiovascular e Fisiopatologia da Circulação (LIM-11), Instituto do Coração (InCor), Faculdade de Medicina da Universidade de São Paulo, São Paulo, BrazilSearch for more papers by this authorCristiano Jesus Correia, orcid.org/0000-0002-1690-9951 Laboratorio de Cirurgia Cardiovascular e Fisiopatologia da Circulação (LIM-11), Instituto do Coração (InCor), Faculdade de Medicina da Universidade de São Paulo, São Paulo, BrazilSearch for more papers by this authorMarina Vidal-dos-Santos, orcid.org/0000-0002-6965-5326 Laboratorio de Cirurgia Cardiovascular e Fisiopatologia da Circulação (LIM-11), Instituto do Coração (InCor), Faculdade de Medicina da Universidade de São Paulo, São Paulo, BrazilSearch for more papers by this authorLucas Ferreira da Anunciação, orcid.org/0000-0002-0398-4370 Laboratorio de Cirurgia Cardiovascular e Fisiopatologia da Circulação (LIM-11), Instituto do Coração (InCor), Faculdade de Medicina da Universidade de São Paulo, São Paulo, BrazilSearch for more papers by this authorRaphael Santos Coutinho e Silva, orcid.org/0000-0001-8818-5043 Laboratorio de Cirurgia Cardiovascular e Fisiopatologia da Circulação (LIM-11), Instituto do Coração (InCor), Faculdade de Medicina da Universidade de São Paulo, São Paulo, BrazilSearch for more papers by this authorLuiz Felipe Pinho Moreira, orcid.org/0000-0003-0179-4976 Laboratorio de Cirurgia Cardiovascular e Fisiopatologia da Circulação (LIM-11), Instituto do Coração (InCor), Faculdade de Medicina da Universidade de São Paulo, São Paulo, BrazilSearch for more papers by this authorHendrik Gerrit Derk Leuvenink, orcid.org/0000-0001-5036-2999 Department of Surgery, University Medical Centre Groningen, University of Groningen, Groningen, The NetherlandsSearch for more papers by this authorAna Cristina Breithaupt-Faloppa, Corresponding Author ana.breithaupt@hc.fm.usp.br orcid.org/0000-0003-0554-0674 Laboratorio de Cirurgia Cardiovascular e Fisiopatologia da Circulação (LIM-11), Instituto do Coração (InCor), Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil Correspondence Ana Cristina Breithaupt-Faloppa, PhD, Laboratório de Cirurgia Cardiovascular e Fisiopatologia da Circulação (LIM/11) - HC-FMUSP, Universidade de São Paulo - Av. Dr. Arnaldo, 455 2º andar - sala 2146 - 01246-903 São Paulo, Brazil. Tel.: +55 1130618647; Fax: +55 1130617178; e-mail: ana.breithaupt@hc.fm.usp.brSearch for more papers by this author Roberto Armstrong-Jr, orcid.org/0000-0002-5228-3992 Laboratorio de Cirurgia Cardiovascular e Fisiopatologia da Circulação (LIM-11), Instituto do Coração (InCor), Faculdade de Medicina da Universidade de São Paulo, São Paulo, BrazilSearch for more papers by this authorFernanda Yamamoto Ricardo-da-Silva, orcid.org/0000-0003-3495-8551 Laboratorio de Cirurgia Cardiovascular e Fisiopatologia da Circulação (LIM-11), Instituto do Coração (InCor), Faculdade de Medicina da Universidade de São Paulo, São Paulo, BrazilSearch for more papers by this authorCristiano Jesus Correia, orcid.org/0000-0002-1690-9951 Laboratorio de Cirurgia Cardiovascular e Fisiopatologia da Circulação (LIM-11), Instituto do Coração (InCor), Faculdade de Medicina da Universidade de São Paulo, São Paulo, BrazilSearch for more papers by this authorMarina Vidal-dos-Santos, orcid.org/0000-0002-6965-5326 Laboratorio de Cirurgia Cardiovascular e Fisiopatologia da Circulação (LIM-11), Instituto do Coração (InCor), Faculdade de Medicina da Universidade de São Paulo, São Paulo, BrazilSearch for more papers by this authorLucas Ferreira da Anunciação, orcid.org/0000-0002-0398-4370 Laboratorio de Cirurgia Cardiovascular e Fisiopatologia da Circulação (LIM-11), Instituto do Coração (InCor), Faculdade de Medicina da Universidade de São Paulo, São Paulo, BrazilSearch for more papers by this authorRaphael Santos Coutinho e Silva, orcid.org/0000-0001-8818-5043 Laboratorio de Cirurgia Cardiovascular e Fisiopatologia da Circulação (LIM-11), Instituto do Coração (InCor), Faculdade de Medicina da Universidade de São Paulo, São Paulo, BrazilSearch for more papers by this authorLuiz Felipe Pinho Moreira, orcid.org/0000-0003-0179-4976 Laboratorio de Cirurgia Cardiovascular e Fisiopatologia da Circulação (LIM-11), Instituto do Coração (InCor), Faculdade de Medicina da Universidade de São Paulo, São Paulo, BrazilSearch for more papers by this authorHendrik Gerrit Derk Leuvenink, orcid.org/0000-0001-5036-2999 Department of Surgery, University Medical Centre Groningen, University of Groningen, Groningen, The NetherlandsSearch for more papers by this authorAna Cristina Breithaupt-Faloppa, Corresponding Author ana.breithaupt@hc.fm.usp.br orcid.org/0000-0003-0554-0674 Laboratorio de Cirurgia Cardiovascular e Fisiopatologia da Circulação (LIM-11), Instituto do Coração (InCor), Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil Correspondence Ana Cristina Breithaupt-Faloppa, PhD, Laboratório de Cirurgia Cardiovascular e Fisiopatologia da Circulação (LIM/11) - HC-FMUSP, Universidade de São Paulo - Av. Dr. Arnaldo, 455 2º andar - sala 2146 - 01246-903 São Paulo, Brazil. Tel.: +55 1130618647; Fax: +55 1130617178; e-mail: ana.breithaupt@hc.fm.usp.brSearch for more papers by this author First published: 04 July 2020 https://doi.org/10.1111/tri.13687Citations: 2 This study was presented at the 19th Congress of the European Society for Organ Transplantation, September 2019, Copenhagen, Denmark; XVI Congresso Brasileiro de Transplantes, October 2019, Campinas, Brazil. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract The viability of donor organs is reduced by hemodynamic and immunologic alterations caused by brain death (BD). Female rats show higher heart inflammation associated with the reduction in female sex hormones after BD. This study investigated the effect of 17β-estradiol (E2) on BD-induced cardiac damage in female rats. Groups of female Wistar rats were assigned: Sham-operation (Sham), brain death (BD), treatment with E2 (50 μg/ml, 2 ml/h) 3 h after BD (E2-T3), or immediately after BD confirmation (E2-T0). White blood cell (WBC) count was analyzed; cytokines and troponin-I were quantified. Heart histopathological changes and expression of endothelial nitric oxide synthase, endothelin-1, intercellular adhesion molecule-1, BCL-2, and caspase-3 were evaluated. Cardiac function was continuously assessed for 6 h by left ventricular pressure-volume loop analysis. E2 decreased the BD-induced median serum concentration of troponin-I (BD:864.2 vs. E2-T0:401.4; P = 0.009), increased BCL-2 (BD:0.086 vs. E2-T0:0.158; P = 0.0278) and eNOS median expression in the cardiac tissue (BD:0.001 vs. E2-T0:0.03 and E2-T3:0.0175; P < 0.0001), and decreased caspase-3 (BD:0.025 vs. E2-T0:0.006 and E2-T3:0.019; P = 0.006), WBC counts, leukocyte infiltration, and hemorrhage. 17β-estradiol treatment was effective in reducing cardiac tissue damage in brain-dead female rats owing to its ability to reduce leukocyte infiltration and prevent cardiomyocyte apoptosis. Introduction Various clinical and experimental studies have demonstrated the negative impact of brain death (BD) on the viability of the organ to be transplanted. The reduced number of suitable organs, mainly heart and lungs, has been a limiting factor to attend the growing number of patients on the waiting lists [1]. Therefore, an understanding of the alterations generated by BD in the donor organs will help in optimization of the clinical management of these patients and assist in obtaining more suitable organs for transplantation. It has been reported that the induction of BD due to increase in the intracranial pressure leads to a hyperdynamic reaction in the heart, causing ventricular function changes, hemodynamic instability, decrease in myocardial isoenzymes, and histological injury [2]. According to the International Society for Heart and Lung Transplantation, donor-recipient gender mismatch is an important factor that impacts the survival of heart transplant patients, especially after the first year of transplantation [3]. Other studies have also suggested donor gender as a factor that influences the outcome of heart transplantation [4, 5]. In this context, experimental studies have shown severe inflammatory response in brain-dead female rats, owing to BD-induced estradiol reduction [6, 7]. In another study, estradiol deficiency was found to be correlated to kidney transplantation failure in female rats [8]. Since the reduction of estradiol concentration by BD is a potential modulator of inflammation, we aimed to investigate whether treatment with 17β-estradiol could be beneficial in female heart donors. Materials and methods Animals Female Wistar rats (7–8 weeks old; n = 32) were obtained from our animal facilities. The rats were allowed free access to water and food before the experimental procedure and housed at 23°C ± 2°C in a 12 h light-dark cycle. All rats received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1996). The Animal Subject Committee of Sao Paulo University Medical School approved the experimental protocol (SDC No. 4350/16/016). Groups and treatment Female rats from maximal estradiol secretion to heat period were assigned to four groups (n = 8): (i) Sham-operated animals (Sham)—rats subjected to trepanation without BD induction, (ii) brain death (BD)—rats subjected to BD, (iii) E2 3 h (E2-T3)—rats administered 17β-estradiol (Sigma-Aldrich, USA) in saline (50 µg/ml to 2 ml/h); 3 h after BD induction, and (iv) E2 6 h (E2-T0)—rats administered 17β-estradiol (Sigma-Aldrich, USA) in saline (50 µg/ml to 2 ml/h) immediately after BD induction. Sham and BD groups received fluid replacement (saline solution, 2 ml/h). Estrous cycle identification and hormone quantification Fluid obtained after vaginal lavage with phosphate-buffered saline (PBS) was placed on slides and stained with crystal violet solution (0.2%). Estrus and proestrus cycle were identified using an optical microscope. The quantification of estradiol and corticosterone was performed by ELISA kit (Cayman Chemical Company, USA), following the manufacturer's protocols. Induction of brain death. Anesthesia was performed in a chamber with 5% isoflurane, followed by intubation and ventilation with a rodent ventilator (Harvard Apparatus, model 683, USA), at a frequency of 70 breaths/min (tidal volume 10 ml/kg) and 2% isoflurane. The carotid artery was cannulated for blood sampling under continuous blood pressure monitoring. Saline solution (2 ml/h), with or without treatment, was infused through cannulation of the jugular vein. BD model was based on the method described by Breithaupt-Faloppa et al. (2016) [6]. Briefly, a catheter Fogarty 4F (Baxter Healthcare Co., USA) was inserted intracranially through a drilled parietal burr hole. BD was induced by rapid balloon inflation (400 μL—saline solution), confirmed by mydriasis, apnea, and absence of reflexes; isoflurane inhalation was interrupted. All animals were monitored for 6 h. White blood cell count (WBC) A 20 μL volume of blood was withdrawn, and WBC was analyzed using an automatic hematologic counter (Mindray BC 28000 Vet, China). Quantification of cytokines and troponin-I The levels of granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory proteins (MIP)-1α, MIP-2, interleukin (IL)-1β, IL-10, monocyte chemoattractant protein-1 (MCP-1), cytokine-induced neutrophil chemoattractant-1 (CINC-1), vascular endothelial growth factor (VEGF), and tumor necrosis factor-α (TNF-α) were quantified in serum samples collected at the end of the experimental protocol, using commercial kit Milliplex® (Merck Millipore, USA). Quantification of the serum levels of troponin-I made by ELISA (Elabscience®, USA). Histopathologic heart examination At the end of the experiment, heart samples were obtained and fixed in 10% formaldehyde for 24 h, embedded in paraffin, sectioned (4 µm), and stained with hematoxylin and eosin. Leukocyte infiltration, edema, and hemorrhage were evaluated by two observers blinded for intervention, and the mean values from each were analyzed by correlation tests to determine interobserver variability. Intercellular myocardial edema and hemorrhage were reported as units per square millimeter, whereas leukocytes infiltration was reported in cells per square millimeter of cardiac tissue. Immunohistochemistry Frozen serial sections of the heart tissue (8 µm) were fixed in acetone (10 min), washed in TRIS-buffered saline Tween-20 (TBST), permeabilized with TBST and Triton X-100, followed by blockade of nonspecific sites with TBST, containing 2% bovine serum albumin (BSA) and then peroxidase with H2O2. Sections were incubated in TBST (complemented with 2% BSA and rabbit antibodies to BCL-2 [1:100; Abcam, Cambridge, MA] and caspase-3 [1:200; Abcam, Cambridge, MA]) for 12 h at 4ºC. After washing with TBST, all sections were incubated for 1 h at 37ºC with anti-rabbit secondary antibodies (1:200) conjugated to peroxidase (HRP; Millipore). Next, the sections were washed with TBST and stained with peroxidase substrate 3-amino-9-ethylcarbazole (AEC; Vector Laboratories, USA) (5-10 min) and counterstained with hematoxylin. Negative control sections were incubated in absence of primary antibodies. To evaluate the expression of intercellular adhesion molecule-1 (ICAM-1), paraffin-embedded tissues were sectioned (4µm), rehydrated, and incubated with citrate buffer (pH 6.0) for 20 min at 100ºC to retrieve antigens. Next, the sections were submitted to blocking of nonspecific sites and endogenous peroxidase, and further incubated with mouse antibodies to rat ICAM-1 (1:50 – 12h at 4ºC) (Cedarlane, Canada). After washing with TBST, the sections were incubated for 2 h at room temperature with anti-mouse secondary antibodies attached to HRP (1:400, Novus Biologicals, USA), rinsed, stained with AEC (5-10 min) (Vector Laboratories, USA), and counterstained with hematoxylin. Negative control sections were incubated in the absence of primary antibodies. The analysis was performed through image acquisition system with a digital camera DS-Ri1 (Nikon, Japan) coupled to a Nikon microscope and analyzed using the NIS-Elements BR software (Nikon, Japan). Hemodynamic measurements: left ventricular pressure-volume loop (LVPV) The right carotid artery was dissected and a 2F microtip pressure-conductance catheter (SPR-838; Millar Instruments, USA) was inserted and placed in the left ventricle (LV). To record signals for 6 h, a pressure-volume conductance system (MPVS-Ultra, Millar Instruments, USA) was connected to a data acquisition system (PowerLab, AD Instruments, USA). After stabilization, data related to ejection fraction (EF), stroke volume (SV), end-diastolic volume (EDV), systolic pressure (SP), stroke work (SW), maximum rate of rise of left pressure (dP/dT max), and time constant of left pressure decay (Tau) were obtained. Statistical analysis All data were presented as mean ± standard error of the mean (SEM) or as median and 95% percentile interval. Normally distributed data were analyzed by ANOVA followed by Dunnett or Sidak’s tests for multiple comparisons, with P-values adjusted to account for multiple comparisons. Abnormally distributed data were analyzed using a Kruskal–Wallis test followed by a post hoc Dunn multiple comparison test also, with P-values adjusted to account for multiple comparisons. Statistical analysis was performed using GraphPad Prism software v.8.3. Results Estradiol and corticosterone concentration The quantification of serum estradiol levels showed that BD group had reduction in estradiol, while the Sham group had significant increase in estradiol after 3 h and 6 h after the beginning of experiment. Compared to the BD group, both the estradiol-treated groups showed an increase in estradiol concentration after the beginning of treatment; the E2-T0 group had changes after 3 h and 6 h, while the E2-T3 group showed an increase in estradiol after 6 h (Fig. 1a). The estradiol infusion was able to maintain the hormone concentration stable during the experimental period. Figure 1Open in figure viewerPowerPoint Serum (a) estradiol and (b) corticosterone concentrations. Sham, false-operated rats; BD, rats submitted to encephalic death; E2-T0, 17β-estradiol (E2) treated rats after confirmation of BD and E2-T3, 17β-estradiol (E2)-treated rats after 3 h of confirmation of BD. Data expressed as mean standard error of the mean (SEM; n = 8) *P < 0.0001 in comparison with BD group. In addition, the serum concentration of corticosterone was evaluated. Compared to the BD group, the Sham group had higher concentration of corticosterone at 3 h and 6 h after the beginning of the experiment. However, no difference was observed in corticosterone levels among BD, E2-T3, and E2-T0 groups (Fig. 1b). WBC count At 3 h and 6 h after the beginning of the experiment, BD group had lower number of lymphocytes, granulocytes, and monocytes than those in the Sham group. However, BD group showed leukocytosis after 3 h, which was maintained until 6 h. In contrast, 17β-estradiol-treated female rats did not develop leukocytosis until the end of the follow-up time (Table 1). Table 1. Total and differential white blood cell counts Leukocytes (cells/mm3) Total Lymphocytes Neutrophils Monocytes Sham 0h 10670 ± 1111 6470 ± 388 3860 ± 778 340 ± 50 3h 28270 ± 2908*,**, *,*** P < 0.05 in comparison with Initial. ** P < 0.05 in comparison with BD. 5630 ± 788**** P < 0.05 in comparison with BD. 21560 ± 2349*,**, *,*** P < 0.05 in comparison with Initial. ** P < 0.05 in comparison with BD. 1100 ± 117*,**, *,*** P < 0.05 in comparison with Initial. ** P < 0.05 in comparison with BD. 6h 18120 ± 3562 3750 ± 577** P < 0.05 in comparison with Initial. 13700 ± 2915** P < 0.05 in comparison with Initial. 670 ± 144** P < 0.05 in comparison with Initial. BD 0h 10110 ± 964 6440 ± 523 3230 ± 443 440 ± 76 3h 14790 ± 2122** P < 0.05 in comparison with Initial. 3510 ± 333** P < 0.05 in comparison with Initial. 10870 ± 1850** P < 0.05 in comparison with Initial. 390 ± 77 6h 13060 ± 1304 3870 ± 431** P < 0.05 in comparison with Initial. 11270 ± 2050** P < 0.05 in comparison with Initial. 440 ± 58 E2-T0 0h 8900 ± 1175 4930 ± 699 4000 ± 485 370 ± 67 3h 9250 ± 1033 2550 ± 174** P < 0.05 in comparison with Initial. 6720 ± 692** P < 0.05 in comparison with Initial. 390 ± 80 6h 8460 ± 1052β 2530 ± 332** P < 0.05 in comparison with Initial. 5430 ± 664 380 ± 81 E2-T3 0h 10500 ± 737 6110 ± 440 4000 ± 485 390 ± 38 3h 10060 ± 902 2970 ± 270** P < 0.05 in comparison with Initial. 6720 ± 692** P < 0.05 in comparison with Initial. 340 ± 70 6h 8770 ± 830 3010 ± 312** P < 0.05 in comparison with Initial. 5430 ± 664 330 ± 56 P ANOVA (group) <0.0001 0.0051 <0.0001 0.0004 P ANOVA (time) <0.0001 <0.0001 <0.0001 0.0046 Data expressed as mean ± SEM (n = 8). Statistical testing consisted of ANOVA followed by Dunnett test for multiple comparisons. SHAM, false-operated rats; BD, brain death; E2-T3, 17β-estradiol (E2)-treated rats after 3h of confirmation of BD; E2-T0, 17β-estradiol (E2)-treated rats after confirmation of BD. * P < 0.05 in comparison with Initial. ** P < 0.05 in comparison with BD. Serum cytokines BD caused an increase in the serum levels of CINC-1, TNF-α, and MIP-1α compared with the Sham group. However, no statistically significant differences were detected in the estradiol-treated groups (Table 2). Table 2. Measurement of inflammatory parameters (pg/ml) Sham BD E2-T0 E2-T3 P value Cytokines CINC-1 24.3 (0.2–1504) 1687 (0.3–2999) 1164 (0.3–1897) 986 (0.1–2614) 0.069 TNF-α 7.78 (2.38–32.79) 28.1 (6.15–37.5) 8.92 (3.28–30.87) 20.4 (4.2–38.79) 0.212 Interleukines IL-1 β 52.9 (9.3–108.2) 71.5 (31.2–267.9) 63.1 (5.2–265.9) 55.7 (3.7–244.1) 0.600 IL-10 242 (75–878) 285 (77–689) 290 (30–706) 228 (122–363) 0.953 Chemokines MIP-1α 555 (330–751) 855 (70–2058) 668 (305–1080) 682 (66–884) 0.776 MCP-1 2638 (953–5599) 7960 (1927–12890) 9969 (3534–18633) 8923 (2486–17536) 0.008 MIP-2 201 (14–787) 180 (25–380) 67 (26–430) 162 (14–732) 0.870 Growth factors G-CSF 4.63 (0.3–10.4) 1.58 (0.3–3.2) 5.56 (0.25 −63.3) 1.66 (0.68–15) 0.350 GM-CSF 0.87 (0.13–3.58) 0.74 (0.13–1.49) 1.28 (0.14–6.04) 0.27 (0.13–1.21) 0.447 VEGF 109 (35–1134) 113 (33–262) 158 (62–280) 200 (41–312) 0.748 Data (pg/ml) are expressed as median and 95% percentile interval (n = 8). Statistical testing consisted of Kruskal‒Wallis test followed by Dunn's test for multiple comparisons. SHAM, false-operated rats; BD, brain death; E2-T3, 17β-estradiol (E2)-treated rats after 3h of confirmation of BD; E2-T0, 17β-estradiol (E2)-treated rats after confirmation of BD. G-CSF, granulocyte-colony stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; MIP, macrophage inflammatory protein; IL, interleukin; MCP-1, monocyte chemoattractant protein-1; CINC-1, cytokine-induced neutrophil chemoattractant-1; VEGF, vascular endothelial growth factor; TNF-α, tumor necrosis factor-α. Histopathological analysis In Fig. 2, 17β-estradiol was able to reduce leukocyte infiltration to the heart and the hemorrhage. Figure 2Open in figure viewerPowerPoint Histopathological analysis of heart tissue. (a) leukocyte infiltration, (b) edema units, and (c) hemorrhage units. Sham, false-operated rats; BD, rats submitted to encephalic death; E2-T0, 17β-estradiol (E2)-treated rats after confirmation of BD and E2-T3, 17β-estradiol (E2)-treated rats after 3 h of confirmation of BD. Formaldehyde-fixed sections of heart tissue were stained with hematoxylin-eosin. Original magnification 40 × for all images. Data expressed as median and 95% percentile interval (n = 8 per group; 1 tissue section per animal, 5 fields) α,βp < 0.05 in comparison with BD group. Endothelial function BD group had expression of eNOS protein decreased while the expression of ICAM-1 increased. In contrast, treatment with 17β-estradiol increased the eNOS expression without altering the expression of ICAM-1. There were no differences in the protein expression of endothelin-1 (Fig. 3). Figure 3Open in figure viewerPowerPoint Quantification of protein expression of eNOS (a), endothelin-1 (b), and ICAM-1 in cardiac tissue. Sham, false-operated rats; BD, rats submitted to encephalic death; E2-T0, 17β-estradiol (E2) treated rats after confirmation of BD and E2-T3, 17β-estradiol (E2)-treated rats after 3 h of confirmation of BD. Data expressed as median and 95% percentile interval (n = 5 per group; 1 tissue section per animal, 5 fields). α,β,θp < 0.05 in comparison with BD group. Quantification of troponin-I serum concentration A significant increase in the level of serum troponin-I was observed in the BD group compared with Sham group (Fig. 4). In contrast, treatment with 17β-estradiol, immediately after BD, reduced the troponin-I levels. Figure 4Open in figure viewerPowerPoint Serum troponin-I concentration. Sham, false-operated rats; BD, rats submitted to encephalic death; E2-T0, 17β-estradiol (E2)-treated rats after confirmation of BD and E2-T3, 17β-estradiol (E2)-treated rats after 3 h of confirmation of BD. Data expressed as median and 95% percentile interval (n = 8). β,θp < 0.05 in comparison with BD group. BCL-2 and caspase-3 expression In Fig. 5a, there was a significant upregulation in the expression of the apoptotic protein, caspase-3 in BD group, which was reversed by 17β-estradiol treatment. In addition, there was upregulation in the expression of anti-apoptotic protein, BCL-2 in E2-T0 group (Fig. 5b). Figure 5Open in figure viewerPowerPoint Immunostaining for apoptotic protein in cardiac tissue. Apoptotic protein, caspase-3 (a) and anti-apoptotic protein, BCL-2 (b) Sham, false-operated rats; BD, rats submitted to encephalic death; E2-T0, 17β-estradiol (E2)-treated rats after confirmation of BD and E2-T3, 17β-estradiol (E2)-treated rats after 3 h of confirmation of BD. Original magnification images: 40 × for caspase-3 and 20 × for BCL-2. Data expressed as median and 95% percentile interval (n = 5 per group; 1 tissue section per animal, 5 fields). β,θP < 0.05 in comparison with BD group. LVPV analyses All parameters were analyzed continuously during the 6 h of experiment (Table 3). All groups submitted to BD presented with a decreased EF after 6 h compared to the initial value. At the end of follow-up time, BD group showed reduced EF values compared with the Sham group. In both the estradiol-treated groups, significant increases of EDV were observed after 6 h of treatment when compared to the initial values. Furthermore, compared to the initial values, SW was reduced in BD, E2-T3, and E2-T0 groups after the 6 h of experiment. Other hemodynamic measurements (SVSP, dP/dT max, and Tau) did not differ among the groups during the follow-up time. Table 3. Left ventricular pressure-volume analysis Sham BD E2-T0 E2-T3 P ANOVA EF (%) 0h 60 ± 5 63 ± 3 68 ± 2 70 ± 2 <0.0001 3h 55 ± 4 45 ± 7** P < 0.05 in comparison with Initial. 50 ± 4** P < 0.05 in comparison with Initial. 52 ± 4** P < 0.05 in comparison with Initial. 6h 50 ± 5**** P < 0.05 in comparison with BD. 28 ± 3** P < 0.05 in comparison with Initial. 35 ± 6** P < 0.05 in comparison with Initial. 31 ± 4** P < 0.05 in comparison with Initial. SV (µL) 0h 131 ± 18 132 ± 15 165 ± 20 154 ± 15 0.9169 3h 123 ± 11 122 ± 22 135 ± 17 145 ± 22 6h 133 ± 7 109 ± 21 135 ± 19 118 ± 19 EDV (µL) 0h 185 ± 19 194 ± 21 159 ± 23 188 ± 26 0.0124 3h 228 ± 22 195 ± 12 231 ± 35 200 ± 45 6h 255 ± 22 269 ± 16 266 ± 25** P < 0.05 in comparison with Initial. 328 ± 48** P < 0.05 in comparison with Initial. SP (mmHg) 0h 101 ± 7 108 ± 6 108 ± 4 90 ± 6 0.0733 3h 96 ± 8 95 ± 6 96 ± 9 100 ± 5 6h 83 ± 4 96 ± 6 85 ± 7 93 ± 3 SW (mmHg/µL) 0h 12847 ± 3806 10961 ± 649 13490 ± 1444 11630 ± 2018 <0.0001 3h 11125 ± 1954 7936 ± 1848 9447 ± 1982** P < 0.05 in comparison with Initial. 7662 ± 2085** P < 0.05 in comparison with Initial. 6h 10853 ± 2136 7669 ± 1774 8734 ± 1397** P < 0.05 in comparison with Initial. 6068 ± 1578** P < 0.05 in comparison with Initial. dP/dT max (mmHg/s) 0h 5461 ± 629 6179 ± 715 6092 ± 304 5754 ± 345 <0.0001 3h 5170 ± 723 4842 ± 449 5081 ± 592 5643 ± 425 6h 4427 ± 625 4201 ± 467 4217 ± 531 3763 ± 382 Tau (ms) 0h 12 ± 2 11 ± 1 14 ± 3 10 ± 1 0.0007 3h 14 ± 3 16 ± 3 18 ± 3 17 ± 2 6h 10 ± 1 14 ± 1 21 ± 3 17 ± 2 Data are expressed as mean ± SEM (6 rats per group). Statistical testing consisted of ANOVA followed by Sidak’s test for multiple comparisons. Ejection fraction (EF), stroke volume (SV), end-diastolic volume (EDV), systolic pressure (SP), stroke work (SW), maximum rate of rise of left pressure (dP/dT max) and time constant of left pressure decay (Tau). * P < 0.05 in comparison with Initial. ** P < 0.05 in comparison with BD.
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