Evaluation of the therapeutic effects of oestradiol on the systemic inflammatory response and on lung injury caused by the occlusion of the proximal descending aorta in male rats.

OBJECTIVES: Ischaemia and reperfusion-induced microvascular dysfunction is a serious problem encountered during a variety surgical procedures, leading to systemic inflammation and affecting remote organs, specially the lungs. 17ß-Oestradiol reduces pulmonary repercussions from various acute lung injury forms. Here, we focused on the 17ß-oestradiol therapeutic effects after aortic ischaemia and reperfusion (I/R) by evaluating lung inflammation. METHODS: Twenty-four Wistar rats were submitted to I/R by insufflation of a 2-F catheter in thoracic aorta for 20 min. Reperfusion took 4 h and 17ß-oestradiol (280 µg/kg, i.v.) was administered after 1 h of reperfusion. Sham-operated rats were controls. Bronchoalveolar lavage was performed and lung samples were prepared for histopathological analysis and tissue culture (explant). Interleukin (IL)-1ß, IL-10 and tumour necrosis factor-α were quantified. RESULTS: After I/R, higher number of leukocytes in bronchoalveolar lavage were reduced by 17ß-oestradiol. The treatment also decreased leukocytes in lung tissue. I/R increased lung myeloperoxidase expression, with reduction by 17ß-oestradiol. Serum cytokine-induced neutrophil chemoattractant 1 and IL-1ß increased after I/R and 17ß-oestradiol decreased cytokine-induced neutrophil chemoattractant 1. I/R increased IL-1ß and IL-10 in lung explants, reduced by 17ß-oestradiol. CONCLUSIONS: Our results showed that 17ß-oestradiol treatment performed in the period of reperfusion, modulated the systemic response and the lung repercussions of I/R by thoracic aortic occlusion. Thus, we can suggest that 17ß-oestradiol might be a supplementary approach leading the lung deterioration after aortic clamping in surgical procedures.

Comparison of acute kidney injury following brain death between male and female rats

Abstract Background Clinical reports associate kidneys from female donors with worse prognostic in male recipients. Brain Death (BD) produces immunological and hemodynamic disorders that affect organ viability. Following BD, female rats are associated with increased renal inflammation interrelated with female sex hormone reduction. Here, the aim was to investigate the effects of sex on BD-induced Acute Kidney Injury (AKI) using an Isolated Perfused rat Kidney (IPK) model. Methods Wistar rats, females, and males (8 weeks old), were maintained for 4h after BD. A left nephrectomy was performed and the kidney was preserved in a cold saline solution (30 min). IPK was performed under normothermic temperature (37°C) for 90 min using WME as perfusion solution. AKI was assessed by morphological analyses, staining of complement system components and inflammatory cell markers, perfusion flow, and creatinine clearance. Results BD-male kidneys had decreased perfusion flow on IPK, a phenomenon that was not observed in the kidneys of BD-females (p< 0.0001). BD-male kidneys presented greater proximal (p= 0.0311) and distal tubule (p= 0.0029) necrosis. However, BD-female kidneys presented higher expression of eNOS (p= 0.0060) and greater upregulation of inflammatory mediators, iNOS (p= 0.0051), and Caspase-3 (p= 0.0099). In addition, both sexes had increased complement system formation (C5b-9) (p=0.0005), glomerular edema (p= 0.0003), and nNOS (p= 0.0051). Conclusion The present data revealed an important sex difference in renal perfusion in the IPK model, evidenced by a pronounced reduction in perfusate flow and low eNOS expression in the BD-male group. Nonetheless, the upregulation of genes related to the proinflammatory cascade suggests a progressive inflammatory process in BD-female kidneys.

Long-term lung inflammation is reduced by estradiol treatment in brain dead female rats.

OBJECTIVES: Lung transplantation is limited by the systemic repercussions of brain death (BD). Studies have shown the potential protective role of 17ß-estradiol on the lungs. Here, we aimed to investigate the effect of estradiol on the long-lasting lung inflammatory state to understand a possible therapeutic application in lung donors with BD. METHODS: Female Wistar rats were separated into 3 groups: BD, subjected to brain death (6h); E2-T0, treated with 17ß-estradiol (50 µg/mL, 2 mL/h) immediately after brain death; and E2-T3, treated with 17ß-estradiol (50 µg/ml, 2 ml/h) after 3h of BD. Complement system activity and macrophage presence were analyzed. TNF-α, IL-1ß, IL-10, and IL-6 gene expression (RT-PCR) and levels in 24h lung culture medium were quantified. Finally, analysis of caspase-3 gene and protein expression in the lung was performed. RESULTS: Estradiol reduced complement C3 protein and gene expression. The presence of lung macrophages was not modified by estradiol, but the release of inflammatory mediators was reduced and TNF-α and IL-1ß gene expression were reduced in the E2-T3 group. In addition, caspase-3 protein expression was reduced by estradiol in the same group. CONCLUSIONS: Brain death-induced lung inflammation in females is modulated by estradiol treatment. Study data suggest that estradiol can control the inflammatory response by modulating the release of mediators after brain death in the long term. These results strengthen the idea of estradiol as a therapy for donor lungs and improving transplant outcomes.

Estradiol prevented intestinal ischemia and reperfusion-induced changes in intestinal permeability and motility in male rats.

OBJECTIVES: Ischemia and reperfusion (I/R) in the intestine could lead to severe endothelial injury, compromising intestinal motility. Reportedly, estradiol can control local and systemic inflammation induced by I/R injury. Thus, we investigated the effects of estradiol treatment on local repercussions in an intestinal I/R model. METHODS: Rats were subjected to ischemia via the occlusion of the superior mesenteric artery (45 min) followed by reperfusion (2h). Thirty minutes after ischemia induction (E30), 17ß-estradiol (E2) was administered as a single dose (280 µg/kg, intravenous). Sham-operated animals were used as controls. RESULTS: I/R injury decreased intestinal motility and increased intestinal permeability, accompanied by reduced mesenteric endothelial nitric oxide synthase (eNOS) and endothelin (ET) protein expression. Additionally, the levels of serum injury markers and inflammatory mediators were elevated. Estradiol treatment improved intestinal motility, reduced intestinal permeability, and increased eNOS and ET expression. Levels of injury markers and inflammatory mediators were also reduced following estradiol treatment. CONCLUSION: Collectively, our findings indicate that estradiol treatment can modulate the deleterious intestinal effects of I/R injury. Thus, estradiol mediates the improvement in gut barrier functions and prevents intestinal dysfunction, which may reduce the systemic inflammatory response.

17ß-Estradiol Treatment Protects Lungs Against Brain Death Effects in Female Rat Donor.

BACKGROUND: Brain death (BD) affects the viability of lungs for transplantation. A correlation exists between high-lung inflammation after BD and the decrease in female sex hormones, especially estradiol. Therefore, we investigated the effects of 17ß-estradiol (E2) treatment on the lungs of female brain dead rats. METHODS: Female Wistar rats were divided into 4 groups: BD (submitted to BD for 6 h), sham (false operated), E2-T0 (treated with E2 immediately after BD; 50 µg/mL, 2 mL/h), and E2-T3 (treated with E2 after 3 h of BD; 50 µg/mL, 2 mL/h). Lung edema, hemorrhage, and leukocyte infiltration were analyzed. Adhesion molecules were evaluated, and analysis of NO synthase gene and protein expression was performed using real-time PCR and immunohistochemistry, respectively. Release of chemokines and matrix degradation in the lungs was analyzed. RESULTS: BD increased leukocyte infiltration, as shown by intravital microscopy (P = 0.017), bronchoalveolar lavage cell count (P = 0.016), the release of inflammatory mediators (P = 0.02), and expression of adhesion molecules. BD also increased microvascular permeability and the expression and activity of matrix metalloproteinase-9 in the lungs. E2 treatment reduced leukocyte infiltration, especially in the E2-T3 group, release of inflammatory mediators, adhesion molecules, and matrix metalloproteinase activity in the lungs. CONCLUSIONS: E2 treatment was successful in controlling the lung inflammatory response in females submitted to BD. Our results suggest that E2 directly decreases the release of chemokines, restraining cell traffic into the lungs. Thus, E2 has a therapeutic potential, and its role in improving donor lung quality should be explored further.

Estradiol prevented intestinal ischemia and reperfusion-induced changes in intestinal permeability and motility in male rats

OBJECTIVES: Ischemia and reperfusion (I/R) in the intestine could lead to severe endothelial injury, compromising intestinal motility. Reportedly, estradiol can control local and systemic inflammation induced by I/R injury. Thus, we investigated the effects of estradiol treatment on local repercussions in an intestinal I/R model. METHODS: Rats were subjected to ischemia via the occlusion of the superior mesenteric artery (45 min) followed by reperfusion (2h). Thirty minutes after ischemia induction (E30), 17β-estradiol (E2) was administered as a single dose (280 μg/kg, intravenous). Sham-operated animals were used as controls. RESULTS: I/R injury decreased intestinal motility and increased intestinal permeability, accompanied by reduced mesenteric endothelial nitric oxide synthase (eNOS) and endothelin (ET) protein expression. Additionally, the levels of serum injury markers and inflammatory mediators were elevated. Estradiol treatment improved intestinal motility, reduced intestinal permeability, and increased eNOS and ET expression. Levels of injury markers and inflammatory mediators were also reduced following estradiol treatment. CONCLUSION: Collectively, our findings indicate that estradiol treatment can modulate the deleterious intestinal effects of I/R injury. Thus, estradiol mediates the improvement in gut barrier functions and prevents intestinal dysfunction, which may reduce the systemic inflammatory response.

Long-term lung inflammation is reduced by estradiol treatment in brain dead female rats

OBJECTIVES: Lung transplantation is limited by the systemic repercussions of brain death (BD). Studies have shown the potential protective role of 17β-estradiol on the lungs. Here, we aimed to investigate the effect of estradiol on the long-lasting lung inflammatory state to understand a possible therapeutic application in lung donors with BD. METHODS: Female Wistar rats were separated into 3 groups: BD, subjected to brain death (6h); E2-T0, treated with 17β-estradiol (50 μg/mL, 2 mL/h) immediately after brain death; and E2-T3, treated with 17β-estradiol (50 μg/ml, 2 ml/h) after 3h of BD. Complement system activity and macrophage presence were analyzed. TNF-α, IL-1β, IL-10, and IL-6 gene expression (RT-PCR) and levels in 24h lung culture medium were quantified. Finally, analysis of caspase-3 gene and protein expression in the lung was performed. RESULTS: Estradiol reduced complement C3 protein and gene expression. The presence of lung macrophages was not modified by estradiol, but the release of inflammatory mediators was reduced and TNF-α and IL-1β gene expression were reduced in the E2-T3 group. In addition, caspase-3 protein expression was reduced by estradiol in the same group. CONCLUSIONS: Brain death-induced lung inflammation in females is modulated by estradiol treatment. Study data suggest that estradiol can control the inflammatory response by modulating the release of mediators after brain death in the long term. These results strengthen the idea of estradiol as a therapy for donor lungs and improving transplant outcomes.

Treatment with 17ß-estradiol protects donor heart against brain death effects in female rat.

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 17ß-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. 17ß-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.

Hypertonic saline reduces cell infiltration into the lungs after brain death in rats.

BACKGROUND: Lung transplantation is a treatment method for end stage lung disease, but the availability of donor lungs remains a major constraint. Brain death (BD) induces hemodynamic instability with microcirculatory hypoperfusion and increased inflammation, leading to pulmonary dysfunction. Hypertonic saline solution (HSS) is a volume expander possessing immunomodulatory effects. This study evaluated the influence of HSS on pulmonary dysfunction and inflammation in a rat model of BD. METHODS: BD was induced by inflation of an intracranial balloon catheter. Rats were divided into [1]: Sham, without BD [2]; NS, NaCl treatment (0.9%, 4 mL/kg, i.v.) immediately after BD [3]; HSS1, HSS treatment (NaCl 7.5%, 4 mL/kg, i.v.) immediately after BD; and [4] HSS60, HSS treatment 60 min post BD. All groups were analyzed after 360 min. RESULTS: Animals subjected to BD exhibited increased exhaled O2 and decreased CO2.The number of leukocytes in the lungs was significantly increased in the NS group (p = 0.002) and the HSS treatment was able to reduce it (HSS1, p = 0.018 and HSS60 = 0.030). In parallel, HSS-treated rats showed reduced levels of ICAM-1 expression, which was increased in the NS compared to Sham group. Lung edema was found increased in the NS group animals compared to Sham and no effect of the HSS treatment was observed. There were no differences among the groups in terms of TNF-α, VEGF, and CINC-1 lung concentrations. CONCLUSIONS: HSS is capable of reducing inflammatory cell infiltration into the lung after BD induction, which is associated with the reduction of ICAM-1 expression in organ vessels.

The influence of female sex hormones on lung inflammation after brain death - an experimental study.

Organ donor's age negatively influences graft survival of organs, increasing risk of complications. Aging occurs in both men and women; however, the menopause marks a decrease in sex hormones and a sudden increase in the process of vascular aging. We investigated sex hormones' influence on the lung inflammatory process induced by BD in female rats. Wistar rats were grouped as: female rats from high estradiol to heat period (non-OVx) and ovariectomized (OVx) female rats. Ovariectomy was carried out 10 days before BD. BD was induced using intracranial balloon rapid inflation. Serum hormones and inflammatory mediators were quantified, leukocytes and platelets counted and lung samples were collected for RT-PCR, immunohistochemical, and histological analysis. Female sex hormones and corticosterone were reduced 6 h after BD in non-OVx group. The infiltration of leukocytes in female non-OVx lungs was higher compared to OVx. G-CSF, VEGF, and CINC-1 were found increased in non-OVx group serum in comparison to OVx. Lung mediators were increased in non-OVx rats compared to controls. The acute reduction of sex hormones induced by BD appears to have a worse effect on lung inflammation than a reduction that has happened over a prolonged period of time, allowing a physiological adaptation prior to BD.

Estradiol mediates the long-lasting lung inflammation induced by intestinal ischemia and reperfusion.

BACKGROUND: Lung inflammation is one of the main consequences of intestinal ischemia reperfusion (intestinal IR) and, in severe cases, can lead to acute respiratory distress syndrome and death. We have previously demonstrated that estradiol exerts a protective effect on lung edema and cytokine release caused by intestinal IR in male rats. MATERIALS AND METHODS: We investigated the role of estradiol on the generation of interleukin (IL)-1ß, IL-10, vascular endothelial growth factor (VEGF), and cytokine-induced neutrophil chemoattractant 1 (CINC-1) in a female rat model of intestinal IR. Blood and bone marrow leukocytes were also quantified. Seven-days-ovariectomized rats were subjected to intestinal IR by occlusion of the superior mesenteric artery for 45 min. After reperfusion of the tissue for 2 h, the rats were sacrificed. Lung tissue was collected, cultured for 24 h and assayed. RESULTS: We observed a significant increase in serum levels of IL-10, CINC-1, uric acid and circulating, but not bone marrow, leukocyte numbers. In addition, intestinal IR induced a significant increase in the ex-vivo lung levels of IL-1ß, IL-10, and VEGF. Treatment with 17ß-estradiol before the induction of intestinal IR prevented the systemic release of IL-10, CINC-1, and uric acid, but it did not affect the leukocytosis. In addition, 17ß-estradiol significantly prevented the ex-vivo release of IL-1ß and VEGF from lung tissue. CONCLUSIONS: We demonstrated that intestinal IR interferes with lung homeostasis, priming the tissue to generate proinflammatory mediators for at least 24 h postischemia. Furthermore, our data confirm that the inflammatory responses caused by intestinal IR are estradiol mediated.

Estradiol Modulates Local Gut Injury Induced by Intestinal Ischemia-Reperfusion in Male Rats.

Intestinal ischemia and reperfusion (I/R) triggers a systemic inflammatory response characterized by leukocyte mobilization from the bone marrow, release of cytokines to the circulation, and increased microvascular permeability, leading to high mortality. Females have shown attenuated inflammatory response to trauma when compared with males, indicating a role for female sex hormones in this process. Here, we have evaluated the effect of estradiol on the local gut injury induced by I/R in male rats. I/R was induced by the clamping of the superior mesenteric artery for 45 min, followed by 2 h of reperfusion. A group received 17ß-estradiol (280 µg/kg, i.v., single dose) at 30 min of ischemia. Morphometric analysis of the gut showed I/R induced a reduction of villous height that was prevented by estradiol. White blood cells, notably granulocytes, were mobilized from the circulation to the intestine by I/R, which was also prevented by estradiol treatment. Groups had the intestine wrapped in a plastic bag to collect intestinal fluid, where leukocytes count, TNF-α, and IL-10 levels were increased by I/R. Serum chemokines (CINC-1, MIP-1α, MIP-2), ICAM-1 expression in the mesenteric tissue, and neutrophils spontaneous migration measured in vitro were also increased after I/R. Estradiol treatment reduced leukocytes numbers and TNF-α on intestinal fluid, serum chemokine release and also downregulated MIP-1α, MIP-2 gene expression, and spontaneous in vitro neutrophil migration. In conclusion, estradiol blunts intestinal injury induced by I/R by modulating chemokines release and leukocyte trafficking.