Targeting brain death-induced injury
van Erp, Anne Cornelie
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The effect of the Human
Chorionic
Gonadotropin-related peptide (EA230) on
brain death-induced
inflammation in rats
Anne C. van Erp Rolando A. Rebolledo Petra J. Ottens Suzanne Veldhuis Rutger J. Ploeg Henri G.D. Leuvenink In preparation3
CHAPTER
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ABBREVIATIONS
ALT Alanine Transaminase AST Aspartate Transaminase
AQGV Ala-Gln-Gly-Val (also called EA230)
BD Brain Death
CT Threshold Cycle
EA230 Ala-Gln-Gly-Val (also called AQGV) FBS Fetal Bovine Serum
HAES Polyhydroxyethyl Starch hCG Human Chorionic Gonadotropin
IL Interleukin
LDH Lactate Dehydrogenase LPS Lipopolysaccharides MAP Mean Arterial Pressure
MCP-1 Monocyte Chemotactic Protein-1
NA Noradrenalin
PBS Phosphate Buffered Saline PMN Polymorphonuclear TNF-α Tumour Necrosis Factor-α
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ABSTRACT
Introduction
Donation after brain death (BD) is the main source of organs for transplantation. However, BD-induced inflammation is inversely correlated to transplantation outcomes. EA230, an oligopeptide of the β-loop of human chorionic gonadotropin (hCG), has shown strong immunosuppressive effects in animal models of septic and haemorrhage shock. We hypothesized that EA230 would attenuate BD-induced inflammation and improve kidney and liver function.
Methods
BD was induced in mechanically ventilated rats through inflation of a Fogarty catheter in the epidural space. EA230 (10 mg/kg), EA230 (30 mg/kg), or vehicle (saline) was administered intraperitoneally 30 min prior to BD induction. Sham-operated rats serves as controls and were ventilated for an experimental duration of 30 min. After 4 h of BD, serum, kidney, and liver tissue were collected. RT-qPCR, routine biochemistry, and immunohistochemistry were performed.
Results
Plasma creatinine, AST, total bilirubin, and IL-6; renal and hepatic gene expression levels of IL-6 and MCP-1; and polymorphonuclear influx in the liver and kidney were all significantly higher in brain-dead compared to sham-operated rats (p < 0.05). No significant differences were found between EA230- and PBS-treated animals. In vitro studies showed that pre- or post-treatment of LPS-activated rat leukocytes with EA230 did not affect IL-6 levels. Conclusion
BD resulted in a significant inflammatory response in the liver and kidney, which could not be attenuated by EA230 treatment. We propose that the ineffectiveness of EA230 is not dose- or time-dependent but might be related to the BD-induced adrenal insufficiency or dysfunctional pituitary stimulation.
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INTRODUCTION
Organ transplantation has become the treatment of choice for most patients with end stage liver and kidney failure. However, for years there has been a global shortage of suitable organs1. As a result, on average 12 patients die each day while waiting for an organ graft in Europe alone2. Most organs worldwide are obtained from brain-dead organ donors. However, brain death (BD) negatively affects organ quality resulting in impaired graft function and patient outcome when compared to living donation3. Therefore, research has focussed on optimizing outcomes of transplantations using BD donor grafts.
BD results in hemodynamic instability, hormonal impairment, and a cascade of inflammatory events in the donor3-6. Hemodynamic instability is caused by a sudden increase in intracranial pressure during BD, resulting in ischemia of the cerebrum, brain stem, and spinal cord. When systemic hypertension fails to maintain cerebral perfusion, a sympathetic catecholamine storm facilitates a rise in mean arterial pressure (MAP) from hypotensive to hypertensive pressures4. During and following this period, compromised organ reperfusion and ischemia can affect the donor organs7,8. Furthermore, ischemia of the brain causes failure of the hypothalamus and pituitary, resulting in systemic hormone dysregulation5,9. Finally, BD is associated with systemic as well as organ-specific pro-inflammatory changes based on activation of the vascular endothelium, the coagulation and complement system, and the innate and adaptive immune system3. This pro-inflammatory environment is evidenced by increased expression of cytokines such as IL-6 and TNF-α, adhesion molecules, and organ-infiltrating leukocytes3,10,11.
Targeting inflammation in the brain-dead donor is potentially clinically relevant, as increased plasma levels of pro-inflammatory cytokine interleukin (IL)-6 are associated with worse transplantation outcomes12-15. High donor IL-6 levels were correlated to lower six-month hospital-free recipient survival14. Following lung transplantation, donor IL-6 levels were inversely correlated to increased recipient survival15. Furthermore, immunological factors such as rejection with or without delayed-graft function are risk factors for lower graft survival following deceased-donor transplantation16. In line with this idea, compounds able to suppress the immune system of the brain-dead donor are potentially beneficial therapeutic strategies to improve transplantation outcomes. Unfortunately, randomized controlled trials do not support the routine use of immunosuppressive drugs methylprednisolone and cyclophosphamide, as these compounds were unable to improve post-transplant graft function and survival rates17-21. This underlines the need for alternative strategies to target BD-induced inflammation.
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β-subunit of human chorionic gonadotropin (hCG) were proven to exert immunosuppressive effects22,23. hCG is a hormone secreted by the placenta during human pregnancy that has been linked to the immune system because symptoms of Th1-mediated autoimmune diseases (cellular immune diseases), such as rheumatoid arthritis and multiple sclerosis, decline during pregnancy24. In mice, a peptide fraction of the β-loop of hCG inhibited the onset of autoimmune type I diabetes in previously non-diabetic mice24. In a mouse model of LPS-induced septic shock, various oligopeptides of this β-subunit inhibited inflammation, severity of disease, and mortality in the liver25. Of the different oligopeptides, the synthetic tetrapeptide Ala-Gln-Gly-Val (AQGV, also called EA230), showed strong immunosuppressive effects on both early and late inflammation26,27 (Fig S1). The safety, tolerability,
pharmacokinetics, and pharmacodynamics of EA230 have also been successfully tested in phase I and II clinical trials in humans28,29. Phase III studies are currently under way30. The aim of this present study was to investigate whether pre-treatment with EA230 could attenuate BD-induced inflammation as well as liver and kidney damage in an already established BD model in rats.
Figure 1. Structure of the β-loop of human chorionic gonadotropin (hCG), with the amino acid
sequence of the second loop shown on the right. The arrows indicate the preferred cleavage site for the various oligopeptides23.
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MATERIALS AND METHODS
Animals
Adult, male Fisher rats (F344, 250-280 g, Harlan, United Kingdom) were used. The rats were bred under specific pathogen-free conditions and acclimated in the animal facility of the University Medical Centre of Groningen for at least one week after arrival. Rats had access to pelleted food and sterilized water ad libitum.
EA230
The selected oligopeptide Ala-Gln-Gly-Val (AQGV, EA230) was synthesized (Ansynth BV, Roosendaal, the Netherlands) using fluorencylmethoxycarbonyl/tert-butyl-based methodology with a 2-chlorotritylchloride resin as the solid support. EA230 was dissolved in phosphate buffered saline (PBS) at a concentration of 1 mg/mL and stored at -20°C. Brain-death model
The BD model used in this experiment was based on the gradual onset brain death model developed by Kolkert et al.31 Rats were anesthetized with 5% isoflurane in a mixture with oxygen (1L/min). During BD induction, anaesthesia was maintained with O2/isoflurane (2%). A cannula was placed in the right femoral artery to monitor blood pressure. The right femoral vein was cannulated to provide intravenous access. Animals were intubated via a tracheostomy and continuously ventilated mechanically during the experiment. A hole was drilled frontolateral to the bregma after which a 4F Fogarty balloon catheter was inserted into the extradural space. BD was induced gradually in a period of 30 minutes by inflation of the balloon with 0.5 mL PBS, using a syringe pump. After start of the induction, a hypotensive period and a subsequent short peak in blood pressure were followed by a rapid fall in blood pressure. Next, blood pressure slowly started to increase due to catecholamine-release. Inflation of the balloon was stopped when blood pressure rose to its basal level, while pressure in the balloon was maintained for the remainder of the experiment. Brain death could be confirmed about 30 minutes after onset of BD by the absence of corneal reflexes and a positive apnea test. During the 4 hr BD period, the MAP was maintained above 80 mmHg (considered normotensive). If necessary, infusion with saline or when needed with the colloid polyhydroxyethyl starch (HAES, 100 g/L in saline) was started to maintain a normotensive MAP. Unresponsiveness to HAES administration indicated the start of an intravenous noradrenaline (NA) drip (1mg/mL) to maintain a normotensive MAP. The body temperature and end tidal CO2 were monitored and maintained at 37°C and between 20-22 mmHg, respectively. Esmeron (0.6 mg/kg) was administered 15 minutes before the end of BD. The rat was then turned in supine position and the abdomen was opened. At 4 hrs of BD, 5 mL of blood and all of the urine were collected, after which all abdominal
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organs were flushed with 50 mL cold saline. After the flush-out, the liver and kidney were harvested, tissue samples snap frozen in liquid nitrogen, and stored at -80°C or fixated in 4% paraformaldehyde. Centrifuged blood samples and urine were also snap frozen.
Rats were randomly divided into one of six groups, each group consisting of eight animals. Sham-operated rats served as controls accounting for the surgical procedure. Sham animals were treated according to the BD procedure, up until insertion of the Fogarty catheter balloon. After a hole was drilled, these animals were ventilated for half an hour under anesthesia before they were sacrificed. In both groups, after cannulation of the femoral artery and vein, rats were injected with PBS, EA230 (10 mg/kg) or EA230 (30 mg/kg) intravenously, 30 min before the start of the BD induction.
The animals were randomly assigned to one of six experimental groups (n = 8 per group): 1. Sham + PBS
2. Sham + EA230 10 mg/kg 3. Sham + EA230 30 mg/kg 4. Brain death + PBS
5. Brain death + EA230 10 mg/kg 6. Brain death + EA230 30 mg/kg Plasma determinations
Plasma levels of creatinine, lactate dehydrogenase (LDH), alanine transaminase (ALT), aspartate transaminase (AST) and bilirubin were determined at the clinical chemistry lab of University Medical Centre Groningen according to standard procedures.
Plasma levels of IL-6 were determined by IL-6 enzyme linked immunosorbent assay (R&D Systems Europe Ltd. Abingdon, Oxon OX143NB, UK) according to the manufacturer’s instructions. All samples were analysed in duplicate and read at 450nm.
RNA isolation and cDNA synthesis
Total RNA was isolated from whole kidneys and livers using TRIzol (Life Technologies, Gaithersburg, MD). Absence of DNA contamination in the RNA samples was verified by means of a RT-PCR reaction without adding reverse transcriptase, using GAPDH primers. For cDNA synthesis, 1 μl T11VN Oligo-dT (0,5 μg/μl) and 1 μg mRNA were incubated for 10 min at 70°C and cooled directly afterwards. cDNA was synthesized by adding a mixture containing 0.5 μl RNaseOUT® Ribonuclease inhibitor (Invitrogen, Carlsbad, USA), 0.5 μl RNase water (Promega), 4 μl 5 x first strand buffer (Invitrogen), 2 μl DTT (Invitrogen), 1 μl dNTP’s, and 1 μl M-MLV reverse transcriptase (Invitrogen, 200U). The mixture was kept at 37°C for 50 min. Reverse transcriptase was inactivated by incubating the mixture at 70°C for 15 min. Samples were stored at -20°C.
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Real-Time PCR
Fragments of several genes were amplified with primer sets outlined in Table 1. Pooled cDNA obtained from brain-dead rats was used as an internal reference. Gene expression levels were quantified by normalization against the mRNA levels of β-actin. Real-time PCR was carried out in reaction volumes of 15 μl containing 10 μl of SYBR Green mastermix (Applied biosystems, Foster City, USA), 0.4 μl of each primer (50 μM), 4.2 μl of nuclease free water and 10 ng of cDNA. All samples were analysed in triplicate.
Table 1. Primer sequences used for Real-Time PCR
Gene Primers Amplication size (bp)
IL-6 5′-CCAACTTCCAATGCTCTCCTAATG-3′ 5′-TTCAAGTGCTTTCAAGAGTTGGAT-3′ 89 MCP-1 5′-CTTTGAATGTGAACTTGACCCATAA-3′ 5′-ACAGAAGTGCTTGAGGTGGTTGT-3′ 78 TNF-α 5′-GGCTGCCTTGGTTCAGATGT-3′ 5′-CAGGTGGGAGCAACCTACAGTT-3′ 79
Thermal cycling was performed on the Taqman Applied Biosystems 7900HT Real Time PCR System with a hot start at 50°C for 2 min, followed by 10 min at 95°C. The second stage was started at 95°C for 15 s (denaturation step) and followed by 60 s at 60°C (annealing step and DNA synthesis). The latter stage was repeated 40 times. The third stage was included to detect formation of primer dimers (melting curve) and began with 15 s at 95°C. Primers were designed with Primer Express software (Applied Biosystems) and primer efficiencies were tested by a standard curve for the primer pair by means of the amplification of serially diluted cDNA samples (10 ng, 5 ng, 2.5 ng, 1.25 ng and 0.625 ng) obtained from brain-dead rats. PCR efficiency was found to be 1.8 < ε < 2.0. Real time PCR products were checked for product specificity on a 1.5% agarose gel. Results were expressed as 2-ΔΔCT (CT: Threshold Cycle).
Immunohistochemistry
To detect polymorphonuclear (PMN) cells in kidney and liver, immunohistochemistry was performed on 5 μm tissue cryosections. Sections were fixated for 10 min using acetone and then stained with HIS-48 mAb (supernatant, two times diluted), using an indirect immunoperoxidase technique. Endogenous peroxidase was blocked using H2O2 0.01% in PBS for 30 min. After thorough washing, sections were incubated with horseradish peroxidase-conjugated rabbit anti-mouse IgG as a secondary antibody for 30 min, followed by incubation with a tertiary goat anti-rabbit IgG antibody for 30 min (both from Dako, Glostrup, Denmark). The reaction was visualized using 9-amino-ethylcarbazole as chromogen
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and H2O2 as substrate. Sections were counter stained using Mayer hematoxylin solution (Merck, Darmstadt, Germany). Negative antibody controls were performed. Localization of immunohistochemical staining was assessed by means of light microscopy. For each tissue section, positive cells per renal glomerulus and renal interstitium in the cortex and in the liver were counted in 1microscopic fields at 20x magnification.
In vitro stimulation of rat leukocytes with lipopolysaccharide
Heparinized blood (3-4 mL) was drawn from the inferior vena cava of adult, male Fisher rats (F344, 250-280 g, Harlan, United Kingdom) and mixed with 45 mL of ammonium chloride solution. After storage at 4°C for 15 min, the solution was centrifuged at 1000G for 5 min. The leukocytes in the pellet were separated from the supernatant with the lysed red blood cells and then mixed with RPMI-1640 +10% Fetal Bovine Serum (FBS) and counted with a cell-counter. Lipopolysaccharides (LPS) from Escherichia coli 0111:B4 (L3491, Sigma-Aldrich) was diluted in PBS at a concentration of 1 mg/mL. EA230 was diluted in phosphate buffered saline at a concentration of 10 mg/mL.
The leukocytes were added at 1 x 106/mL/well or 2 x 106/mL/well in 48 well plates with RPMI-1640 + 10% FBS medium. Then, 100 ng/mL, 500 ng/mL, or 1000 ng/mL LPS was added. EA230 was added before, during, or after LPS stimulation in concentrations of 20 μg/ mL or 50 μg/mL. Five, 12, 18 or 24 hours after LPS administration (see Fig 2), supernatant was collected from each well and stored at -20°C until cytokine levels were measured. Leukocytes with medium alone and leukocytes with medium and LPS or EA230, were used as controls and were collected at the same time point as the other samples.
Plasma levels of IL-6 were determined by IL-6 enzyme linked immunosorbent assay (R&D Systems Europe Ltd. Abingdon, Oxon OX143NB, UK) according to the manufacturer’s instructions. All samples were analyzed in duplicate and read at 450nm.
Statistical analysis
Due to the two-factorial design of the experiment, the two-way ANOVA test was done to analyze the results (SPSS Statistics 20). After data transformations, data were normally distributed and variances of the dependent variable equal across groups according to Levene’s test of equality of error variances (indicated by p > 0.05). This allowed us to use the two-way ANOVA test despite the small group sizes. If there was a significant difference either between groups (BD vs. sham) or treatment (PBS vs. EA230), we tested for individual group differences using Bonferroni’s post-tests. For liver TNF-a gene expression, we found a positive interaction term. For this group, we continued testing between groups using the Mann-Whitney test.
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For these parameters, the Kruskal-Wallis test was performed to analyse between the six experimental groups, followed by the Mann-Whitney test to compare between two groups individually (Prism 5.0 GraphPad). All statistical tests were 2-tailed and p < 0.05 was regarded as significant. Results are presented as mean ± SD (standard deviation).
Figure 2. General overview of in vitro experimental setup. Orange bars represent different LPS
concentrations (1000 ng/mL, 500 ng/mL, and 0 ng/mL, represented from dark to light orange, respectively). Green bars represent two different EA230 concentrations (50 ug/mL and 20 ug/mL, represented by dark and light green, respectively).
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RESULTS
Brain death induction
The MAP in the rat increased slightly immediately after the start of BD induction, followed by a drop and subsequent increase; a profile which is characteristic for this BD model31. This
pattern of the MAP during the BD induction was seen uniformly and consistently amongst all BD groups, with a mean time to declare BD of 31.9 minutes (Fig 3). After completing the induction, the MAP of all brain-dead animals was at or above 80 mmHg throughout the experiment. In saline-treated brain-dead rats, infusion of 0.81 ± 0.59 ml 10% HAES was required to maintain stable blood pressures. In EA230 (30 mg/kg)-treated brain-dead rats, infusion of 1.13 ± 0.35 ml 10% HAES was required to maintain stable blood pressures (p = 0.315). Saline-treated brain-dead rats were administered 0.83 ± 0.95 mL of NA while EA230-treated brain-dead rats received 0.69 ± 0.83 mL NA (p = 0.874).
Figure 3. Mean arterial pressure (MAP) during brain death induction. Following balloon insertion,
the MAP shortly rises after insertion of the balloon, followed by quick drop in MAP and a subsequent hypotensive period. After about 30 minutes, the blood pressure became normal (above 80 mmHg) again; this period marked the beginning of the in total 4 hr brain death period.
Organ function
Renal function was assessed measuring plasma creatinine levels. Plasma creatinine levels significantly increased in brain-dead animals compared to sham-operated animals (71.2± 8.33 vs. 33.9 ± 3.00, p < 0.001, Fig 1). Amongst the six groups, no significant differences were found between saline and EA230 treatment groups (Fig 4).
Cellular liver damage and liver function were determined measuring AST, ALT, LDH, and direct and total bilirubin levels. Plasma AST, ALT and LDH levels significantly increased in brain-dead animals compared to sham-operated animals (AST: 108 ± 17.2 vs. 64.4 ± 4.07,
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p < 0.001; ALT: 71.8 ± 10.9 vs. 51.9 ± 3.77, p = 0.005; LDH: 299 ± 151 vs. 150 ± 5.87, p = 0.003, Fig 4). Plasma direct and total bilirubin levels in EA230-treated BD animals showed no differences compared to saline-treated BD animals. Plasma total bilirubin levels in saline-treated BD animals significantly increased compared to saline-treated sham animals (2.24 ± 0.91 vs. 0.95 ± 0.05, p = 0.006, Fig 4). Plasma direct bilirubin levels in saline-treated brain-dead and sham animals were not significantly different. Amongst the six groups, no significant differences in AST, ALT, and LDH were found between saline and EA230 treatment groups (Fig 4).
Figure 4. Increased levels of plasma inflammatory marker IL-6 and hepatic and renal injury markers following BD were unaffected by EA230 treatment. Plasma levels of A. aspartate aminotransferase (AST), B. alanine aminotransferase (ALT), C. lactate dehydrogenase (LDH), D. creatinine, and E. total and F. direct bilirubin, following 4 hrs of brain death in rats pre-treated with EA230 (10 or 30 mg/kg) or phosphate buffered saline (PBS) solution. Results are presented as mean ± SD, n = 8 per group (**p < 0.01, ***p < 0.001).
Inflammatory status Plasma levels of IL-6
The systemic inflammatory status of the rats was assessed looking at plasma levels of pro-inflammatory cytokine IL-6. Plasma IL-6 levels significantly increased in brain-dead animals compared to sham-operated animals (820 ± 110 vs. 6.53 ± 11.3, p < 0.001, Fig 5). Amongst the six groups, no significant differences were found between saline and EA230 treatment groups (Fig 5).
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Figure 5. Increased levels of plasma inflammatory marker IL-6 was unaffected by EA230 treatment.
Plasma levels of interleukin-6 (IL-6) following 4 hrs of brain death in rats pre-treated with EA230 (10 or 30 mg/kg) or PBS solution. Results are presented as mean ± SD, n = 8 per group (*** p < 0.001).
Renal and hepatic gene expression levels
The hepatic inflammatory status was assessed looking at gene expression of pro-inflammatory cytokines and acute phase reactants IL-6, tumour necrosis factor-6 (TNF-α) and monocyte chemotactic protein-1 (MCP-1). Gene expression of IL-6 and MCP-1 significantly increased in brain-dead animals compared to sham-operated animals (IL-6: 0.74 ± 0.09 vs. 0.01 ± 0.00, p < 0.001; MCP-1: 1.19 ± 0.46 vs. 0.46 ± 0.08, p < 0.001, Fig 6). Gene expression of TNF-α significantly decreased in saline-treated brain-dead compared to sham animals (15.5 ± 6.70 vs. 42.7 ± 14.2, p < 0.001, Fig 6). Amongst the six groups, no significant differences were found between saline- and EA230- treated groups (Fig 3), besides an increase in TNF-α gene expression in EA230 (30 mg/kg)- versus saline-treated brain-dead animals (29.0 ± 12.1 vs. 42.7 ± 14.2, p = 0.03, Fig 6).
In the kidney, gene expression of IL-6, MCP-1, and TNF-α significantly increased in brain-dead compared to sham-operated animals (IL-6: 0.95 ± 0.30 vs. 0.01 ± 0.00, p < 0.001; MCP-1: 4.22 ± 0.21 vs. 0.49 ± 0.16, p < 0.001; TNF-α: 6.63 ± 0.92 vs. 2.80 ± 0.70, p = 0.009, Fig 6). Amongst the six groups, no significant differences were found between saline- and EA230- treated groups (Fig 6).
Polymorphonuclear influx
The influx of PMN cells in the kidney and liver was determined using tissue cryosections stained with HIS-48 mAb. The number of positive cells per microscopic fieldin the liver significantly increased in brain-dead compared to sham animals (liver: 23.6 ± 1.67 vs. 7.98 ± 1.00, p < 0.001, Fig 7). In the kidney, there was a significant increase in PMN influx in both the renal interstitium and the glomeruli (interstitium (saline-treated sham versus brain-dead animals): 3.85 ± 0.49 vs. 2.77 ± 0.60, p < 0.001; glomeruli: 1.30 ± 0.10 vs. 0.99 ± 0.14,
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p = 0.002, Fig 7). Amongst all groups, no significant differences were found between saline and EA230 treatment groups (Fig 7).
Figure 6. Increased hepatic and renal inflammatory gene expression following brain death was not attenuated by EA230. Hepatic (A-C) and renal (D-F) gene expression of inflammation-related genes A,D. interleukin-6 (IL-6), B,E. monocyte chemotactic protein-1 (MCP-1), and C,F. tumour necrosis
factor- α (TNF-α) following 4 hs of brain death in rats pre-treated with EA230 (10 or 30 mg/kg) or phosphate buffered saline (PBS). Results are presented as mean ± SD, n = 8 per group (* p < 0.05, **
p < 0.01, *** p < 0.001).
Figure 7. Increased leukocyte infiltration in the kidney and liver following brain death was not altered by EA230 treatment. A. Hepatic and renal B. glomerular and C. interstitial leukocyte infiltration
following 4 hrs of brain death in rats pre-treated with EA230 (10 or 30 mg/kg) or phosphate buffered saline (PBS). Results are presented as mean ± SD, n = 8 per group (** p < 0.01, *** p < 0.001).
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In vitro, LPS-stimulated rat leukocytes and EA230 treatment
A set of in vitro experiments were performed using rat leukocytes to determine whether timing or dosage of EA230 treatment before or an LPS stimulus would affect the level of inflammation, as measured by pro-inflammatory cytokine IL-6. The optimal dosage and timing of LPS stimulation until measurement of IL-6 levels in this in vitro setting were determined to be 500 ng/mL and 12 hours with an average IL-6 concentration of 1.54 x 103 ± 85.7 pg/mL. Next, the optimal dosage and timing of EA230 treatment in this in vitro setting were tested. Lowest IL-6 levels were found when EA230 was added 30 minutes after LPS addition. The optimal EA230 concentration was found to be 20 μg/mL, as this resulted in IL-6 levels of 270 ± 31.0 pg/mL (Fig 8).
After determining the optimal dosages of EA230 and LPS, we investigated whether timing of EA230 in regard to the LPS stimulus had an effect on the anti-inflammatory effects of EA230 as measured by leukocyte IL-6 production. Treatment with EA230 before the LPS addition (pre-treatment) versus treatment with EA230 after LPS addition (post-treatment) did not result in significantly different IL-6 levels (p = 0.18). Furthermore, no significant different were found when pre-treatment with EA230 was compared to post-treatment after 30 minutes, 30 minutes and 4 hrs, 4 hrs, or at 4 hrs with a double dose of EA230 (p = 0.41). As a control for this in vitro experiment, we tested for IL-6 levels in control samples with LPS only, EA230 only, or leukocytes only. Levels of IL-6 in LPS control samples were significantly higher than samples with EA230 or leucocytes alone (p = 0.05, Fig 8).
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Figure 8. Timing and dosage of EA230 therapy did not alter LPS-induced interleukin-6 (IL-6) levels in rat leukocytes. IL-6 levels in rat leukocytes treated in vitro with lipopolysaccharide (LPS) and/or
EA230. A. Optimal dosage and timing of LPS stimulation was found to be 500 ng/mL LPS and 12 hrs of incubation time. B. Optimal timing and dosage of EA230 treatment was found to be 20 μg/mL added 30 min after LPS addition. C. Control IL-6 levels following LPS treatment were significantly higher than groups with EA230 alone or leukocytes alone. D. No significant differences in IL-6 levels when EA230 treatment was given before or after the LPS stimulus. E. No significant difference in IL-6 levels when EA230 treatment was given before, or 30 min, 30 min and 4 hrs, and 4 hrs (single or double dose) after the LPS stimulus. See also Figure S3 for a graphical representation of the different setups.
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DISCUSSION
In this study, effects of the immunosuppressive drug EA230 on hemodynamics, inflammation, organ function, and leukocyte infiltration were studied following BD in rats.
BD resulted in significant inflammation, hemodynamic instability, and tissue injury in the liver and kidney, all of which were unaffected by EA230 treatment. First, we confirmed that renal and hepatic function was impaired following BD, as is in line with prior BD animal studies32-34. We also observed increased plasma levels of IL-6, an important inflammatory marker that can increase the expression of acute phase genes35. In this way, an IL-6-induced acute phase response intends to restore disturbances in the physiological homeostasis and can result in both local and systemic immune responses, including the accumulation and activation of PMNs and mononuclear cells35. Indeed, we observed local inflammation in the liver and kidney following BD, as was seen in prior BD studies10,11,14,36,37. Particularly gene expression of IL-6 and MCP-1 were significantly upregulated in the liver and kidney. Particularly MCP-1 is of interest, as this cytokine is involved in the recruitment of monocytes and T-cells to inflamed tissues. Indeed, we observed increased leukocyte invasion in the liver as well as in the interstitium and the glomeruli of the kidney, a finding that corresponds to the results obtained by Nijboer et al.38,39. In contrast with prior studies40,41, we observed a decrease in TNF-α gene expression in the liver of brain-dead rats. However, whether this decrease reflects inflammation or not is unclear, as the role of TNF and the TNF-receptors in the pathogenesis of disease has been shown to be both protective and pathogenic42,43. Furthermore, the expression of TNF-receptors can be modulated by many agents, including cytokines like IL-6, hormones like thyroid stimulating hormone, and TNF itself42. Given that all other inflammatory markers in our study were increased following BD, this decrease in TNF-α gene expression likely reflects a pathogenic, inflammatory response.
Combining all these results, pre-treatment of brain-dead rats with EA230 did neither alter inflammation nor improve liver and kidney function. This is in contrast with other animal models, where EA230 lowered inflammation and improved survival rates. In a model of haemorrhagic shock and resuscitation, rats treated with EA230 60 minutes after haemorrhage shock showed decreased levels of inflammatory cytokines25. In a mouse model of ischemia-reperfusion injury, administration of EA230 prior to and at the end of the ischemic period, showed significantly improved survival rates and lowered serum IL-6, IL-10, and TNF-α levels26. These studies suggest that timing of EA230 treatment, i.e. post-treatment versus pre-treatment, might determine the effectiveness of this compound. Therefore, we initiated a set of in vitro experiments in leukocytes exposed to an LPS stimulus, to investigate whether timing or dosage of EA230 treatment might explain the ineffectiveness of EA230 in our model (see Fig S3). Unfortunately, our data showed no significant differences between
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the leukocytes treated with EA230 before (pre-treatment) or after the LPS stimulus (post-treatment). Furthermore, no significant differences were found when leukocytes were treated at multiple time points after LPS addition, as well as with a double dose of EA230. Together, these data suggest that the timing and dosage of EA230 therapy were likely not responsible for the ineffectiveness of this compound in our model.
Our results suggest that the ineffectiveness of EA230 might be related to the hemodynamic, hormonal or inflammatory changes specific to the BD condition. A previous study by van der Zee et al. suggests that LQGV, a peptide closely related to EA230, exerted its anti-inflammatory effects by stimulating the adrenal gland to produce corticosteroids22. However, following BD, 87% of brain-dead patients suffered from adrenal insufficiency44,45. Furthermore, adrenocorticotropic hormone concentrations were lower than the normal clinical range in these patient44,45. Finally, a BD study in non-human primates showed that cortisol levels increased 30 minutes after BD induction but then decreased until below baseline levels after six hrs10. Therefore, a lack of pituitary stimulation of the adrenal gland or the incidence of adrenal insufficiency after BD might explain why EA230 was unable to attenuate inflammation in our model. More research into the working mechanism of EA230 will need to be conducted to elucidate why EA230 was uneffective in the BD setting. In conclusion, we observed a clear inflammatory response as well as organ dysfunction following BD in the liver and kidney, which could not be attenuated by the immunosuppressive drug EA230. We propose that the lack of immunosuppressive qualities of EA230 might be related to the hemodynamic, hormonal, or inflammatory changes of the BD condition. A better understanding of the working mechanism of EA230 in the BD setting might explain the inefficacy of EA230 in our model.
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