Novel 24-h ovine model of brain death to study the profile of the endothelin axis during cardiopulmonary injury
© Watts et al. 2015
Received: 28 May 2015
Accepted: 13 November 2015
Published: 24 November 2015
Upregulation of the endothelin axis has been observed in pulmonary tissue after brain death, contributing to primary graft dysfunction and ischaemia reperfusion injury. The current study aimed to develop a novel, 24-h, clinically relevant, ovine model of brain death to investigate the profile of the endothelin axis during brain death-associated cardiopulmonary injury. We hypothesised that brain death in sheep would also result in demonstrable injury to other transplantable organs.
Twelve merino cross ewes were randomised into two groups. Following induction of general anaesthesia and placement of invasive monitoring, brain death was induced in six animals by inflation of an extradural catheter. All animals were supported in an intensive care unit environment for 24 h. Animal management reflected current human donor management, including administration of vasopressors, inotropes and hormone resuscitation therapy. Activation of the endothelin axis and transplantable organ injury were assessed using ELISA, immunohistochemistry and standard biochemical markers.
All animals were successfully supported for 24 h. ELISA suggested early endothelin-1 and big endothelin-1 release, peaking 1 and 6 h after BD, respectively, but there was no difference at 24 h. Immunohistochemistry confirmed the presence of the endothelin axis in pulmonary tissue. Brain dead animals demonstrated tachycardia and hypertension, followed by haemodynamic collapse, typified by a reduction in systemic vascular resistance to 46 ± 1 % of baseline. Mean pulmonary artery pressure rose to 186 ± 20 % of baseline at induction and remained elevated throughout the protocol, reaching 25 ± 2.2 mmHg at 24 h. Right ventricular stroke work increased 25.9 % above baseline by 24 h. Systemic markers of cardiac and hepatocellular injury were significantly elevated, with no evidence of renal dysfunction.
This novel, clinically relevant, ovine model of brain death demonstrated that increased pulmonary artery pressures are observed after brain death. This may contribute to right ventricular dysfunction and pulmonary injury. The development of this model will allow for further investigation of therapeutic strategies to minimise the deleterious effects of brain death on potentially transplantable organs.
KeywordsBrain death Organ transplantation Haemodynamics Pulmonary circulation Ventricular Function Right Endothelin-1 Receptors Endothelin Sheep
After brain death (BD), the lungs are particularly susceptible to injury in the peri-transplant period secondary to direct trauma, soiling with blood or gastric contents, iatrogenic injury, infection and inflammation [1–3]. Details of the specific mechanisms of catecholamine- and cytokine-induced donor organ injury after BD are yet to be fully elucidated [2, 4, 5]. Peri-transplant injury contributes to the ongoing shortage of transplantable lungs; this is highlighted by American data reporting an average rate of lungs transplanted per donor of 0.37 .
Endothelins, their precursors, receptors and associated signalling pathways are collectively referred to as the endothelin axis [7, 8]. Endothelin-1 (ET-1) is a potent vasoconstrictor, smooth muscle cell and fibroblast mitogen and a stimulator of inflammatory cell infiltration [9–11]. Once released, ET-1 stimulates matrix metalloproteinase (MMP) expression in pulmonary tissue, resulting in protein hydrolysis and interstitial oedema [2, 8]. Our group first demonstrated that the endothelin axis was upregulated after BD in rats, and that this correlated with pulmonary injury . Upregulation of endothelin receptors “primes” the lungs for post-transplant injury  and may partly explain the relationship between endothelin expression and primary graft dysfunction that has been observed in human lung allograft recipients .
Haemodynamic instability has limited the duration of previous BD animal studies and supportive measures used to extend these models to clinically relevant timeframes are difficult to apply to small animals [13–15]. Interventions utilised in human BD donors, such as fluid or vasoactive agent administration, may have significant effects on genomic expression of inflammatory mediators [16, 17], further limiting the ability of small animal models to replicate comprehensive, modern, intensive care monitoring and management. To begin to address these issues, porcine models have been extended to 24 h [18, 19]. Zhai et al. investigated hepatic injury after BD in BaMa miniature pigs . Although this is a valid extended model, the animals were small compared to humans (average of 25 kg), and the use of other clinically relevant interventions, such as vasopressors, inotropes and hormone resuscitation, were not reported. The model published by Sereinigg et al. was developed to more closely reflect clinical experience with BD donors, including the use of vasoactive agents, however this publication did not specifically include a control group for comparison .
No animal model can absolutely replicate all aspects of human pathophysiology . For example, controversy exists regarding rodent modelling of human disease, with evidence both supporting and refuting similarities of inflammatory genomic responses to injury between the two species [16, 21]. Both pigs and sheep have been effectively utilised as large animal models of human pathology, with each offering notable benefits [22, 23]. Ovine models have been highlighted as particularly suitable for investigating human lung disease [17, 23–25]. Furthermore, sheep models have provided detailed insight into the endothelin axis and its contribution to pulmonary haemodynamics, as well as the role of ET-1 in lung inflammation [23, 26–28]. Therefore, based on these considerations, we have developed a 24-h ovine model to investigate the role of the endothelin axis in BD-related pulmonary inflammation. Additionally, the results of comprehensive investigation of the effects of BD on ovine haemodynamics and systemic markers of transplantable organ injury are presented.
This study was conducted with the approval of the Queensland University of Technology Animal Ethics Committee, approval number 0900000319. All experiments were performed in accordance to NHMRC Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and the Animal Care and Protection Act 2001 (QLD).
Medications for the protocol
Initial infusion rate
Lignocaine 1 %
3–5 mL subcutaneously
Over central venous access insertion sites
Administered six hourly during protocol
If further boluses needed, dosed at 0.5 mg/kg
Adjusted to surgical plane
Adjusted to surgical plane
Used only if required (if alfaxalone exceeded 250 mg/h)
Titrated to CVP 8–12 mmHg. Boluses if needed for low urine output (<0.5 mL/kg/h) or hypotension (MAP < 60 mmHg). Initial fluid of choice
Normal saline 0.9 %
Boluses if needed for low urine output (UO < 0.5 mL/kg/h) or hypotension (MAP < 60 mmHg)
Dextrose 5 % or dextrose 4 % in saline 0.18 %
Utilised for hypoglycaemia (BSL < 6 mmol/L)
Vasopressors, inotropes and cardiovascular support
Utilised in emergency situations for hypotension only
Utilised in emergency situations for bradycardia (HR < 60 bpm) only
Adjusted to MAP > 60 mmHg
Adjusted to MAP > 60 mmHg
Adjusted to MAP > 60 and HR > 60 bpm. Utilised only if considered bradycardia as cause of hypotension
For hypertension (SBP > 180 mmHg) if necessary
5 mg/kg over 2 h
Infusion for appropriate dysrhythmias (e.g. atrial fibrillation) if necessary. Could be repeated
Bolus for BSL > 16 mmol/L. Infusion adjusted to BSL 6–10 mmol/L, tested hourly once infusion commenced
Dextrose 50 %
For management of hypoglycaemia (BSL < 3.5 mmol/L). Please also note that dextrose 5 % could be used for ongoing maintenance per above
If urine output >300 mL/h for two consecutive hours
Hormone resuscitation at 12 h
0.5–4.0 U/h (initial dose 2.0 U/h)
Adjusted to SVR 800–1200 dyn.s.cm−5
Adjusted to potassium 3.5–5.0 mmol/L
Calcium chloride 10 %
Administered to keep ionised calcium > 1.05 mmol/L
Allowed for management of dysrhythmias (e.g. atrial fibrillation)
Another burr hole was created on the contralateral side in animals allocated to BD, followed by the extradural placement of a 16 Fr Foley catheter. Brain death was induced by normal saline inflation of the catheter to increase intracranial pressure (ICP) above the mean arterial pressure (MAP) for greater than 30 min . Commencement of inflation served as the BD induction time (BIT). Confirmation of brain death was achieved by continuously negative cerebral perfusion pressure (defined as MAP-ICP) for greater than 30 min, loss of pupillary and corneal reflexes and lack of respiratory efforts. Protocol start time was deemed once BD was confirmed in animals allocated to this group. Due to variability in duration required for induction and confirmation of BD, haemodynamic results are reported as time from BIT. Haemodynamic deterioration was managed with intravenous fluid and vasopressors or inotropes as appropriate.
Twelve hours after PST, hormone therapy was commenced with vasopressin, methylprednisolone and liothyronine in all animals. This time point was chosen to reflect the clinical realities of delays in diagnosis and confirmation of brain death, family consent for organ transplantation, and the change from lifesaving to organ preserving treatment . After completion of the 24-h protocol, the animals were sacrificed using sodium pentobarbitone.
Sample retrieval and storage
Blood was collected from the peripheral arterial line at baseline (prior to BIT), 1, 6, 12, 18 and 24 h after confirmation of BD. Blood samples were then centrifuged, supernatant transferred into vials (Eppendorf, North Ryde, Australia) and stored at −80 °C until analysis. After animals were euthanised, the lungs were removed en bloc and samples taken from both lower lobes. These were fixed in 10 % phosphate-buffered formalin, embedded in paraffin and mounted on slides for histological analysis.
Histological and tissue analysis
Samples were taken from the right lower lobe to assess for wet:dry weight ratio, as an indicator of inflammatory oedema. These were dehydrated in an oven at 45 °C for 48 h, at which time they were reweighed and the ratio calculated.
Haematoxylin and eosin staining of lung specimens was performed to allow morphologic assessment of tissue samples. Inflammation was graded semi-quantitatively as previously reported .
Immunohistochemical staining was employed to assess the patency of the endothelin axis using standard methods (see also Additional file 1) . Monoclonal anti-ET-1 (Sigma Aldrich, St Louis, MO), polyclonal anti-ETRA, anti-ETRB and anti-MMP-2 (Merck Millipore, Billerica, MA), polyclonal anti-MMP-9 (Biorbyt, San Francisco, CA) and polyclonal anti-TIMP-1 (tissue inhibitor of metalloproteinase-1) and anti-TIMP-2 (Bioss, Woburn, MA) were selected as primary antibodies.
Immunohistochemistry and histological scoring was performed independently by two investigators (RW and IB) and results compared. Disagreements in scoring were resolved by using the lowest score (i.e. indicating less injury). Slides were assessed in random order and the assessors were blinded to group of allocation.
Systemic concentrations of ET-1 and pro-endothelin-1 (big ET-1) were assessed in EDTA plasma using commercially available sandwich ELISA kits (BiomedicaGruppe, Austria). Absorbance was read at 450 nm with reference 630 nm on a 96-well plate spectrophotometer (FluoStar Omega, BMG LabTech, Germany). The results from one animal in the BD group were excluded due to technical reasons preventing accurate spectrophotometric analysis.
Biomarkers of organ injury were assayed using the COBAS Integra 400 chemical analyser (Roche Diagnostics, Dee Why, Australia), following manufacturer’s instructions. Reagent cassettes were calibrated using the calibrator for automated systems (CFAS, Roche Diagnostics). Precision and accuracy of assays were confirmed using standard quality controls (Precinorm Clin Chem Multi 1 and 2, Roche Diagnostics). All tests were performed in duplicate, averaged and compared to CFAS in order to interpolate sample concentrations.
Analysis of biochemical data was performed using Prism 6 (GraphPad Software Inc., USA). All regression analyses were conducted using R software (www.r-project.org). A two-sided statistical significance level of <0.05 was adopted. Results are reported as mean ± standard deviation. Two-way repeated-measures analysis of variance (ANOVA) was used to test for significant differences in dependent variables. Student’s t test was used to compare changes in physiological variables at specified time points. Fisher’s exact test was used to compare semi-quantitative assessment of tissue samples. For continuous physiological variables, a regression model was used to examine the changes in variables over time. A mixed model with a random intercept for each sheep to account for repeated responses from the same animal was used .
All 12 animals survived the 24-h protocol and induction of BD was successful in all animals allocated to this group. Summary tables detailing ventilation, haemodynamics, fluid balance, biochemistry and histology can be found in Additional file 1.
Animal management and point of care testing
Time to confirmation of brain death after inflation of the extradural catheter was 50 ± 22 min. During this time, the highest ICP achieved was 237 ± 79 mmHg, with a resultant CPP of −117 ± 34 mmHg. At completion of the study, ICP was 87 ± 12 mmHg with a CPP of −6.5 ± 12 mmHg.
Histological and tissue analysis
There was no statistically significant difference in lung wet:dry ratio between groups, with an average ratio of 3.48 vs 3.39 (non-BD vs BD, p = 0.68).
Semi-quantitative assessment of lower lobe pulmonary samples demonstrated increased inflammation in BD animals (non-BD animals: none-mild inflammation (0 or +); BD animals: moderate-severe inflammation changes (++ or +++), including increased interstitial oedema and inflammatory cell infiltration, p = 0.014).
The endothelin axis was detectable by immunohistochemical staining (examples can be found in Additional file 1). Staining of ET-1 was localised to bronchiolar epithelium and perivascular smooth muscle in both BD and non-BD animals, with no appreciable difference in expression noted. With regard to the endothelin receptors, ETRA was stained minimally in both groups within bronchiolar epithelium and smooth muscle, and ETRB was well localised to airway columnar epithelium. There was no difference in expression of either receptor between groups. Overall, there were no differences in intensity of staining for MMP-2 or MMP-9 between groups, with MMP-2 slightly expressed within vascular endothelial and bronchiolar epithelial cells and MMP-9 able to be identified within bronchiolar epithelium and perivascular smooth muscle.
Low intensity of staining for TIMP-1 and -2 was observed for both groups, with no detectable difference. Staining of TIMP-1 was primarily localised within the columnar epithelia in the bronchioles with some staining within the alveolar parenchyma. Similarly to TIMP-1, TIMP-2 expression was mainly observed in bronchiolar epithelia, though some staining in the alveolar parenchyma and pulmonary blood vessels was noted.
This is the first report to document a 24-h, clinically relevant, ovine model of brain death and assess systemic and pulmonary endothelin expression. Histological analysis indicated increased inflammation in the BD lung tissue, consistent with previous literature [1, 8]. The components of the endothelin axis were identifiable by immunohistochemical staining, with no demonstrable difference found between groups. This is in contradistinction to previous observations in rodents . ELISA did suggest an early elevation and then resolution of both ET-1 and big ET-1 in plasma after brain death; this may reflect an early release with rapid clearance , however no ongoing systemic expression was detected. Observed peak concentrations obtained at 1 and 6 h for ET-1 and big ET-1, respectively, may reflect significant peaks that occurred earlier but were not captured by the sampling time in this study. Oishi et al. previously demonstrated that ET-1 peaks as early as 30 min in BD canines, however this elevation was still detectable at 60 min . Another complicating factor in comparing these results to the current study is the nature of sampling; whilst Oishi’s group sampled coronary venous sinus blood (to detect cardiac generated ET-1), our study analysed arterial blood. Systemic levels of ET-1 indicate a spillover effect  and therefore may only be detectable in very low concentrations when assessed in this manner due to mixing of blood returning to the pulmonary circulation. Secondly, pulmonary ETRB may continue to serve its usual function of clearing circulating ET-1, concealing any detectable elevations in mixed central venous blood . Both of these factors may have contributed to our observed results and provide opportunities for future study.
Data is accumulating of the role of ET-1 in brain death and organ donation; activation of the endothelin axis has been demonstrated early in BD-related pulmonary inflammation , it contributes to complications associated with human lung donation [12, 36, 37] and it may contribute to the altered cardiopulmonary haemodynamics observed in the current study, similar to other forms of pulmonary hypertension. As a potent mitogen, ET-1 stimulates smooth muscle hyperplasia and leads to airway remodelling and oedema . Furthermore, ET-1-initiated cell signalling leads to short- and long-term injury, fibrosis and, ultimately, allograft rejection . Thus, endothelin-1 may induce an inflammatory response that continues to manifest itself long after any detectable elevations in plasma concentrations have resolved. Inflammatory cells recruited to the lung by endothelin signalling and increased ET-1 receptors in allograft tissue  may be further activated after transplant by ischemia reperfusion injury  and the inflammatory state of the recipient .
Activation of the sympathetic nervous system during brain death results in dramatic increases in vascular resistance and arterial pressure and contributes to systemic inflammation . Novitzky et al. observed that the resulting increase in SVRI and left atrial pressure leads to transfer of up to 72 % of total blood volume to the lower resistance pulmonary vasculature; mPAP and PVRI were also noted to increase with induction of BD . This has been hypothesised to contribute to pulmonary capillary injury after BD. Bittner and colleagues demonstrated that, when observed for 6 h, the PVRI decreased below baseline after the initial sympathetic storm of BD, resulting in increased pulmonary flow and vascular congestion, contributing to increased extravascular lung water content . These authors reported that the decrease in PVRI was secondary to sympathetic failure and increased vascular distensibility . The data over the first 6 h in the present study supports these observations, replicating the early increase in SVRI and MAP, with reduction in PVRI after an early peak. Cardiac index increased from baseline, reaching a maximal value after the SVRI had dropped, resulting in a hyperdynamic circulation. This may maintain the observed reduction in PVRI via distension of pulmonary vessels and pulmonary capillary reserve recruitment , in addition to the loss of sympathetic vasoconstriction.
Pulmonary interstitial oedema did not differ between groups. The lack of oedema between the groups may be due to resolution of excess lung water by the end of the protocol. Skilled management likely influenced this outcome; fluid balance was similar between groups, thus preventing iatrogenic pulmonary oedema. The influence of duration of care was posited by Avlonitis et al., who observed that, in a prolonged rat model of brain death (15 min vs 5 h), longer duration of care was associated with better oxygenation and reduced post-transplant PVR despite greater exposure to inflammatory cytokines . This group postulated that the improvement in donor oxygenation at 4 h reflected clearance of neurogenic pulmonary oedema . This finding was replicated in our study, demonstrating impaired oxygenation for the first 2 h, with subsequent recovery to levels similar to controls. Duration of BD donor care also influences recipient survival in humans; time from BD to cold preservation greater than 10 h is associated with a survival advantage at both 5 and 10 years . Hormone resuscitation may have also contributed to the observed effect; methylprednisolone administration reduces extravascular lung water in BD donors . Furthermore, dopamine stimulates alveolar fluid clearance and is another possible mechanism to explain our findings .
Whilst the absolute values of mPAP did not greatly exceed the defined cut-off for pulmonary hypertension (>25 mmHg)  in the present study, the increased pressure was significantly greater than baseline and does reflect greater resistance that needs to be overcome by a damaged myocardium. The observation that mPAP was elevated at 24 h suggests that the effect of BD on pulmonary pressures may be greater than previously identified. Extended elevations in right ventricular afterload may contribute to the previously identified right ventricular ischaemia and fibrosis, further priming the right ventricle for acute failure in the recipient. Optimisation of ventilation, oxygenation and pH prevented contributions of these factors to the observed increase in pulmonary pressures in the current study.
Administration of catecholamines to the BD animals did represent a difference in care between the two groups. However, this is unlikely to explain the observed findings, because elevated pulmonary pressures remained unchanged when doses of vasopressors were decreased after commencement of hormone therapy. The use of vasoactive agents is common in BD donors  and has been demonstrated to reduce inflammation associated with hypotension and resultant poor tissue perfusion . Previous studies of noradrenaline and dopamine infusions in sheep do not support that these agents were causative of the observed alterations in pulmonary pressures [43, 44]. In a study of noradrenaline infusion in healthy and endotoxaemic sheep, Lange et al. observed an increase in PVRI in endotoxaemic sheep only . Dopamine infusion in sheep has been associated with an increase in mPAP at rates significantly higher than the doses used in the current study [44, 45]. Although ET-1 can both stimulate  and be stimulated by catecholamines [47, 48], comparison of ET-1 and big ET-1 concentrations over time with the average hourly dose of vasoactive agents did not reveal any correlation in the current study and, therefore, does not account for the observed results.
The current study also confirms that BD induces injury in other transplantable organs in sheep. Elevation of hepatic transaminases indicates hepatocellular injury consistent with previous animal models [49, 50]. Serum cholestatic enzyme activities did not increase over time and suggest hepatic injury was not associated with biliary obstruction.
All BD animals required management of haemodynamic collapse with vasopressors. Haemodynamic support was reduced after hormone therapy was initiated. This is in part due to the inclusion of vasopressin but may also reflect improved haemodynamics directly due to hormonal administration. Thyroid hormone may play a role in regulating anaerobic metabolism and cardiovascular stability post-BD, however the benefits of its routine administration remains controversial [15, 51]. The current trial included hormonal resuscitation consistent with local protocols .
Limitations of the study
Several important limitations have been noted in this study. As previously identified, plasma sampling times may have missed very early peaks in ET-1 or big ET-1. More frequent sampling around the induction of BD in future studies will better characterise the time course of ET-1. Small numbers of animals in each group raise the possibility of type 2 error, although pre-clinical animal models have used similar numbers [18, 19]. Myoglobin and CK-MB were chosen to assess for cardiac injury in the current study. Although troponin may reflect cardiac function in the donor, the correlation between troponin levels and recipient outcome remains controversial . Recently published guidelines continue to include CK as a biomarker in assessment of potential heart transplantation donors [54, 55]. Although NT-proBNP has been noted as a potential marker for assessing cardiac function in potential donors , it is yet to be included among standard tests for donors . Inflammatory cytokines have been well characterised in other animal models of BD. An ongoing challenge in developing new models is a relative paucity of validated, species-specific analytical methods. Our group continues to develop and validate ovine-specific tests [17, 58], and the presented model will provide a platform to further investigate cytokine expression after BD in future studies.
The present model replicated the clinical realities leading to delays in organ retrieval upon BD. Haemodynamic disturbances which occur in BD animals have limited the duration of previous studies. By utilising complete haemodynamic monitoring and support in the same fashion as is applied to human donors, it is possible to maintain a BD sheep for 24 h. Whilst sheep undergoing BD demonstrate complex haemodynamic changes similar to those seen in humans, our data also suggests that early haemodynamic and inflammatory derangements may improve over time with aggressive donor management. This reduces the urgency for organ retrieval and supports such timeframes as are frequently encountered in daily clinical practice. However, significant increases in pulmonary blood pressure may be noted up to 24 h after brain death. Big ET-1 and ET-1 are detectable early after BD and may contribute to the inflammatory cascade that primes allografts for post-transplant dysfunction. Endothelin-1 may also be a key factor in the induction of right ventricular dysfunction observed in cardiac transplantation. Further investigation, targeting the endothelin axis, may provide a novel management option in order to improve the condition of transplantable hearts and lungs, increasing the number and quality of allografts available.
arterial blood gas
analysis of variance
- Big ET-1:
brain death induction time
body surface area
calibrator for automated systems
creatine kinase MB fraction
cerebral perfusion pressure
endothelin A receptor
endothelin B receptor
- FiO2 :
fraction of inspired oxygen
left ventricular stroke work index
mean arterial pressure
mean pulmonary artery pressure
- P(A-a)O2 :
alveolar-arterial oxygen gradient
- PaCO2 :
arterial carbon dioxide partial pressure
- PaO2 :
arterial oxygen partial pressure
pulmonary arterial occlusion pressure
protocol start time
pulmonary vascular resistance index
right ventricular stroke work index
stroke volume index
- SvO2 :
mixed central venous oxygen saturation
systemic vascular resistance index
tris-buffered saline with triton
tissue inhibitor of metalloproteinases
This study was supported by funding provided by Roche Australia’s Cellcept Australia Research Grant and The Prince Charles Hospital Foundation New Investigator Grant. Prof. John Fraser is supported by the Health Research Fellowship funded by Queensland Health. We kindly thank Jurox Australia for donation of alfaxalone for anaesthesia and also Dr. Kirby Pasloske for technical animal anaesthetic advice. Special thanks to Elissa Milford, Scott Temple, Ben Anderson, Michael Manning, Jonathon Taylor and Sue Christie for their assistance with the animal protocols. Thank you to Margaret Passmore for her guidance with the histological investigations and to Daniel Kilburn for assistance with slide preparation. We also thank Phillip Rumballe from The Prince Charles Hospital Pharmacy Department and the staff of the Medical Engineering Research Facility for their assistance.
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