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There is currently some anecdotal evidence that the peripheral wounds of patients who suffer concurrent brain trauma, such as those in road traffic accidents, heal better than those of patients without brain trauma. It has been suggested that the event of brain trauma itself causes many changes in the human body, some of which may lead to the observed improved peripheral wound healing.
This project aims to identify some of the peripheral changes associated with brain trauma that may influence peripheral wound healing. This information can then be used to design experiments to analyse the effect of brain trauma on peripheral wound healing.
For this project, we will be using samples of rabbit tissue obtained from animals being used for a project investigating the effects of brain death on lung transplantation. This project is conducted by Prof John Fraser and his team at the University of Queensland. We will receive skin, dorsal root ganglion and spinal tissue samples from brain dead and sham treated rabbits at zero and four hours post-brain death. In addition we will have access to blood samples from these rabbits. Blood, neural and skin tissue will be analysed for changes in inflammatory markers, apoptotic markers, neuronal signalling markers and markers of innervation.
By comparing the changes observed in the sham controls and brain-injured rabbits we aim to identify potential specific factors that may lead to improved peripheral healing after brain trauma.
Burden of Burns Injury
An Australian Institute of Health and Welfare study examining hospital separations due to injury and poisoning found that in 2004-05 burns injuries accounted for 1.8% of all community injury cases1. Furthermore, the age-standardised rate of hospitalised burns and scalds cases was found to be 27.2 per 100,000 in the Australian population1.
Burn injuries form life-long scars due to the imperfect healing process, and the high rates of burning and scalding occurring in the 0-4 years age group means many patients live with the burden of scarring for the vast majority of their lives1. This study aims to provide insight into the systemic burn healing process, and consequently to lead to improved treatment and outcomes for burn patients.
The CNS and Peripheral Injury Communication
To date, there have been anecdotal reports that patients who suffer brain trauma at the same time as other peripheral injuries will heal the peripheral injuries with significantly less scarring than would be expected. This has been suggested to be due to changes that may occur in response to the brain trauma that also influence the peripheral wound healing response. However, to date there is little or no evidence to support this, or to suggest a potential mechanism by which this could occur2.
Previous research, including within this research group, have demonstrated systemic changes in innervation in response to localised injury, as well as changes in the CNS in the long-term after peripheral burn injury2-7. This suggests an interaction between the CNS and the periphery in the injury response, although again the mechanism has not yet been elucidated. This project aims to determine whether brain trauma affects the skin, and peripheral nerves, in a manner which would be expected to influence healing. The findings of this project will provide the basis for further investigation to determine the influence of the CNS and of CNS trauma on the peripheral wound healing response.
This area of research remains unexplored to date, with only anecdotal support. Therefore there is an urgent need to collect the data that can either verify or dismiss the anecdotal evidence and determine whether CNS injury has an impact on peripheral healing, and if so, by what mechanism.
Previous studies have examined the systemic effects of brain death and trauma8-14. These findings will provide the basis for our initial studies in determining the specific inflammatory and other markers to measure.
From the previous studies of brain trauma and systemic changes, particularly in cytokines, a number of potential markers to be investigated in this project have been identified. In particular, the levels of interleukin-1Î± and interleukin-1Î², interleukin-6, interleukin-8, C-Reactive Protein, TNF-Î±, neuropeptide Y, Activin A, basic fibroblast growth factor, Metallothionein-1+2 and chondroitin sulphate proteoglycans are all significantly altered by brain trauma, whilst also being critical to the burn injury response2-4,12,13,15-19. These markers can be analysed from blood samples. In addition, the dorsal root ganglion and peripheral nerves in skin tissue samples will be examined, for example using the pan-neuronal marker PGP9.5 to investigate peripheral nerve changes. .
Ethics approval has been obtained by Prof. Fraser for the original project "Effect of brain death to immunogenicity of Transplanted lungs", approved by the University Animal Ethics Committee of QUT Council in November 2009 (Reference number 08 0000 0195). An amendment to allow tissue sharing without increasing numbers or interventions has been submitted to QUT ethics committee.
It is important to note no additional animals or procedures will be involved for the purposes of this project. Tissues will simply be collected from animals being euthanased as part of the original study.
Trauma to the central nervous system promotes peripheral wound healing
Determine whether trauma to the central nervous system affects the levels of specific serum cytokines
Determine whether trauma to the central nervous system directly affects the skin within a 4 hour time-frame
Determine whether trauma to the central nervous system affects peripheral, and specifically cutaneous innervation
The tissue samples and blood analyses will be obtained from Prof J Fraser's group at QUT. The analysis of the skin and neural tissue collected will be done using fixed tissue and immunohistochemistry for markers of apoptosis, innervation and signalling.
A brief description of the project conducted by Prof Fraser's group is outlined below as well as details of post-tissue collection procedures that will be carried out within the Burn injury research unit (BIRU) at UWA.
Several weeks prior to transplantation, arterial blood is collected from the donor and recipient rabbits. For the transplantation, donor rabbits are anaesthetised and receive pain relief. Donor rabbits are then assigned as either 'brain dead' or 'non-brain dead' (sham) donors. The chosen method of inducing brain death is similar to the method of brain death in human donors where swelling compresses the brain9,14. This utilises a small balloon placed through a hole in the skull and inflated. This compresses the base of the brain where it exits the skull, causing impaired blood flow to and from the brain. The impaired blood flow causes brain death.
Four hours post brain-death, the right lung is removed from the donor and placed on ice for two hours, to simulate a typical human transplantation interval. The donor rabbit is then euthanased. The recipient rabbit is then anaesthetised and receives pain relief. The recipient rabbits' right lung is then removed and the donor lung implanted. The recipient rabbit remains anaesthetised for four hours after the transplantation to allow for data collection. The recipient rabbit is then euthanased and both lungs removed for further analysis.
At various points throughout the procedure, blood, tissue and bronchoalveolar lavage (BAl) samples are taken from both donor and recipient rabbits
For the purposes of this project, all analysis will be conducted on donor animals, with either sham or brain dead animals.
Blood samples obtained will be analysed for multiple cytokine levels, which will include IL-1Î± and IL-1Î², IL-8, C-Reactive Protein, and TNF-Î±. This will determine the effect of brain death on these proteins in the blood when compared to sham controls. Significant differences may indicate potential proteins that may mediate the hypothesised amelioration of scarring caused by brain trauma.
At time of brain death, or sham operation (0 hours), and at time of euthanasia (4 hours post-brain death), tissue samples will also be collected for this project including skin samples. Samples of dorsal root ganglia and spinal cord tissue will also be collected at time of euthanasia (4 hours). These will be fixed using Zamboni's fixative and then subsequently paraffin embedded and processed.
Paraffin embedded tissue will be sectioned, and sections analysed for markers of innervation, apoptosis and inflammatory markers using immunohistochemistry. A brief protocol for standard immunohistochemistry using the PGP9.5 pan-neuronal marker antibody will be carried out.
Sections are deparaffinized using xylene and ethanol washes and then undergo antigen retrieval using 1mg/mL trypsin at 37ËšC. Sections are washed in 0.1M phosphate buffered saline containing 10 mM Tris (TPBS), followed by application of a blocking solution containing 10% Donkey serum in TPBS, 0.3% Triton X and 0.1% Sodium Azide. The primary antibody, Protein Gene Product 9.5 (Rabbit, Chemicon, VIC, Australia), is then applied at 1:100 diluted in blocking solution. Sections are incubated in a humidified box for 48 hours at 4ËšC and then washed in TPBS. The secondary antibodies, either FITC (Jackson Immuno Research Laboratories, West Grove, PA, USA, donkey anti-goat) or CY3 (Jackson Immuno Research Laboratories, West Grove, PA, USA, donkey anti-rabbit) are diluted (each 1:750) in 5% Donkey serum with TPBS, 0.3% Triton X and 0.1% sodium azide and applied to the sections which are incubated in the dark, at 4ËšC for 24 hours. Sections are then washed in TPBS, dried, and mounted with ProLong gold Anti-fade (Molecular Probes, Eugene, OR, USA). Control sections are processed in parallel without the addition of primary antibody.
Data will be analysed using R, version 2.8.1. Appropriate tests will be performed, with Wilcoxon Signed Rank test on non-normal distributions, or alternatively the Wilcoxon Rank Sum test for unpaired comparisons. Results will be considered significant at the 0.05 level.
Introduction, Methods and Literature Review
- Blood Markers
Data Analysis and Write-Up