Completed Early Career Research Fellowship:
Edwin Yan
Host Organisation: Alfred Health
TAC Funding: $326,270
Mentors: Professor Jeffrey Rosenfeld, Dr James Bourne & Associate Professor
Cristina Morganti-Kossmann
Fellowship Project:
Establishment a model of traumatic brain injury (TBI) in non-human primate Marmoset and examining the changes in neurotoxin quinolinic acid concentration
following TBI
Aims:
This project investigated whether activation of the kynurenine pathway in the injured
brain of both humans and rats contributes to the secondary brain damage that evolves
after a TBI. Specifically, this project focused on the production of neurotoxins and
potential therapeutical intervention after TBI. 3 studies were undertaken as part of this
Fellowship: 1. Investigating neurotoxins in cerebrospinal fluid of TBI patients; 2. in
post-mortem human brains after TBI; 3. Validation of the human findings in a rat
model.
Methods:
Study 1: Tryptophan metabolism in human CSF is enhanced by post-traumatic
hypoxia.
Severe TBI patients with implantation of a cerebro-ventricular catheter for CSF
drainage were enrolled in this study. Each day after TBI, CSF was collected into bags
kept at 4°C and changed every 24 h until the catheter was removed (n=25). Control
CSF samples were obtained from non-TBI, non-cancer, or non-neurodegenerative
disease patients undergoing neurosurgery (n=15). The following tryptophan
metabolites, tryptophan (TRY), kynurenine (KYN), kynurenic acid (KYNA), 3hydroxykynurenine (3-OHK) and 3-hydroxyanthranilic acid (3-OHAA) were
measured by high performance liquid chromatography (HPLC), while quinolinic acid
(QUIN), was measured by gas chromatography-mass spectroscopy (GC-MS).
Patient’s neurological outcomes were determined by Glasgow Outcome Score
Extended (GOSE) and correlated with neurotoxin QUIN concentrations.
Study 2: TBI enhances the expression of kynurenine pathway (KP) enzymes in
post mortem human brain.
Human brain tissues were obtained through the NTFB funded by the VNI. We
analysed 21 post-mortem TBI brain samples and 13 brains frominstant non-TBI death.
Fresh frozen brain tissues were collected for determining the mRNA levels of KP
enzymes (IDO (indoleamine 2,3-dioxygenase), KAT (kynurenine aminotransferase),
KYNOH-ase (kynurenine-3-hydroxylase), 3-HAO (3-hydroxyanthranilic acid
oxygenase) and QPRT-ase (quinolinic acid phosphoribosy transferase)). Additional
brain tissues from the same individuals were fixed in 40% formaldehyde, and then
processed for paraffin wax embedding. The tissues were cut into 10 μm sections for
immunohistochemistry to identify the localization and distribution of the key enzymes
of the KP (IDO and KYNOH-ase). The QUIN containing cells will also be identified
by immunohistochemistry.
Study 3: Tryptophan metabolism increases in rat brain after TBI, and is
exacerbated by post-TBI hypoxia.
Diffuse TBI was induced by dropping a 450 grams weight from 2 meters through a
vertical tube onto a metal disk fixed onto rat (325-375 grams) skull between bregma
and lambda under isoflurane anaesthesia. Four groups of animals were used: (i), 30
min hypoxia only (n=30); (b), TBI only (n=30); (c), TBI followed by a 30 min
hypoxia (n=30); and (d), sham operated (n=30). Hypoxia will be induced immediately
following TBI by ventilating with a 12% O2 / 88% N2 gas mixture, while for
normoxic animals a 22% O2 / 78% N2 gas mixture was used. Animals from
eachgroup were euthanized at 2, 12, and 24 hours, 2, and 5 days after treatments (n=6
per time point). Fresh brain tissues were collected from frontal, parietal and occipital
cortex for tryptophan metabolite measurements using HPLC and GC-MS. Enzyme
expressions of KP (same as in Study 2) were analysed in the same regions described
above using real-time-PCR with specific primers for rat. Oxidative stress was
determined by measuring malondialdehyde (MDA) levels in brain homogenates.
Results:
This project has established whether activation of the kynurenine pathway in the
injured brain of humans is contributing to secondary brain damage that evolves after
TBI. The first study has demonstrated for the first time: (a), the alterations of
tryptophan metabolism over 5 days following severe TBI; (b), the effect of post-TBI
hypoxia in exacerbating tryptophan metabolism and enhancing neurotoxic QUIN
production; and (c), strong correlation between increased QUIN production after TBI
and severe neurological outcomes. Through the recently established Neurotrauma
Tissue / Fluid Bank (NTFB), we have the unique opportunity to obtain human TBI
tissue for in situ detection of molecules of interest. Therefore, Study 2 has provided
results for the first time on the level of activation of the kynurenine pathway in human
injured brain and the identification of the cellular source of the enzymes involved in
this pathway. Study 3 has attempted to use a standardised, reproducible animal model
of diffuse TBI in rat. The choice of a diffuse rat brain injury model in combination
with post-traumatic hypoxia is clinically relevant since clinical data have shown that
majority of severe TBI patients have diffuse type of brain injury. Unfortunately, rats
have lacked in QUIN production although the evidences showed the kynurenine
pathway was activated following TBI. We have also attempted to measure QUIN
concentration in tissues of focal brain injury in mice. The results also showed no
changes. These collective evidences have suggested the rodents may not be suitable
for investigation of kynurenine pathway after brain injury.
Nevertheless, this project has provided further understanding of the kynurenine
pathway in the post-TBI human brain, which has demonstrated the potential of
blocking neurotoxin QUIN production to reduce secondary brain damage. The better
understanding in tryptophan metabolism and enzyme expression following TBI will
lead to develop such therapeutical treatment.
Conclusions:
In this project, we have demonstrated significant increase in QUIN and other
intermediate molecule productions. Most importantly, this study has shown for the
first time that QUIN increases for at least 15-fold in CSF after TBI. These increases in
neurotoxin may contribute to the on-going brain damage after TBI. This study has
demonstrated that patients with higher QUIN concentration in the brain soon after
TBI had worse neurological outcome at 6 months post-injury. This project had a well
understanding on how neurotoxic molecule QUIN been produced after TBI, which
may lead to therapeutical protocols aimed at specifically blocking the production of
this toxin.
Feedback on the Fellowship from Edwin Yan:
“I do like to express my sincerely appreciation for TAC and VNI to award this
research fellowship. It has tremendously enhanced my research career in traumatic
brain injury, advanced my track record and competitiveness in successful in research
grants. Through this scheme I have met many internationally recognised researchers
in the field of neurotrauma and built numerous collaborations. I also would like to
thank all the project managers involved during the period of this fellowship, thank
you for your help, patient and encouragement. Finally, I would like to repeat that I
had excellent experience and time as a research fellow of TAC”
Publications
GENG G, JOHNSTON LA, YAN E, BRITTO JM, SMITH DW, WALKER DW, EGAN GF.
Biomechanisms for modelling cerebral cortical folding. Med Image Anal. 2009;13:920-930.
YAN EB, BABURAMANI AA, WALKER AM, WALKER DW. Changes in cerebral blood flow,
cerebral metabolites and breathing movements in the sheep foetus following asphyxia produced by
occlusion of the umbilical cord. Am J Physiol Regul Integr Comp Physiol. 2009;297:R60-69.
HELLEWELL SC, YAN EB, BYE N, AGYAPOMAA D, MORGANTI-KOSSMANN MC. Posttraumatic hypoxia exacerbates brain tissue damage: Analysis of axonal injury and glial responses. J
Neurotrauma 27:1997-2010.
MORGANTI-KOSSMANN MC, SEMPLE B, ZIEBELL J, YAN E, BYE N, KOSSMANN T.
Modulation of immune response by head injury. In: BERCZI I., ARNASON B, Editors. Neuroimmune
Biology: The Brain and host defence. Manitoba, Canada. Elsevier Science; 2010. p.193-210.
MORGANTI-KOSSMANN MC, YAN E, BYE N. Animal models of traumatic brain injury: Is there an
optimal model to reproduce human brain injury in the laboratory? Injury. 2010;41 Suppl 1:S10-3.
YAWNO T, YAN EB, WALKER DW, HIRST JJ. Neuroactive steroids induce changes in foetal sheep
behaviour during normoxic and asphyxic insult. Stress. 2011;14( 1):1-5.
BYE N, CARRON S, HAN X, AGYAPOMAA D, NG SY, YAN E, ROSENFELD JV, MORGANTIKOSSMANN MC. Neurogenesis and glial proliferation are stimulated following diffuse traumatic
brain injury in adult rats. J Neurosci Res 2011:89:986-1000.
Presentations
ALWIS, COLEMAN, PARKINGTON, ZHANG, MORGANTI-KOSSMANN, YAN. Diffuse
traumatic axonal injury induces sensorimotor deficit, memory loss and hippocampal axonal
hyperexcitability. Trauma Melbourne; 2009 November 20-21, Melbourne, Australia.
KENNA, YAN, GRAY, BOCKING, BRIEN, WALKER, HARDING. Physiologic responses to daily
alcohol infusion in the ovine foetus and ewe during late gestation. Society for Gynaecologic
Investigation; 2009 March 17-21, Glasgow, Scotland.
YAN, GUILLEMIN, WALKER, MORGANTI-KOSSMANN. Increase tryptophan metabolism after
traumatic brain injury. International Society for Tryptophan Research; 2009 July 9-11, Florence, Italy.
YAN, HELLEWELL, AGYAPOMAA, MORGANTI-KOSSMANN. Post-trauma hypoxia exacerbates
neurological deficits, neuroinflammation, and axonal damage in a rat model of diffuse axonal injury.
International Neurotrauma Society; 2009 September 7-11, Santa Barbra, USA.
BABURAMANI, YAN, WALKER. Prolonged decrease in cerebral blood flow following a transient
severe hypoxia in late gestation foetal sheep. Foetal and Neonatal Physiological Society; 2009
September 27-30, Lake Arrowhead, USA.
HELLEWELL, YAN, AGYAPOMAA, MORGANTI-KOSSMANN. Post-traumatic hypoxia
exacerbates brain damage in an animal model of diffuse axonal injury. Australian Neuroscience
Society; 2009 January 27-30, Canberra, Australia.
GENG, JOHNSTON, YAN, BRITTO, WALKER, EGAN. Diffusion MR anisotropy predictors of
cerebral cortical folding in a foetal sheep model. Australian Neuroscience Society; 1-3 February 2010,
Sydney, Australia.
ALWIS, COLEMAN, PARKINGTON, ZHANG, MORGANTI-KOSSMANN, YAN. Diffuse
traumatic axonal injury induces sensorimotor deficit, memory loss and hippocampal axonal
hyperexcitability. Australian Neuroscience Society; 1-3 February 2010, Sydney, Australia.
YAN, FRUGIER, LIM, TAN, ROSENFELD, WALKER, GUILLEMIN, MORGANTI-KOSSMANN.
Enhanced activation of kynurenine pathway and increased neurotoxin quinolinic acid production
following traumatic brain injury in humans. International Neurotrauma Society, 27-30 May 2011,
Shanghai, China.
HELLEWELL, YAN, BELLANDER, AGYAPOMAA, MORGANTI-KOSSMANN. Post-traumatic
hypoxia exacerbates neurological deficit, neuroinflammation and cerebral metabolism in rats with
diffuse traumatic brain injury “From Roadside to Recovery – Successful Systems of Trauma Care”
Conference, 18-19 November 2011, Melbourne, Australia.
YAN, SATGUNASEELAN, HELLEWELL, BYE, ROSENFELD, COOPER, MORGANTIKOSSMANN. A hypoxic insult in patients with traumatic brain injury enhances cerebral inflammatory
cytokines, serum biomarkers, and blood brain barrier dysfunction leading to unfavourable outcome.
“From Roadside to Recovery – Successful Systems of Trauma Care” Conference, 18-19 November
2011, Melbourne, Australia.