Traumatic Hypoxia and Cerebral Inflammation
Role of Post-traumatic Hypoxia in the Exacerbation of Cerebral Inflammation
Elicited by Brain Injury
Chief Investigator: Associate Professor Cristina Morganti-Kossmann
Associate Investigators: Professor Jamie Cooper, Professor Jeffrey Rosenfeld
Lead Organisation: Bayside Health
TAC Neurotrauma Funding: $232,794
Project Dates: 1 March 2007 – 28 February 2010
Background:
Patients with severe traumatic brain injury (TBI) often suffer from respiratory distress
immediately after an accident, an event which decreases the amount of oxygen in the
blood stream. This insult is named hypoxia. The brain is particularly vulnerable to
hypoxia especially after trauma due to its functional high oxygen consumption.
Epidemiological studies on thousands of TBI victims concluded that the combination
of TBI and a post-traumatic hypoxic insult is associated with worse neurological
outcome. Much of our research has focussed on monitoring the inflammatory
response occurring in the injured brain since this is a pathological phenomenon that
has been recognised to exacerbate brain damage. Intrinsic proteins of the nervous
system (Myelin Basic Protein, Neuronal Specific Enolase and S100) are readily
detectable in the blood stream following head trauma due to the diffusion from the
injured brain to the blood following the rupture of the brain vessels. These proteins
can be used as diagnostic/prognostic markers of brain damage correlating with
neurological outcome scores. By combining both clinical and animal studies our lab is
able to identify and validate these results in human TBI. Once validated these markers
may have an immediate impact for monitoring patients during their treatment or
become useful during the testing of novel therapeutics in clinical trials.
Aims:
The overall aim was to establish whether a hypoxic insult following TBI enhances
pathological events leading to additional brain damage and poor neurological
outcome. We investigated the extent of the inflammatory response by examining the
production of soluble mediators in human cerebrospinal fluid (the fluid in which the
brain is embedded) and rat brain tissue as well as the level of cellular activation
within the brain of injured rats. The measurement of brain injury markers in the blood
stream allowed us to establish correlations between their levels and the neurological
outcome scores using the extended Glasgow Outcome Scale (GOSE) assessed at 6
months following TBI.
Methods:
Over 50 patients with severe TBI were recruited at the Alfred hospital following the
implantation of brain catheters used to treat brain oedema (fluid accumulation in the
brain). These catheters allow the collection of cerebrospinal fluid for 6 consecutive
days after TBI. Simultaneously blood samples were taken on the same days. Patients
were grouped based on the occurrence of a hypoxic insult or normal oxygenation as
reported by paramedics at the accident scene.
The rat model of diffuse TBI is well established in our laboratory and others worldwide. Animals were divided into those treated to brain trauma alone or in combination
with a 30 minute hypoxic insult immediately after TBI. For control purposes, two
groups were generated, one with hypoxia alone and another with anaesthesia and
surgical procedures but without trauma (sham).
In humans, the concentration of 9 immune mediators or cytokines was measured in
the brain fluid for 6 consecutive days whereas in the rat these mediators were
measured in brain tissue samples at 5 time points over 4 days. Additionally rat brain
sections were stained to quantify the infiltration of blood cells into the brain tissue and
examine axonal damage, glial activation, size of the ventricles and brain oedema. In
both humans and animals the neurological deficit was assessed to determine post-TBI
and hypoxia outcome. In the patients the extended Glasgow Outcome Scale at 6
months post-TBI was used, whereas in rats a variety of established tests including the
rotarod, sticky tape removal, beam walk and open field tests were employed for 14
days post-injury.
Results:
Systemic hypoxia in rats was confirmed by the reduction of oxygen by 50% and blood
oxygenation (pO2) by 75% in arterial blood during the exposure to low (12%)
Oxygen. This also coincided with an increase in lactate blood levels and reduction in
mean arterial blood pressure. With the sensorimotor tests rats subjected to combined
TBI and hypoxia showed a more severe deficit in the rotarod during the 6 to 8 days
post-injury. In the open field test the distance travelled was significantly reduced in
rats subjected to TBI and Hypoxia (TBI+Hx) as compared to TBI alone.
In the rat brain, the concentration of pro-inflammatory cytokines interleukin-1 (IL-1),
IL-6, and tumor necrosis factor (TNF) was found exacerbated in the TBI+Hx group as
compared to TBI alone at specific early timepoints (2-24 hours), while IL-2, IL-4 and
IL-10 did not change in any of the groups. Brain oedema increased in both TBI and
TBI+Hx rats but there were no differences between treatments. The enlargement of
the ventricle size occurred in all TBI groups at 1 and 7 days but was more pronounced
at 1 day in TBI+Hx rats. Axonal damage detected with the marker amyloid precursor
protein (APP) showed the presence of axonal swelling and bulb formation which was
exacerbated following TBI+Hx at 1 and 7 days in the corpus callosum and brain stem
which are regions rich in myelin and axonal bundles. Similarly, the infiltration of
blood macrophages and the activation of glial cells were also more pronounced in the
brain of TBI+Hx rats as compared to TBI rats.
In the human study, all cytokines were found increased after TBI in the cerebrospinal
fluid (CSF). However differences between cytokine levels were only found when
patients with focal brain injury were compared with diffuse brain injury. In the latter,
injury markers interleukin-2 and interleukin-4 (IL-2 and IL-4) were found more
elevated than in the focal group. Overall, two injury markers, myelin basic protein
(MBP) and S100 protein were found more elevated in the serum of TBI+Hx group
over the TBI alone group. Also, the increased concentration of S100 correlated to a
poorer Neurological outcome (lower GOSE score) in the TBI+Hx group. NSE levels
showed the weakest correlations to outcome as compared to the other markers S-100
and MBP.
Generally, stronger correlations were found when comparing diffuse with focal brain
injured patients, with an increased presence of S100 protein coinciding with a lower
GOSE score in the diffuse TBI group. MBP correlated with outcome in both focal and
diffuse TBI; however it showed a stronger correlation in the focal group. In the focal
TBI patients all three injury markers were found in blood serum at higher levels than
the focal TBI patients.
Conclusions:
We found that post-traumatic hypoxia has a significant impact in exacerbating
neurological deficit, secondary brain damage and neuroinflammation particularly in
the animal experiments, where it was possible to standardise the severity and type of
brain trauma.
In the patient study more reliable cytokine data were obtained by comparing different
patterns of brain damage (focal versus diffuse) rather than examining the effect of
post-traumatic hypoxia. All markers were more elevated in the focal TBI group over
diffuse group. Two specific injury markers were found more elevated in the TBI and
hypoxia group whereby one of them strongly correlated with a poorer neurological
outcome. In future studies, a larger patient sample size would be useful to validate
these preliminary results.
Publications:
MORGANTI-KOSSMANN MC, SATGUNASEELAN L, BYEN, NGUYEN P, KOSSMANN T. Role
of the inflammatory process in traumatic brain damage. In: KILPATRICK T, WESSELINGH S,
RANSOHOFF R, editors. Neuroinflammation in the brain. Cambridge University Press. 2008.
MORGANTI-KOSSMANN MC, SATGUNASEELAN L, BYE N, KOSSMANN T. Modulation of
immune response by head injury. Injury. 2007;38(12):1392-1400.
HELLEWELL S, YAN E, AGYAPOMAA D, BYE N, MORGANTI-KOSSMANN MC. Posttraumatic hypoxia exacerbates brain tissue damage: Analysis of axonal injury and glial responses. J
Neurotrauma. 2010;27(11):1997-2010.
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.
Presentations:
SATGUNASEELAN L, YAN E, BYE B, NGUYEN P, AGYAPOMAA D, KOSSMANN T,
ROSENFELD JV, COOPER J, MORGANTI-KOSSMANN MC. The effect of post-traumatic hypoxia
on neuroinflammation, protein maker release and brain metabolism after traumatic brain injury.
Neurosurgical Society Australasia Annual Conference; 2007, Gold Coast, Australia.
SATGUNASEELAN L, NGUYEN P, AGYAPOMAA D, KOSSMANN T, MORGANTIKOSSMANN MC. The effect of post-traumatic hypoxia (PTH) on neuroinflammation, protein marker
release and brain metabolism after traumatic brain injury (TBI). 7th International Brain Research
Organization World Congress; 2007 July 12-16, Melbourne, Australia.
YAN EB, WALKER DW, ROSENFELD J, SEIFMAN M, COOPER J, MORGANTI-KOSSMANN
MC. Hypoxia following traumatic brain injury alters the tryptophan metabolism and melatonin
productions in the patients. Sixth Australian Conference on the Kynurenine Pathway of Tryptophan
Metabolism; 2007, Sydney, Australia.
SATGUNASEELAN L, YAN EB, NGUYEN P, AGYAPOMAA D, KOSSMANN T, MORGANTIKOSSMANN MC. Monitoring the neuroinflammatory response in traumatic brain injury using
microdialysis: an experimental model. International Conference on Clinical Microdialysis; 2007,
Cambridge, UK.
SATGUNASEELAN L, NGUYEN P, BYE N, AGYAPOMAA D, KOSSMANN T, MORGANTIKOSSMANN MC. The diagnostic role of protein markers and cytokines in focal and diffuse traumatic
brain injury (TBI). 7th International Brain Research Organization World Congress; 2007 July 12-16,
Melbourne, Australia.
HELLEWELL SC, YAN EB, AGYAPOMAA D, MORGANTI-KOSSMANN MC. Post-traumatic
hypoxia worsens neuropathological damage in an animal model of diffuse axonal injury. Trauma
Melbourne; 2008 November 21-22, Melbourne, Australia.
YAN EB, WALKER DW, AGYAPOMMA D, GUILLEMIN GJ, MORGANTI-KOSSMANN MC.
Increase tryptophan metabolism after traumatic brain injury. Trauma Melbourne; 2008 November 2122, Melbourne, Australia.
SATGUNASEELAN L, YAN EB, AGYAPOMAA D, NGUYEN P, KOSSMAN T, COOPER DJ,
VALLANCE S, MORGANTI-KOSSMANN MC. Measurement of brain specific proteins in traumatic
brain injury (TBI)- A role for outcome prediction? Trauma Melbourne; 2008 November 21-22,
Melbourne, Australia.
YAN EB, SATGUNASELLAN L, NGUYEN P, BYE N, WALKER DW, AGYAPOMAA D,
ROSENFELD J, SEIFMAN M, KOSSMANN T, MORGANTI-KOSSMANN MC. The effect of posttraumatic hypoxia on neuroinflammation, tryptophan oxidation and melatonin production after
traumatic brain injury Australian neuroscience Society 28th Annual Meeting; 2008, Hobart, Australia.
YAN EB, HELLEWELL SC, AGYAPOMAA D, MORGANTI-KOSSMANN MC. Post-trauma
hypoxia exacerbates neurological deficits, neuroinflammation, and axonal damage in a rat model of
diffuse axonal injury. Trauma Melbourne; 2009 November 19-21, Melbourne, Australia.
ALWIS DS, COLEMAN HC, PARKINGTON HC, ZHANG AM, MORGANTI-KOSSMANN MC,
EDWIN B, YAN EB. Diffuse traumatic axonal injury induces sensorimotor deficit, memory loss and
hippocampal axonal hyperexcitability. Trauma Melbourne; 2009 November 19-21, Melbourne,
Australia.
YAN E, HELLEWELL S, AGYAPOMAA D, MORGANTI-KOSSMANN MC. Posttrauma hypoxia
exacerbates neurological deficits, neuroinflammation and axonal damage in a rat model of diffuse
axonal injury. Joint National/International Neurotrauma Conference; 2009 September 7-11, Santa
Barbara, USA.
HELLEWELL SC, YAN EB, AGYAPOMMAA D, MORGANTI-KOSSMANN MC. Post-traumatic
hypoxia exacerbates brain damage in an animal of diffuse axonal injury. ANS Meeting; 2009 January
27-30, Canberra, Australia.
ALWIS, COLEMAN, PARKINGTON, ZHANG, MORGANTI-KOSSMANN, YAN. Diffuse
traumatic axonal injury induces sensorimotor deficit, memory loss and hippocampal axonal
hyperexcitability. Australian Neuroscience Society; 2010 February 1-3, Sydney, Australia.