Molecular mechanisms of traumatic brain injury: the missing link in management

Our understanding of the molecular mechanisms of traumatic brain injury (TBI) has improved over the last decade, but a gap still exists between these advances and their translation into direct clinical care. About 0.5–1 million patients present to hospitals in the UK with TBI. It is the leading cause of disability in people under 40, and severely disables 150–200 people per million annually [1, 2]. In the US, TBI affects 1.4 million people, at an estimated annual cost of $56 billion [3]. Diseases of the nervous system (International Classification of Diseases-revision 9) accounted for 8.4% of the total health and social services net public expenditure for 1992 and 1993 in England [4]. The purpose of this review is to look at genetic and molecular influences after an acute head injury and the long term outcome.

Although our ability to assess and predict neurological outcome following TBI has improved, most of the prognostic tools are still poorly validated and therefore rarely used [5]. Understanding the molecular mechanisms and integrating these into clinical practice will help us to predict outcomes more accurately, and will also pave the way for newer treatment modalities and further research.

Current understanding of the basic molecular mechanisms resulting in neurological damage following TBI has sparked several significant attempts to synthesise drugs (e.g. Selfotel) [6]. So far these attempts have universally met with little success clinically, but they have provided some insights for future research [6]. Such research has been hampered by a lack of translation of results from animal models into humans. Despite this it is likely that such work, both in animal models and observational studies in patients with acute TBI will continue to shed light in this important subject.

Acute TBI is characterised by two injury phases, primary and secondary. The primary brain injury is the direct injury to the brain cells incurred at the time of the initial impact. This results in a series of, biochemical processes which then result in secondary brain injury. The primary aim for the acute management of TBI is to limit the secondary injury. The secondary brain injury is caused by a dynamic interplay between ischaemic, inflammatory and cytotoxic processes. Studies with microdialysis techniques have shown that one of the most significant factors causing secondary brain injury is the excessive release of excitotoxins such as glutamate and aspartate that occurs at the time of the primary brain injury. These excitotoxins act on the N-methyl-D-aspartate channel, altering cell wall permeability with an increase in intracellular calcium and sodium and activation of calcineurin and calmodulin. This ultimately, leads to destruction of the axon [7, 8]. Potassium is also released from the cells and absorbed by the astrocytes, in an attempt to restrict the ionic imbalance causing swelling of the cells and ultimately cell death.

There is a complex cascade of cellular inflammatory response following TBI which propagates secondary brain damage. This inflammatory process lasts from hours to days contributing continuously to secondary brain damage. The inflammatory response resulting from an acute TBI is not limited to the brain and multiple organ dysfunction syndromes are commonly seen. The major molecules in the brain involved in this cascade are growth factors, catecholamines, neurokinins, cytokines and chemokines [9].

Interleukins (IL) are proinflammatory cytokines, the levels of interleukins seen in intracerebral bleeds, and clinical signs of inflammation at admission, have correlated well with the magnitude of perilesional oedema and mortality [10, 11]. There is a rise in IL-6 and 10 in children following a TBI. The increased level of IL-10 was directly related to mortality in TBI [12]. The rise in inflammatory cytokines (e.g. IL-6) following TBI is a double edged sword; both neurotoxicity and neuroprotection may be induced by it. Inflammatory cytokines facilitate neurotoxicity by encouraging excitotoxicity and the inflammatory response, but simultaneously they facilitate the neurotrophic mechanisms and induction of cell growth factors which are neuroprotective [13]. It has also been shown by Vuylsteke et al that there is an increased gradient of inflammatory marker IL-8 in the brain after cardiopulmonary bypass, which is attenuated by hypothermia [14]. This gradient continued into the postoperative period.

The primary insult also results in an immediate disturbance of the cerebral circulation, resulting in cerebral ischaemia and which contributes significantly to about 90% of deaths after closed head injuries. [15]. Ischaemic brain damage is perpetuated by factors such as hypotension, hypoxia, raised intracranial pressure, oedema, focal tissue compression, damage to microvasculature, and in late phases, vasospasm in the remaining vessels [16, 17]. The time sequence after TBI can be arbitrarily divided into an early (phase 1, immediate, with hypoperfusion), intermediate (phase 2, on days 1–3, when hyperaemia can be seen) and a late vasospastic phase (phase 3, on days 4–15, with a marked reduction in blood flow) [17]. These different phases are associated with marked regional variations in cerebral blood flow, with a reduction in blood flow to the surrounding of the ischaemic core, which does not respond to augmentation of cerebral perfusion pressure [18].

Programmed cell death (which is often referred to as apoptosis although strictly speaking this refers to the distinct morphological changes after programmed cell death) is a genetic mechanism by which cells are eliminated during development, and is the physiological mechanism by which cells are normally removed in the adult animal [19]. This involves specific genes and proteins which were first described in neuronal development of the round worm [20]. Following TBI there is increased expression of two main sets of genes which are genes encoding for the caspase family of cysteine proteases [including interleukin-1β converting enzyme (ICE) and cpp32] and a family of genes that are homologous to the oncogene Bcl-2 that either promote or suppress cell death. The Bcl-2 gene family controls both caspase dependent and independent apoptosis. [19, 21‐23]. The endpoint of all these steps is fragmentation of cellular DNA with collapse of the nuclear structure, followed by the formation of membrane-wrapped apoptotic bodies, cleared by macrophages [24].

Apoptosis is now recognised as an important factor in secondary brain injury [25]. Following TBI, two different types of cells are visible; type 1 and 2 cells. The type 1 cells show a classic necrotic pattern (this follows the primary brain injury) and type 2 cells shows a classic apoptotic pattern on microscopy [25, 19]. Cells undergoing apoptosis die without membrane rupture and therefore elicit less inflammatory reactions. This is in contrast to the cells undergoing necrosis [26]. There is therefore a suggestion that neuronal apoptosis after TBI may be a protective response by the brain in order to remove injured tissue cells whilst having little effect on remaining brain tissue [27]. Apoptotic cells have been identified within contusions in the acute post-traumatic period, and in regions remote from the site of injury days and weeks after trauma.

Pharmacological strategies to reduce apoptotic cell death have been investigated, [28] For example, rats treated with the caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (DEVD) demonstrate a 30% reduction in lesion volume measured 3 weeks after TBI when compared with vehicle-treated controls [19].

Recent advances in the management of severe acute TBI has resulted in improved outcomes for patients who might previously have had poor outcomes. In particular the management of such patients in specialist units has had a significant impact, although the definitive factors contributing to improved outcomes remain elusive. [29]. In recent years there has been increasing interest in elucidating the long term problems experienced by patients following TBI. Further, there have been reports of people developing dementia-like symptoms following relatively minor head injuries (Brain injury with a GCS greater than 13 and without loss of consciousness, as well as an increased incidence of post traumatic stress disorders and depression [30]. TBI causes a generalised atrophy of brain which is proportional to the severity of the injury. [31]. The mechanisms for this are yet to be fully determined. In rats it has been shown that there are multiple antibodies to the amyloid precursor protein and amyloid precursor protein-like proteins for up to six months, which predisposes them to degeneration of the striatum and corpus callosum. This degeneration then leads to progressive brain atrophy and calcifications [32]. In moderate to severe TBI there is a high incidence of hippocampal atrophy which predisposes patients to cognitive decline. When anoxic brain damage was compared to TBI there was no overwhelming evidence of localised nerve damage. This supports the theory that the final mechanism for neurological injury is the same irrespective of the type of initial insult [33].

Surprisingly humans are made up of only 20,000 – 25,000 protein-coding genes, and these genes have profound implications on our survival [34]. The genetic constituents not only modify the risk of development of disease and its severity, but also the ability of an organ to repair, heal and function after an injury. In head injured patients the outcomes are variable and cannot easily be predicted. This variability cannot be fully explained by clinical features or by the character of the injury [35]. One of the mechanisms which could explain this is genetic polymorphism. This may also contribute to variability in outcome in the acute response, and functional recovery. A greater understanding of the genetics could aid in the prediction of outcomes and could be targeted for treatment strategies.

Studies in animals using cDNA microarray hybridization technique have shown differential regulation of 86 genes (seven classes) which take part in the physiological and pathological response to TBI. The key classes they encompass include transcription factors, signal transduction genes and inflammatory proteins [36]. Such changes in gene expression are interlinked with both disease processes (for example IL-6 and haemoxygenase-1), and outcome in TBI.

A variety of pharmacological agents have been trialed, all of which have shown promising results in animal models, but when translated into the clinical setting have universally failed to influence outcome following TBI. These agents include Selfotel, Cerestat, CP 101–606, D-CPP-ene, Steroids, tirilazad, PEG-SOD, IGF-1/growth hormone, Nimodipine, Bradycor, Dexanabinol, SNX-III, and anticonvulsants (such as Valproate and Magnesium Sulphate). The neuroprotective actions of these agents result from a variety of mechanisms of action, including antagonism of glutamate (Selfotel and CP 101–606), and free radical scavenging (PEG-SOD) [6].

Dexanabinol is a synthetic chemical analogue of the active component of marijuana. It is a non-competitive inhibitor of the NMDA receptor, a free radical scavenger and antioxidant, and an inhibitor of the pro-inflammatory cytokine TNF alpha [6].

Steroids are used with good effect in the treatment of brain oedema associated with brain tumours, and have been shown in laboratory studies to reduce free radical production and have a protective effect on the brain. However, several clinical studies in TBI have shown no clear beneficial effect on outcome or intracranial pressure [6].

This review emphasises that the molecular mechanisms underlying secondary brain damage following TBI are complex. Our understanding of these mechanisms has increased significantly in recent years, but is far from complete. Advances in the acute management of TBI, is likely to be dependant both on an improved understanding of these mechanisms, as well as the translation of such knowledge into the development of new molecules and techniques to improve the clinical outcome.