Traumatic Penumbra Research Articles

Persistently “Leaky” Nav Channels In Traumatized Axons: Lowered Barriers To Nav1.6 Voltage Sensor Motions In Blebbed Plasma Membrane As A Possible Explanation

Mechanical trauma of CNS nodes of Ranvier generates axolemmal blebs. The nodal Na+-channel, Nav1.6, leaks Na+ after traumatic brain injury but its molecular “lesion” is not understood. We found that traumatic stretch of Na+-dye loaded HEK-Nav1.6 cells causes an immediate TTX-sensitive Na+-leak. Also, using oocytes, we measured Nav1.6 current in cell-attached patches before and after pipette aspiration (which causes blebs) and observed irreversibly left-shifted g(V) and availability(V). To determine if intact cell Nav1.6 operation left-shifts with trauma, within-cell comparisons of HEK-Nav1.6 cell INa before and after traumatic stretch would be ideal, but this is impracticable. Instead, perforated patch recordings (multi-pulse protocols) of INa were obtained in a given HEK-Nav1.6 cell which was then swollen grossly (distilled water, 90-180 s) and returned to normal saline. After 5 min (for cell morphology and [Na+]ext to regularize) INa was re-measured. The resulting pattern of INa changes at various voltages showed that, post-osmotrauma, both g(V) and availability(V) were left-shifted at least 5 mV and this was irreversible (10 min experiments). Time controls showed no left-shift. A simple explanation is that, post-trauma, abnormally fluid disorganized bilayer of blebbed membrane presents abnormally low energy barriers to Nav1.6 voltage sensor motions. Smaller depolarizations are thus required to elicit sensor repacking than in stiffer pre-bleb bilayer. To the extent that trauma-induced blebbing was non-uniform, left-shift would be “smeared”. For mildly traumatized axons of the traumatic penumbra, such left-smeared Nav1.6 window current, by leaving no “safe” voltage, should prove even more excitotoxic than maximal left-shifting. Positive feedback in free-running axons would persistently elicit Na+-leak as window currents from variously traumatized areas triggered each other. Supported by CIHR and HSFO.

Causal Role of Apoptosis-Inducing Factor for Neuronal Cell Death Following Traumatic Brain Injury

Traumatic brain injury (TBI) consists of two phases: an immediate phase in which damage is caused as a direct result of the mechanical impact; and a late phase of altered biochemical events that results in delayed tissue damage and is therefore amenable to therapeutic treatment. Because the molecular mechanisms of delayed post-traumatic neuronal cell death are still poorly understood, we investigated whether apoptosis-inducing factor (AIF), a pro-apoptotic mitochondrial molecule and the key factor in the caspase-independent, cell death signaling pathway, plays a causal role in neuronal death following TBI. Using an in vitro model of neuronal stretch injury, we demonstrated that AIF translocated from mitochondria to the nucleus of neurons displaying axonal disruption, chromatin condensation, and nuclear pyknosis in a caspase-independent manner, whereas astrocytes remained unaffected. Similar findings were observed following experimental TBI in mice, where AIF translocation to the nucleus coincided with delayed neuronal cell death in both cortical and hippocampal neurons. Down-regulation of AIF in vitro by siRNA significantly reduced stretch-induced neuronal cell death by 67%, a finding corroborated in vivo using AIF-deficient harlequin mutant mice, where secondary contusion expansion was significantly reduced by 44%. Hence, our current findings demonstrate that caspase-independent, AIF-mediated signaling pathways significantly contribute to post-traumatic neuronal cell death and may therefore represent novel therapeutic targets for the treatment of TBI.

Changes of Cerebral Blood Flow during the Secondary Expansion of a Cortical Contusion Assessed by14C-Iodoantipyrine Autoradiography in Mice Using a Non-Invasive Protocol

Although changes of cerebral blood flow (CBF) in and around traumatic contusions are well documented, the role of CBF for the delayed death of neuronal cells in the traumatic penumbra ultimately resulting in secondary contusion expansion remains unclear. The aim of the current study was therefore to investigate the relationship between changes of CBF and progressive peri-contusional cell death following traumatic brain injury (TBI). CBF and contusion size were measured in C57Bl6 mice under continuous on-line monitoring of (ETp)CO2 before, and at 15 min and 24 h following controlled cortical impact by 14C-iodoantipyrine autoradiography (IAP-AR; n = 5-6 per group) and by Nissl staining, respectively. Contused and ischemic (CBF < 10%) tissue volumes were calculated and compared over time. Cortical CBF in not injured mice varied between 69 and 93 mL/100mg/min depending on the anatomical location. Fifteen minutes after trauma, CBF decreased in the whole brain by approximately 50% (39 +/- 18 mL/100mg/min; p < 0.05), except in contused tissue where it fell by more than 90% (3 +/- 2 mL/100mg/min; p < 0.001). Within 24 h after TBI, CBF recovered to normal values in all brain areas except the contusion where it remained reduced by more than 90% (p < 0.001). Contusion volume expanded from 24.9 to 35.5 mm3 (p < 0.01) from 15 min to 24 h after trauma (+43%), whereas the area of severe ischemia (CBF < 10%) showed only a minimal (+13%) and not significant increase (22.3 to 25.1 mm3). The current data therefore suggest that the delayed secondary expansion of a cortical contusion following traumatic brain injury may not be caused by a reduction of CBF alone.

Evaluation of the apoptosis-related proteins of the BCL-2 family In the traumatic penumbra area of the rat model of cerebral contusion, treated by hyperbaric oxygen therapy: a quantitative immunohistochemical study

The growth and progression of traumatic brain injury (TBI) lesions depend significantly on developments in the traumatic penumbra area, perilesional region, where delayed neuronal death occurs. Recent data supports the important role of apoptosis in delayed cell death in TBI. Previously we demonstrated a significant reduction of apoptosis in traumatic penumbra in animals treated by hyperbaric oxygen (HBO). In this study we evaluate the expression of apoptosis-related proteins of the Bcl-2 family (Bcl-2, Bax and Bcl-xL) in the traumatic penumbra area in correlation with the extent of apoptosis in the rat model of focal cerebral contusion, treated by HBO. Sprague-Dawley rats underwent cortical dynamic deformation, some with subsequent hypoxemia. A group of both hypoxemic and non-hypoxemic animals was treated by HBO. The pathological study was based on immunohistochemical staining of the brain sections for Bcl-2, Bax and Bcl-xL with quantitative evaluation of staining by image analysis. The expression of Bcl-2 in hypoxemic animals was lower than in non-hypoxemic animals, but a significant increase in Bcl-2 expression was seen in both groups after HBO treatment. Bcl-xL also demonstrated an increase after HBO treatment but less significant. Staining for Bax protein did not demonstrate significant change after treatment. These data correlate well with the reduction of TUNEL-positive cells in traumatic penumbra after HBO treatment. We concluded that the apoptotic mechanisms are important in delayed cell death in TBI and that post-traumatic hypoxemia increases the intensity of apoptosis, probably through a decrease in Bcl-2 and Bcl-xL expression which normally repress apoptosis. The beneficial effect of HBO treatment in our model of brain contusion correlates well with the increased expression of anti-apoptotic proteins (Bcl-2 and Bcl-xL) following treatment and the appropriate decrease in the extent of apoptosis. In light of these results, the usage of HBO is justified as neuroprotective treatment in TBI.

Incidence and mechanisms of cerebral ischemia in early clinical head injury.

Antemortem demonstration of ischemia has proved elusive in head injury because regional CBF reductions may represent hypoperfusion appropriately coupled to hypometabolism. Fifteen patients underwent positron emission tomography within 24 hours of head injury to map cerebral blood flow (CBF), cerebral oxygen metabolism (CMRO2), and oxygen extraction fraction (OEF). We estimated the volume of ischemic brain (IBV) and used the standard deviation of the OEF distribution to estimate the efficiency of coupling between CBF and CMRO2. The IBV in patients was significantly higher than controls (67 +/- 69 vs. 2 +/- 3 mL; P < 0.01). The coexistence of relative ischemia and hyperemia in some patients implies mismatching of perfusion to oxygen use. Whereas the saturation of jugular bulb blood (SjO2) correlated with the IBV (r = 0.8, P < 0.01), SjO2 values of 50% were only achieved at an IBV of 170 +/- 63 mL (mean +/- 95% CI), which equates to 13 +/- 5% of the brain. Increases in IBV correlated with a poor Glasgow Outcome Score 6 months after injury (rho = -0.6, P < 0.05). These results suggest significant ischemia within the first day after head injury. The ischemic burden represented by this "traumatic penumbra" is poorly detected by bedside clinical monitors and has significant associations with outcome.

Release of excitatory amino acids in the penumbra of a focal cortical necrosis.

A cortical tissue necrosis from focal trauma expands between 30% and 300% from its initial size within 24 h, depending on the species studied. To shed light on the pathophysiological processes in the penumbra 1 zone after a focal cortical lesion, the release of excitatory amino acids into the traumatic penumbra zone 1 was measured throughout the entire period of necrosis expansion. A microdialysis probe was inserted at an oblique angle into the cortex of Sprague-Dawley rats 2 mm below the brain surface. One day later, a highly standardized cortical freezing lesion was induced at the brain cortex above the microdialysis probe. Dialysate was continuously collected prior to, during, and up to 24 h after trauma and analyzed for primary amino acids. In each animal, it was confirmed histologically that the tip of the microdialysis probe was localized in the gray matter in close proximity to the primary lesion. Following induction of the trauma, a statistically significant sharp increase of the dialysate level of aspartate, glutamate, glycine, and serine was observed. Thereafter, the dialysate levels of these amino acids returned to baseline levels without any further increase throughout the remaining observation period. This process ranged in time from a few minutes to a few hours. The level of alanine in the dialysate was essentially not altered throughout the experiment. Although the early post-traumatic increase of the excitatory neurotransmitters aspartate and glutamate may well contribute to the secondary lesion growth of a cortical necrosis after trauma, glutamate receptor targeted therapeutic intervention may be in view of these findings of limited use when initiated post trauma.

Current aspects of pathophysiology and cell dysfunction after severe head injury.

Traumatic brain injury is a major health problem in all developed countries. The main aim of this review is to provide a short update on the most recent advances in our knowledge of the brains response to mechanical injuries, focusing on metabolic, cellular, subcellular, and molecular events that take place in severe head injuries. Knowledge of these events is essential for a better understanding of new pharmacological avenues and non-pharmacological strategies, such as moderate hypothermia, which are being developed to improve the outcome of this silent epidemic. We will focus on several topics that we consider to be the most significant: diffuse axonal injury, ischemia and the cascades it generates, metabolic derangements, excitotoxicity, oxidative stress, and other phenomena that have been included in the term tertiary injuries. Recent evidence has clearly demonstrated that traumatic brain lesions are highly dynamic and that the different lesions observed after closed head injury are not single events but processes set in motion by the mechanical impact. These processes are not finished until an unpredictable time after injury. We will discuss recent evidence showing that in diffuse axonal injury, primary immediate damage can coexist with axons that, although initially intact, may be evolving towards secondary disconnection. The concept of ischemic penumbra and the more recent concept of traumatic penumbra are discussed, together with recent experimental and clinical data that shed light on the non-ischemic forms of brain hypoxia. The role of excitotoxicity in mechanically-induced cell death and the molecular events that excessive release of glutamate induce, including apoptosis and delayed inflammatory processes, are reviewed. Finally, new knowledge on how central nervous system cells regulate their volume, the new family of channel water molecules known as aquaporins and their possible role in the physiopathology of the swollen brain are discussed. Basic and clinical investigations are still needed to translate the huge amount of pathophysiological knowledge acquired in the last decade into effective treatments for these patients.

The penumbra zone of a traumatic cortical lesion: a microdialysis study of excitatory amino acid release.

A cortical tissue necrosis from focal trauma expands to 150% of its initial volume within 24 hrs. It is currently unknown, whether this phenomenon is part of the primary traumatic lesion or if it involves secondary mechanisms such as the release of excitatory amino acids into the traumatic penumbra zone. A microdialysis probe was inserted for that purpose in an oblique angle into the cortex of Sprague-Dawley rats, approximately 2 mm below the brain surface. One day later a highly standardized cortical freezing lesion was induced at the brain cortex above the microdialysis probe. Dialysate was collected prior to, during, and after trauma in 10 min intervals. In each animal, it was confirmed histologically, that the tip of the microdialysis probe was localized in the grey matter in close vicinity to the primary lesion. Following induction of the trauma a statistically significant increase of the dialysate level of aspartate, glutamate, glycine, and serine was observed, whereas that of alanine was not altered throughout the experiment. The posttraumatic increase of the excitatory neurotransmitters aspartate and glutamate indicates that these amino acids are involved in the secondary lesion growth after trauma. Confirmation of this hypothesis would require that specific antagonization of these excitotoxic amino acids is inhibiting growth of the lesion.

PIAL CIRCULATION AND SPREADING DEPRESSION OF ACTIVITY IN THE CEREBRAL CORTEX

PIAL CIRCULATION AND SPREADING DEPRESSION OF ACTIVITY IN THE CEREBRAL CORTEX