Accepted Manuscript Title: Cortical Spreading Depression in Traumatic Brain Injuries: is there a role for astrocytes? Author: Daniel Torrente Ricardo Cabezas Marco Fidel Avila Luis Miguel Garc´ıa-Segura George E. Barreto Rubem Carlos Ara´ujo Guedes PII: DOI: Reference:
S0304-3940(13)01139-7 http://dx.doi.org/doi:10.1016/j.neulet.2013.12.058 NSL 30293
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
11-10-2013 21-12-2013 23-12-2013
Please cite this article as: D. Torrente, R. Cabezas, M.F. Avila, L.M. Garc´iaSegura, G.E. Barreto, R.C.A. Guedes, Cortical Spreading Depression in Traumatic Brain Injuries: is there a role for astrocytes?, Neuroscience Letters (2014), http://dx.doi.org/10.1016/j.neulet.2013.12.058 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cortical Spreading Depression in Traumatic Brain Injuries: is there a role for
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astrocytes?
Daniel Torrente1, Ricardo Cabezas1, Marco Fidel Avila1, Luis Miguel García-Segura2,
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Ph.D., George E. Barreto1, Ph.D., Rubem Carlos Araújo Guedes3 *, Ph.D.
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1. Departamento de Nutrición y Bioquímica, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá D.C., Colombia. 2. Instituto Cajal, CSIC, Avenida Doctor Arce 37, 28002, Madrid, Spain
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3. Departamento de Nutrição, Centro de Ciências da Saúde, Universidade Federal de Pernambuco, Recife-PE, Brazil.
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* Corresponding author: Rubem Carlos Araujo Guedes. Departamento de Nutrição, Centro de Ciencias da Saúde, Universidade Federal de Pernambuco, Rua Professor Moraes Rego,
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S/N Cidade Universitária, 50670-901 Recife, PE, Brazil Email: [email protected]
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Abstract Cortical spreading depression (CSD) is a presumably pathophysiological phenomenon that
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interrupts local cortical function for periods of minutes to hours. This phenomenon is important due to its association with different neurological disorders such as migraine,
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malignant stroke and traumatic brain injury (TBI). Glial cells, especially astrocytes, play an important role in the regulation of CSD and in the protection of neurons under brain
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trauma. The correlation of TBI with CSD and the astrocytic function under these conditions
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remain unclear. This review discusses the possible link of TBI and CSD and its implication for neuronal survival. Additionally, we highlight the importance of astrocytic function for
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brain protection, and suggest possible therapeutic strategies targeting astrocytes to improve the outcome following TBI-associated CSD.
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Keyword: TBI, CSD, Astrocytes, Neurons.
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1. Introduction Cortical spreading depression (CSD) is a pathophysiological phenomenon characterized by
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a depolarization wave in the brain cells [1,2] interrupting local cortical function for periods of minutes to hours [3,4]. It has been established that the CSD phenomenon only occurs in
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the Central Nervous System (CNS) and involves neurons and glial cells [5]. The rate of propagation through the cerebral cortex is approximately 1–5mm/min [6]. For many years
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it was believed that CSD was an artifact without importance for human neurological
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conditions. This has changed due to the clinical relevance of this phenomenon in different neurological disorders such as migraine, malignant stroke and traumatic brain injury (TBI)
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[7].
Neurons play an important role in the propagation of the wave and previous studies focused
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in elucidating the neuronal triggering mechanism of CSD and its implication on
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neurological disorders [8-9]. Nevertheless, glial cells, especially astrocytes, are essential
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during the treatment of neurological disorders. In this aspect, recent studies demonstrate the importance of astrocytic functions for the CSD propagation wave and protection of neuronal population [10-12].
The relation between astrocytic functions and CSD pathologies related to brain trauma, such as TBI, is not well known. In this review, we discuss the role of astrocytes in the CSD propagation and the relevance of targeting astrocytes in treatments related to CSD pathologies such as TBI and others.
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2. Astrocyte involvement in CSD pathology Glial cells, especially astrocytes, play a major role in the CNS mainly supporting neurons
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population by providing antioxidant protection, substrates for neuronal metabolism, and glutamate clearance [13, 14]. The connection between neuron and astrocytes is well known
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and has gained growing importance [15,16]. Nevertheless, the astrocytic functions in CSD under pathologies, such as TBI, might elucidate mechanisms of CSD and discover potential
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therapeutic targets in brain trauma.
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Astrocytes are known to help in the regulation of excitatory amino acid levels, energy metabolism, ionic balance and regulation of oxidative stress in neurons under normal and
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pathological conditions [16-18]. Moreover, astrocytes in CSD limit the activity of the propagating wave. For example, CSD appears more difficult to evoke in brains with higher
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ratios of glia cells to neurons [19 24]. This might be due to that during CSD, K+ and other
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ions are released into the extracellular space and astrocytes may play an important role
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regulating the capture of K+. Some mechanisms of how astrocytes could regulate K+ from the extracellular space include active transport via Na+/K+-ATPase, H+/K+ exchange and inwardly rectifying K+ channels [20,21]. In addition, glutamate is also released into the extracellular space during CSD leading to excitotoxicity [22]. In this context, if astrocytes are not able to regulate K+ and glutamate uptake, the accumulation into the extracellular compartment may amplify CSD propagation and subsequently induce neuronal damage [23]. This mechanism is more evident during energy failure in astrocytes using selective Krebs cycle inhibitors, such as fluorocitrate, and thereafter exposing the brain to a CSD wave. Neuronal damage is increased in energy deprived brain areas compared to those with normal energy supply in astrocytes [24].
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It is proposed that after TBI depolarization waves such as CSD could exacerbate brain damage. This is due to the CSD association with metabolic changes in brain activity similar to those that occur in TBI [7].These changes include cerebral blood flow modifications,
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protein synthesis modifications and increase in glucose, ATP and O2 consumption [7,25,26]. Energy failure due to mitochondrial impairment in TBI could be related to an
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increase in energy consumption in CSD [27]. For example, it is known that after a CSD
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episode, glucose consumption increases in the affected brain area in order to regulate the ionic misbalance created by the depolarization wave [28]. These facts suggest that
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astrocytes are an important checkpoint for the regulation of TBI and CSD outcome in
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neurons.
3. Protecting astrocytic function for neuronal survival in CSD pathologies
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A physiological astrocytes-neuronal crosstalk is an important asset for brain normal
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functioning [29]. Some of these functions include ionic buffer, energy support, excitatory
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amino acid uptake, cerebral blood flow control and reactive oxygen species (ROS) management [30]. The proper functions of these tasks are essential for neuronal survival under stress conditions. Both in TBI and CSD-induced astrocytic dysfunction could lead to neuronal death [24,31]. In the following paragraphs we discuss the possible astrocytes functions that may play a key role in CSD pathophysiology, especially in a TBI paradigm that might affect brain´s recovery and reduce neurodegeneration.
3.1 Energy support
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Astrocytes are the most important cell type that can metabolize glucose in the brain, as they give support to neurons by reducing glucose to lactate and releasing the lactate to the extracellular space [39]. Nevertheless, astrocytes need glucose from the blood to produce
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ATP and fulfill its functions. During states of energy impairment, astrocytes develop some strategies to keep ATP production. Some of these strategies include glucose transporters
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upregulation, glycogen breakdown and oxidation of metabolic intermediates such as lactate,
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pyruvate, glutamate or acetate [33,34] (Fig. 1A) in order to avoid mitochondrial impairment, which is a common early event that could induce cell death [27]. Not only
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energy withdrawal is observed in a TBI event, but also a reduction in oxygen supply in the brain damaged area [35]. It is known that oxygen and glucose deprivation (OGD) in the
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substantia nigra evokes spreading depression [36], suggesting a high probability of evoking
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CSD following TBI. Moreover, it was recently shown that CSD increased both oxygen and
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energy consumption in rat frontal cortex [37], demonstrating that, besides energy depletion caused by TBI, both mechanisms could lead to cell death due to metabolic impairment. The
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importance of astrocytes in this metabolic condition is observed when astrocytic metabolic dysfunction induces permanent damage to neuronal population [24]. More importantly, astrocytes under glucose starvation condition have shown protective mechanisms to the normal neuronal function [38]. In this regard, some clinical strategies exist to maintain the ATP levels in the CNS [39-42], and these methods are mainly focused on external energy supply and reduction of metabolic rate. For example, clinically induced hypothermia demonstrates improvement in neurological outcomes following cardiac arrest [39] and is widely accepted as a standard method by which the body can protect the brain [41]. Also, the use of glyceryl triacetate (GTA) and lactate benefit neuronal outcome, although the GTA is more effective than lactate treating TBI [40]. Finally, Shao et al (2010) have
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suggested the importance of protecting mitochondrial functions in the brain as a successful strategy against pathologies like TBI, including the maintenance of mitochondrial
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membrane potential and decreasing ROS production [42].
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3.2 Excitatory amino acid uptake
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The uptake of the excitatory amino acid glutamate is the most important function of astrocytes [16]. Glutamate clearance occurs by the conversion of glutamate to glutamine by
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the enzyme glutamine synthetase [43] (Fig. 1B). In CSD glutamate triggers this
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phenomenon and glutamine, a precursor of glutamate, increases CSD wave propagation [44,45]. Furthermore, it is known that excitatory amino acid transporters (EAAT) play a
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key role in the clearance of glutamate. In this aspect, the anti-sense knock-down of
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glutamate transporter GLT-1 in mice leads to increased glutamate levels and posterior neuronal and mice death [46,47]. There are 5 types of the EAAT in mammals (EAAT1-5).
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The EAAT1 and EAAT2 are primarily, but not exclusive, expressed on astrocytes [46,47]. In the brain, the astrocytic EAAT2 has been estimated to be responsible for near 90% of the total glutamate uptake [48,49]. This suggests that the manipulation of the EAAT2 represent a potential therapeutic target in pathologies related with excitotoxicity, as this can be considered as the main pathologic feature in TBI and CSD. Currently, there are drug trials for increasing the expression, affinity or activity of this group of transporters including ceftriaxone [49], Riluzole [50] and Parawixin1 [51,52]. However, further research is needed to evaluate their effects in TBI and CSD. Moreover, combinations of treatments using these drugs may have a synergetic effect in these pathologies. For example, the
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combined use of ceftriaxone and parawixin1 might increase both the expression and activity of the EAAT2 and could be a promising strategy in the reduction of the
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excitotoxiticy caused by CSD and TBI.
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3.3 Ionic buffering
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In TBI-CSD association there is a massive disturbance of the ionic homeostasis balance, characterized by an increase in the extracellular K+ and the intracellular Na+ and Ca+ [53-
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55]. This change in ionic balance in neuron populations could lead to an excessive release
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of excitatory amino acids and death due to excitotoxiticy. On the other hand, astrocytes present some mechanisms that could maintain the balance of this ionic homeostasis
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especially of K+, thus protecting neurons. These mechanisms include K+ uptake from the
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extracellular space by the Na+ /K+ pump, the inwardly rectifying potassium (IKR) channels, responsible for regulation of baseline K+ and H+/K+ exchange through the hydrogen
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potassium ATPase. Of these 3 mechanisms, the Na+ /K+ pump is the most important for the regulation of K+ [56,57]. It has been shown that the Na+ /K+ pump determines the rate of K+ recovery following excessive neuronal release by regulating the changes in intracellular Na+ [56] (Fig 1C). In this aspect, this pump must be accompanied by an increase in intracellular Na+, suggesting that the Na+ /K+ pump could be a suitable target in the K+ and Na+ homeostasis due to the increase of the intracellular Na+ after CSD and TBI.
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3.4 ROS management Under pathological conditions, such as TBI and CSD, oxidative damage might become a
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major source of cell injury due to the multiple pathways that could increase ROS production under stress conditions, including the damage of the cell membrane by lipid
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peroxidation, protein and DNA oxidation and inhibition of the mitochondrial electron-
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transport chain [58,59].
The increase in extracellular glutamate and intracellular Ca2+ leads to self-digesting
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intracellular molecules that involve the overproduction of ROS and activation of cell death signaling pathways through effectors such as caspases 3 and 9 [60]. Furthermore, during
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brain pathologies, the consequent mitochondrial dysfunction becomes a massive source of ROS such as superoxide, peroxide, or hydroxyl radicals [59,60]. In the mitochondria, the
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radical superoxide can be produced by respiratory complexes I and III, both in normal
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conditions and during cell damaging processes [61]. Superoxide, highly unstable, is
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transformed into hydrogen peroxide (H2O2) by the mitochondrial superoxide dismutase 2 (SOD2). As an important antioxidant defense, astrocytes have various enzymes and compounds, such as GSSG-GSH, SODs (1,2 and 3), catalase, glutathione peroxidase and ascorbate [62,63] (Fig. 1D). Recent evidence has shown that neurons cocultured with astrocytes present higher levels of glutathione compared to neurons cultured alone, suggesting that astrocytes may provide supplementary antioxidant defenses to neurons, for example in the form of glutathione precursors [62]. Administration of arginine, a precursor of nitric oxide (NO), which is a potent oxidative radical, increases CSD propagation [64], demonstrating a possible implication of ROS in this pathological mechanism [65]. It is possible that the improvement of antioxidant mechanisms in astrocytes is a powerful
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strategy for reducing cell death during CSD and associated pathologies. In this aspect, different antioxidant mechanisms have been used to control oxidative damage in TBI, including the overexpression of SOD 2 [65], lipid peroxidation inhibitors (U-78517F and
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U-83836E) [66] and paracrine factors of mesenchymal stem cells that decreased the superoxide production [67]. On the other hand, unexpectedly, topical application of 5%
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conditioned medium from adipose tissue-isolated mesenchymal cells increased the velocity
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of wave propagation following KCl-induced CSD. In contrast, 10% conditioned medium was able to reduce the velocity of propagation, suggesting a possible neuroprotective effect
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(Fig. 2). All of these treatments have shown a positive effect for ROS production in
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astrocytes, therefore reflecting an important decrease in cell death. 4. Conclusions
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Astrocytes present an important role in TBI associated with CSD and might be primary
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targets for future investigations in the treatment of TBI, and associated pathologic
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condition, represented by CSD. In this aspect, the improvement of astrocytic functions could be a protecting strategy in neurological diseases related to CSD by decreasing neuronal damage after these insults.
Acknowledgments
This work was supported in part by grants PUJ IDs 4327, 5024 and 4367, and PROLAB IBRO/LARC/CNPq grant to GEB, and by the Brazilian Agencies CNPq (INCT de Neurociencia Translacional No. 573604/2008-8), MS/SCTIE/DECIT (No. 17/2006), and IBN-Net/CNPq. RCAG is Research Fellow from CNPq (no. 301190/2010-0).
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Figures Legends Fig. 1. Mechanisms of protection in astrocytes under pathological condition. A. Main
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metabolic pathways for energy production in astrocytes, and ATP-related production in different mechanisms of protection. Glucose is the main substrate for astrocytes and could
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be used for ATP production in Krebs cycle or metabolized to lactate for neuron consumption. Different elements could decrease or inhibit energy production witch ROS
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from mitochondrial impairment seems to be an important one. B. Excitatory amino acids
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are recycled in astrocytes by the EAAT2. This transport is fundamental for a reduction of excitotoxicity. Additionally, astrocytes carry out the glutamate-glutamine cycle for the
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reuse of glutamine by neurons. This mechanism is highly ATP dependent. C. Membrane proteins associated with the regulation of ion misbalance generated by TBI and CSD. The
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reestablishment of ion might be generated by the Na+ /K+ pump that depends on ATP
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consumption (Blue, Red and Yellow represent K+, Ca2+ and Na+, respectively). D.
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Mitochondrial impairment is the main source of ROS production. On the other hand, SODs, GPX and Catalase are the key enzymes that regulate the accumulation of free radicals in cell.
Fig. 2. CSD-velocity in adult male rats after topical cortical application of CM-hMSC during the 15 min immediately preceding CSD elicitation with KCl. CM-hMSC was topically applied to a circular area (3 to 4mm diameter) of the parietal cortical surface (recording place) on the intact dura-mater. CM-hMSC was used with the following concentrations (%) 5 and 10; Control: baseline CSD velocities before topical treatment.
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CM: CSD velocities obtained after topical application. Recovery: CSD velocities after cortical removal of the CM-hMSC. Five rats were use in each CM-hMSC concentration (n=5). Data are expressed as mean±SEM; * indicates significant different between Control
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and CM and between Control and Recovery (p
S0304-3940(13)01139-7 http://dx.doi.org/doi:10.1016/j.neulet.2013.12.058 NSL 30293
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
11-10-2013 21-12-2013 23-12-2013
Please cite this article as: D. Torrente, R. Cabezas, M.F. Avila, L.M. Garc´iaSegura, G.E. Barreto, R.C.A. Guedes, Cortical Spreading Depression in Traumatic Brain Injuries: is there a role for astrocytes?, Neuroscience Letters (2014), http://dx.doi.org/10.1016/j.neulet.2013.12.058 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cortical Spreading Depression in Traumatic Brain Injuries: is there a role for
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astrocytes?
Daniel Torrente1, Ricardo Cabezas1, Marco Fidel Avila1, Luis Miguel García-Segura2,
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Ph.D., George E. Barreto1, Ph.D., Rubem Carlos Araújo Guedes3 *, Ph.D.
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1. Departamento de Nutrición y Bioquímica, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá D.C., Colombia. 2. Instituto Cajal, CSIC, Avenida Doctor Arce 37, 28002, Madrid, Spain
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3. Departamento de Nutrição, Centro de Ciências da Saúde, Universidade Federal de Pernambuco, Recife-PE, Brazil.
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* Corresponding author: Rubem Carlos Araujo Guedes. Departamento de Nutrição, Centro de Ciencias da Saúde, Universidade Federal de Pernambuco, Rua Professor Moraes Rego,
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S/N Cidade Universitária, 50670-901 Recife, PE, Brazil Email: [email protected]
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Abstract Cortical spreading depression (CSD) is a presumably pathophysiological phenomenon that
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interrupts local cortical function for periods of minutes to hours. This phenomenon is important due to its association with different neurological disorders such as migraine,
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malignant stroke and traumatic brain injury (TBI). Glial cells, especially astrocytes, play an important role in the regulation of CSD and in the protection of neurons under brain
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trauma. The correlation of TBI with CSD and the astrocytic function under these conditions
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remain unclear. This review discusses the possible link of TBI and CSD and its implication for neuronal survival. Additionally, we highlight the importance of astrocytic function for
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brain protection, and suggest possible therapeutic strategies targeting astrocytes to improve the outcome following TBI-associated CSD.
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Keyword: TBI, CSD, Astrocytes, Neurons.
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1. Introduction Cortical spreading depression (CSD) is a pathophysiological phenomenon characterized by
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a depolarization wave in the brain cells [1,2] interrupting local cortical function for periods of minutes to hours [3,4]. It has been established that the CSD phenomenon only occurs in
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the Central Nervous System (CNS) and involves neurons and glial cells [5]. The rate of propagation through the cerebral cortex is approximately 1–5mm/min [6]. For many years
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it was believed that CSD was an artifact without importance for human neurological
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conditions. This has changed due to the clinical relevance of this phenomenon in different neurological disorders such as migraine, malignant stroke and traumatic brain injury (TBI)
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[7].
Neurons play an important role in the propagation of the wave and previous studies focused
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in elucidating the neuronal triggering mechanism of CSD and its implication on
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neurological disorders [8-9]. Nevertheless, glial cells, especially astrocytes, are essential
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during the treatment of neurological disorders. In this aspect, recent studies demonstrate the importance of astrocytic functions for the CSD propagation wave and protection of neuronal population [10-12].
The relation between astrocytic functions and CSD pathologies related to brain trauma, such as TBI, is not well known. In this review, we discuss the role of astrocytes in the CSD propagation and the relevance of targeting astrocytes in treatments related to CSD pathologies such as TBI and others.
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2. Astrocyte involvement in CSD pathology Glial cells, especially astrocytes, play a major role in the CNS mainly supporting neurons
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population by providing antioxidant protection, substrates for neuronal metabolism, and glutamate clearance [13, 14]. The connection between neuron and astrocytes is well known
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and has gained growing importance [15,16]. Nevertheless, the astrocytic functions in CSD under pathologies, such as TBI, might elucidate mechanisms of CSD and discover potential
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therapeutic targets in brain trauma.
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Astrocytes are known to help in the regulation of excitatory amino acid levels, energy metabolism, ionic balance and regulation of oxidative stress in neurons under normal and
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pathological conditions [16-18]. Moreover, astrocytes in CSD limit the activity of the propagating wave. For example, CSD appears more difficult to evoke in brains with higher
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ratios of glia cells to neurons [19 24]. This might be due to that during CSD, K+ and other
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ions are released into the extracellular space and astrocytes may play an important role
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regulating the capture of K+. Some mechanisms of how astrocytes could regulate K+ from the extracellular space include active transport via Na+/K+-ATPase, H+/K+ exchange and inwardly rectifying K+ channels [20,21]. In addition, glutamate is also released into the extracellular space during CSD leading to excitotoxicity [22]. In this context, if astrocytes are not able to regulate K+ and glutamate uptake, the accumulation into the extracellular compartment may amplify CSD propagation and subsequently induce neuronal damage [23]. This mechanism is more evident during energy failure in astrocytes using selective Krebs cycle inhibitors, such as fluorocitrate, and thereafter exposing the brain to a CSD wave. Neuronal damage is increased in energy deprived brain areas compared to those with normal energy supply in astrocytes [24].
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It is proposed that after TBI depolarization waves such as CSD could exacerbate brain damage. This is due to the CSD association with metabolic changes in brain activity similar to those that occur in TBI [7].These changes include cerebral blood flow modifications,
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protein synthesis modifications and increase in glucose, ATP and O2 consumption [7,25,26]. Energy failure due to mitochondrial impairment in TBI could be related to an
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increase in energy consumption in CSD [27]. For example, it is known that after a CSD
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episode, glucose consumption increases in the affected brain area in order to regulate the ionic misbalance created by the depolarization wave [28]. These facts suggest that
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astrocytes are an important checkpoint for the regulation of TBI and CSD outcome in
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neurons.
3. Protecting astrocytic function for neuronal survival in CSD pathologies
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A physiological astrocytes-neuronal crosstalk is an important asset for brain normal
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functioning [29]. Some of these functions include ionic buffer, energy support, excitatory
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amino acid uptake, cerebral blood flow control and reactive oxygen species (ROS) management [30]. The proper functions of these tasks are essential for neuronal survival under stress conditions. Both in TBI and CSD-induced astrocytic dysfunction could lead to neuronal death [24,31]. In the following paragraphs we discuss the possible astrocytes functions that may play a key role in CSD pathophysiology, especially in a TBI paradigm that might affect brain´s recovery and reduce neurodegeneration.
3.1 Energy support
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Astrocytes are the most important cell type that can metabolize glucose in the brain, as they give support to neurons by reducing glucose to lactate and releasing the lactate to the extracellular space [39]. Nevertheless, astrocytes need glucose from the blood to produce
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ATP and fulfill its functions. During states of energy impairment, astrocytes develop some strategies to keep ATP production. Some of these strategies include glucose transporters
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upregulation, glycogen breakdown and oxidation of metabolic intermediates such as lactate,
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pyruvate, glutamate or acetate [33,34] (Fig. 1A) in order to avoid mitochondrial impairment, which is a common early event that could induce cell death [27]. Not only
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energy withdrawal is observed in a TBI event, but also a reduction in oxygen supply in the brain damaged area [35]. It is known that oxygen and glucose deprivation (OGD) in the
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substantia nigra evokes spreading depression [36], suggesting a high probability of evoking
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CSD following TBI. Moreover, it was recently shown that CSD increased both oxygen and
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energy consumption in rat frontal cortex [37], demonstrating that, besides energy depletion caused by TBI, both mechanisms could lead to cell death due to metabolic impairment. The
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importance of astrocytes in this metabolic condition is observed when astrocytic metabolic dysfunction induces permanent damage to neuronal population [24]. More importantly, astrocytes under glucose starvation condition have shown protective mechanisms to the normal neuronal function [38]. In this regard, some clinical strategies exist to maintain the ATP levels in the CNS [39-42], and these methods are mainly focused on external energy supply and reduction of metabolic rate. For example, clinically induced hypothermia demonstrates improvement in neurological outcomes following cardiac arrest [39] and is widely accepted as a standard method by which the body can protect the brain [41]. Also, the use of glyceryl triacetate (GTA) and lactate benefit neuronal outcome, although the GTA is more effective than lactate treating TBI [40]. Finally, Shao et al (2010) have
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suggested the importance of protecting mitochondrial functions in the brain as a successful strategy against pathologies like TBI, including the maintenance of mitochondrial
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membrane potential and decreasing ROS production [42].
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3.2 Excitatory amino acid uptake
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The uptake of the excitatory amino acid glutamate is the most important function of astrocytes [16]. Glutamate clearance occurs by the conversion of glutamate to glutamine by
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the enzyme glutamine synthetase [43] (Fig. 1B). In CSD glutamate triggers this
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phenomenon and glutamine, a precursor of glutamate, increases CSD wave propagation [44,45]. Furthermore, it is known that excitatory amino acid transporters (EAAT) play a
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key role in the clearance of glutamate. In this aspect, the anti-sense knock-down of
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glutamate transporter GLT-1 in mice leads to increased glutamate levels and posterior neuronal and mice death [46,47]. There are 5 types of the EAAT in mammals (EAAT1-5).
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The EAAT1 and EAAT2 are primarily, but not exclusive, expressed on astrocytes [46,47]. In the brain, the astrocytic EAAT2 has been estimated to be responsible for near 90% of the total glutamate uptake [48,49]. This suggests that the manipulation of the EAAT2 represent a potential therapeutic target in pathologies related with excitotoxicity, as this can be considered as the main pathologic feature in TBI and CSD. Currently, there are drug trials for increasing the expression, affinity or activity of this group of transporters including ceftriaxone [49], Riluzole [50] and Parawixin1 [51,52]. However, further research is needed to evaluate their effects in TBI and CSD. Moreover, combinations of treatments using these drugs may have a synergetic effect in these pathologies. For example, the
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combined use of ceftriaxone and parawixin1 might increase both the expression and activity of the EAAT2 and could be a promising strategy in the reduction of the
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excitotoxiticy caused by CSD and TBI.
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3.3 Ionic buffering
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In TBI-CSD association there is a massive disturbance of the ionic homeostasis balance, characterized by an increase in the extracellular K+ and the intracellular Na+ and Ca+ [53-
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55]. This change in ionic balance in neuron populations could lead to an excessive release
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of excitatory amino acids and death due to excitotoxiticy. On the other hand, astrocytes present some mechanisms that could maintain the balance of this ionic homeostasis
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especially of K+, thus protecting neurons. These mechanisms include K+ uptake from the
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extracellular space by the Na+ /K+ pump, the inwardly rectifying potassium (IKR) channels, responsible for regulation of baseline K+ and H+/K+ exchange through the hydrogen
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potassium ATPase. Of these 3 mechanisms, the Na+ /K+ pump is the most important for the regulation of K+ [56,57]. It has been shown that the Na+ /K+ pump determines the rate of K+ recovery following excessive neuronal release by regulating the changes in intracellular Na+ [56] (Fig 1C). In this aspect, this pump must be accompanied by an increase in intracellular Na+, suggesting that the Na+ /K+ pump could be a suitable target in the K+ and Na+ homeostasis due to the increase of the intracellular Na+ after CSD and TBI.
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3.4 ROS management Under pathological conditions, such as TBI and CSD, oxidative damage might become a
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major source of cell injury due to the multiple pathways that could increase ROS production under stress conditions, including the damage of the cell membrane by lipid
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peroxidation, protein and DNA oxidation and inhibition of the mitochondrial electron-
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transport chain [58,59].
The increase in extracellular glutamate and intracellular Ca2+ leads to self-digesting
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intracellular molecules that involve the overproduction of ROS and activation of cell death signaling pathways through effectors such as caspases 3 and 9 [60]. Furthermore, during
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brain pathologies, the consequent mitochondrial dysfunction becomes a massive source of ROS such as superoxide, peroxide, or hydroxyl radicals [59,60]. In the mitochondria, the
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radical superoxide can be produced by respiratory complexes I and III, both in normal
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conditions and during cell damaging processes [61]. Superoxide, highly unstable, is
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transformed into hydrogen peroxide (H2O2) by the mitochondrial superoxide dismutase 2 (SOD2). As an important antioxidant defense, astrocytes have various enzymes and compounds, such as GSSG-GSH, SODs (1,2 and 3), catalase, glutathione peroxidase and ascorbate [62,63] (Fig. 1D). Recent evidence has shown that neurons cocultured with astrocytes present higher levels of glutathione compared to neurons cultured alone, suggesting that astrocytes may provide supplementary antioxidant defenses to neurons, for example in the form of glutathione precursors [62]. Administration of arginine, a precursor of nitric oxide (NO), which is a potent oxidative radical, increases CSD propagation [64], demonstrating a possible implication of ROS in this pathological mechanism [65]. It is possible that the improvement of antioxidant mechanisms in astrocytes is a powerful
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strategy for reducing cell death during CSD and associated pathologies. In this aspect, different antioxidant mechanisms have been used to control oxidative damage in TBI, including the overexpression of SOD 2 [65], lipid peroxidation inhibitors (U-78517F and
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U-83836E) [66] and paracrine factors of mesenchymal stem cells that decreased the superoxide production [67]. On the other hand, unexpectedly, topical application of 5%
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conditioned medium from adipose tissue-isolated mesenchymal cells increased the velocity
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of wave propagation following KCl-induced CSD. In contrast, 10% conditioned medium was able to reduce the velocity of propagation, suggesting a possible neuroprotective effect
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(Fig. 2). All of these treatments have shown a positive effect for ROS production in
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astrocytes, therefore reflecting an important decrease in cell death. 4. Conclusions
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Astrocytes present an important role in TBI associated with CSD and might be primary
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targets for future investigations in the treatment of TBI, and associated pathologic
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condition, represented by CSD. In this aspect, the improvement of astrocytic functions could be a protecting strategy in neurological diseases related to CSD by decreasing neuronal damage after these insults.
Acknowledgments
This work was supported in part by grants PUJ IDs 4327, 5024 and 4367, and PROLAB IBRO/LARC/CNPq grant to GEB, and by the Brazilian Agencies CNPq (INCT de Neurociencia Translacional No. 573604/2008-8), MS/SCTIE/DECIT (No. 17/2006), and IBN-Net/CNPq. RCAG is Research Fellow from CNPq (no. 301190/2010-0).
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Figures Legends Fig. 1. Mechanisms of protection in astrocytes under pathological condition. A. Main
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metabolic pathways for energy production in astrocytes, and ATP-related production in different mechanisms of protection. Glucose is the main substrate for astrocytes and could
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be used for ATP production in Krebs cycle or metabolized to lactate for neuron consumption. Different elements could decrease or inhibit energy production witch ROS
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from mitochondrial impairment seems to be an important one. B. Excitatory amino acids
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are recycled in astrocytes by the EAAT2. This transport is fundamental for a reduction of excitotoxicity. Additionally, astrocytes carry out the glutamate-glutamine cycle for the
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reuse of glutamine by neurons. This mechanism is highly ATP dependent. C. Membrane proteins associated with the regulation of ion misbalance generated by TBI and CSD. The
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reestablishment of ion might be generated by the Na+ /K+ pump that depends on ATP
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consumption (Blue, Red and Yellow represent K+, Ca2+ and Na+, respectively). D.
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Mitochondrial impairment is the main source of ROS production. On the other hand, SODs, GPX and Catalase are the key enzymes that regulate the accumulation of free radicals in cell.
Fig. 2. CSD-velocity in adult male rats after topical cortical application of CM-hMSC during the 15 min immediately preceding CSD elicitation with KCl. CM-hMSC was topically applied to a circular area (3 to 4mm diameter) of the parietal cortical surface (recording place) on the intact dura-mater. CM-hMSC was used with the following concentrations (%) 5 and 10; Control: baseline CSD velocities before topical treatment.
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CM: CSD velocities obtained after topical application. Recovery: CSD velocities after cortical removal of the CM-hMSC. Five rats were use in each CM-hMSC concentration (n=5). Data are expressed as mean±SEM; * indicates significant different between Control
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and CM and between Control and Recovery (p
