Brain Injury and Cellular Responses
1. Introduction: Brain Injury and Cellular Responses
Mechanisms causing damage to the central nervous system (CNS) are numerous and complex, ranging from those associated with age-related neurodegeneration to the acute mechanisms of traumatic brain injury (TBI), ischemic stroke, and radiation exposure. In all cases, however, astrocytes play a central role in the compensatory responses that nature has designed to protect against the loss of terminally differentiated, nonreplicating neurons.
Like aging, acute injuries can result in a long-term progression of pathogenic changes that alter brain functions for years afterwards [1]. Specifically, following an initial TBI, secondary events can occur that extend both the area of as well as the intensity of the injury. Loss of vascular integrity resulting in a breakdown of the blood brain barrier (BBB) causes exposure of the CNS to exogenous immune cell types, abnormal levels of cytokines, and other cellular mediators and ionic disruption that can lead to a cascade of pathogenesis [2–7]. Loss of BBB integrity is also observed following ischemic stroke, radiation exposure, and in certain neurodegenerative disorders, due to the loss of neurovascular functions [8–11]. Secondary damage due to vascular and metabolic imbalances leads to increased glutamate release and subsequent excitotoxicity, mitochondrial dysfunction, and excessive production of reactive oxygen species (ROS), as well as disruption of glucose metabolism/release, and further alterations of ion concentrations [12–14]. Glutamate is thought to be a central mediator in this constellation of secondary injury events. An increase of extracellular glutamate activates N-methyl-D-aspartate receptors (NMDARs) in neurons, allowing calcium influx [15]. The resulting calcium excitotoxicity affects mitochondrial functions, causing a disruption of energy balance and production of excessive ROS, ultimately causing acute necrotic cell death and/or delayed apoptotic cell death [15–18]. Further damage can occur due to prolonged neuroinflammatory and related Hindawi immune responses that exacerbate the injury [19, 20] and may underlie long-term pathogenesis.
Although the initiating events of CNS damage may
differ, similar patterns of secondary injuries are observed
[10, 21, 22]. This implies that understanding of the mechanisms
underlying the CNS response to any injury may allow
the development of treatments for other diseases or disorders.
Historically, treatments for acute or chronic damage to
the nervous system have focused on neuronal responses
and survival. This was due to the neurons’ perceived importance
in cognition and their postmitotic status which prevents
their replacement when damaged [23, 24]. However,
more attention is now being paid to the impact of nonneuronal
cell types that function to mitigate damage and promote
neuronal function and repair following tissue injury. In
recent years, there has been a greater appreciation of the role
of astrocytes in brain function and survival. The perceived
value of astrocytes has risen from their initially defined role
of “brain glue” to current findings that astrocytes are critical
for modulating synaptic transmissions, managing energy
metabolism, water, and ion homeostasis, and protection of
neurons from oxidative stress under both mild and catastrophic
conditions [25–29]. Here, we review the role of
astrocytes in the protection of neurons from the consequences
of initial and secondary injury processes (Figure 1).
2. Astrocytes: Origin, Morphology,
and Activation
Astrocytes are members of a larger family of nonneural, glial
cells which include oligodendrocytes and Schwann cells, both
of which form myelin and microglia, which are specialized
macrophages that aid in immunity. Astrocytes and the other
cells of the glial family are defined, in part, by their inability
to produce an action potential upon stimulation [30]. Astrocytes
are embryonically derived from progenitor cells of
neuroepithelium which differentiate to function in their traditional
roles as support cells. They provide nutrients and
remove end products of metabolism [31]. Astrocytes exhibit
spongiform morphology, with processes in close contact with
neuronal synapses and other components of the CNS [32].
Recent advances in our understanding of astrocytes, discussed
below, reveal the astrocyte to have essential roles in
synaptic function and nervous system repair [33, 34].
Astrocytes, the most abundant nonneuronal cell type
in the brain, consist of two main subclasses: protoplasmic
and fibrous [35]. Protoplasmic astrocytes display a stellate
appearance in the grey matter, and fibrous astrocytes primarily
exist as long, thin, fibrocyte-like cells in the white matter
of the CNS [36]. Each subtype has a distinctive profile of gene
expression, as reflected in their expression of specific receptors
and proteins [37, 38]. These two types of astrocytes
display differences in their development and their expression
of receptors and proteins [37, 38]. However, both subtypes
express glial fibrillary acidic protein (GFAP), the main astrocytic
intermediate filament, as well as calcium-binding S100B
protein (S100B) [39, 40].
Activation of astrocytes can occur in response to a variety
of injuries to the brain and in response to inflammation or
pathological neurodegeneration [35]. The activated state,
astrogliosis or reactive astrogliosis, is believed to have multiple
functions in the brain and has been the topic of controversy
for over 20 years [32, 35, 41]. While in some cases,
astrocyte activation has been linked to repair and return to
homeostasis, and in other cases, astrocyte activation has been
related to the formation of scar tissue and the inhibition of
neuronal axon outgrowth [35]. Induction of the reactive state
of astrocytes can occur through multiple mechanisms including
the presence of amyloid beta peptides (Aβ peptides) to
neuronal damage or neurodegeneration, the release of proinflammatory
cytokines by microglia and macrophages, or in
response to acute injury to cells of the CNS [42–44]. The time
course of astrocyte reactivity is heterogeneous and may
depend on the location and type of injury [45]. In certain
murine models of mild CNS injury, astrocyte reactivity is
transient [46]. However, other studies indicate long-lasting
increases in astrocyte reactivity occurring after either moderate
or severe CNS injury from TBI or by radiation [47, 48].
Mild perturbations of the CNS can be adequately repaired,
and homeostasis can be maintained with cooperation among
glial cells. However, under more severe conditions, astrocytes
remain in a state of reactivity indicating an inability to
adequately repair. Similarly, astrocytes in postmortem Alzheimer’s
patients appear to maintain themselves in a continuous
reactive state, consistent with chronic inflammation
observed in this disease [49]. Thus, astrocyte reactivity persistence
may indicate the presence of unresolved dysfunction
in the CNS.
The primary alterations in the transformation of normal
astrocytes to reactive astrocytes include hypertrophy of
their main cellular processes, proliferation, and alterations
in protein expression [32, 50, 51]. Fibrous and protoplasmic
astrocytes display differences in the length of their processes
following mechanical injury. In a murine model of axonal
injury, fibrous astrocytes displayed condensed, retracted processes
[46]. In contrast, protoplasmic astrocytes displayed
increased length and branch complexity of their processes
after injury [32, 52]. This may be a reflection of their
functions within the brain, but more research is required to
understand the significance of these changes. Of greater
interest are their different sensitivities to damage. Research
of brain ischemia and cortical lesions has shown that protoplasmic
astrocytes may either die or differentiate into fibrous
astrocytes after brain injury caused by ischemia and cortical
lesions [52, 53]. This suggests that the differences between
astrocyte types are fluid and dependent on environmental
conditions. Significantly, protoplasmic astrocytes promote
the differentiation of neural stem cell (NSC) into neurons
via their secretion of brain-derived neurotrophic factor
(BDNF) secretion [54]. Also, while both protoplasmic and
fibrous astrocytes aid in motor neuron neurite outgrowth,
protoplasmic astrocytes produced factors in the extracellular
matrix that aided in axonal growth of V2a interneurons,
while extracellular matrix produced by fibrous astrocytes
had more factors that inhibited axon growth of V2a interneurons,
suggesting that the actions of the protoplasmic and
fibrous astrocytes are selective for specific neurons [55].
Thus, the differentiation or death of protoplasmic astrocytes
2 Oxidative Medicine and Cellular Longevity
Glutamate
1
Astrocyte
GLT-1
ROS
2
Antioxidant gene
regulation
Glutathione
precursors
3
Damaged Neuron
mitochondria
Healthy
mitochondria
Glucose
Lactate
Glycogen
store
EAAT2
GLUT1
4
Fe2+
5
T cell
Monocyte
Ferritin
TRPC
DMT1
7 6
Mitophagy
DNA-damaging
events
HR and NHEJ
Nrf2
Nucleus
Nucleus
Cell cycle
pause
O O O OH
CH3
CH2OH
O O HO
H
NH3
− −
+
H H
H
HO
H
H
OH
OH
OH
O
Figure 1: Schematic of mechanisms of neuroprotective effects of astrocytes. There are at least seven distinct mechanisms by which astrocytes
protect neurons from damage. (1) Protection against glutamate toxicity occurs through astrocyte uptake of extracellular glutamate through
the excitatory amino acid transporter 2 (EAAT2) and the glutamate transporter 1 (GLT-1). (2) Protection against redox stress through the
activation of Nrf2 and regulation of antioxidant genes; protection of the neurons is also advanced by the export of glutathione precursors
to help neurons synthesize glutathione. (3) Mediation of mitochondrial repair mechanisms by which astrocytes received damaged
mitochondria from neurons for mitophagy and in return deliver healthy mitochondria to the neurons. (4) Protection against glucoseinduced
metabolic stress, which required astrocytes to take up extracellular glucose for storage as glycogen; the glycogen can be released to
neurons as lactate for their metabolism at a later time. (5) protection against iron toxicity, in which astrocytes take up free iron from the
extracellular space via transient receptor potential canonical (TRPC) channels and divalent metal transporter (DMT1); the iron is then
stored in complex with ferritin. (6) Modulation of the immune response in the brain occurs by astrocyte inhibition of both T cell and
monocyte activation; the mechanisms for these actions are not fully known. (7) Maintenance of tissue homeostasis in the presence of
DNA damage, where astrocytes can effectively repair their DNA through both homologous recombination (HR) or nonhomologous end
joining (NHEJ), following pause of the cell cycle.
Oxidative Medicine and Cellular Longevity 3
may have a significant impact on replenishing neurons and
regrowth of neuronal axons in the CNS following injury
depending upon the site of injury.
Reactive astrocytes perform a variety of tasks in response
to injury which can be beneficial or deleterious to the surrounding
neurons, depending on the circumstances of the
injury. Reactive astrocytes can form scars after CNS trauma.
In some cases, scars can be viewed as initially beneficial since
they limit immune cell invasion, decrease neuroinflammation,
and maintain ion homeostasis in damaged brain tissue
[56, 57]. Ablation of proliferating reactive astrocytes after
moderate closed cortical impact (CCI) in mice produced
increased inflammation and neuronal death, suggesting that
the overall value of astrocyte reactivity is for the protection
of neurons postinjury [58]. Evidence indicates interference
in the development of the astroglial scar results in increased
neuronal cell death and decreased modulation of inflammation
[59]. However, there is controversy over its long-term
impact of the scar tissue on repair and functional recovery
[60, 61]. Prior evidence suggests that glial scar formation prevents
or inhibits axonal regrowth of neurons [62]. This has
been attributed to astrocyte expression of chondroitin sulfate
proteoglycan, a known inhibitor of neuronal axons during
embryogenesis [63]. However, in murine models where
astrocyte scar formation is impaired, there was demonstrated
to be less neuronal axon regrowth and remodeling [64, 65].
Using transgenic murine models, one research group demonstrated
that the formation of an astrocytic scar actually
improved neuron axonal regrowth, provided that brainderived
neurotrophic factor (BDNF) and neurotrophin-3
(NT3) were added [64]. Together, these studies suggest that,
in contrast to initial hypotheses, the presence of astrocytic
scars alone does not prevent axonal regrowth, but rather that
the lack of adequate growth factors may be the problem.
The beneficial nature of gliosis may become detrimental
when damage is too severe for homeostasis to be reestablished.
For the purposes of this review, we will focus mostly
on the mechanisms by which astrocytes protect neurons
under basal conditions and after injury. This will involve
focusing on the astrocyte’s ability to collect and transport
vital nutrients, neurotransmitters, and ions in the brain, to
release antioxidants during redox stress, to repair mitochondria
and DNA after injury.
3. Astrocyte Defense against Glutamate Toxicity
Glutamate is the most abundant excitatory neurotransmitter
in the brain, with actions mediated through a diverse family
of receptors to modulate synaptic transmission and aid in
plasticity [66, 67]. In normal synaptic communication, neurons
release measured quanta of glutamate into the synaptic
cleft. However, following physical trauma, radiation exposure,
and chronic neurodegenerative disorders, including
Alzheimer’s disease, excessive glutamate is released or fails
to be taken up for days after injury [68–71]. The cause of
glutamate dysregulation in TBI and neurodegeneration is
not completely understood, but elevations in free glutamate
are linked to poor clinical outcome [71]. Recent evidence
indicates that glutamate is released by dying or damaged
neurons, possibly via the cystine glutamate antiporter
[72, 73]. Excessive extracellular glutamate leads to excitatory
neuronal cell death attributed to overstimulation of NMDAR
and subsequent overproduction of ROS in neurons [74, 75].
Under conditions of normal neuronal activity, astrocytes
are responsible for the uptake of excess glutamate from the
synaptic cleft. Following uptake, astrocytes process the glutamate
into glutamine and return it to neurons for reuse [76].
Consistent with this role, astrocytes highly express the excitatory
amino acid transporter 2 (EAAT2) and the glutamate
transporter 1 (GLT-1) which are responsible for the active
uptake of glutamate [77]. Glutamate homeostasis is a critical
function of astrocytes in the brain, as demonstrated experimentally
by the neurotoxicity that results from inhibition of
the astrocyte glutamate transporters [78, 79].
Following tissue injury, astrocytes can actively take
up excessive glutamate from the extracellular (nonsynaptic)
space and buffer its potential excitotoxic actions on neurons.
The reduction of extracellular glutamate by astrocytes
decreases the subsequent lesion size, mitigates neuronal
mortality, and improves CNS function postinjury [80].
Under conditions of severe injury, extent of damage and 2
types of injury to the astrocytes themselves can impact the
ability of astrocytes to protect neurons from glutamate toxicity
[81]. For example, astrocytes injured by radiation or more
severe forms of TBI display reduced glutamate uptake
activity as compared to the uninjured condition, allowing
increased neuronal uptake of glutamate and a greater extent
of neuronal cell death and seizure activity [70, 81, 82]. The
mechanism for radiation inhibition of astrocyte uptake of
glutamate is thought to be related to ROS inhibition of the
astrocytic glutamate transporter via oxidation of protein
sulfhydryl groups critical for function [83, 84]. At least three
potential mechanisms have been proposed for increased
extracellular glutamate expression and subsequent excitotoxicity
in TBI [85, 86]. These mechanisms may occur in
tandem and are not exclusive. In the first potential mechanism,
tumor necrosis factor-α (TNF-α), the proinflammatory
factor released during brain damage, downregulates glutamate
uptake by astrocytes and suppresses conversion of
glutamate to glutamine [87]. In the second possible mechanism,
TBI- and ischemia-induced efflux of glutamate from
injured astrocytes may occur in response to thrombin, which
is released after BBB disruption [88]. In a third potential
mechanism, ischemia and glucose deprivation may induce
altered glutamate release by astrocytes [89]. Under normal
circumstances, glutamate uptake occurs against its gradient
and must be actively transported into the astrocyte via
EAATs. However, under acidic conditions, this transporter
is reversed and expels glutamate [89]. Thus, more severe
neuronal injuries and/or chronic disruptions lead to cell
death when the astrocytes themselves exacerbate glutamate
imbalance as they fail to maintain homeostasis.
4. Redox Stress Reduction by Astrocytes
Basal levels of ROS in the brain can result from normal cellular
functions and metabolic activity. While the production of
ROS is a natural consequence of mitochondrial respiration,
4 Oxidative Medicine and Cellular Longevity
overproduction of ROS following injury exceeds the capacity
of natural cellular antioxidant mechanisms, resulting in the
pathological modification of proteins, lipids, and nucleic
acids [90–93]. To combat these processes, the brain utilizes
multiple pathways for antioxidant defense including superoxide
dismutases (SOD), catalases and glutathione detoxification
pathways, and thioredoxin detoxification pathways
[94]. These mechanisms are utilized to different degrees by
different cell types.
A hallmark of glutamate excitotoxicity is increased
intracellular redox stress. Excessive glutamate activation of
NMDAR causes Ca2+ influx into the cytosol of neurons
[95]. The excessive intracellular Ca2+ can translocate into
the mitochondrial matrix where it leads to the collapse of
mitochondrial membrane potential with loss of ATP production
and, ultimately, cell death [22, 74]. To prevent
this, many cell types upregulate uncoupling proteins (UCPs),
which aid in removal of intracellular Ca2+ and prevention of
Ca2+ entry into the mitochondria [96, 97]. UCPs decrease the
levels of hydrogen protons in the mitochondrial intermembrane
space and therefore the mitochondrial electrochemical
proton gradient, by leaking them into the mitochondrial
matrix [98, 99]. Since the electrochemical proton gradient is
necessary for ATP synthase function, a decrease in hydrogen
protons decreases ATP production [100]. The increase of
hydrogen protons in the mitochondrial matrix also causes
diminished entry of positively charged molecular calcium
[101]. In the short term, the activity of UCP may benefit
the neurons for immediate survival, but in the long term, it
is detrimental, since this process inhibits ATP production
[102, 103]. Catastrophic calcium entry due to acute or
chronic brain injury can overcome the UCP system, leading
to the production of ROS which causes further mitochondrial
dysfunction and cell death [22, 104–106]. This mitochondrial
membrane depolarization and increase in ROS induced by
high Ca2+ levels can cause apoptosis by facilitating the release
of cytochrome C through the mitochondrial transition pore
and activation of caspase 3 [107, 108].
Astrocytes normally display a higher basal level of
glutathione (0.91 ± 0.08mM) as compared to neurons
(0.21 ± 0.02 mM), suggesting that under normal conditions,
they are capable of detoxification of higher amounts of reactive
oxygen and nitrogen species [109, 110]. Astrocytes also
have a greater inducible expression of glutathione in response
to oxidative stress [111, 112]. The ROS-inducible transcription
factor nuclear factor E2-related factor 2 (Nrf2) regulates
the glutathione system, as well as the thioredoxin system and
SOD [113–115]. Under basal conditions, Nrf2 is constitutively
produced and ubiquitinated for degradation by binding
to the Kelch-like ECH-associated protein 1 (Keap1) in the
cytoplasm [116]. Under conditions of increased oxidative
stress, Keap1 binding to Nrf2 is inhibited [117], allowing
Nrf2 to escape degradation and instead to translocate to
the nucleus where it interacts with the antioxidant
response element (ARE) in gene promoters that activate
the expression of oxidative stress response genes. Previous
research indicated that astrocytes display higher basal and
stimulated levels of ARE binding by NRF2 as compared to
neurons [118].
Interestingly, Nrf2-induced expression and downstream
upregulation of antioxidant defenses in astrocytes confer
enhanced resistance to oxidative stress for both astrocytes
and neurons [119, 120]. As stated above, the enhanced Nrf2
within astrocytes effectively upregulates antioxidant genes
for the protection of the astrocytes [121]. However, Nrf2
expression in astrocytes was also demonstrated to increase
neuronal survival in a murine model of amyotrophic lateral
sclerosis (ALS) and in vitro in acute hydrogen peroxide exposure
[122, 123]. The mechanism by which Nrf2 upregulation
in astrocytes allows protection of neurons is complex, and
further research is required for a full understanding. However,
two mechanisms have been proposed for astrocyte
protection of neurons in response to ROS. In the first mechanism,
Nrf2 induces glutathione secretion from astrocytes
into the extracellular matrix where it is cleaved to one of its
precursors (CysGly, γGluCys, or cysteine) which are then
taken up and used by neurons for glutathione resynthesis
for their own detoxification [21, 124, 125]. In the second
mechanism, the increased levels of Nrf2 induce the upregulation
of the EAAT3 in astrocytes. As described above,
this neurotransmitter transporter is critical for the removal
of extracellular glutamate which after injury can induce
neuronal excitotoxicity. Thus, the removal of extracellular
glutamate protects neurons via a second independent
mechanism [126]. This redox buffering capacity of astrocytes
was demonstrated to be necessary for neuronal homeostasis
under normal basal conditions [127].
5. Astrocyte Defense against Mitochondrial
Dysfunction in Neurons
As describe in Section 4, brain injury can lead to Ca2+-
induced mitochondrial dysfunction, including overproduction
of ROS, loss of mitochondrial membrane potential and
pH gradient, and failure to generate required amounts of
ATP [128]. Recently, the transfer of mitochondria from one
cell type to another has been described as a mechanism for
the replacement and repair of damaged mitochondria. The
benefits of mitochondrial transfer were initially shown in
cell culture studies in which human mesenchymal stem
cells (hMSC) repaired the aerobic respiration of A549-
transformed lung epithelial cells that contained mutated
mitochondria [129]. Mutant A549 cells which received mitochondria
from hMSCs displayed improved ATP production,
increased lactate uptake, and higher levels of oxygen consumption,
a marker of electron transport chain activity
[129]. This study provided compelling evidence for mitochondrial
transfer and demonstrated the benefits of this
activity as an effective means for protecting vulnerable cell
types. The mechanisms by which mitochondria and other
organelles are trafficked between different cell types are still
not well understood. One proposed mechanism for organelle
transfer involves the creation of tunneling nanotubes (TNTs)
[130, 131]. TNTs are created by a cell after it is subjected to
stress and has been demonstrated to occur during neuronal
development [130, 132]. Of special interest, neurons are
capable of guiding the formation of astrocyte TNTs during
periods of high synaptic activity and thus, high energy
Oxidative Medicine and Cellular Longevity 5
demand [132]. Transference of healthy mitochondria from
astrocytes to neurons in a murine model of stroke was
observed in vivo [133]. Further, it was noted in this model
that astrocytes only transferred healthy mitochondria to
damaged neurons in a calcium-dependent manner, suggesting
neuronal activity was necessary for transference [133].
Conversely, in a separate model, it was demonstrated that
retinal ganglion cells are capable of shedding damaged mitochondria
and that the shed mitochondria were shown to be
taken up by adjacent astrocytes where they underwent mitophagy
[134]. Thus, evidence suggests that mitochondrial
transfer provides means to deliver healthy mitochondria
to injured neurons and for the elimination of damaged
mitochondria involved in the overproduction of ROS.
6. Astrocyte Protection against Glucose-Induced
Metabolic Stress
The brain is highly metabolically active, utilizing fully 25% of
the body’s glucose [28]. Accordingly, efficient glucose uptake
and distribution throughout the brain is critical for cognition
and survival. Disruptions in the delivery of glucose to the
brain induce neuronal cell death. Under normal conditions,
the BBB acts as a selective barrier to control entry of glucose
into the brain; however, this barrier is often disrupted in
brain injury [135]. Endothelial cells of the BBB and astrocytes
express glucose transporter 1 (GLUT1), a facilitated glucose
transporter, to aid in glucose entry into the brain [136].
Astrocytic endfeet encircles endothelial cells of the blood
brain barrier and mediates the uptake of glucose [137–139].
Once past the BBB, glucose is taken up by all cell types of
the CNS. In astrocytes, glucose is converted into glycogen
and stored [140]. In times of need, astrocytes mobilize
their glycogen to make lactate available for neuronal use.
This is especially important when energy demand is high
but neuronal glucose supply is low, such as under hypoglycemic
conditions [141–143]. While neurons express glucose
transporter 3 (GLUT3), a high affinity glucose transporter,
they have been shown to prefer lactate as an energy substrate
during times of high synaptic activity [144–146]. Glutamate
induces the rapid uptake of glucose in astrocytes. Because
extracellular glutamate is released during neurotransmission,
this indicates that glutamate-stimulated glycogen production
in astrocytes is linked to neuronal activity [147, 148].
Insulin and insulin-like growth factor 1 (IGF-1) increase
glycogen storage in many cell types of the body but have no
impact on astrocytes, since they do not express the insulin/
IGF-1-responsive glucose transporter 4 (GLUT4) which is
expressed primarily in adipose and striated muscle [149].
However, selective ablation of insulin receptors in mouse
astrocytes in vivo results in a significantly lower cerebral glucose
levels, suggesting that astrocytes are also responsive to
metabolic conditions in the rest of the body [150]. This
indicates a central role for astrocytes in monitoring neuronal
metabolic activity and maintaining whole brain energy
balance in a manner that is responsive to insulin release in
the blood, but in a manner that is different from the regulation
that occurs in other tissues.
Acute brain damage, including radiation, TBI, and ischemic
stroke, can produce sudden damage to the BBB which
can lead to a disruption in the supply of glucose as well as
imbalances in extracellular ions. Of particular importance
in BBB permeability is increased extracellular potassium that
must be removed from the extracellular space [151, 152]. The
increase in extracellular potassium may be due to multiple
factors including direct cellular injury and secondary mechanisms
that compromise potassium buffering by astrocytes
[153–155]. While glial cells are capable of buffering normal
increases in extracellular potassium, they become overwhelmed
under conditions of more severe injuries and the
potassium overload can cause death of neurons [155, 156].
Both initial disruption of the BBB and the need to maintain
ion homeostasis produce a rapid depletion of glucose and
metabolic emergency [151, 152, 157]. Hypo- and hyperglycemic
conditions both induce greater cell death in neurons
than astrocytes [158–160]. Astrocyte survival in hypoglycemic
conditions may rely on several factors including
glycogen storage within the astrocytes, alternative energy
metabolism of fatty acids, and utilization of antioxidant
systems to manage increased oxidative stress [161–163].
In vitro research also demonstrates that astrocytes can
improve neuronal survival under situations of glucose disruption
by upregulating their respective monocarboxylate
transporters (MTCs) which transfers lactate from astrocytes
to neurons [164, 165].
While astrocytes may increase their release of lactate after
TBI, there is some controversy regarding the possible benefit
of this release, as neurons appear less capable of taking up the
lactate depending on their level of damage [166, 167].
Increased release of lactate by astrocytes may contribute to
lactic acidosis which can exacerbate ischemia-induced oxidative
stress [168]. High lactate levels in the cerebrospinal fluid
(CSF) of TBI patients have been linked worse clinical outcomes,
which is blamed on neuronal mitochondrial dysfunctions,
neuronal inability to uptake lactate, and subsequent
necrosis in the brain [169]. Increased lactate was also seen
in patients after they had seizures caused by severe TBI, with
astrocytes potentially releasing lactate as an energy source for
these overactive neurons [170, 171]. Under normal homeostasis
and conditions of mild-to-moderate injury, astrocytes
act to maintain neuronal survival by providing energy
resources and maintaining the energy balance of the extracellular
environment of the brain, but these actions can produce
further damage if the CNS is already severely compromised.
7. Astrocyte Mitigation of Iron Toxicity
Astrocytes are responsible for the transfer through the
BBB of a variety of nutrients required for brain tissue
homeostasis, including iron [172]. Iron performs multiple
functions within the brain, serving as an essential cofactor
in several enzymatic reactions including those involved
in the remyelination of neurons after injury [173, 174].
Iron levels are tightly regulated in the brain via specific
transport proteins and metabolic pathways, but dysregulation
can occur under pathological conditions [175].
Iron deficiency in the brain, due to causes such as dietary
6 Oxidative Medicine and Cellular Longevity
insufficiency or anemia, can produce cognitive impairments
[176, 177]. However, an excess of iron, due to TBI,
hemorrhagic stroke, or neurodegenerative diseases, causes
neurotoxicity [175, 177, 178].
When present at high levels, ferrous iron (Fe2+) interacts
with hydrogen peroxide to generate toxic levels of hydroxyl
radicals through the Fenton reaction [179]. Neuronal susceptibility
to iron-mediated necrotic, apoptotic, and autophagic
cell death is likely due to their inability to effectively buffer
the resulting ROS to combat redox stress [180]. This is in
marked contradiction to astrocytes which are highly effective
at detoxification of ROS [181, 182, 183]. Excess iron induces
lipid peroxidation, protein and DNA oxidation, and cell
death in neurons [175, 184]. Disruptions in free iron handling
within the CNS have been observed after acute injuries
such as TBI as well as in chronic neurodegenerative disorders
[185, 186]. Iron and other transition metals within the brain
bind to Aβ a peptide that accumulates in Alzheimer’s disease,
causing greater neuronal death and toxicity than Aβ alone
[187, 188]. Similarly, in a murine model, it was demonstrated
that TBI results in an increase in iron deposition in the brain
starting as early as four hours postinjury and extending for at
least three weeks after initial damage [189]. These findings
support the proposal that acute deregulation of iron homeostasis
may participate in long-lasting pathogenic effects that
underly neuronal damage and death [185, 190] with associated
cognitive impairment.
Astrocytes utilize several distinctly different mechanisms
to directly regulate free iron in the CNS. As discussed above,
astrocytes utilize parallel mechanisms including increased
expression of Nrf2, glutathione, and catalase to combat redox
stress that is likely one of the consequences of excessive free
iron [183, 191]. Astrocytes may also protect neurons from
iron-induced cell damage under normal and pathological
conditions by sequestering free iron through transient receptor
potential canonical (TRPC) channels and divalent metal
transporter (DMT1), respectively [192, 193]. TRPC channels
are best known for their proposed role in calcium influx after
activation, though they transport multiple cation types across
the cell membrane [194, 195]. In a cell culture model, it was
demonstrated that overexpression of TPRC6 can increase
basal levels of intracellular iron as well as increasing iron
presence after stimulation, suggesting that iron transfer
through TRPC channels may occur under basal conditions
[196]. In contrast, DMT1 expression is controlled by proinflammatory
cytokines. The proinflammatory cytokine tumor
necrosis factor alpha (TNF-α), lipopolysaccharide, and
interleukin-6 (IL-6) increase DMT1 expression in astrocytes
while simultaneously decreasing ferroportin 1 (FPN-1)
expression [197, 198]. FPN-1 is an iron efflux transporter
so the result of this activity then is to increase total iron
uptake and storage in astrocytes after injury. Excess iron in
the microenvironment of astrocytes upregulates the expression
of ferritin, a rapidly inducible protein which binds and
neutralizes ferrous iron, thus preventing its effects on oxidative
stress [199]. Ferritin functions by first converting ferrous
iron to its less reactive state of ferric iron then nucleating this
ferric iron (Fe3+) and storing it within ferritin’s iron core
[200]. Together, the upregulation of iron transporters plus
the upregulation of ferritin allows astrocytes to act as iron
stores, resulting in reduced free ferrous iron in the microenvironment
where it may contribute to neuronal toxicity.
8. Modulation and Regulation of Immune
Responses in the CNS
Immunological activity in the CNS is relevant for the prevention
of pathogenic infection as well as responses to injury
such as stroke and TBI when the BBB is compromised
[201]. Astrocytes play a complex role in responding to such
CNS insults, and their inflammatory status as well as regulation
of immune cells is controversial. Astrogliosis is the
defensive reaction of astrocytes to trauma, ischemic damage,
inflammation, or pathological neurodegeneration [35]. During
astrogliosis, astrocytes increase at the site of the lesion,
exhibit altered morphology with increased thicknesses of
cellular processes, and display changes in gene expression
related to altered function [32, 35]. The increase in astrocytes
at the site of injury is believed to be due to proliferation
of astrocytes adjacent to the lesion and not due to astrocyte
migration from neighboring areas of the brain [35].
Astrocytes can be activated to a proinflammatory or
anti-inflammatory phenotype with an associated alteration
in their secretome [202–205]. The overall “defensive
response” of astrocytes following injury is highly complex
and has been shown in some studies to exacerbate inflammation
while generally, it is found to mitigate it [35].
The proinflammatory activation of astrocytes and their 3
expression of proinflammatory cytokines are dependent
upon the nature of the stimulation they receive and their
location in the brain [206]. The activation of proinflammatory
astrocytes can occur through interactions with microglia
and the response to cytokines such as IL-1, IL-6, oncostatin
M, leukemia inhibitory factor (LIF), and transforming
growth factor-α (TGF-α) and in response to overt physical
damage following brain injury, from interaction with Aβ
plaques or as a result of calcium-dependent phosphatase
calcineurin activation [35, 41, 207]. Under normal circumstances,
astrocytes aid in the morphological and physiological
development of neurons and synaptogenesis [208].
However, cell culture studies suggest that inappropriate activation
or overactivation of astrocytes can induce the production
of TNF-α and other cytotoxic factors that inhibit neurite
growth and synapse formation [209]. Additionally, exposure
of astrocytes in cell culture to cytokines, such as interferon-γ
(IFN-γ), can induce their production of nitric oxide which
drives the formation of its toxic metabolite, peroxynitrite
[210]. In cell culture, this does not harm astrocytes but can
lead to mitochondrial dysfunction and eventual cell death
in cocultured neurons [211].
Astrocytes can also modulate the immune system to
reduce inflammation. Normal human astrocytes were shown
to suppress both monocyte and T cell activation in cell cultures
[205]. It was found that astrocytes reduced monocyte
activation, not by secreting IL-10, but by blocking CD80
induction on the monocytes through an undefined mechanism
[205]. Astrocytes can also function in a manner to
recruit and direct white blood cells, both leukocytes and
Oxidative Medicine and Cellular Longevity 7
monocytes, to an area of injury, while at the same time
protecting healthy tissue from inflammatory consequences
of white blood cell invasion [56, 212, 213]. Importantly,
ablation of activated astrocytes in a murine model of spinal
cord injury resulted in greater inflammation, increased neuronal
degeneration, and negatively impacted subsequent
motor function, suggesting that the activated astrocytes control
the extent and location of inflammation following injury
[214]. The mechanisms of suppression of inflammation by
astrocytes require further investigation.
9. Tissue Homeostasis under Conditions of
DNA Damage
DNA repair and synthesis are necessary for normal tissue
homeostasis. DNA repair in neurons has been demonstrated
to occur in a nonuniform and, in some cases, inefficient
manner. Due to a decreased antioxidant response, neurons
display increased chromosomal and mitochondrial DNA
lesions that can result in cell death [215]. As compared to
astrocytes, neurons are slower in rejoining DNA double
strand breaks following radiation exposure, and they display
greater cell death after episodes of DNA damage [216]. Interestingly,
DNA damage in neurons can induce the production
of cell cycle enzymes, cyclin B, cyclin E, and proliferating cell
nuclear antigen (PCNA) [217, 218]. But this cell cycle progression
precedes apoptotic cell death rather than survival
and proliferation in neurons [219, 220]. Administration of
cell cycle inhibitors after traumatic brain injury was shown
to decrease neuronal cell death [221]. The reason for this
behavior could be related to the hypothesis that slower
cycling cells repair DNA more efficiently [222]. In contrast,
a murine model of stroke indicated that a pause of several
days occurred in the cell cycle of astrocytes before they
continue to proliferate after exposure to injury [223]. Such
differences in cell cycle control may mean the difference
between life and death at the cellular level. An ability to repair
effectively before allowing for cell proliferation may explain
astrocyte survival postinjury [222].
Neurons do display a limited ability to repair DNA in
both wild type and 7,8-dihydro-8-oxoguanine glycosylase
(OGG1) deficient mice in response to ischemia [224].
OGG1, a DNA glycosylase involved in base excision repair,
protects neuronal mitochondrial DNA from oxidative damage
under ischemic conditions [224]. However, the effectiveness
of this repair has been called into question and may
depend on the location of DNA damage within the chromosome
of the mitochondria. Studies of DNA damage from
radiation in NTERA-2-derived neurons showed that DNA
was efficiently repaired for transcribed genes but inefficiently
repaired in nontranscribed areas, suggesting that chromosomal
organization plays an important role in the effectiveness
of DNA repair mechanisms in neurons [225].
In contrast to neurons, astrocytes display robust DNA
repair capacities for both nuclear and mitochondrial DNA
[226]. In cell culture assays, astrocytes exposed to menadione,
an agent which induces oxidative stress, displayed a
lower mitochondrial DNA strand break frequency and more
efficient DNA repair as compared to all other cell types of the
brain [227]. The mechanisms induced to repair DNA in
astrocytes are multifaceted and include upregulation of both
primary double strand break pathways: nonhomologous end
joining and homologous recombination [228, 229]. Accordingly,
astrocytes are better able to protect themselves after
DNA damage as compared to neurons. To do this, they
utilize a hierarchy of mechanisms. Protection of astrocyte
DNA enables prevention of mutations and subsequent loss
of function or induction of cell transformation and carcinogenesis.
This resilience allows astrocytes to respond to and
aid in the protection of neurons and other cell types after
brain damage.
10. Conclusions
Astrocytes are highly involved in the maintenance and protection
of the CNS microenvironment under normal and
pathophysiological conditions. Brain damage can begin with
mechanical damage to cells, as in TBI, or through oxidative
stressors, as in radiation or in neurodegenerative diseases.
While the cause of the damage differs, the consequences are
similar with an unbalance of extracellular nutrients and ions,
damage to the BBB, and excessive release of excitatory neurotransmitters.
The resulting conditions can damage mitochondria
leading to the production of dangerous levels of
ROS that will in turn exacerbate DNA damage and increase
inflammation, ultimately leading to cell death. Astrocyte
maintenance of the ionic and metabolic environment protects
neurons occurring through multiple mechanisms.
Astrocytes take up and sequester excess neurotransmitters,
ions, and metabolic products to restore the homeostatic
balance. Astrocytes also take up and process damaged mitochondria
from neurons and transfer healthy mitochondria
back to injured neurons. Astrocytes are capable of producing
a robust antioxidant response to protect themselves and also
neurons, through the release of glutathione precursors to
neurons. Their role in scar formation allows astrocytes to
regulate and contain the immune responses in a manner that
controls neuroinflammation. Further understanding of the
endogenous protective mechanisms provided by astrocytes
may provide new insights that could lead to the development
of novel treatment options for the protection of susceptible
cells, such as neurons, under conditions of acute
injury or pathology.
