Brain Injury and Cellular Responses

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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.

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