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4 Proliferation of Reactive Astrocytes After FIS

4 Proliferation of Reactive Astrocytes After FIS

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Astrocytes as a Target for Ischemic Stroke



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also with substantial tissue shrinking [45]. Spontaneous recovery from brain injury

in the chronic phase of ischemia may involve reactive astrogliosis [79]. While it is

well known that the common feature of reactive astrocytes is increased GFAP

expression levels, whether reactive astrogliosis is merely through the upregulation

of GFAP or through cell proliferation is less understood. Bromodeoxyuridine (Brdu)

labeling and immunostaining are the general approaches used to assess the spatial

and temporal changes of reactive astrocyte proliferation [44–46, 73, 75, 80]. Li

et al. [68] studied the dynamic change of proliferation of astrocytes and microglia

after FIS. They designed a “time-block” protocol to label proliferating cells at different times after PT. Mice were administered with Brdu at days 1, 3, 4, 5, 9, 11, and

13 post-PT for 2 consecutive days and transcardially perfused after 1 day from the

last injection. The proliferation of reactive astrocytes was studied using GFAP and

Brdu double staining. Brdu+ cells and GFAP+ astrocytes dramatically increased

from day 2 post PT and reached their peak at day 4; however, overall, the

GFAP+Brdu+ proliferating astrocytes only accounted for a small percentage of total

Brdu+ cells, which reached a peak value of about 6 % from days 3 to 4 post PT and

then declined sharply. Based on the labeling protocol, the GFAP+Brdu+/Brdu+

ratio at each time point represents the relative rate of the generation of reactive

astrocytes. On the other hand, the GFAP+Brdu+/GFAP+ ratio also reached the

highest level within days 3–4 post PT. These data demonstrated that FIS increases

the population of proliferating reactive astrocytes in a highly temporal dependent

manner. The results indicate that glial scar is formed largely from the existing astrocytes through the upregulation of GFAP, rather than from newly generated astrocytes through proliferation. Although proliferation rate reduces dramatically after 8

days following PT, the morphology of reactive astrocytes maintains straight processes pointing to ischemic core for a prolonged time based on the GFAP expression [68, 73, 75]. This phenomenon suggests that the expression of certain genes is

irreversible in the penumbra during recovery from focal ischemic injury, and glial

scar formation causes substantial tissue remodeling and permanent and persistent

structural changes. Although the approach might underestimate the total number of

proliferating cells since the injection protocol may not label all the proliferating

cells [81], the GFAP+Brdu+/Brdu+ ratio should represent the ratio of newly generated reactive astrocytes out of the total proliferating cells.



2.5



Signaling Pathways of Reactive Astrogliosis After Ischemia



Advancements in genetics and molecular biology have helped to extensively characterize reactive astrocytes after brain injury. A plethora of molecular markers and

signaling pathways associated with reactive astrogliosis have been identified [80,

82–85]. The following is a brief summary of the major signaling pathways that have

been identified to regulate reactive astrogliosis in ischemic stroke.

Intermediate filaments. The intermediate filament GFAP is expressed both in

protoplasmic and fibrous astrocytes. Following ischemic stroke, the peri-lesional



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astrocytes become reactivated and dramatically upregulate their GFAP in a temporally

and spatially dependent manner. Genetic deletion of GFAP is neuroprotective

against metabolic insult and excitotoxicity which is attributed to increased production of the glial-derived neurotrophic factor (GDNF) by GFAP knockout astrocytes

[86]. GFAP−/− mice have exhibited a more significant decrease in cortical cerebral

blood flow, and a relatively larger infarct volume than WT mice after MCAo, indicating that GFAP-null mice have a high susceptibility to cerebral ischemia, which

indicates that astrocytes and GFAP play an important role in the progression of

brain damage after ischemia [87]. However, studies using GFAP−/−/Vimentin−/− double knockout mice show that in acute ischemia, double deletion of GFAP and

vimentin results in decreased glutamate uptake, increased susceptibility to oxidative

stress, and significantly increased infarct volumes (two to three times compared

with that of WT mice). Hence, the deletion of GFAP and vimentin in the acute stage

disrupts glial scar formation and promotes neuronal regeneration after ischemia

[88]. Whereas in chronic stages of stroke, attenuation of astroglial reactivity by

GFAP and vimentin deletion impaired axonal remodeling and functional recovery

[89]. Together, these studies suggest that reactive astrocytes have a protective role

in brain ischemia. GFAP and vimentin are essential to retaining the beneficial

function of reactive astrocytes in ischemic as well as recovering nervous tissues.

Thus, manipulation of astrocytic reactivity may represent a therapeutic target for

neurorestorative strategies in ischemic stroke.

p38 MAPK pathway. Mitogen-activated protein kinases (MAPK) are a family of

enzymes transducing a wide range of extracellular signals such as inflammation,

growth factors, and toxic stimuli as well as integrating corresponding cellular

responses [90]. Among p38, MAPKs is of particular importance since it transduces

cellular inflammation [91]. p38 MAPK can become activated in neurons, microglia,

and astrocytes after ischemic stroke [92–94]. A recent study using an astrocytespecific p38 MAPK knockout mouse model demonstrated that delayed activation of

p38 MAPK following ischemic injury corresponds with upregulation of GFAP in

the penumbra region [95], suggesting an important role of p38 MAPK in signaling

reactive astrogliosis following ischemic stroke. Furthermore, conditional deletion

of using GFAP-Cre/LoxP p38 MAPK mice resulted in a significant reduction of

reactive astrogliosis in the penumbra following ischemia compared to WT animals,

but had no significant effect on motor functional recovery after ischemia. p38

MAPK signaling may be a critical transducing pathway to modulate reactive astrogliosis in ischemic stroke.

Notch signaling pathway. Notch signaling is a highly conserved pathway critical for

the maintenance and self-renewal of progenitor cells and to inhibit precocious neurogenesis [96]. In ischemic stroke, Notch 1 signaling was activated in astrocytes in

the peri-infarct region by 24 h [97], and conditional deletion of Notch 1 from GFAP

positive cells resulted in a decreased number of proliferating astrocytes and an

increased number of invading CD45+ immune cells following ischemic injury [98].

Further study showed that Notch1–STAT3–ETBR axis connects a signaling network that promotes reactive astrocyte proliferation after a stroke [99]. Genetic fate

mapping analysis revealed that a subpopulation of reactive astrocyte activated



Astrocytes as a Target for Ischemic Stroke



121



Notch1 signaling in response to injury and became proliferative [97, 100]. A recent

study also showed that Notch1 signaling is important in reactive astrocyte differentiation to neurons in an ischemic brain [101]. These studies suggest that Notch 1

plays different roles including neurogenesis, reactive astrocyte proliferation, and

transdifferentiation after brain injury. Some outstanding questions which will be

mandatory in future studies are: Why does only a subset of reactive astrocytes

express Notch 1 signaling and become proliferative? What is the spatiotemporal

pattern of Notch 1 signaling after ischemia? Does inhibiting Notch 1-induced astrocyte proliferation affect functional recovery? Answering these questions will better

define the role of Notch 1 in reactive astrogliosis and provide novel therapeutic

insights for stroke therapy.

STAT3 signaling. Signal transducer and activator of transcription 3 (STAT3) is a

member of the Jak-STAT family and activated by hormones, growth factors, or cytokines [102]. The role of STAT3 in reactive astrogliosis has been extensively studied

in spinal cord injury models [81, 103–105]. In ischemic stroke, reperfusion-induced

increases in ROS and inflammatory cytokines are known to be powerful stimulants

of STAT3 signaling [106, 107], but it remains a matter of extensive debate regarding

cell type specific activation of STAT3 in the post ischemic brain. Some reports suggest neurons are the predominant source of activated STAT3 [108, 109], while others have shown that STAT3 is also activated in astrocytes along with neurons [110,

111]. More recently, it suggests that STAT3 can regulate reactive astrogliosis

induced by neurotoxic insults where activation of STAT3 in astrocytes and subsequent increases in GFAP expression can be induced by direct neuronal death even

in the absence of astrocyte damage [112, 113]. It appears that its role in driving

reactive astrogliosis in ischemic stroke remains to be explored.

Transforming growth factor-Beta (TGF-β) signaling. TGF-β is an injury-related

peptide and has been shown to increase immunoreactivity in both the infarct and

penumbra in stroke patients [114] as well as animal models of FIS [115–117].

TGF-β has been mostly regarded as neuroprotective particularly against the

N-methyl-D-aspartate receptor (NMDA)-induced excitotoxicity [116, 118]. It is

suggested that the protective action of TGF-β is mostly mediated by astrocytes [119,

120]. Most recent data suggests that disrupting astrocytic FGF receptors results in

reduced scar size, increased astrocyte activation, and inflammation, but did not

affect infarct volume [121, 122]. These studies suggest that TGF-β signaling in

astrocytes can limit neuroinflammation, reactive astrogliosis in the peri-infarct cortex and preserve brain function during the subacute period after stroke.

Despite the extensive characterization of reactive gliosis, high heterogeneity and

the context-dependent nature of reactive astrogliosis in the injured brain (e.g., acute

phase vs. chronic phase, white matter vs. gray matter, penumbral region vs. distant

region) complicate our understanding of signaling pathways for regulating this process [3, 85, 123]. Recent studies on the genome-wide transcriptome profiles of pure

glial cells using microarray and RNA-Seq technologies provide an unprecedented

opportunity to study glia phenotypes and functions in health and disease [8, 80, 124,

125]. Data from these studies open a window for a comprehensive view of complex

mechanisms by which astrocytes contribute to brain protection and repair in ischemia.



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Especially when using RNA-Seq, a large number of signaling pathways that trigger

reactive astrogliosis and molecular changes of astrocytes can be identified. Changes

in transcriptome profiles at different times after ischemic stroke will help to identify

novel molecular pathways by which astrocytes respond to ischemic insults at different stages [80]. These data will shed new light on the roles of astrocytes in brain

protection and repair and provide information for astrocyte-targeting therapeutic

strategies for ischemic therapy.



2.6



Stem-Cell-like Properties of Reactive Astrocytes

and Endogenous Neuronal Differentiation of Reactive

Astrocytes After Ischemic Stroke



Growing evidence has shown that ischemic stroke dramatically increases neurogenesis in the subventricular zone (SVZ) and subgranular layers in dentate gyrus [126].

Endogenous progenitor cells proliferate and differentiate into neurons in response

to ischemic insult; the newly generated neurons migrate to the damaged region

thereby integrating into the neural network [127–129]. These studies suggest that

endogenous neurogenesis contributes to brain repair and spontaneous recovery after

ischemia. As we already discussed, normally quiescent astrocytes become reactive

and resume proliferation after ischemia. This implies that reactive astrocytes might

be in an immature state due to a certain degree of dedifferentiation induced by ischemia. Studies have demonstrated that proliferating astrocytes in specific brain

regions act as adult neural stem cells under normal conditions [130, 131]. In addition to many changes in gene profile, reactive astrocytes also exhibit stem cell-like

properties [132–136]. These studies suggest that reactive astrocytes may attempt to

reconstitute neurons after ischemia. Indeed, they express neural stem-cell-related

proteins such as nestin and Sox2 [136], doublecortin (DCX), an immature neural

stem cell marker [101, 137], and oligo 2, a transcription factor that regulates neuroglia fate decision [138]. It shows that a sonic hedgehog (SHH) signal can directly

act on the astrocytes and is necessary and sufficient to elicit the stem cell response

both in vitro and in vivo [133]. Stroke also elicits a latent neurogenic program in

striatal astrocytes in a mouse model [101, 139]. It is reported that Notch 1 signaling

is reduced in reactive astrocytes after stroke and attenuated Notch 1 signaling is

necessary for neurogenesis by striatal astrocytes [101]; furthermore, blocking Notch

1 signaling triggers astrocytes in the striatum and the medial cortex to enter a neurogenic program even in the absence of a stroke, indicating that attenuated Notch 1

signaling is necessary for neurogenesis by striatal astrocytes. After a prolonged time

(e.g., 13–16 weeks) following ischemia, reactive astrocyte-derived neurons in striatum not only express mature neuronal markers but also are able to fire the action

potential and exhibit synaptic activity, suggesting the integration into their local

neural network [139]. These studies provide a molecular base for how reactive

astrocytes acquire stem cell lineage and become neurons after a stroke.



Astrocytes as a Target for Ischemic Stroke



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Reactive astrocytes also affect the migration of neural progenitor cells after ischemia.

Young et al. [140] reported for the first time that ischemic stroke causes substantial

reactive astrogliosis in SVZ and the hypertrophic reactive astrocytes and their tortuous processes disrupt the neuroblast migratory scaffold and cause SVZ reorganization after a stroke [140]; thus, reactive astrocytes in SVZ might also play a

modulating role in neurogenesis thereby affecting stroke recovery.



2.7



Direct Astrocyte-to-Neuron Conversion After Ischemic

Stroke



The fact that reactive astrocytes express neural stem markers and can be transformed

into mature neurons after a prolonged time following ischemia indicate that reactive

astrocytes have the intrinsic potential to generate neurons and reactive astrocytes-toneuron conversion can be facilitated under permissive conditions such as overexpression of neurogenic factors. It has been recently reported that astrocytes indeed

can be converted into neuroblasts and neurons in vitro and in vivo by forced expression of a single transcriptional factor such as Sox2 [141, 142], Neurog-2 [143–145],

NeuroD1 [146], Ascl1 [147], or a combination of multiple transcriptional factors

such as Ascl1, Lmx1B, Nurr1, Oct4, Sox2, or Nanog [148, 149]. Astrocyteconverted neurons express mature neuronal markers such as Map2 and NeuN [141,

146]. Electrophysiological recording showed that cultured mouse astrocyteconverted neurons by NeuroD1 are glutamatergic; furthermore, astrocyte-converted

neurons by NeuroD1 in mouse brain can exhibit synaptic currents and integrate into

neural circuit in vivo [146]. In vitro direct reprogramming study of postnatal astrocytes showed that astrocyte-to-neuron conversion is swift, but different proneural

factors can elicit distinct transcriptional programs. Both Neurog-2 and Ascl1 rapidly

elicited neurogenic programs to induce glutamatergic and GABAergic neuronal

conversion but followed distinct paths with a few common genes in the neurogenic

cascades [145]. The study of shared target genes will allow identifying a particularly important subset of downstream targets capable of directly converting astrocytes into functional neurons.

Although, reactive astrocyte-to-neuron conversion by forced expression of transcription factors has not been reported in the context of strokes, these studies suggest that recruiting reactive astrocytes and directly converting them into neurons

might be a promising strategy for brain repair after ischemic stroke. Since reactive

astrocytes exhibit different proliferating rates at different times after FIS [68], there

might be an optimal timing to convert them into neurons using genetic manipulation

techniques such as astrocyte-specific inducible transgenic mice and viral transduction. Because stroke causes a large damage in the cortex, it is also interesting to

study whether reactive astrocytes in the cortex have the similar capacity of neuronal

transformation to striatal astrocytes. Future study is also needed to explore whether

any small molecular drugs targeting the signaling pathway of endogenous neuronal



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