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Figure 9.5 Proposed pathogenetic scheme whereby various signaling factors lead to reactive astrocytosis. Cytokines and ONS from microglial sources activate intracellular signaling kinases (PKC, PI3K and MAPKs), and such kinases further stimulate transcription factors (NF-kB and STAT3) resulting in reactive astrocytosis.

(e.g. dinitrophenol, triethyl tin, hexachlorophene, isoniazid), severe hypothermia, Reye’s Syndrome, acute hepatic encephalopathy (acute HE), as well as in the early phase of ischemic stroke and traumatic brain injury (TBI). The signaling pathways involved in such swelling process are presented below.

9.3.1Oxidative/Nitrosative Stress (ONS)

As noted above, ONS is an important early signaling event in many neurological disorders. ONS has been viewed as a particularly important signaling event in hyperammonemia/acute hepatic encephalopathy.151,152 Increased free-

radical formation, including nitric oxide, was observed in hyperammonemic rat brain,153,154 as well as in ammonia-treated astrocyte cultures.155 Cultured

astrocytes exposed to ammonia induce lipid peroxidation and oxidation of cellular proteins.156–158 A variety of morphological abnormalities are produced in astrocyte cultures exposed to ammonia,91 and such e ects are attenuated by

treatment of cultures with antioxidants.159

Free radicals have been shown to cause cell swelling in brain slices,160–162 as well as in cultured astrocytes.163–166 We recently demonstrated that astrocyte cultures exposed to oxidants/nitric oxide donors or to a pathophysiological concentration of ammonia (known to induce free radicals) resulted in cell

swelling, and such swelling was significantly attenuated by antioxidants or NOS inhibitors.167,168

Increased free radical production was also observed in brain after ischemia, as well as in cultured astrocytes after ischmic insult,169,170 and trauma.171–174

Inhibition of ONS significantly reduced cell swelling in cultured astrocytes after ischemia and trauma.171,173,174 These studies strongly suggest that ONS is an

important early event in cell swelling.

9.3.2Cytokines

Proinflammatory cytokines, likely derived from activated microglia, have been considered as a major driving force in cell swelling/brain edema after ischemia and acute HE. Various cytokines have been shown to be synthesized in brain after injury.175 Recombinant human IL-1-b infusion in brain was reported to aggravate the cytotoxic brain edema after middle cerebral artery occlusion.176 Elevated levels of TNFa, IL-1b and IL-6 were found in blood of patients with

acute HE,177–179 and induction of endotoxemia was shown to exacerbate cytotoxic brain edema in an experimental model of hyperammonemia.180 Additionally, it has recently been shown that cultured astrocyte exposed to cytokines (TNFa, IL-1b, IL-6 and IFN-g) stimulate cell swelling.181 Taken together, these studies suggest an important role of cytokines in the development of astrocyte swelling/brain edema in various neurological conditions. While the means by which cytokines cause astrocyte swelling/brain edema is unclear, cytokines are well known to induce ONS as well as to activate signaling kinases and transcription factors (e.g. MAPKs and NF-kB). It is likely that the activation of these factors contributes to the cell swelling/brain edema in these conditions.

Signaling Kinases

9.3.3Protein Kinase C (PKC)

Activation of PKC has been shown to facilitate the swelling induced by hypoosmotic stress in astrocyte cultures.182 Cultured astrocytes exposed to arachidonic acid (a well-known free radical inducer) were also shown to induce cell swelling, and such cell swelling was mediated, at least in part, through activation of PKC.183 Additionally, ATP and thrombin-induced PKC activation were shown to contribute to cell swelling in cultured astrocytes.184,185 Activation of PKC and the subsequent increase in astrocyte swelling were also observed in a rat model of acute hemophilus influenzae meningitis.186 While these studies support the involvement of PKC in cell swelling mechanisms, some reports indicate that activation of PKC decreases brain edema in rats after middle cerebral artery occlusion followed by reperfusion,187 and decreases cell swelling in cultured astrocytes after cerebral ischemia/reoxygenation.188 These contrasting results may reflect di erences in timing and the experimental models employed.

9.3.4Phosphatidylinositol 3-Kinase (PI3K)

Activation of PI3Ks was demonstrated in astrocytes after middle cerebral artery occlusion in transgenic mice expressing the human vascular endothelial growth factor, and it was shown that inhibition of PI3K significantly reduced cytotoxic brain edema.189 Activation of PI3K was also documented in perivascular astrocytes after cerebral ischemia in mice and such activation was shown to contribute to cytotoxic brain edema.190

9.3.5Protein Kinase G (PKG)

PKG, also known as cGMP-dependent protein kinase, is a serine/threonine kinase that phosphorylates other proteins known to be involved in cell cycle regulation.244 Activation of PKG has recently been shown to be involved in cell swelling of cultured astrocytes after ammonia treatment, and inhibition of such activation blocked ammonia-induced astrocyte swelling.191

9.3.6Mitogen-activated Protein Kinases (MAPKs)

Activation of MAPKs was observed in brains after acute HE.192,193 Addi-

tionally, cultured astrocytes exposed to ammonia have been shown to increase the phosphorylation (activation) of ERK1/2, p38 and JNK,192–194 and inhibi-

tion of ERK1/2, p38 and JNK activity significantly blocked ammonia-induced astrocyte swelling.167

Activation of ERK1/2, p38 and JNK was also observed in rat brain after TBI,195 as well as in cultured astrocytes after in vitro trauma,174 and inhibition of such activation reduced the trauma-induced swelling in cultured astrocytes.174 Similar findings were also observed in rats subjected to middle cerebral artery occlusion.196 Activation of MAPKs was also reported after hypoosmotic swelling in cultured astrocytes.197

While the means by which MAPKs mediate astrocyte swelling is unclear,

recent studies have suggested that they are involved in the activation of ion transporting systems (ITSs), i.e. NKCC1,198,199 NHE1200–202 and NCX.203,204

These ITSs have been implicated in the astrocyte swelling/brain edema associated with ischemia,205 trauma206,207 and ammonia neurotoxicity.208 The

involvement of ITSs in astrocyte swelling is elaborated in greater detail below.

Transcription Factors

9.3.7Signal Transducer and Activator of Transcription 3 (STAT3)

Sinn et al. (2007)209 found decreased STAT3 phosphorylation and increased brain edema following intracerebral hemorrhage in rats. Conversely, increased STAT3 phosphorylation and decreased brain edema were observed when rats were treated with geranylgeranylacetone, an activator of STAT3. We recently found that STAT3 is inactivated in ammonia-treated astrocyte cultures and that over-expression of STAT3 in cultures treated with the phophatase inhibitor sodium orthovanadate increased STAT3 phosphorylation as well as reduced cell swelling after ammonia treatment (unpublished observations). It

is noteworthy that factors known to induce cell swelling (ONS, activation of MAPKs, NF-kB and p53) become activated after STAT3 inactivation.210–212

These findings suggest the interesting possibility that STAT3 inactivation may be a crucial event in the evolution of astrocyte swelling/brain edema in various neurological conditions.

9.3.8Nuclear Factor Kappa B (NF-jB)

Activation of NF-kB has been implicated in the formation of brain edema after ischemia.213 We recently documented the activation of NF-kB in ammonia-

treated astrocyte cultures, as well as in the cerebral cortex of mice with acute HE.214,215 Such activation was mediated, at least in part, through the activation

of MAPKs.214 Additionally, treatment of astrocyte cultures with BAY 11-7082, an inhibitor of NF-kB, reduced cell swelling.214 Similar findings were also observed in astrocyte cultures derived from transgenic mice that have a functional inactivation of astrocytic NF-kB.215 We also found that transgenic mice having a functional inactivation of astrocytic NF-kB did not exhibit brain edema after thioacetamide-induced acute HE.215

While the means by which activation of NF-kB contributes to cell swelling

remains to be determined, it is known that inhibition of NF-kB significantly reduces the activity of inducible nitric oxide synthase,214,216,217 an enzyme implicated in cell swelling mechanisms.214,218,219 Additionally, NF-kB was

found to increase the activity of cyclooxygenase 2 (COX2), and the inhibition of COX2 was shown to reduce cell swelling in cultured retinal Mu¨ller cells.220 Inhibition of NF-kB significantly reduced COX2 activity as well as ammoniainduced astrocyte swelling.218

9.3.9p53

p53 is a tumor suppressor protein and transcription factor that is a key mediator of stress responses.221,222 Activated p53 triggers a number of signaling pathways that may lead to cell cycle arrest, apoptosis and DNA repair.223,224

It also mediates mitochondrial dysfunction, including decreases in the mitochondrial membrane potential and reduction in complex IV activity.225–227 Increased p53 expression was observed in rat brain cytoplasmic extract after

acute ammonia administration.228 It was recently found that astrocyte cultures exposed to ammonia stimulate p53 (phospho-p53ser392) and that treatment of

cultures with pifithrin-a (PFT), an inhibitor of p53, attenuated the ammoniainduced astrocyte swelling.229,230 Additionally, Yan et al. (2008)231 reported

that rats treated with PFT had less brain edema after subarachnoid hemorrhage.

Swelling E ectors

Activation of the above-noted signaling factors ultimately converge on systems that immediately bring about cell swelling. These include various ion channels, exchangers and transporters, as well as the water channel protein aquaporin-4 (AQP-4).

9.3.10Ion Channels/Transporters/Exchangers

Ion channels, exchangers and transporters (ion transporting systems, ITSs) are key factors involved in the maintenance of ionic balance in cells.232–235 These

ITSs include the Na–K–Cl cotransporter (NKCC), volume-sensitive osmolyte anion channels, Na1/Ca21 exchanger, the Na1/H1 exchanger, the SUR1regulated non-selective cation channel (NCCa-ATP) and the transient receptor

Figure 9.6 Schematic representation of signaling pathways in astrocyte swelling in di erent neurological conditoins. Cytokines and ONS from microglial or peripheral sources activate intracellular signaling kinases (PKC, PI3K, PKG and MAPKs), which then stimulate various transcription factors (NF-kB and p53). Activation of these signaling factors subsequently converge to activate ion transporting systems (ITSs) as well as AQP4 ultimately to result in cell swelling.

potential (TRP) ion channels.232–235 A disturbance in one or more of these systems may result in loss of ion homeostasis and cell swelling. NKCC1, in particular, plays an important role in the maintenance of intracellular levels of Na1, K1 and Cl–, and a defect in this system results in astrocyte swelling

in many neurological disorders including ischemic stroke, TBI and acute HE.89,205,236 Additionally, activation of NCX, NHE and NCCa-ATP was also shown to induce cell swelling/brain edema after ischemia and TBI.205,234

9.3.11Aquaporin-4 (AQP-4)

Aquaporins are integral membrane proteins that form pores and regulate water movement across biological membranes.237,238 AQP-4 is the predominant water

channel in brain,239 and it is particularly enriched in astrocytes. AQP-4 in astrocytes has been shown to play a prominent role in the development of cell swelling/brain edema following ischemia, TBI and acute HE.240,241 Conversely,

AQP-4-null mice have been shown to be more resistant to cytotoxic brain edema.242,243 A schematic diagram illustrating the involvement of signaling

systems in cell swelling is shown in Figure 9.6.

9.4 Conclusions and Perspectives

A growing body of evidence indicates that astrocytes play crucial roles in the normal function of the CNS, including the regulation of synaptic activity, modulation of excitatory and inhibitory neurotransmission, support of energy metabolism, maintenance of extracellular pH and ion homeostasis, detoxification processes and maintenance of blood-brain barrier (BBB) integrity. Astrocytes are also key players in almost all pathological processes a ecting the CNS. Their dramatic transformation into hypertrophic cells, a process referred to as reactive astrocytosis (astrogliosis), constitutes an event that follows all destructive lesions in the CNS. These activated cells play vital roles in restoring the composition of the extracellular milieu relative to appropriate levels of excitatory and inhibitory amino acid neurotransmitters, ions and pH, as well

as eliminating free radicals, generating growth factors necessary for repair, improving the operation of the BBB and providing a barrier to confine the lesion.

However, reactive astrocytes have also been implicated in deleterious consequences following CNS injury, in particular, by creating an obstacle to axonal regeneration. While there is evidence to support this claim, the issue remains unsettled. It should be recalled that impediments to axonal regeneration are found in oligodendrocytes, and when lesions occur in the spinal cord, they are often invaded by meningeal-derived cells that do indeed provide a barrier to regeneration.48 The so-called glial scar also contains microglia and macrophages, which are capable of generating toxic cytokines and free radicals. It remains to be proven whether the astrocytic glial ‘‘scar’’ alone is capable of obstructing the process of regeneration.

Thus, whether reactive astrocytosis is beneficial or detrimental remains a controversial issue. In the authors’ opinion, the beneficial e ects far outweigh the potential detrimental e ects of astrogliosis. We propose that reactive astrocytosis is a critical and necessary response in the early stages of destructive CNS processes. However, it may be harmful in latter stages by possibly contributing to an inhibition of axonal regeneration.

While the signaling pathways involved in reactive astrocytosis remain incompletely understood, it appears that the release of factors derived largely from microglia (cytokines, free radicals, ATP and others) can activate a number of signaling pathways in astrocytes culminating in astrogliosis. These include the signaling kinases, PKC, PI3K and MAPKs, as well as the activation of the transcription factors STAT3 and NF-kB.

The other astrocytic alteration following many CNS injuries is cell swelling. No beneficial consequences have ever been proposed for this astroglial response as it always appears to have detrimental e ects, in particular by contributing to the formation of cytotoxic brain edema and increased intracranial pressure. Additionally, swollen astrocytes display degenerative ultrastructural changes that may result in defective astroglial functions.

Many of the identified signaling pathways that contribute to reactive astrocytosis are also involved in the mechanism of cell swelling. These include PKC, PI3K and MAPKs, as well as the transcription factor NF-kB. On the other hand, STAT3, which is activated in reactive astrocytes, appears to be inactivated in swollen astrocytes. Additionally, activation of p53 and PKG has been shown to contribute to cell swelling/brain edema in di erent neurological conditions, although their role in reactive astrogliosis has not been investigated.

The aim of this article was to review normal and pathological properties of astrocytes, focusing on the signaling mechanisms involved in the formation of reactive astrocytes and cell swelling. It is clear that much work is still necessary to achieve a more comprehensive assessment of signaling pathways involved in these critical astrocytic responses to CNS injury. A fuller understanding of the signaling mechanisms involved in these responses may provide novel therapeutic targets for ameliorating the ravages brought about by neurologic disease.