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with AG490 in cultured astrocytes, was shown to inhibit reactive astro-
cytosis.140 A similar reduction of astrogliosis was observed in STAT3 deleted astrocytes in mice after spinal cord injury.141,142
9.2.6Nuclear Factor Kappa B (NF-jB)
NF-kB is a protein complex that controls the transcription of many genes, especially those involved in immune responses, inflammation and cell proliferation.143 NF-kB is normally present in the cytoplasm in an inactive form where it is bound to a member of the IkB family of inhibitory proteins. Upon activation by extracellular stimuli (e.g. cytokines, free radicals and intracellular signaling kinases), IkB inhibitors are modified through site-specific phosphorylation thereby allowing NF-kB to translocate to the nucleus and bind to target DNA elements where it activates the transcription of many genes.
Increased NF-kB was first identified in reactive astrocytes after kainic acidinduced seizures in rats,144 and has subsequently been found in reactive astrocytes in scrapie-infected mice.145 Amyloid precursor protein stimulates NF-kB activation in cultured astrocytes and such activation was shown to promote reactive astrocytosis.101 Recent studies have also shown that cultured astrocytes exposed to a mixture of gangliosides (inducers of inflammatory processes in astrocytes146) develop reactive astrocytosis as demonstrated by increased GFAP expression. A ganglioside mixture also increased iNOS expression and NF-kB activation in cultured astrocytes, and these changes were blocked by inhibiting NF-kB.146
Increased GFAP, as well as activation of astrocytic NF-kB, was observed in rat brain after cerebral ischemia induced by bilateral common carotid artery occlusion, and inhibition of NF-kB prevented the formation of astrogliosis.147 Ischemia-like injury induced by the alpha(2)-adrenoceptor agonist clonidine,
or by adenosine A2a receptor activation in cultured astrocytes, was shown to induce astrogliosis,148,149 and such an e ect was partly mediated by activation of NF-kB.150
While the precise means by which activation of NF-kB contributes to increased GFAP expression and subsequent astrocytosis is unclear, it has been demonstrated that inhibition of NF-kB reduced levels of GFAP mRNA and protein in cultured human astrocytes.150
In summary, the above findings suggest that ONS and activation of signaling kinases, including PKC, PI3K and MAPKs, as well as the activation of the transcription factors STAT3 and NF-kB contributes to the development of reactive astrocytosis. A schematic diagram illustrating signaling systems involved in reactive astrocytosis is shown in Figure 9.5.
9.3 Signaling Systems in Astrocyte Swelling
As |
noted above, |
astrocyte swelling represents an |
important |
component |
of |
the cytotoxic |
brain edema following various |
forms of |
intoxication |
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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
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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
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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