Differential Response of Neural Cells to Trauma-Induced Swelling In Vitro.

Brain edema and the associated increase in intracranial pressure are major consequences of traumatic brain injury (TBI) that accounts for most early deaths after TBI. We recently showed that acute severe trauma to cultured astrocytes results in cell swelling. We further examined whether trauma induces cell swelling in neurons and microglia. We found that severe trauma also caused cell swelling in cultured neurons, whereas no swelling was observed in microglia. While severe trauma caused cell swelling in both astrocytes and neurons, mild trauma to astrocytes, neurons, and microglia failed to cell swelling. Since extracellular levels of glutamate are increased in brain post-TBI and microglia are known to release cytokine, and direct exposure of astrocytes to these molecules are known to stimulate cell swelling, we examined whether glutamate or cytokines have any additive effect on trauma-induced cell swelling. Exposure of cultured astrocytes to trauma caused cell swelling, and such swelling was potentiated by the exposure of traumatized astrocytes to glutamate and cytokines. Conditioned medium (CM) from traumatized astrocytes had no effect on neuronal swelling post-trauma, while CM from traumatized neurons and microglia potentiated the effect of trauma on astrocyte swelling. Further, trauma significantly increased the Na-K-Cl co-transporter (NKCC) activity in neurons, and that inhibition of NKCC activity diminished the trauma-induced neuronal swelling. Our results indicate that a differential sensitivity to trauma-induced cell swelling exists in neural cells and that neurons and microglia are likely to be involved in the potentiation of the astrocyte swelling post-trauma.

Role of Matricellular Proteins in Disorders of the Central Nervous System.

Matricellular proteins (MCPs) are actively expressed non-structural proteins present in the extracellular matrix, which rapidly turnover and possess regulatory roles, as well as mediate cell-cell interactions. MCPs characteristically contain binding sites for other extracellular proteins, cell surface receptors, growth factors, cytokines and proteases, that provide structural support for surrounding cells. MCPs are present in most organs, including brain, and play a major role in cell-cell interactions and tissue repair. Among the MCPs found in brain include thrombospondin-1/2, secreted protein acidic and rich in cysteine family (SPARC), including Hevin/SC1, Tenascin C and CYR61/Connective Tissue Growth Factor/Nov family of proteins, glypicans, galectins, plasminogen activator inhibitor (PAI-1), autotaxin, fibulin and perisostin. This review summarizes the potential role of MCPs in the pathogenesis of major neurological disorders, including Alzheimer's disease, amyotrophic lateral sclerosis, ischemia, trauma, hepatic encephalopathy, Down's syndrome, autism, multiple sclerosis, brain neoplasms, Parkinson's disease and epilepsy. Potential therapeutic opportunities of MCP's for these disorders are also considered in this review.

Neuronal Cell Death Induced by Mechanical Percussion Trauma in Cultured Neurons is not Preceded by Alterations in Glucose, Lactate and Glutamine Metabolism.

Traumatic brain injury (TBI) is a devastating neurological disorder that usually presents in acute and chronic forms. Brain edema and associated increased intracranial pressure in the early phase following TBI are major consequences of acute trauma. On the other hand, neuronal injury, leading to neurobehavioral and cognitive impairments, that usually develop months to years after single or repetitive episodes of head trauma, are major consequences of chronic TBI. The molecular mechanisms responsible for TBI-induced injury, however, are unclear. Recent studies have suggested that early mitochondrial dysfunction and subsequent energy failure play a role in the pathogenesis of TBI. We therefore examined whether oxidative metabolism of (13)C-labeled glucose, lactate or glutamine is altered early following in vitro mechanical percussion-induced trauma (5 atm) to neurons (4-24 h), and whether such events contribute to the development of neuronal injury. Cell viability was assayed using the release of the cytoplasmic enzyme lactate dehydrogenase (LDH), together with fluorescence-based cell staining (calcein and ethidium homodimer-1 for live and dead cells, respectively). Trauma had no effect on the LDH release in neurons from 1 to 18 h. However, a significant increase in LDH release was detected at 24 h after trauma. Similar findings were identified when traumatized neurons were stained with fluorescent markers. Additionally (13)C-labeling of glutamate showed a small, but statistically significant decrease at 14 h after trauma. However, trauma had no effect on the cycling ratio of the TCA cycle at any time-period examined. These findings indicate that trauma does not cause a disturbance in oxidative metabolism of any of the substrates used for neurons. Accordingly, such metabolic disturbance does not appear to contribute to the neuronal death in the early stages following trauma.

Sulfonylurea receptor 1 contributes to the astrocyte swelling and brain edema in acute liver failure.

Astrocyte swelling (cytotoxic brain edema) is the major neurological complication of acute liver failure (ALF), a condition in which ammonia has been strongly implicated in its etiology. Ion channels and transporters are known to be involved in cell volume regulation, and a disturbance in these systems may result in cell swelling. One ion channel known to contribute to astrocyte swelling/brain edema in other neurological disorders is the ATP-dependent, nonselective cation (NCCa-ATP) channel. We therefore examined its potential role in the astrocyte swelling/brain edema associated with ALF. Cultured astrocytes treated with 5 mM ammonia showed a threefold increase in the sulfonylurea receptor type 1 (SUR1) protein expression, a marker of NCCa-ATP channel activity. Blocking SUR1 with glibenclamide significantly reduced the ammonia-induced cell swelling in cultured astrocytes. Additionally, overexpression of SUR1 in ammonia-treated cultured astrocytes was significantly reduced by cotreatment of cells with BAY 11-7082, an inhibitor of NF-κB, indicating the involvement of an NF-κB-mediated SUR1 upregulation in the mechanism of ammonia-induced astrocyte swelling. Brain SUR1 mRNA level was also found to be increased in the thioacetamide (TAA) rat model of ALF. Additionally, we found a significant increase in SUR1 protein expression in rat brain cortical astrocytes in TAA-treated rats. Treatment with glibenclamide significantly reduced the brain edema in this model of ALF. These findings strongly suggest the involvement of NCCa-ATP channel in the astrocyte swelling/brain edema in ALF and that targeting this channel may represent a useful approach for the treatment of the brain edema associated with ALF.

Brain edema in acute liver failure: role of neurosteroids.

Brain edema is a major neurological complication of acute liver failure (ALF) and swelling of astrocytes (cytotoxic brain edema) is the most prominent neuropathological abnormality in this condition. Elevated brain ammonia level has been strongly implicated as an important factor in the mechanism of astrocyte swelling/brain edema in ALF. Recent studies, however, have suggested the possibility of a vasogenic component in the mechanism in ALF. We therefore examined the effect of ammonia on blood-brain barrier (BBB) integrity in an in vitro co-culture model of the BBB (consisting of primary cultures of rat brain endothelial cells and astrocytes). We found a minor degree of endothelial permeability to dextran fluorescein (16.2%) when the co-culture BBB model was exposed to a pathophysiological concentration of ammonia (5mM). By contrast, lipopolysaccharide (LPS), a molecule well-known to disrupt the BBB, resulted in an 87% increase in permeability. Since increased neurosteroid biosynthesis has been reported to occur in brain in ALF, and since neurosteroids are known to protect against BBB breakdown, we examined whether neurosteroids exerted any protective effect on the slight permeability of the BBB after exposure to ammonia. We found that a nanomolar concentration (10nM) of the neurosteroids allopregnanolone (THP) and tetrahydrodeoxycorticosterone (THDOC) significantly reduced the ammonia-induced increase in BBB permeability (69.13 and 58.64%, respectively). On the other hand, we found a marked disruption of the BBB when the co-culture model was exposed to the hepatotoxin azoxymethane (218.4%), but not with other liver toxins commonly used as models of ALF (thioacetamide and galactosamine, showed a 29.3 and 30.67% increase in permeability, respectively). Additionally, THP and THDOC reduced the effect of TAA and galactosamine on BBB permeability, while no BBB protective effect was observed following treatment with azoxymethane. These findings suggest that ammonia does not cause a significant BBB disruption, and that the BBB is intact in the TAA or galactosamine-induced animal models of ALF, likely due to the protective effect of neurosteroids that are synthesized in brain in the setting of ALF. However, caution should be exercised when using azoxymethane as an experimental model of ALF as it caused a severe breakdown of the BBB, and neurosteriods failed to protect against this breakdown.

Endothelial-astrocytic interactions in acute liver failure.

Brain edema and the subsequent increase in intracranial pressure are major neurological complications of acute liver failure (ALF), and swelling of astrocytes (cytotoxic brain edema) is the most prominent neuropathological abnormality in ALF. Recent studies, however, have suggested the co-existence of cytotoxic and vasogenic mechanisms in the brain edema associated with ALF. This review 1) summarizes the nature of the brain edema in humans and experimental animals with ALF; 2) reviews in vitro studies supporting the presence of cytotoxic brain edema (cell swelling in cultured astrocytes); and 3) documents the role of brain endothelial cells in the development of astrocyte swelling/brain edema in ALF.

Role of cerebral endothelial cells in the astrocyte swelling and brain edema associated with acute hepatic encephalopathy.

Brain edema is an important complication of acute hepatic encephalopathy (AHE), and astrocyte swelling is largely responsible for its development. Elevated blood and brain ammonia levels have been considered as major etiological factors in this edema. In addition to ammonia, recent studies have suggested that systemic infection, inflammation (and associated cytokines (CKs)), as well as endotoxin (lipopolysaccharide (LPS)) are also involved in AHE-associated brain edema. As endothelial cells (ECs) are the first resident brain cells exposed to blood-borne "noxious agents" (i.e., ammonia, CKs, LPS) that are present in AHE, these cells may be in a critical position to react to these agents and trigger a process resulting in astrocyte swelling/brain edema. We therefore examined the effect of conditioned media (CM) from ammonia, LPS and cytokine-treated cultured brain ECs on cell swelling in cultured astrocytes. CM from ammonia-treated ECs when added to astrocytes caused significant cell swelling, and such swelling was potentiated when astrocytes were exposed to CM from ECs treated with a combination of ammonia, LPS and CKs. We also found an additive effect when astrocytes were exposed to ammonia along with CM from ammonia-treated ECs. Additionally, ECs treated with ammonia showed a significant increase in the production of oxy-radicals, nitric oxide (NO), as well as evidence of oxidative/nitrative stress and activation of the transcription factor nuclear factor kappa B (NF-κB). CM derived from ECs treated with ammonia, along with antioxidants (AOs) or the NF-κB inhibitor BAY 11-7082, when added to astrocytes resulted in a significant reduction in cell swelling, as compared to the effect of CM from ECs-treated only with ammonia. We also identified increased nuclear NF-κB expression in rat brain cortical ECs in the thioacetamide (TAA) model of AHE. These studies suggest that ECs significantly contribute to the astrocyte swelling/brain edema in AHE, likely as a consequence of oxidative/nitrative stress and activation of NF-κB.

NF-κB in the mechanism of brain edema in acute liver failure: studies in transgenic mice.

Astrocyte swelling and brain edema are major complications of the acute form of hepatic encephalopathy (acute liver failure, ALF). While elevated brain ammonia level is a well-known etiological factor in ALF, the mechanism by which ammonia brings about astrocyte swelling is not well understood. We recently found that astrocyte cultures exposed to ammonia activated nuclear factor-κB (NF-κB), and that pharmacological inhibition of such activation led to a reduction in astrocyte swelling. Although these findings suggest the involvement of NF-κB in astrocyte swelling in vitro, it is not known whether NF-κB contributes to the development of brain edema in ALF in vivo. Furthermore, pharmacological agents used to inhibit NF-κB may have non-specific effects. Accordingly, we used transgenic (Tg) mice that have a functional inactivation of astrocytic NF-κB and examined whether these mice are resistant to ALF-associated brain edema. ALF was induced in mice by treatment with the hepatotoxin thioacetamide (TAA). Wild type (WT) mice treated with TAA showed a significant increase in brain water content (1.65%) along with prominent astrocyte swelling and spongiosis of the neuropil, consistent with the presence of cytotoxic edema. These changes were not observed in Tg mice treated with TAA. Additionally, WT mice with ALF showed an increase in inducible nitric oxide synthase (iNOS) immunoreactivity in astrocytes from WT mice treated with TAA (iNOS is known to be activated by NF-κB and to contribute to cell swelling). By contrast, Tg mice treated with TAA did not exhibit brain edema, histological changes nor an increase in iNOS immunoreactivity. We also examined astrocytes cultures derived from Tg mice to determine whether these cells exhibit a lesser degree of swelling and cytopathological changes following exposure to ammonia. Astrocyte cultures derived from Tg mice showed no cell swelling nor morphological abnormalities when exposed to ammonia for 24h. By contrast, ammonia significantly increased cell swelling (31.7%) in cultured astrocytes from WT mice and displayed cytological abnormalities. Moreover, we observed a lesser increment in iNOS and NADPH oxidase activity (the latter is also known to be activated by NF-κB and to contribute to astrocyte swelling) in astrocyte cultures from Tg mice treated with ammonia, as compared to ammonia-treated WT mice astrocytes. These findings strongly suggest that activation of NF-κB is a critical factor in the development of astrocyte swelling/brain edema in ALF.

Aquaporin-4 in manganese-treated cultured astrocytes.

Manganese in excess is neurotoxic and causes CNS injury resembling that of Parkinson's disease. In brain, astrocytes predominantly take up and accumulate manganese and are thus vulnerable to its toxicity. Manganese was shown to induce cell swelling in cultured astrocytes, and oxidative/nitrosative stress (ONS) mediates such swelling. As aquaporin-4 (AQP4) is important in the mechanism of astrocyte swelling, we examined the effect of manganese on AQP4 protein levels in cultured astrocytes. Treatment of cultures with manganese increased AQP4 protein in the plasma membrane (PM), whereas total cellular AQP4 protein and mRNA levels were unchanged, suggesting that increased AQP4 levels is due to its increased stability and/or increased trafficking to the PM and not to its neosynthesis. AQP4 gene silencing by small interfering ribonucleic acid resulted in a marked reduction in astrocyte swelling by manganese. Antioxidants, as well as an inhibitor of nitric oxide synthase, diminished the increase in AQP4 protein expression, suggesting a role of ONS in the mechanism of AQP4 increase. As ONS is known to activate mitogen-activated protein kinases (MAPKs) and MAPK activation has been implicated in astrocyte swelling, we examined the effect of manganese on the activation of MAPKs and found an increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2, C-Jun amino-terminal kinase (JNK)1/2/3, and p38-MAPK. Inhibitors of ERK1/2 and p38-MAPK (but not of JNK) blocked (40-60%) the manganese-induced increase in AQP4 protein content and astrocyte swelling, suggesting the involvement of these kinases in the increased AQP4 content. Inhibition of oxidative stress or MAPKs may represent potential strategies for counteracting AQP4-related neurological complications associated with manganese toxicity.

Role of mitogen-activated protein kinases in the mechanism of oxidant-induced cell swelling in cultured astrocytes.

Cytotoxic brain edema, usually a consequence of astrocyte swelling, is an important complication of stroke, traumatic brain injury, hepatic encephalopathy, and other neurological disorders. Although mechanisms underlying astrocyte swelling are not fully understood, oxidative stress (OS) has generally been considered an important factor in its pathogenesis. To better understand the mechanism(s) by which OS causes cell swelling, we examined the potential involvement of mitogen-activated protein kinases (MAPKs) in this process. Cultures exposed to theoxidant H(2)O(2) (10, 25, 50 microM) for different time periods (1-24 hr) significantly increased cell swelling in a triphasic manner. Swelling was initially observed at 10 min (peaking at 30 min), which was followed by cell shrinkage at 1 hr. A subsequent increase in cell volume occurred at approximately 6 hr, and the rise lasted for at least 24 hr. Cultures exposed to H(2)O(2) caused the activation of MAPKs (ERK1/2, JNK and p38-MAPK), whereas inhibition of MAPKs diminished cell swelling induced by 10 and 25 microM H(2)O(2). These findings suggest that activation of MAPKs is an important factor in the mediation of astrocyte swelling following oxidative stress.

Ammonia-induced activation of p53 in cultured astrocytes: role in cell swelling and glutamate uptake.

Cytotoxic brain edema, due principally to astrocyte swelling, is a major neurological complication of the acute form of hepatic encephalopathy (HE) (acute liver failure, ALF), a condition likely caused by elevated levels of brain ammonia. Potential mediators of ammonia-induced astrocyte swelling include oxidative/nitrosative stress (ONS), the mitochondrial permeability transition (mPT), mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB), since blockade of these factors reduces the extent of astrocyte swelling. As p53, a tumor suppressor protein and transcription factor, is a downstream target of ONS and MAPKs, we examined its potential role in the mechanism of ammonia-induced astrocyte swelling. Astrocytes exposed to NH(4)Cl (5mM) showed increased phosphorylation (activation) of p53((Ser392)) at 1h and such phosphorylation was significantly reduced by inhibitors of MAPKs (ERK1/2, JNK and p38-MAPK), antioxidants (vitamin E, catalase, PBN, desferoxamine, MnTBAP), as well as by L-NAME, an inhibitor of nitric oxide synthase, indicating a key role of oxidative/nitrosative stress and MAPKs in the ammonia-induced activation of p53. Since p53 is known to induce the mPT and to activate NF-kappaB (factors leading to ONS and implicated in ammonia-induced astrocyte swelling), we examined whether inhibition of p53 activation blocked mPT induction, NF-kappaB activation, as well as cell swelling. Pifithrin-alpha (PFT), an inhibitor of p53, blocked these processes. Impairment of astrocytic glutamate uptake is another important feature of HE and hyperammonemia. We therefore examined the potential role of p53 in the ammonia-induced inhibition of glutamate uptake and found that PFT also reversed the ammonia-induced inhibition of glutamate uptake. Our results indicate that a potentially important downstream target of ammonia neurotoxicity is p53, whose activation contributes to astrocyte swelling and glutamate uptake inhibition, processes likely a consequence of ONS derived from the mPT and activation of NF-kappaB.

Signaling factors in the mechanism of ammonia neurotoxicity.

Mechanisms involved in hepatic encephalopathy (HE) still remain poorly understood. It is generally accepted that ammonia plays a major role in this disorder, and that astrocytes represent the principal target of ammonia neurotoxicity. In recent years, studies from several laboratories have uncovered a number of factors and pathways that appear to be critically involved in the pathogenesis of this disorder. Foremost is oxidative and nitrosative stress (ONS), which is largely initiated by an ammonia-induced increase in intracellular Ca(2+). Such increase in Ca(2+) activates a number of enzymes that promote the synthesis of reactive oxygen-nitrogen species, including constitutive nitric oxide synthase, NADPH oxidase and phospholipase A2. ONS subsequently induces the mitochondrial permeability transition, and activates mitogen-activated protein kinases and the transcription factor, nuclear factor-kappaB (NF-kappaB). These factors act to generate additional reactive oxygen-nitrogen species, to phosphorylate various proteins and transcription factors, and to cause mitochondrial dysfunction. This article reviews the role of these factors in the mechanism of HE and ammonia toxicity with a focus on astrocyte swelling and glutamate uptake, which are important consequences of ammonia neurotoxicity. These pathways and factors provide attractive targets for identifying agents potentially useful in the therapy of HE and other hyperammonemic disorders.

New concepts in the mechanism of ammonia-induced astrocyte swelling.

It is generally accepted that astrocyte swelling forms the major anatomic substrate of the edema associated with acute liver failure (ALF) and that ammonia represents a major etiological factor in its causation. The mechanisms leading to such swelling, however, remain elusive. Recent studies have invoked the role of oxidative stress in the mechanism of hepatic encephalopathy (HE), as well as in the brain edema related to ALF. This article summarizes the evidence for oxidative stress as a major pathogenetic factor in HE/ALF and discusses mechanisms that are triggered by oxidative stress, including the induction of the mitochondrial permeability transition (MPT) and activation of signaling kinases. We propose that a cascade of events initiated by ammonia-induced oxidative stress results in cell volume dysregulation leading to cell swelling/brain edema. Blockade of this cascade may provide novel therapies for the brain edema associated with ALF.

Aquaporin-4 in hepatic encephalopathy.

Brain edema is a critical component of hepatic encephalopathy (HE) associated with acute liver failure and such edema appears to be principally due to astrocyte swelling (cytotoxic edema). Ammonia is believed to represent a major factor responsible for astrocyte swelling, although the mechanisms by which ammonia causes such swelling are not completely understood. Recent studies have implicated potential role of oxidative stress, and the mitochondrial permeability transition (mPT). While it is not known how oxidative stress and the mPT cause astrocyte swelling, it is reasonable to suggest that these events may affect one or more plasma membrane proteins involved in water and ion homeostasis in astrocytes. One such protein strongly implicated in brain edema in other neurological conditions is the water channel protein aquaporin-4 (AQP-4), which is abundantly expressed in astrocytes. This article summarizes the potential role of AQP-4 in brain edema in in vivo models of HE, as well as in ammonia-induced cell swelling in cultured astrocytes. The involvement of AQP-4 in the effects of manganese, another toxin implicated in HE, will also be discussed.

Downregulation of the 18-kDa translocator protein: effects on the ammonia-induced mitochondrial permeability transition and cell swelling in cultured astrocytes.

Hepatic encephalopathy (HE) is a major neurological complication in patients with severe liver disease. While the pathogenesis of HE is unclear, elevated blood and brain ammonia levels are believed to be major etiological factors, and astrocytes appear to be the primary target of its toxicity. A notable feature of ammonia neurotoxicity is an upregulation of the 18-kDa translocator protein (TSPO) (formerly referred to as the peripheral benzodiazepine receptor or PBR), which is found on the outer mitochondrial membrane. However, the precise significance of this upregulation is unclear. To examine its potential role in ammonia-induced astrocyte dysfunction, we downregulated the TSPO using antisense oligonucleotides, and examined whether such downregulation could alter two prominent features of ammonia gliotoxicity, namely, the mitochondrial permeability transition (MPT) and astrocyte swelling. Nontransfected cultures treated with NH4Cl (5 mM; 48 h) showed a significant increase in astrocyte cell volume (37.5%). In cultured astrocytes transfected with TSPO antisense oligonucleotides, such cell swelling was reduced to 17%, but this change was not significantly different from control cell volume. Similarly, nontransfected cultures treated with NH4Cl (5 mM; 24 h) exhibited a 40% decline in the cyclosporin A-sensitive mitochondrial inner membrane potential (DeltaPsi(m)) (P < 0.01) (a measure of the MPT). By contrast, cells transfected with TSPO antisense oligonucleotides did not display a significant loss of the DeltaPsi(m) following ammonia exposure. Our findings highlight the important role of the TSPO in the mechanism of ammonia neurotoxicity.

The mitochondrial permeability transition in neurologic disease.

Mitochondria, being the principal source of cellular energy, are vital for cell life. Yet, ironically, they are also major mediators of cell death, either by necrosis or apoptosis. One means by which these adverse effects occur is through the mitochondrial permeability transition (mPT) whereby the inner mitochondrial membrane suddenly becomes excessively permeable to ions and other solutes, resulting in a collapse of the inner membrane potential, ultimately leading to energy failure and cell necrosis. The mPT may also bring about the release of various factors known to cause apoptotic cell death. The principal factors leading to the mPT are elevated levels of intracellular Ca2+ and oxidative stress. Characteristically, the mPT is inhibited by cyclosporin A. This article will briefly discuss the concept of the mPT, its molecular composition, its inducers and regulators, agents that influence its activity and describe the consequences of its induction. Lastly, we will review its potential contribution to acute neurological disorders, including ischemia, trauma, and toxic-metabolic conditions, as well as its role in chronic neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis.

Manganese induces cell swelling in cultured astrocytes.

Manganese in excess is neurotoxic and causes a CNS disorder that resembles Parkinson's disease (manganism). Manganese highly accumulates in astrocytes, which renders these cells more vulnerable to its toxicity. Consistent with this vulnerability, manganese has been shown to cause histopathological changes in astrocytes (Alzheimer type II change), generates oxidative stress and bring about mitochondrial dysfunction, including the induction of the mitochondrial permeability transition (mPT) in astrocytes. In addition to manganism, increased brain levels of manganese have been found in hepatic encephalopathy, a chronic neurological condition associated with liver dysfunction, wherein Alzheimer type II astrocytic changes are also observed. As low-grade brain edema, possibly secondary to astrocyte swelling, has been reported in hepatic encephalopathy, we hypothesized that manganese may contribute to such edema. We therefore exposed cultured astrocytes to manganese (Mn(3+)) acetate (25 and 50microM) for different time periods and examined for changes in cell volume. Manganese dose-dependently induced astrocyte swelling; such swelling was first observed at 12h (28%), which further increased (54%) at later time points (24-48h). Pretreatment of astrocyte cultures with antioxidants, including vitamin E, the spin trapping agent PBN, and the iron-chelating agent desferroximine, as well as the nitric oxide synthase inhibitor l-NAME, all significantly blocked (50-80%) astrocyte swelling caused by manganese, suggesting that oxidative/nitrosative stress is involved in the mechanism of such swelling. Cyclosporin A, an inhibitor of mPT also blocked (90%) manganese-induced astrocyte swelling. The data indicate that manganese exposure results in astrocyte swelling and such swelling, at least in part, may be caused by oxidative stress and/or mPT. Astrocyte swelling by manganese may represent an important aspect of manganese neurotoxicity, and may be a factor in low-grade brain edema associated with chronic hepatic encephalopathy.

Calcitonin gene-related peptide immunoreactivity in chronic human spinal cord injury.

STUDY DESIGN: Histopathological study of the human spinal cord. SETTING: International Collaboration on Repair Discoveries, Vancouver, BC, Canada. RATIONALE: In animals, primary dorsal root afferent fibers, which are immunoreactive for calcitonin gene-related peptide (CGRP), sprout following spinal cord injury (SCI) into deeper laminas of the dorsal horn below the level of injury. It has been suggested that this aberrant sprouting plays a role in altering cardiovascular control after SCI and could be responsible for life-threatening episodes of autonomic dysreflexia (AD). OBJECTIVES: To observe the changes of CGRP distribution after SCI and compare the differences between normal and injured human spinal cord. METHODS: Upper thoracic segments from individuals with chronic cervical SCI (n=4) and individuals with intact spinal cords (n=5) were processed immunocytochemically to identify CGRP fibers and histologically to identify the severity of degeneration. RESULTS: Semiquantitative analysis showed a significant increase in CGRP immunoreactivity in the dorsal horns of individuals with chronic SCI (P<0.001). Furthermore, one of the SCI individuals in this study displaying significant CGRP sprouting had well documented episodes of AD. CONCLUSIONS: Our observations suggest that SCI in humans results in significant sprouting of CGRP fibers. This aberrant sprouting of sensory fibers could contribute to the abnormal cardiovascular control and pain commonly observed following chronic human SCI.

Inhibition of glutamine transport into mitochondria protects astrocytes from ammonia toxicity.

Hepatic encephalopathy (HE) is a major neurological complication that occurs in the setting of severe liver failure. Ammonia is a key neurotoxin implicated in this condition, and astrocytes are the principal neural cells histopathologically and functionally affected. Although the mechanism by which ammonia causes astrocyte dysfunction is incompletely understood, glutamine, a by-product of ammonia metabolism, has been strongly implicated in many of the deleterious effects of ammonia on astrocytes. Inhibiting mitochondrial glutamine hydrolysis in astrocytes mitigates many of the toxic effects of ammonia, suggesting the involvement of mitochondrial glutamine metabolism in the mechanism of ammonia neurotoxicity. To determine whether mitochondriaare indeed the organelle where glutamine exerts its toxic effects, we examined the effect of L-histidine, an inhibitor of mitochondrial glutamine transport, on ammonia-mediated astrocyte defects. Treatment of cultured astrocytes with L-histidine completely blocked or significantly attenuated ammonia-induced reactive oxygen species production, cell swelling, mitochondrial permeability transition, and loss of ATP. These findings implicate mitochondrial glutamine transport in the mechanism of ammonia neurotoxicity.

Oxidative stress and mitogen-activated protein kinase phosphorylation mediate ammonia-induced cell swelling and glutamate uptake inhibition in cultured astrocytes.

Hepatic encephalopathy (HE) is a major neurological complication in patients with severe liver failure. Elevated levels of ammonia have been strongly implicated as a factor in HE, and astrocytes appear to be the primary target of its neurotoxicity. Mechanisms mediating key aspects of ammonia-induced astrocyte dysfunction such as cell swelling and inhibition of glutamate uptake are not clear. We demonstrated previously that cultured astrocytes exposed to ammonia increase free radical production. We now show that treatment with antioxidants significantly prevents ammonia-induced astrocyte swelling as well as glutamate uptake inhibition. Because one consequence of oxidative stress is the phosphorylation of mitogen-activated protein kinases (MAPKs), we investigated whether phosphorylation of MAPKs may mediate astrocyte dysfunction. Primary cultured astrocytes exposed to 5 mm NH4Cl for different time periods (1-72 h) significantly increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38(MAPK), and c-Jun N-terminal kinase (JNK) 1/2/3, which was inhibited by appropriate MAPK inhibitors 1, 4-diamino-2, 3-dicyano-1, 4-bis (2-aminophenylthio) butadiene (UO126; for ERK1/2), trans-1-(4-hydroxyclyclohexyl)-4-(4-fluorophenyl)-5-(2-methoxypyrimidin-4-yl)imidazole (SB 239063; for p38(MAPK)), and anthra[1,9-cd]pyrazol-6(2H)-one (SP600125; for JNK1/2/3), as well as by antioxidants. Kinase inhibitors partially or completely prevented astrocyte swelling. Although SB239063 and SP600125 significantly reversed glutamate uptake inhibition and ammonia-induced decline in glutamate-aspartate transporter protein levels, UO126 did not, indicating a differential effect of these kinases in ammonia-induced astrocyte swelling and glutamate transport impairment. These studies strongly suggest the involvement of oxidative stress and phosphorylation of MAPKs in the mechanism of ammonia-induced astrocyte dysfunction associated with ammonia neurotoxicity.