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.

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.

Nutritional Interventions that Slow the Age-Associated Decline in Renal Function in a Canine Geriatric Model for Elderly Humans.

OBJECTIVE: To determine the effects of feeding traditional and renal protective foods (RPF) supplemented with functional food bioactives on glomerular filtration rate (GFR), lean body percent (LB%), and selected circulating biomarker and metabolite concentrations in a geriatric dog model. DESIGN: Randomized block design and cross-sectional study. SETTING: Hill's Pet Nutrition, Inc. dog colony. PARTICIPANTS: Eighty-one geriatric dogs (mean age, 10.4; range, 7.9-14.2 years) and 30 mature-adult dogs (mean age, 5.0; range, 3.3-6.9 years). INTERVENTION: Geriatric dogs were fed one of three foods (n = 27 per group) for 6 months: a traditional RPF (control) that was energy dense and mildly protein-restricted, or control food supplemented with increasing amounts of functional food bioactives: fish oil, lipoic acid, fruits and vegetables, and higher quality protein sources [functional foods one (FF1) and two (FF2)]. Geriatric dogs were compared before and after the feeding trial with mature adult dogs. MEASUREMENTS: Renal function was assessed by GFR, LB% was determined by dual energy x-ray absorptiometry, and circulating biomarkers and metabolites were measured in blood. RESULTS: Before the feeding trial, GFR (+28.2%), LB% (+18.6%), and serum total protein (+10.0%) were higher in mature versus healthy geriatric dogs (all P<0.001). Geriatric dogs consuming all three foods increased (P<0.001) GFR over time; group averages ranged from 13.0-16.9%. Dogs fed the highest supplemented level of bioactives (FF2) had lower (P<0.001) symmetric dimethylarginine (SDMA) concentrations (-14.3%). Feeding functional foods did not alter body weight, but increased (P<0.001) serum protein concentration (+6.7%). CONCLUSION: Supplementation with functional food bioactives can temporarily reverse the age-associated decline in renal function and serum total protein.

Overexpression of Bcl-xl protects septal neurons from prolonged hypoglycemia and from acute ischemia-like stress.

Overexpression of Bcl-xl, a member of the Bcl-2 protein family, is reported to protect from a variety of stresses involving delayed cell death. We tested the ability of Bcl-xl overexpression to protect primary cultures of embryonic rat septal neurons subjected to one of four different stresses: 6 h of combined oxygen-glucose deprivation, which produces rapid cell death, or a 24 h exposure to hypoglycemia, hyperglycemia, or 1mM 3-nitropropionic acid (an inhibitor of mitochondrial respiration), which results in a more slowly-developing death. Prior to the stress neurons were transiently transfected to overexpress either green fluorescent protein only or green fluorescent protein along with wild-type Bcl-xl. Immediately after oxygen-glucose deprivation, many neurons expressing green fluorescent protein only showed process blebbing and disintegration, with only 49% of the initial cells remaining intact with processes. Neurons expressing both green fluorescent protein and Bcl-xl showed less damage (68% intact post-stress, P<0.05). This result indicates that Bcl-xl's saving effects are not due solely to blocking delayed (apoptotic) death, because death following oxygen-glucose deprivation was rapid and was not accompanied by increased activation of caspase-3. Bcl-xl expression also significantly protected against the hypoglycemic stress (23% intact 24 h post-stress with green fluorescent protein only, compared with 70% with Bcl-xl and green fluorescent protein), but did not protect from hyperglycemia or 3-nitropropionic acid. Thus Bcl-xl does not protect against all forms of delayed death. Bcl-xl's protective effects may include blocking early damaging events, perhaps by increasing mitochondrial function in the face of low levels of energy substrates. Bcl-xl's protective effects may require an intact electron transport chain.

Hypoglycemia-induced seizures reduce cyclic AMP response element binding protein levels in the rat hippocampus.

Cyclic AMP response element binding protein (CREB) is a transcription factor that has been implicated in the activation of protein synthesis required for long-term memory. Since memory deficits are manifest following seizure, we undertook the present study to investigate the effects of hypoglycemia-induced seizure on CREB-immunoreactive neurons in several brain regions. We induced generalized seizures in male Long Evans rats (n=5) by injecting them with insulin (30 IU/kg, i.p). Animals were recovered by administration of 3 ml of 30% glucose within 5 min of the occurrence of seizure. Control animals (n=3) were injected with saline instead of insulin. All animals were perfused 90 min after recovery and the brains processed for CREB immunohistochemistry. Cell counts were determined for CREB-positive neurons using a computer-assisted program. When compared to control animals there was a 50% decrease (P<0.0001) in CREB-positive neurons in the CA1 region of the experimental animals. In the CA3 and dentate gyrus there was a 36% (P<0.001) and 25% decrease (P<0.001), respectively. Given the importance of hippocampus in memory-related processes and evidence that CREB is critical for memory formation, it is possible that seizures interfere with memory by disrupting CREB-dependent transcription.

Estrogen attenuates over-expression of beta-amyloid precursor protein messager RNA in an animal model of focal ischemia.

Cerebral ischemia is a risk factor for late onset Alzheimer's disease. Since estrogen replacement therapy benefits the outcome of cerebral stroke in post-menopausal women, we designed the present study to investigate the effects of estrogen on the expression of beta-amyloid precursor protein (APP) mRNA following focal ischemia in female rats. Female rats were ovariectomized (OVX) for two weeks. A single dose of 17 beta-estradiol (E2) (100 microgram/kg) was injected s.c. two hours before a unilateral middle cerebral artery (MCA) occlusion. Brain samples were harvested from ischemic core and penumbra of cortices at one hour and twenty-four hours following MCA occlusion. The expression of APP mRNA was assessed by RT-PCR. At one hour after MCA occlusion, OVX rats had a 67.9% (p<0.05) increase in APP mRNA in the penumbra. E2 treatment reduced this APP mRNA over-expression by 26.3% at that region. At twenty four hours following MCA occlusion, OVX rats had increases in APP mRNA of 52.9% and 57.0% (p<0.05) in the core and penumbra, respectively. E2 treatment reduced the APP mRNA over-expression by 61.0% and 48.6% (p<0.05) in these two regions, respectively. These effects appeared to reflect an interaction between hormonal environment and ischemia, since in the absence of MCA occlusion, there were no significant differences in APP mRNA expression among OVX, OVX-E2 treated and intact female rats. The present study demonstrates that estrogen may have an important role in reducing the over-expression of APP mRNA following focal ischemia.

Comparison of the effects of buspirone and chlordiazepoxide on differential reinforcement of low rates of response.

Buspirone is a novel agent which is clinically effective as an anxiolytic but which lacks the muscle relaxant, anticonvulsant and sedative effects of classical anxiolytics. It also lacks the full spectrum of action of classical anxiolytics in animal models of anxiety based on shock and novelty. In the present paper the effects of buspirone and chlordiazepoxide were tested on acquisition of differential reinforcement of low rates of response (DRL). This schedule involves the suppression of behaviour by reward omission and has shown consistent effects with classical anxiolytics. Buspirone was tested at doses of 0.3, 1.1 and 3.3 mg/kg i.p. and chlordiazepoxide at 5 and 20 mg/kg. Buspirone produced effects similar to those of chlordiazepoxide on accuracy of DRL responding. However, the size of the observed effects of buspirone was small even in relation to the 5 mg/kg dose of chlordiazepoxide and did not appear to be directly related to dose. Chlordiazepoxide increased overall rate of responding, while buspirone decreased it. Buspirone appears to show only limited conformity with benzodiazepines in animal models of anxiety and this result appears independent of the reinforcer used in the task.

Dose-response analysis of the effects of buspirone on rearing in rats.

The effects of buspirone were tested on rearing in an open field. Six different doses of buspirone (10, 3.3, 1.1, 0.3, 0.1 and 0.04 mg/kg) and a single dose of chlordiazepoxide (5 mg/kg) were administered i.p. to separate groups of rats. Buspirone produced a dose-dependent decrease in rearing in the range 0.04-10 mg/kg, whereas only the higher doses (10 and 3.3 mg/kg) decreased ambulation significantly. Chlordiazepoxide reduced rearing to an extent equivalent to 1 mg/kg of buspirone. Together with data in the literature, the results suggest that 5-HT1A agonists affect rearing at lower doses than ambulation; that the effects of buspirone in the open field are similar to classical anxiolytics; and that changes in rearing may be more closely related to anxiolytic than muscle relaxant, anti-convulsant and other GABA-mediated effects of the classical anxiolytics.

Effects of buspirone on fixed interval responding in rats.

Buspirone is a novel anxiolytic which does not share the muscle relaxant, anticonvulsant and sedative properties of classical anxiolytics such as the benzodiazepines. Its effects in different animal models of anxiety are also variable. The present experiments investigated the effects of buspirone on a fixed interval 60 s schedule of reinforcement (FI). In experiment 1, four doses of buspirone (10, 3.3, 1.1 and 0.3 mg/kg, i.p.) and two doses of chlordiazepoxide (5 and 20 mg/kg, i.p.) were administered to separate groups of rats throughout acquisition of the FI task. In experiment 2, four doses of buspirone (1.1, 0.3, 0.1 and 0.03 mg/kg, i.p.) and a single dose of chlordiazepoxide (5 mg/kg, i.p.) were used. Chlordiazepoxide generally released responding. At higher doses (1.1 mg/kg and above) buspirone suppressed responding in the later parts of the FI interval. The effects of lower doses were variable but included some response release in the later parts of the FI interval. At no dose did buspirone release responding at the beginning of the FI interval. The experiments show that buspirone differs qualitatively as well as quantitatively from chlordiazepoxide and that current animal models based on behavioural inhibition may need to be used with considerable care if detection of novel anxiolytics is to be ensured.

Comparison of the effects of buspirone and chlordiazepoxide on successive discrimination.

Buspirone is a novel anxiolytic which does not share the muscle relaxant, anticonvulsant and sedative properties of classical anxiolytics such as the benzodiazepines. It has variable effects in conflict tasks based on shock which normally show consistent effects with classical anxiolytics. The present experiment investigated the effects of buspirone on successive discrimination, a conflict task employing omission of reward rather than shock. Buspirone (3.3, 1.1 and 0.3 mg/kg, IP) and chlordiazepoxide (5 and 20 mg/kg, IP) were administered to separate groups of rats throughout acquisition of a visual successive discrimination. Chlordiazepoxide released nonrewarded responding in a dose-related fashion. The effects of buspirone were qualitatively similar in releasing response suppression but were both less in magnitude and less clearly related to dose. The experiment shows that the action of buspirone in successive discrimination tasks does not depend on the use of shock but, rather, appears to be a genuine failure to fully release behavioural inhibition.

The pituitary-adrenal axis and the different behavioral effects of buspirone and chlordiazepoxide.

Benzodiazepines and the novel anxiolytic buspirone share a common capacity to relieve clinical anxiety but do not share any side effects. Anxiety releases stress hormones and, at moderate doses, anxiolytic benzodiazepines block this release. It is interesting, therefore, that buspirone and other 5-HT1A agonists release stress hormones at moderate doses. Both the U-shaped dose-response curve seen with buspirone in some animal tests of anxiety and its slow onset of clinical action could be attributed to this release of stress hormones. Metyrapone (200 mg/kg), an inhibitor of 11-beta-hydroxylase, was used in the present experiments as a form of chemical adrenalectomy and was combined with administration of corticosterone (1 mg) to produce rats with presumed approximately normal corticosterone levels but no capacity to release endogenous corticosterone. This treatment reduced the difference normally observed in the effects of chlordiazepoxide (5 mg/kg) and buspirone (0.37 mg/kg) on a fixed interval schedule particularly in the early part of the interval when release of behavioral inhibition would be expected to contribute most to the effects. These results are consistent with the previous suggestion of Johnston and File (8) that the anxiolytic action of buspirone may be counteracted by activation of the pituitary-adrenal axis. Corticosterone appears to be the most likely critical agent for this antagonist action in the present experiments, although CRF and ACTH are also possibilities. It is likely that there is a mutual functional opposition between endogenous anxiolytic factors and stress hormones.

A procyanidin type A trimer from cinnamon extract attenuates glial cell swelling and the reduction in glutamate uptake following ischemia-like injury in vitro.

Dietary polyphenols exert neuroprotective effects in ischemic injury. The protective effects of a procyanidin type A trimer (trimer 1) isolated from a water soluble cinnamon extract (CE) were investigated on key features of ischemic injury, including cell swelling, increased free radical production, increased intracellular calcium ([Ca(2+)](i)), mitochondrial dysfunction, and the reduction in glutamate uptake. Astrocyte (glial) swelling is a major component of cytotoxic brain edema in ischemia and, along with vasogenic edema, may contribute to increased intracranial pressure, brain herniation, and additional ischemic injuries. C6 glial cultures were exposed to oxygen-glucose deprivation (OGD) for 5 h, and cell swelling was determined at 90 min after the end of OGD. OGD-induced increases in glial swelling were significantly blocked by trimer 1, but not by the major nonpolyphenol fractions of CE including cinnamaldehyde and coumarin. Increased free radical production, a contributing factor in cell swelling following ischemic injury, was also significantly reduced by trimer 1. Mitochondrial dysfunction, another key feature of ischemic injury, is hypothesized to contribute to glial swelling. Depolarization of the inner mitochondrial membrane potential (ΔΨ(m)) was assessed using a fluorescent dye (tetramethylrhodamine ethyl ester [TMRE]), and was significantly attenuated by trimer 1 as was OGD-induced increased [Ca(2+)](i). Taken together with our previous observation that blockers of [Ca(2+)](i) reduce cell swelling, our results indicate that trimer 1 may attenuate cell swelling by regulating [Ca(2+)](i). Trimer 1 also significantly attenuated the OGD-induced decrease in glutamate uptake. In addition, cyclosporin A, a blocker of the mitochondrial permeability pore (mPT), but not FK506 (that does not block the mPT), reduced the OGD-induced decline in glutamate uptake indicating a role of the mPT in such effects. Thus, the effects of trimer 1 in attenuating the reduction in glutamate uptake are likely mediated through their action on the mitochondria.

Mechanisms underlying the protective effects of myricetin and quercetin following oxygen-glucose deprivation-induced cell swelling and the reduction in glutamate uptake in glial cells.

The protective effects of the flavonoid polyphenols, myricetin and quercetin, were investigated on key features of ischemic injury in cultures including cell swelling and the reduction in glutamate uptake. C6 glial cells were exposed to oxygen-glucose deprivation (OGD) for 5 h and cell swelling was determined 90 min after the end of OGD. OGD-induced swelling was significantly blocked by both quercetin and myricetin although higher concentrations were required for quercetin. OGD-induced free radical production, a contributing factor in cell swelling, was significantly reduced by both myricetin and quercetin. However, depolarization of the inner mitochondrial membrane potential (ΔΨ(m)), the blockade of which generally reduces swelling, was significantly diminished by myricetin, but not quercetin. This indicated that quercetin could reduce swelling despite its inability to prevent depolarization of ΔΨ(m) possibly through other signaling pathways. Increased intracellular calcium ([Ca²+](i)) is an important characteristic of ischemic injury and is implicated in swelling. Both myricetin and quercetin attenuated the increase in [Ca²+](i). Further, a reduction in [Ca²+](i), through the use of nifedipine, nimodipine, verapamil, dantrolene, or BAPTA-AM, significantly reduced OGD-induced cell swelling indicating that one possible mechanism by which such flavonoids attenuate cell swelling may be through regulating [Ca²+](i). OGD-induced decrease in glutamate uptake was attenuated by myricetin, but not quercetin. Cyclosporin A, a blocker of the mitochondrial permeability transition (mPT) pore, but not FK506 (that does not block the mPT), attenuated the decline in glutamate uptake after OGD, indicating the involvement of the mPT in glutamate uptake. Our results indicated that while blockade of ΔΨ(m) may be sufficient to reduce swelling, it may not be a necessary factor, and that flavonoids reduce cell swelling by regulating [Ca²+](i). The differential effects of myricetin and quercetin on OGD-induced reduction on glutamate uptake may be due to their differential effects on mitochondria.

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.

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.

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.

Astrocytes protect neurons from ammonia toxicity.

Ammonia is a neurotoxin that is implicated in the CNS dysfunction associated with hepatic encephalopathy, urea cycle disorders, Reye's syndrome and other neurological conditions. While in vivo studies suggest that astrocytes are the principal target of ammonia toxicity, recent in vitro investigations suggest that neurons may also be directly affected by ammonia. To further examine the issue of neural cell sensitivity to ammonia, pure rat cortical neuronal cultures, as well as co-cultures of neurons and astrocytes, were exposed to 5 mM NH4Cl for 48 h. Cultures were examined for morphological changes by light microscopy, measures of cell death, free radical production and changes in the mitochondrial inner membrane potential. Ammonia caused extensive degenerative changes in pure cultured neurons, while such neuronal changes were minor in the co-cultures. Similarly, processes of pure cultured neurons displayed a significant loss of the mitochondrial inner membrane potential, as compared to neurons in co-cultures. Cell death (LDH release) in ammonia-treated neuronal cultures was twice as great as untreated controls, while in co-cultures ammonia did not significantly increase cell death. Free radical production at 3 min was increased (69%, P<0.05) in pure neuronal cultures but not in co-cultures. The neuroprotective effects observed in co-cultures may have been mediated by the astrocyte's ability to scavenge free radicals, by their detoxification of ammonia and/or by their neurotrophic actions. The neuroprotective action of astrocytes may explain the failure to detect significant pathological changes in neurons in ammonia toxicity in vivo.

Effects on free radical generation by ligands of the peripheral benzodiazepine receptor in cultured neural cells.

The effect of peripheral benzodiazepine receptor (PBR) ligands on free radical production was investigated in primary cultures of rat brain astrocytes and neurons as well as in BV-2 microglial cell lines using the fluorescent dye dichlorofluorescein-diacetate. Free radical production was measured at 2, 30, 60 and 120 min of treatment with the PBR ligands 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195), 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one (Ro5-4864) and protoporphyrin IX (PpIX) (all at 10 nm). In astrocytes, all ligands showed a significant increase in free radical production at 2 min. The increase was short-lived with PK11195, whereas with Ro5-4864 it persisted for at least 2 h. PpIX caused an increase at 2 and 30 min, but not at 2 h. Similar results were observed in microglial cells. In neurons, PK11195 and PpIX showed an increase in free radical production only at 2 min; Ro5-4864 had no effect. The central-type benzodiazepine receptor ligand, clonazepam, was ineffective in eliciting free radical production in all cell types. As the PBR may be a component of the mitochondrial permeability transition (MPT) pore, and free radical production may occur following induction of the MPT, we further investigated whether cyclosporin A (CsA), an inhibitor of the MPT, could prevent free radical formation by PBR ligands. CsA (1 micro m) completely blocked free radical production following treatment with PK11195 and Ro5-4864 in all cell types. CsA was also effective in blocking free radical production in astrocytes following PpIX treatment, but it failed to do so in neurons and microglia. Our results indicate that exposure of neural cells to PBR ligands generates free radicals, and that the MPT may be involved in this process.

Differential response of neural cells to trauma-induced free radical production in vitro.

CNS trauma has been associated with an increase in free radical production, but the cellular sources of this increase or the mechanism involved in the production of free radicals are not known. We, therefore, investigated the effects of trauma on free radical production in cultured neurons, astrocytes and BV-2 microglial cells. Free radicals were measured with the fluorescent dye DCFDA following in vitro trauma. At 30 and 60 min following trauma, there was a 132% and 64% increase, respectively, in free radical production in neurons when compared to controls. In astrocytes, there was a 94% and 133% increase at 30 and 60 min, respectively. Microglial cells, however, displayed no significant increase in free radicals at 30, 60 or 120 min following trauma. Since trauma can induce the mitochondrial permeability transition (MPT), a process associated with mitochondrial dysfunction, we further investigated whether cyclosporin A (CsA), an agent known to block the MPT, could prevent free radical formation following trauma. In neurons CsA did not block free radical production at 30 min but blocked it by 90% at 60 min. In contrast, in astrocytes CsA completely blocked free radical production at 30 min but did not block it at 60 min. Our results indicate that a differential sensitivity to trauma-induced free radical production exists in neural cells; that the MPT may be involved in the production of free radical post-trauma; and that the CsA-sensitive phase of free radical production is different in neurons and astrocytes.