Interaction between cytokines and ammonia in the mitochondrial permeability transition in cultured astrocytes.

Hepatic encephalopathy (HE) is the major neurological complication occurring in patients with acute and chronic liver failure. Elevated levels of blood and brain ammonia are characteristic of HE, and astrocytes are the primary target of ammonia toxicity. In addition to ammonia, recent studies suggest that inflammation and associated cytokines (CKs) also contribute to the pathogenesis of HE. It was previously established that ammonia induces the mitochondrial permeability transition (mPT) in cultured astrocytes. As CKs have been shown to cause mitochondrial dysfunction in other conditions, we examined whether CKs induce the mPT in cultured astrocytes. Cultures treated with tumor necrosis factor-α, interleukin-1ß, interleukin-6, and interferon-γ, individually or in a mixture, resulted in the induction of the mPT in a time-dependent manner. Simultaneous treatment of cultures with a mixture of CKs and ammonia showed a marked additive effect on the mPT. As oxidative stress (OS) is known to induce the mPT, so we examined the effect of CKs and ammonia on hemeoxygenase-1 (HO-1) protein expression, a marker of OS. Such treatment displayed a synergistic effect in the upregulation of HO-1. Antioxidants significantly blocked the additive effects on the mPT by CKs and ammonia, suggesting that OS represents a major mechanism in the induction of the mPT. Treatment of cultures with minocycline, an antiinflammatory agent, which is known to inhibit OS, also diminished the additive effects on the mPT caused by CKs and ammonia. Induction of the mPT in astrocytes appears to represent a major pathogenetic factor in HE, in which CKs and ammonia are critically involved.

Marked potentiation of cell swelling by cytokines in ammonia-sensitized cultured astrocytes.

BACKGROUND: Brain edema leading to high intracranial pressure is a lethal complication of acute liver failure (ALF), which is believed to be cytotoxic due to swelling of astrocytes. In addition to the traditional view that elevated levels of blood and brain ammonia are involved in the mechanism of brain edema in ALF, emerging evidence suggests that inflammatory cytokines also contribute to this process. We earlier reported that treatment of astrocyte cultures with a pathophysiological concentration of ammonia (5 mM NH4Cl) resulted in the activation of nuclear factor-kappaB (NF-κB) and that inhibition of such activation diminished astrocyte swelling, suggesting a key role of NF-κB in the mechanism of ammonia-induced astrocyte swelling. Since cytokines are also well-known to activate NF-κB, this study examined for additive/synergistic effects of ammonia and cytokines in the activation of NF-κB and their role in astrocyte swelling. METHODS: Primary cultures of astrocytes were treated with ammonia and cytokines (TNF-α, IL-1, IL-6, IFN-γ, each at 10 ng/ml), individually or in combination, and cell volume was determined by the [3H]-O-methylglucose equilibration method. The effect of ammonia and cytokines on the activation of NF-κB was determined by immunoblots. RESULTS: Cell swelling was increased by ammonia (43%) and by cytokines (37%) at 24 h. Simultaneous co-treatment with cytokines and ammonia showed no additional swelling. By contrast, cultures pretreated with ammonia for 24 h and then exposed to cytokines for an additional 24 h, showed a marked increase in astrocyte swelling (129%). Treatment of cultures with ammonia or cytokines alone also activated NF-κB (80-130%), while co-treatment had no additive effect. However, in cultures pre-treated with ammonia for 24 h, cytokines induced a marked activation of NF-κB (428%). BAY 11-7082, an inhibitor of NF-κB, completely blocked the astrocyte swelling in cultures pre-treated with ammonia and followed by the addition of a mixture of cytokines. CONCLUSION: Our results indicate that ammonia and a mixture of cytokines each cause astrocyte swelling but when these agents are added simultaneously, no additive effects were found. On the other hand, when cells were initially treated with ammonia and 24 h later given a mixture of cytokines, a marked potentiation in cell swelling and NF-κB activation occurred. These data suggest that the potentiation in cell swelling is a consequence of the initial activation of NF-κB by ammonia. These findings provide a likely mechanism for the exacerbation of brain edema in patients with ALF in the setting of sepsis/inflammation.

Freeway safety as a function of traffic flow.

In this paper, we present evidence of strong relationships between traffic flow conditions and the likelihood of traffic accidents (crashes), by type of crash. Traffic flow variables are measured using standard monitoring devices such as single inductive loop detectors. The key traffic flow elements that affect safety are found to be mean volume and median speed, and temporal variations in volume and speed, where variations need to be distinguished by freeway lane. We demonstrate how these relationships can form the basis for a tool that monitors the real-time safety level of traffic flow on an urban freeway. Such a safety performance monitoring tool can also be used in cost-benefit evaluations of projects aimed at mitigating congestion, by comparing the levels of safety of traffic flows patterns before and after project implementation.