COX-2 inhibitors are contraindicated for treatment of combined injury.

Casualties of radiation dispersal devices, nuclear detonation or major ionizing radiation accidents, in addition to radiation exposure, may sustain physical and/or thermal trauma. Radiation exposure plus additional tissue trauma is known as combined injury. There are no definitive therapeutic agents. Cyclooxygenase-2 (COX-2), an inducible enzyme expressed in pathological disorders and radiation injury, plays an important role in inflammation and the production of cytokines and prostaglandin E(2) (PGE(2)) and could therefore affect the outcome for victims of combined injury. The COX-2 inhibitors celecoxib and meloxicam were evaluated for their therapeutic value against combined injury in mice. In survival studies, the COX-2 inhibitors had no beneficial effect on 30-day survival, wound healing or body weight gain after radiation injury alone or after combined injury. Meloxicam accelerated death in both wounded and combined injury mice. These drugs also induced severe hepatic toxicity, exaggerated inflammatory processes, and did not enhance hematopoietic cell regeneration. This study points to potential contraindications for use of COX-2 inhibitors in patients undergoing therapy for radiation injury and combined injury.

The biokinetics of uranium migrating from embedded DU fragments.

Military uses of depleted uranium (DU) munitions have resulted in casualties with embedded DU fragments. Assessment of radiological or chemical health risks from these fragments requires a model relating urinary U to the rate of migration of U from the fragments, and its accumulation in systemic tissues. A detailed biokinetic model for U has been published by the International Commission on Radiological Protection (ICRP), but its applicability to U migrating from embedded DU fragments is uncertain. Recently, Pellmar and colleagues (1999) conducted a study at the Armed Forces Radiobiology Research Institute (AFRRI) on the redistribution and toxicology of U in rats with implanted DU pellets, simulating embedded fragments. This paper compares the biokinetic data from that study with the behavior of commonly studied forms of U in rats (e.g., intravenously injected U nitrate). The comparisons indicate that the biokinetics of U migrating from embedded DU is similar to that of commonly studied forms of U with regard to long-term accumulation in kidneys, bone, and liver. The results provide limited support for the application of the ICRP's model to persons with embedded DU fragments. Additional information is needed with regard to the short-term behavior of migrating U and its accumulation in lymph nodes, brain, testicles, and other infrequently studied U repositories.

Biological effects of embedded depleted uranium (DU): summary of armed forces radiobiology research institute research.

The Persian Gulf War resulted in injuries of US Coalition personnel by fragments of depleted uranium (DU). Fragments not immediately threatening the health of the individuals were allowed to remain in place, based on long-standing treatment protocols designed for other kinds of metal shrapnel injuries. However, questions were soon raised as to whether this approach is appropriate for a metal with the unique radiological and toxicological properties of DU. The Armed Forces Radiobiology Research Institute (AFRRI) is investigating health effects of embedded fragments of DU to determine whether current surgical fragment removal policies remain appropriate for this metal. These studies employ rodents implanted with DU pellets as well as cultured human cells exposed to DU compounds. Results indicate uranium from implanted DU fragments distributed to tissues far-removed from implantation sites, including bone, kidney, muscle, and liver. Despite levels of uranium in the kidney that were nephrotoxic after acute exposure, no histological or functional kidney toxicity was observed. However, results suggest the need for further studies of long-term health impact, since DU was found to be mutagenic, and it transformed human osteoblast cells to a tumorigenic phenotype. It also altered neurophysiological parameters in rat hippocampus, crossed the placental barrier, and entered fetal tissue. This report summarizes AFRRI's depleted uranium research to date.

Electrophysiological changes in hippocampal slices isolated from rats embedded with depleted uranium fragments.

Although nephrotoxicity is considered to be the most serious consequence of uranium exposure, several studies have previously suggested the potential for neurotoxicity. In Operation Desert Storm, U.S. military personnel were wounded by fragments of depleted uranium (DU). This study was initiated to test the potential for DU fragments to cause electrophysiological changes in the central nervous system. Rats were surgically implanted with pellets of DU or tantalum (Ta) as a control metal. After 6, 12 and 18 months rats were euthanized, hippocampi removed and electrophysiological potentials analyzed by extracellular field potential recordings. Six months after implantation, synaptic potentials in DU-exposed tissue were less capable of eliciting spikes (E/S coupling). At 12 months, amplitudes of synaptic potentials were significantly increased in tissue from DU treated rats compared to Ta controls. E/S coupling was reduced. The differences between the electrophysiological measurements in DU-treated and control tissue were no longer evident at the 18 month time point. An analysis of the changes in the synaptic potentials and E/S coupling over the three time points suggests that by 18 months, the effects of aging and DU exposure converge, thereby obscuring the effects of the metal. Since kidney toxicity was not evident in these animals, effects secondary to nephrotoxicity are unlikely. This study raises the possibility that physiological changes occur in the brain with chronic exposure to DU fragments, which could contribute to neurological deficits.

Distribution of uranium in rats implanted with depleted uranium pellets.

During the Persian Gulf War, soldiers were injured with depleted uranium (DU) fragments. To assess the potential health risks associated with chronic exposure to DU, Sprague Dawley rats were surgically implanted with DU pellets at 3 dose levels (low, medium and high). Biologically inert tantalum (Ta) pellets were used as controls. At 1 day and 6, 12, and 18 months after implantation, the rats were euthanized and tissue samples collected. Using kinetic phosphorimetry, uranium levels were measured. As early as 1 day after pellet implantation and at all subsequent sample times, the greatest concentrations of uranium were in the kidney and tibia. At all time points, uranium concentrations in kidney and bone (tibia and skull) were significantly greater in the high-dose rats than in the Ta-control group. By 18 months post-implantation, the uranium concentration in kidney and bone of low-dose animals was significantly different from that in the Ta controls. Significant concentrations of uranium were excreted in the urine throughout the 18 months of the study (224 +/- 32 ng U/ml urine in low-dose rats and 1010 +/- 87 ng U/ml urine in high-dose rats at 12 months). Many other tissues (muscle, spleen, liver, heart, lung, brain, lymph nodes, and testicles) contained significant concentrations of uranium in the implanted animals. From these results, we conclude that kidney and bone are the primary reservoirs for uranium redistributed from intramuscularly embedded fragments. The accumulations in brain, lymph nodes, and testicles suggest the potential for unanticipated physiological consequences of exposure to uranium through this route.

Regional differences in glial cell modulation of synaptic transmission.

Metabolic integrity of glial cells in field CA1 of the guinea pig hippocampus is critical to maintenance of synaptic transmission (Keyser and Pellmar [1994] Glia 10:237-243). To determine if this tight glial-neuronal coupling is equally important in other brain regions, we compared the effect of fluoroacetate (FAC), a glial specific metabolic blocker, on synaptic transmission in field CA1 to synaptic transmission in area dentata (DG). FAC was significantly more effective in decreasing synaptic potentials in CA1 than in DG. A similar regional disparity in the FAC-induced decrease in ATP levels was evident. Isocitrate, a glial specific metabolic substrate, prevented the FAC-induced synaptic depression in both CA1 and DG. The results suggest that glia of CA1 and dentate respond differently to metabolic challenge. Modulation of this glial-neuronal coupling could provide a regionally specific mechanism for synaptic plasticity. Additionally, site-specific glial-neuronal interactions can impact on a variety of physiological and pathophysiological conditions.

Use of brain slices in the study of free-radical actions.

To understand the neuropathological roles of free radicals we investigate their actions in a model neuronal system, the hippocampal brain slice. Free radicals can be generated through a number of methods: hydrogen peroxide to produce hydroxyl radicals, dihydroxyfumarate to generate superoxide and ionizing radiation producing a variety of radical species. We find that free radicals have a number of profound effects in this system, which can be prevented by free-radical scavengers and antioxidants. With exposure to free radicals, the ability to generate spikes and synaptic efficacy are impaired. Decreased spike generating ability is correlated with lipid peroxidation. No change in membrane potential, membrane resistance, or many of the potassium currents can account for the effect on spike generation. Protein oxidation is likely to underlie synaptic damage. Both inhibitory and excitatory synaptic potentials are reduced by free-radical exposure. Presynaptic mechanisms are implicated. Lower concentrations of radicals prevent the maintenance of long-term potentiation, perhaps through oxidation of the NMDA receptor. The actions of the free radicals are often reversible because of the presence of repair mechanisms, such as glutathione, in hippocampal slices. The brain slice preparation has allowed us to begin to understand the electrophysiological and biochemical consequences of free-radical exposure.

Neurophysiological consequences of nitroxide antioxidants.

Nitroxides are antioxidant compounds that have been shown to provide radioprotection in vivo and in vitro. Radioprotection in vivo is limited by toxicity, which appears to be neurologic in nature. To further evaluate the toxicity of these compounds, three representative nitroxides, Tempol, Tempamine, and Tempo, were examined in slices of guinea pig hippocampus. Each nitroxide increased the population spike and caused potentiation of excitatory postsynaptic potential--spike coupling. Repetitive activity and epileptiform activity were observed at the highest concentrations of Tempo and Tempamine. Tempol was the least toxic compound in this system, followed by Tempamine and Tempo. Additional studies are necessary to further define the effects of nitroxides on the central nervous system and to develop strategies to mitigate these effects.

Radiation chemistry of the hippocampal brain slice.

The in vitro hippocampal brain slice is a 0.4 mm thick neural network that can be used to study brain responses to radiation and related injuries. This preparation is unique in that it responds to ionizing radiation within minutes after exposure without complications from changes in vascularity, blood flow, blood pressure, etc. Electrophysiological studies have shown that x- and gamma-rays alter synaptic transmission and spike generation, elements of normal brain activity. To evaluate the role of hydroxyl free radicals (OH) in these changes, slices were exposed to dilute H2O2 solutions. EPR spin trapping experiments verified that OH is produced. Neural responses, while similar, were not identical to those due to radiation, possibly because of a different distribution of OH. Although H2O2 is freely diffusible, it produces OH at specific sites where, e.g. iron reduces it. In contrast, x- and gamma-rays produce OH more uniformly throughout the tissue. H2O2 may provide a better model for high-LET radiation where yields of radical products of water radiolysis are decreased and peroxide reactions predominate.

Free radicals enhance basal release of D-[3H]aspartate from cerebral cortical synaptosomes.

Excessive generation of free radicals has been implicated in several pathological conditions. We demonstrated previously that peroxide-generated free radicals decrease calcium-dependent high K(+)-evoked L[3H]-glutamate release from synaptosomes while increasing calcium-independent basal release. The present study evaluates the nonvesicular release of excitatory amino acid neurotransmitters, using D-[3H]aspartate as an exogenous label of the cytoplasmic pool of L-glutamate and L-aspartate. Isolated presynaptic nerve terminals from the guinea pig cerebral cortex were used to examine the actions and interactions of peroxide, iron, and desferrioxamine. Pretreatment with peroxide, iron alone, or peroxide with iron significantly increased the calcium-independent basal release of D-[3H]aspartate. Pretreatment with desferrioxamine had little effect on its own but significantly limited the enhancement by peroxide. High K(+)-evoked release in the presence of Ca2+ was enhanced by peroxide but not by iron. These data suggest that peroxide increases nonvesicular basal release of excitatory amino acids through Fenton-generated hydroxyl radicals. This release could cause accumulation of extracellular excitatory amino acids and contribute to the excitotoxicity associated with some pathologies.

Synaptic transmission in the hippocampus: critical role for glial cells.

The importance of glial cells in controlling the neuronal microenvironment has been increasingly recognized. We now demonstrate that glial cells play an integral role in hippocampal synaptic transmission by using the glial-specific metabolic blocker fluoroacetate (FAC) to selectively inhibit glial cell function. FAC inhibits evoked intracellular postsynaptic potentials (PSPs; IC50 = 39 microM) as well as population PSPs (IC50 = 65 microM) in field CA1 of the guinea pig hippocampal slice. Spontaneous synaptic transmission is concurrently decreased. These effects are time and dose dependent. ATP concentrations in glial but not neuronal elements are also significantly reduced with FAC treatment. Simultaneous application of the metabolic substrate isocitrate with FAC prevents both the reduction in glial ATP concentrations and the decrease in evoked PSPs. Given that isocitrate is selectively taken up by glia, these data further support a glial specific metabolic action of FAC. Additionally, FAC has no postsynaptic effects as peak responses to iontophoretically applied glutamate are unchanged. However, the decay of both iontophoretic and evoked PSPs are prolonged following FAC treatment suggesting inhibition of glutamate uptake may contribute to the FAC-induced depression of synaptic potentials. These results show, for the first time, that glial cells are critical for maintenance of synaptic transmission and suggest a role for glial cells in the modulation of synaptic efficacy.

Effect of oxidative stress on excitatory amino acid release by cerebral cortical synaptosomes.

Previous studies in our laboratory have suggested that an oxidation reaction is responsible for the actions of free radicals to decrease synaptic potentials. Recently we observed that free radicals both decreased depolarization-induced vesicular release and enhanced basal, nonvesicular release of the excitatory amino acid, [3H]L-glutamate. In order to evaluate the contribution of oxidative reactions to this latter effect, we evaluated the actions of the oxidizing agent chloramine-T on synaptosomal release of excitatory amino acids, using [3H]D-aspartate as the exogenous label. Basal and depolarization evoked [3H]D-aspartate release were calcium-independent and nonvesicular. Chloramine-T pretreatment significantly increased basal release, while having no effect on high K(+)-evoked release. These data suggest that an oxidative process can mimic the free radical increase of basal release, as well as the decrease in synaptic potentials. On the other hand, the calcium-independent-evoked release may involve a different mechanism. Our results demonstrate that under basal, nondepolarizing conditions, oxidative stress exerts an adverse effect on the presynaptic nerve terminal, resulting in an increased release of potentially damaging excitatory amino acid neurotransmitters.

Gamma radiation (5-10 Gy) impairs neuronal function in the guinea pig hippocampus.

Guinea pigs were exposed to 5 and 10 Gy gamma radiation. Hippocampal brain slices were isolated 30 min, 1 day, 3 days and 5 days after irradiation or sham irradiation and the electrophysiological characteristics of the neural tissue were evaluated. Both radiation doses elicited significant changes that were dependent on dose, dose rate and time. Synaptic efficacy decreased soon after exposure to 5 Gy at dose rates of both 1 and 20 Gy/min. Recovery occurred by 5 days. Ten grays at 20 Gy/min potentiated the postsynaptic potential 1 day after irradiation. By 3 days, synaptic efficacy was decreased and did not recover. The ability of the synaptic potentials to generate spikes was potentiated within 30 min after exposure to 5 Gy at 1 Gy/min and persisted through 3 days, with recovery at 5 days. At the 20 Gy/min dose rate, a similar potentiation did not result with 10 Gy and occurred only at 3 days after irradiation with 5 Gy. Rather, within 30 min and after 5 days, spike generation was significantly depressed by these exposures. Both synaptic efficacy and spike generation contribute to the net input-output relationship of the neuronal population. This relationship was profoundly decreased within 30 min with recovery at 1 day and subsequent decline with the higher dose rate in a dose-dependent manner. These persistent changes in neuronal function are likely to be a consequence of the actions of ionizing radiation on the physiological processes that influence the neuronal environment.

Role of glutathione in repair of free radical damage in hippocampus in vitro.

Depletion of glutathione (GSH), an intrinsic antioxidant, increases vulnerability to free radical damage in a number of cell systems. This study investigates the role of GSH in limiting electrophysiological damage and/or recovery from free radical exposure in slices of guinea pig hippocampus. Synaptic potentials (PSPs) and population spikes (PSs) were recorded from field CA1. Free radicals were generated from 0.006% peroxide through the Fenton reaction. Analysis of the input-output curves showed that peroxide treatment decreased PSPs and impaired ability of the PSPs to generate PSs as previously reported. Recovery was nearly total within a half hour. Treatment with 5 mM buthionine sulfoximine (BSO) for 2 h depleted hippocampal GSH to 79.2% of control values. The extent of free radical damage was not increased. Recovery, however, was only partial. GSH was further depleted by oxidation with diamide or covalent bonding with dimethyl fumarate (DMF) immediately before and during the peroxide treatment. Neither diamide nor DMF treatment in BSO-incubated tissue enhanced peroxide-induced electrophysiological deficits. Following these treatments, however, tissue showed little recovery from free radical damage. We conclude that glutathione is essential for repair processes in hippocampal neurons exposed to oxidative damage.

Peroxide effects on [3H]L-glutamate release by synaptosomes isolated from the cerebral cortex.

Basal (non-depolarized) and high K(+)-stimulated [3H]L-glutamate release in the presence and absence of Ca2+ were assessed using presynaptic nerve terminals (synaptosomes) isolated from the cerebral cortex of the guinea pig. Basal glutamate release was found to be Ca(2+)-independent and was significantly increased following treatment with hydrogen peroxide (H2O2). On the other hand, depolarization-induced release had both a Ca(2+)-dependent and Ca(2+)-independent component. Both components of stimulated release were suppressed by H2O2. In fact, Ca(2+)-dependent evoked release was virtually eliminated by H2O2 pretreatment. The data suggest that H2O2 exerts a differential effect on the neurochemical mechanisms involved in basal and stimulated glutamate release at the presynaptic nerve terminal.

Electrophysiological consequences of exposure of hippocampal slices to dihydroxyfumarate, a generator of superoxide radicals.

In an effort to understand the damaging actions of free radicals to neuronal electrophysiology, the superoxide generator, dihydroxyfumarate (DHF), was evaluated in slices of guinea pig hippocampus. Using field potential recording techniques, population spikes and population synaptic potentials were recorded in field CA1. Slices were exposed to 3 mM DHF either alone or in the presence of a protectant. DHF did not alter the ability of the afferent volley to generate a synaptic potential, but it did impair the ability of the synaptic potential to elicit a population spike. In addition, DHF induced lipid peroxidation as measured by the thiobarbituric acid assay. Superoxide dismutase (SOD) provided no protection. Instead, SOD treatment promoted DHF damage to synaptic potentials. Catalase alone mitigated the actions of DHF, but only in SOD plus catalase was the DHF-induced electrophysiological deficit and lipid peroxidation completely antagonized. The iron chelator, Desferal, did not protect but promoted synaptic damage. Desferal may be ineffective because of the nitroxide radical formed upon its reaction with DHF. The hydroxyl radical scavenger, dimethylsulfoxide, prevented lipid peroxidation and reduced the DHF-induced deficit but did not completely prevent the impairment of spike generation. These data suggest that DHF exerts its actions through generation of hydrogen peroxide which would further react with tissue iron to produce hydroxyl radicals.

Fatty acids modulate excitability in guinea-pig hippocampal slices.

A variety of fatty acids produced sustained changes in excitability in the guinea-pig hippocampal slice. Although each fatty acid was unique, a general pattern was evident. During a 30-min exposure, the synaptic potential was minimally affected, although population spike amplitude showed significant increases. With wash, synaptic efficacy increased. The increase in the synaptic potential was significant with arachidonic acid (100 microM), oleic acid (100 microM), myristic acid (250 microM) and capric acid (250 microM). Also with wash, the coupling between the synaptic potential and the population spike was reduced significantly for most of the fatty acids tested: arachidonic acid (50 microM, 100 microM), linoleic acid (100 microM) oleic acid (100 microM), stearic acid (100 microM), myristic acid (250 microM) and capric acid (250 microM, 500 microM). The fatty acids may influence neuronal excitability, in part, through a direct membrane action. The observed synaptic enhancement is consistent with a role for a fatty acid in long-term potentiation. In addition, fatty acid exposure mimics the effects of X-radiation. We suggest that free radical-induced release of fatty acids contributes to electrophysiological damage in a number of pathological states.

Free radicals accelerate the decay of long-term potentiation in field CA1 of guinea-pig hippocampus.

Free radicals have been implicated in a number of pathological conditions. To evaluate the neurophysiological consequences of free radical exposure, slices of hippocampus isolated from guinea-pigs were exposed to hydrogen peroxide which reacts with tissue iron to generate hydroxyl free radicals. Long-term potentiation, a sustained increase in synaptic responses, was elicited in field CA1 by high frequency stimulation of an afferent pathway. We found that 0.002% peroxide did not directly affect the responses evoked by stimulation of the afferent pathway but did prevent maintenance of long-term potentiation. Short-term potentiation and paired-pulse facilitation were not affected by peroxide treatment. Peroxide was less effective if removed following high frequency stimulation and was ineffective if applied only after high frequency stimulation. Input/output analysis showed that the increase in synaptic efficacy was reduced with peroxide treatment. Changes in the enhanced ability of the synaptic potential to generate a spike were less apparent. These data show that the interference of free radicals with long-term potentiation may contribute to pathological deficits. It is possible that intracellular calcium regulation is disrupted by peroxide treatment. A number of second messenger systems involved with long-term potentiation are potential targets for free radical attack.

Time- and dose-dependent changes in neuronal activity produced by X radiation in brain slices.

A new method of exposing tissues to X rays in a lead Faraday cage has made it possible to examine directly radiation damage to isolated neuronal tissue. Thin slices of hippocampus from brains of euthanized guinea pigs were exposed to 17.4 ke V X radiation. Electrophysiological recordings were made before, during, and after exposure to doses between 5 and 65 Gy at a dose rate of 1.54 Gy/min. Following exposure to doses of 40 Gy and greater, the synaptic potential was enhanced, reaching a steady level soon after exposure. The ability of the synaptic potential to generate a spike was reduced and damage progressed after termination of the radiation exposure. Recovery was not observed following termination of exposure. These results demonstrate that an isolated neuronal network can show complex changes in electrophysiological properties following moderate doses of ionizing radiation. An investigation of radiation damage directly to neurons in vitro will contribute to the understanding of the underlying mechanisms of radiation-induced nervous system dysfunction.