abstract
Nerve agent-induced seizures often lead to irreversible brain damage. While seizures can be controlled if treated soon after onset, medical care for some battlefield casualties may be delayed beyond the therapeutic window of opportunity, i.e., after secondary pathways of neuronal injury have been activated and anticonvulsants can no longer stop seizure activity. Therefore, there is a need for adjunct drugs that can be administered one or more hours after nerve agent exposure to prevent or alleviate the seizure-related brain damage.
INTRODUCTION
Organophosphorus nerve agents are the principal chemical warfare agents known to produce brain injury. They exert their biological effects by inhibiting the enzyme acetylcholinesterase (AChE), thereby blocking hydrolysis of the neurotransmitter acetylcholine (ACh) resulting in greatly increased post synaptic ACh levels. This causes a spectrum of effects, including miosis, excess secretions, nausea, vomiting, and muscle fasciculations. At moderate to high doses, nerve agents also cause seizures and associated convulsions. If left untreated, seizures rapidly progress to status epilepticus (SE) and cause irreversible seizure-related brain damage (SRBD) (Solberg, 1997; Shih, 2003). U.S. military forces have adopted a regimen of pretreatment with the carbamate pyridostigmine bromide (PB) and post exposure treatment with atropine sulfate (AS) and the oxime pyridine-2-aldoxime methylchloride (2-PAM); this regimen greatly reduces morbidity and mortality. These drugs do not prevent nerve agent-induced seizures in all casualties, and the regimen now includes diazepam to terminate seizures and prevent SRBD (Shih, 2003).
Even with diazepam, the treatment regimen has limitations. The decision to include diazepam was based on animal data showing that it could terminate nerve agent-induced seizures/convulsions and enhance survival when given in conjunction with carbamate pretreatment (e.g., PB), atropine (i.e., AS) and oxime (e.g., 2-PAM, HI-6) therapy (Lipp, 1972; Lipp, 1973; McDonough, 1989; McDonough, 1993; Shih, 1990). However, diazepam is unable to afford absolute protection against SRBD. Although neuropathology is reduced in diazepam-treated animals, the incidence and degree of protection afforded by diazepam is not complete (Clement, 1993; Hayward, 1990; McDonough, 1989; McDonough, 1995; Baze, 1993).
Battlefield nerve agent exposure levels are likely to be sufficient to induce seizures in troops that are not in full protective ensemble at the time of the attack (Fanzone, 2002). In such cases, the therapeutic window for arresting seizures with anticonvulsants and for preventing or reducing neuropathology is less than one hour following onset. By this time, seizures have progressed to SE and are refractory to anticonvulsant therapy; in addition, secondary pathways of neuronal injury have been activated (Lipp, 1972, 1973; Shih, 1990; Shih, 1991; Capacio, 1991; Philippens, 1992; Sparenborg, 1993; McDonough, 1993; Harris, 1994; Shih, 1999; Lallement, 2000; McDonough, 2000). The current regimen is thus unlikely to be effective in preventing SRBD if treatment is delayed for more than 1 hr following exposure. Prompt treatment of battlefield nerve agent casualties can be expected to be problematic. Confusion and high mobility of a battlefield scenario may cause delays in locating and evacuating casualties. Some casualties may not be received at the battalion aid station for one or more hours after nerve agent exposure. Thus, it is likely that the administration of anticonvulsants to some victims undergoing seizures may be delayed beyond the therapeutic window of opportunity to terminate seizures and prevent irreversible brain damage. It also is possible that some victims may undergo silent seizures, i.e. without convulsions (DeLorenzo, 1998). For these victims, treatment would almost certainly be delayed beyond the therapeutic window. If treatment is delayed, those soldiers who experience seizures and survive are more likely to develop irreversible brain damage and less likely to return to duty. In light of the above considerations, there is a clear need for drugs that will prevent or minimize brain damage when administered one or more hours after nerve agent exposure.
NEUROLOGICAL MECHANISMS OF INJURY
Nerve agent-induced SRBD is the result of a complex, multi-phasic response of individual neurons to numerous extra- and intracellular events. Following inhibition of AChE and accumulation of ACh at cholinergic synapses, seizures are triggered by hyperstimulation of cholinergic receptors on postsynaptic membranes (Lallement, 1992; McDonough, 1993; Tonduli, 1999). Subsequently, there is recruitment of, and excessive stimulation by, the glutamatergic neurotransmitter system. Glutamate is the major excitatory neurotransmitter in the brain and is responsible for sustaining soman-induced seizures and promoting the development of SE (Olney, 1983; Wade, 1987; Braitman, 1989; Sparenborg, 1992; Fosbraey, 1990; Solberg, 1997). Excessive stimulation of glutamate ionotropic receptors (see below) causes large pathological elevations in the concentrations of intracellular sodium and especially calcium, and prolonged depolarization of postsynaptic membranes. This initiates a deleterious cascade of pathological processes, most of which center around a prolonged increase in intracellular free calcium or delayed calcium overload, and leads to excitotoxic cell death (Olney, 1983; Choi, 1988; Shih, 1993; Solberg, 1997). [Figure 1] provides a simplified overview of this cascade.
A transient elevation in intracellular free calcium is a ubiquitous signalling mechanism and regulator of intracellular processes ranging from cell growth and metabolism to cell death (Verkhratsky, 2003; Parekh, 2003; Carafoli, 2002). Increased cytosolic free calcium is also a critical neuronal mediator of learning and memory (Bliss, 1993). However, when normal homeostatic control of intracellular calcium is lost and a sustained elevation occurs, this delayed calcium overload triggers neuronal pathology by necrosis or apoptosis (a form of programmed cell death) (Randal, 1992; Orrenius, 1994; Orrenius, 1992; Nicotera, 2003; Nicholls, 2004). In neurons, the majority of calcium influx occurs through N-methyl-D-aspartate (NMDA) ionotropic glutamate receptors as well as through voltage-gated calcium channels (e.g., L-type). Calcium influx also occurs through alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid/kainate (AMPA/KA) receptors, another class of glutamate ionotropic receptors, but to a much lesser extent (for review, see Jayakar, 2004). Excessive stimulation of NMDA receptors is the first step in glutamate excitotoxicity (Olney, 1983; Choi, 1988). Release of intracellular stores is also responsible for increased cytosolic free calcium. The endoplasmic reticulum (ER) releases calcium following binding of the second messenger, inositol triphosphate (IP3), to its ionotropic receptors. Calcium is also released from the ER via ryanodine receptors, i.e., ion-gated calcium channels that are responsive to calcium itself (Verkhratsky, 2003). The ER plays a critical role in normal calcium homeostasis. Excessive release or impaired uptake of calcium has been implicated in pathology resulting from calcium overload (Randal, 1992; Verkhratsky, 2003). Brain mitochondria are important for calcium buffering as cytosolic concentrations rise, and their ability to sequester calcium is ATP-dependent (Kulak, 2004). However, when calcium overload occurs, mitochondria undergo a permeability transition characterized by loss of mitochondrial transmembrane potential, curtailment of ATP synthesis, mitochondrial swelling, release of stored calcium, and neuronal death by necrosis (Duchen, 2000; Halestrap, 2002; Chang, 2002; Mattson, 2003).
It is widely acknowledged that the majority of soman-induced SRBD results from glutamate excitotoxicity and the delayed calcium overload that ensues (Olney, 1983; Braitman, 1989; Sparenborg, 1992; Solberg, 1997). When delayed calcium overload occurs in neurons, a pathological sequence is initiated that is characterized by activation of several potentially damaging enzymes. These include oxygenases, phospholipases, and nitric oxide synthase (NOS), which produce reactive oxygen species (ROS), i.e., superoxide radical, hydrogen peroxide, hydroxyl radical, nitric oxide, and peroxynitrite). ROS-induced neuronal injury includes direct damage to cell membranes, DNA and intracellular proteins, and it induces mitochondrial release of cytochrome c, and caspase activation (for review see Mattson, 2003). Release of cytochrome c, caspase activation, and DNA fragmentation are molecular hallmarks of apoptosis (Hou, 2002; Mattson, 2003; Nicholls, 2004). Cysteine proteases called calpains are also activated by sustained elevations in intracellular free calcium. Calpains degrade various intracellular proteins, including those of the cytoskeleton, membrane channels, and metabolic enzymes, and cause neuronal death by necrosis (Hou, 2002; Mattson, 2003; Nicholls, 2004). It should be noted that necrosis produces localized inflammation, while apoptosis is not associated with inflamation. The culmination of these events may result in cell death hours or days after the initial insult (see for example, Orrenius, 1992; Orrenius, 1994; Nicotera, 2003).
CANDIDATE NEUROPROTECTANT COMPOUNDS
Much of our research on neuroprotective agents derives from the consensus that nerve agent-induced seizures lead to the development of glutamate-mediated excitotoxicity (Olney, 1983; Choi, 1987; Braitman, 1989; Lallement, 1991; Sparenborg, 1992; Solberg, 1997; Shih, 1997; McDonough, 1997) in which delayed calcium overload is the intracellular trigger of the final sequences leading to cell death (Olney, 1983; Choi, 1987; Randal, 1992; Verkhratsky, 2003; Nicholls, 2004). Classes of drugs that have been tested for their abilities to ameliorate nerve agent-induced SRBD by specifically mitigating delayed calcium overload [Table 1] include NMDA receptor antagonists, glycosphingolipids which reduce intracellular calcium by blocking the translocation of protein kinase C (PKC) (Manev, 1993; Tubaro,1993; Otani, 2002; Monnet, 2003; Chaban, 2004); ryanodine receptor antagonists at the ER (Niebauer; 1999; Ballough, 2003; Krause, 2004); and poly(ADP-ribose) polymerase (PARP) inhibitors, that indirectly lower intracellular calcium by preventing ATP depletion. Increased ATP availability facilitates calcium efflux by the plasma membrane Ca2+ ATPase, calcium sequestration by the mitochondria, and indirectly enhances Na+/Ca2+ exchange by maintaining Na+/K+ ATPase functionality (Kulak, 2004).
There is an obvious need for a neuroprotectant drug that is capable of blocking delayed calcium overload resulting from nerve agent-induced seizures and is effective when administered 1-4 hours after exposure. Several candidate neuroprotectants have shown promise in animal models. When given in conjunction with PB, atropine methylnitrate (AMN) and 2-PAM, the non-competitive NMDA receptor antagonist dizocilpine (MK-801) was reported to reduce nerve agent-induced SRBD in the piriform cortex, amygdala, hippocampus, and thalamus (Sparenborg, 1992). These are among the most severely damaged brain regions in SRBD resulting from soman (Petras, 1981; Lemercier, 1983; McLeod, 1984; Pazdernik, 1985; Carpentier,1990; Petras, 1994; Ballough, 1995; Ballough, 1998). In the Sparenborg study, MK-801 (0.5, 1.0 or 5 mg/kg, i.p.) reduced brain damage and diminished or arrested seizures, in guinea pigs, when administered as a pretreatment 30 min prior to soman, and the effects were dose-dependent. The anticonvulsant profile of MK-801 against soman-induced seizures was definitively characterized by Shih (Shih, 1990). He showed that the anticonvulsant effect of MK-801 is four times greater than that of diazepam, but at doses of 1 mg/kg or higher, MK-801 potentiated the lethal effects of soman. Concern was raised for the use of NMDA antagonists as treatments to counter neuropathology resulting from glutamate excitotoxicity when it was reported that MK-801 induces neuronal degeneration in the posterior cingulate, retrosplenial cortices, and other corticolimbic regions (Olney, 1989; Fix, 1993). The proposed mechanism by which this occurs is disinhibition of multiple converging excitatory pathways (Corso, 1997). Specifically, excessive blockage of glutamatergic pathways leads to excessive stimulation of cholinergic function (Olney, 1991). This is supported by the findings that neurotoxicity by MK-801 is augmented when cholinergic receptors (i.e., muscarinic) are activated (Wozniak, 1998).
Memantine is an uncompetitive NMDA receptor antagonist (Bormann, 1989) that has been tested for its anticonvulsant effects against soman-induced seizures. It has been suggested that memantine's pharmacokinetics make it a safer candidate than MK-801 (Chen, 1992). McLean (McLean, 1992) reported that memantine alone (18 mg/kg, sc) blocked the onset of soman-induced seizures and was able to terminate seizures when administered 15 minutes after soman injection. Their findings, however, are not in agreement with those of Shih (Shih, 1999), who report that memantine by itself is completely ineffective as an anticonvulsant against soman-induced seizures. The latter authors pointed to a need for electroencephalographic (EEG) monitoring when determining anticonvulsant efficacy, and suggested that the former workers may have mistaken diminished convulsive behavior as evidence of reduced seizure activity. Neither study addressed possible neuroprotective effects of memantine, i.e., reduced neuropathology independent of anticonvulsant activity. Irrespective of the above discrepancies, the neuroprotective benefit of memantine in other models of excitotoxicity is widely accepted (Parsons, 1999; Lipton, 2004). For example, in a rat model of stroke, memantine, given 2 hours after the ischemic event, reduced brain damage by approximately 50% (Chen, 1998). Memantine is well tolerated and does not produce neurotoxicity at therapeutic dosages. It was recently approved by the FDA for treatment of Alzheimer's disease.
Proof of concept that neuroprotection is possible following nerve agent-induced seizures and SE, irrespective of a drug's anticonvulsant activity, has been demonstrated using a nonpsychotropic derivative of tetrahydrocannabinol, dexanabinol (HU-211) (Filbert, 1999). HU-211 has been reported to inhibit NMDA receptors, act as an antioxidant and free radical scavenger, suppress nitrous oxide (NO) and tumor necrosis factor-α (TNF-α) generation and stabilize calcium levels (Shohami, 1993; Biegon, 1995; Lavie, 2001). HU-211 is generally well tolerated in humans ( Darlington, 2003). When HU-211 (25 mg/kg, i.p.) was administered 5 minutes after onset of soman-induced seizures i.e., in conjunction with HI-6 and AMN pre- and post-treatment, respectively, temporal lobe lesion volume/necrosis (assessed at 28 hours after seizure onset) was reduced by 86%, compared with unprotected soman-positive controls [Figure 2]. Importantly, HU-211 had no effect on the strength or duration of seizure activity, as determined by quantitative EEG analysis. Significant neuroprotection was also observed when HU-211 administration was delayed 40 minutes after seizure onset. Neuroprotection by HU-211 was most evident in the piriform cortex and contiguous temporal lobe structures, e.g., amygdala, entorhinal, and perirhinal cortices, but did not extend to the thalamus. Co-administration of HU-211 and diazepam at 40 minutes after seizure onset did not augment the neuroprotection obtained with diazepam alone (data not shown). As regards the mechanisms of neuroprotection by these two drugs, it is important to differentiate between protection obtained by anticonvulsant effects vs. that produced by interfering with delayed calcium overload. In the above studies, HU-211 was protective despite the continued presence of undiminished seizures and SE, while diazepam attenuated (without stopping) seizure intensity and thereby reduced the initial insult. The anticonvulsant action of diazepam, via agonistic modulation of GABA A receptors, is well known and does not require elaboration. These mechanisms are non-overlapping and neuroprotective effects should be additive or synergistic.
Of the NMDA receptor antagonists, the most promising neuroprotectant candidate to date is gacyclidine (GK-11). When GK-11 (0.01 - 0.1 mg/kg, i.v.) was given to rats 10 minutes after soman exposure (in conjunction with PB pretreatment, and AS, 2-PAM and diazepam post-treatments, 1 minute after soman injections), it completely blocked SRBD when assessed 3 weeks after exposure (Lallement, 1997). In a more realistic battlefield scenario, GK-11 was administered 45 minutes after an 8-LD50 soman exposure in nonhuman primates. Animals also received PB pretreatment, followed by AS, 2-PAM, and diazepam post-treatments (one minute after soman exposure) equivalent to a single autoinjector of each in man. When brain pathology was assessed 3 weeks after exposure, all three GK-11-treated primates showed little or no evidence of pathology in the frontal and entorhinal cortices, amygdala, caudate nucleus, hippocampus, thalamus, midbrain, pons, medulla, and cerebellum, compared with the only surviving soman-treated animal (1 of 3) that received AS, 2-PAM and diazepam but not GK-11 (Lallement, 1998). In a study that approximates casualty management following a terrorist attack, soman-intoxicated primates (2.0 LD50) did not receive PB pretreatment and received delayed AS, 2-PAM, and diazepam treatments (one man-equivalent of each, see above) 30 minutes post-exposure; this was followed by GK-11 (0.1 mg/kg, i.v.). In this study, the addition of GK-11 restored normal EEG activity and completely prevented neuropathology (assessed 5 weeks after exposure), compared with subjects that received AS/2-PAM/diazepam alone (Lallement, 1999). GK-11 has a binding affinity for NMDA receptors that is only one-tenth that of MK-801. In addition, it binds to non-NMDA receptors when interaction with NMDA receptors is prevented. For these reasons, it is considered substantially less neurotoxic than MK-801 (Hirbec, 2001). It is currently being evaluated in human clinical trials for a different neuroprotective indication (Hirbec, 2001; Lepeintre, 2004).
In an effort to avoid neurotoxicity associated with NMDA receptor antagonism and mitigate delayed calcium overload that has already become established, drugs that target events subsequent to calcium overload have been tested against soman-induced SRBD. Intracerebroventricular (i.c.v.) infusion in rats of GM1 monosialoganglioside (5 mg/kg/day, for 5 days prior to and 27 hours after soman exposure), in rats, markedly reduced cross-sectional areas of soman-induced temporal lobe necrosis, i.e., 85.9% lesion reduction in the piriform cortex and contiguous structures, compared with unprotected soman-positive controls (Ballough, 1998). In this study, all rats were pretreated with PB before soman exposure, and AMN and 2-PAM post-treatments. Considerable neuroprotection was also obtained with the water-soluble GM1 derivative WILD20. As an adjunct to HI-6 pretreatment and AMN post-treatment, WILD20 (2.5 mg/kg, ip) reduced volumetric temporal lobe necrosis by 75.2% (data not shown). Neuroprotection by these two compounds occurs although neither seizure intensity nor duration (assessed via EEG monitoring) were diminished.
The mechanism by which GM1 and WILD20 exert their neuroprotective effects involves inhibition of PKC translocation to the plasma membrane (Vaccarino, 1987;Manev, 1993; Costa, 1994; Tubaro, 1993; Otani, 2002; Monnet, 2003; Chaban, 2004). It has been shown that PKC activation and translocation enhance glutamate excitotoxicity (Wagey, 2001; Koponen, 2003). Furthermore, it has been reported that protein kinase C’s (PKC) role in the excitotoxic process is to prolong NMDA receptor activation and possibly inhibit calcium extrusion mechanisms (Manev, 1993; Costa, 1994; Zhang, 1996). In addition, WILD20 is reported to reduce inflammatory response by its inhibitory effects on specific leukocytes, i.e., neutrophils (Tubaro, 1994). Despite our promising results with GM1 and WILD20, further studies have been discontinued due to concerns of possible contamination by prions associated with bovine spongiform encephalopathy, i.e., mad cow disease (Mattei, 2002).
Recent studies indicate that PARP inhibition is neuroprotective following neuropathological insults involving excitotoxicity, e.g., cerebral ischemia and traumatic brain injury (Eliasson, 1997; Mandir, 2000; Whalen, 2000; Abdelkarim, 2001; Kamanaka, 2004; Sharma, 2004; Nakajima, 2005; Ying, 2005). PARP is an abundant nuclear enzyme that is activated by ROS-induced DNA strand breaks (reviewed in Szabo, 1998; Ying, 2005). With moderate insults, it facilitates DNA repair by utilizing cellular nicotinamide adenine dinucleotide (NAD+) to form poly (ADP-ribose). Excessive PARP activation leads to NAD+ depletion, metabolic inhibition, i.e., glycolysis block, ATP insufficiency, and cell death by necrosis (Szabo, 1998; Abdelkarim, 2001; Virag, 2002). Neurons are especially vulnerable to metabolic insufficiency resulting from PARP over-activation, since glucose is normally the only metabolic substrate and the dependency on glycolysis is exceptionally high (Ying, 2005). In excitotoxic models, over-activation of PARP is closely linked to calcium-induced NOS activation which leads to the production of NO; the detrimental effects of NO are mostly mediated through peroxynitrite that forms when NO reacts with superoxide (Szabo, 1998; Park, 2004; Wang, 2004).
In 1999, Meier et al., reported reduced lesion volumes and increased survival in soman-exposed rats that received the PARP inhibitor benzamide (Meier, 1999). Further investigation of the neuroprotective efficacy of PARP inhibition warrants consideration. Subsequent studies might do well to include several new-generation PARP inhibitors that have shown increased efficacy, e.g., ONO-1924H, DR2313 and FR247304 (Kamanaka., 2004; Iwashita, 2004; Nakajima, 2005).
Another drug that has shown neuroprotective efficacy against soman-induced SRBD is dantrolene (Ballough, 2003). Dantrolene is a ryanodine receptor antagonist that prevents the release of calcium from the ER and is FDA-approved for use in malignant hyperthermia. While some neuroprotection is produced by diazepam alone ( 20 mg/kg, i.m., 40 min after seizure onset), this protection is significantly augmented in the dorsal and lateral cortices of rats by co-administration of 10 mg/kg, i.v., dantrolene (Ballough, 2003). Insolubility problems associated with dantrolene makes administraton of a full dosage in a single injection difficult. To overcome solubility problems and achieve the desired 10 mg/kg dantrolene dosage, 4 separate i.v. injections were performed between 40 minutes and 8 hours after seizure onset, with a total injection volume approximating 1 mL per rat. A unique formulation of dantrolene (Lyotropic Therapeutics, Inc.) as a nano-crystal dispersion has been utilized to obviate solubility problems. With this formulation, it is possible to administer a much higher dosage of dantrolene in a much lower injection volume. This is critical in as much as the concentration of dantrolene reaching the brain is lowered by liver enzymes when dantrolene is administered by i.p. injection. The nano-crystal formulation of dantrolene minimizes the effects of the liver enzymes.
The results with the nano crystal formulation of dantrolene corroborated and extended those of the previous study. Where the former study was unable to demonstrate significant protection in the piriform cortex.i.e., the most severely damaged region, 40 mg/kg, i.p. dantrolene (nano-crystal dispersion) plus diazepam (20 mg/kg, i.m.) reduced piriform cortical necrosis by an additional 15.6% more than that seen with diazepam alone (unpublished). In these experiments, all soman-exposed rats also received HI-6 (125 mg/kg, i.p., 30 min prior to soman) and AMN (2 mg/kg, i.m., < 1 min following soman) to protect against the peripheral effects of soman and ensure survival. Neuroprotection by dantrolene in the above experiments occurred without changes in seizure intensity or duration, i.e., dantrolene produced no discernable effects on the electrocorticographic (ECoG) profiles of soman-exposed rats. These findings are consistent with those of Niebauer and Gruenthal (1999) who examined the effects of dantrolene on hippocampal neuronal damage associated with 140 minutes of limbic SE in the rat. Administration of dantrolene within 15 minutes after onset of SE is associated with a significant reduction in the amount of neuronal injury in all hippocampal subregions. When dantrolene administration was delayed until 140 minutes after onset of SE, protection was seen in the CA3 pyramidal cell subregion only (Niebauer and Gruenthal, 1999).
CONCLUSION
Ideally, there is a need for an FDA-approved adjunct therapy that is safe to use on victims in a far-forward situation and those for whom the therapeutic window of opportunity to arrest seizures has passed (i.e., > 40 minutes following nerve agent exposure). Therapy should improve neuronal survival and neurologic functioning when administered with standard treatments and should have demonstrated efficacy when given one or more hours after nerve agent exposure. Because of the many pathways leading to irreversible neuronal cell death in nerve agent-induced excitotoxicity, a "cocktail" mixture may be necessary. The present report identifies several neuroprotective strategies with proven efficacy in the alleviation of brain damage resulting from soman-induced seizures and status epilepticus. The mechanisms of action converge on the central theme of blocking the excitotoxic cascade and resultant delayed calcium overload. Of the neuroprotectant drugs discussed, two have received FDA approval for other indications, i.e., dantrolene and memantine, while several others are in clinical trials.
ACKNOWLEDGEMENTS
For their invaluable efforts in various aspects of our experiments using GM1 ganglioside, HU-211 and dantrolene, the authors wish to express sincere gratitude to the following workers. Special thanks are extended to Jeffry Forster, Michael Jaworski, Joseph Jaworski, Blair Hontz and Denise Fath for their expert technical assistance. In addition, the authors wish to extend thanks to LTC C. Dahlem Smith, Dr. Larry Mitcheltree and MAJ J. Scot Estep for their expert pathological evaluations of H&E stained brain sections. For his efforts on technical report from which this review was developed, we extend our thanks to Dr. Alan Feister (SAIC, Frederick, Maryland). The provisions of GM1 ganglioside by FIDIA Labs (Terme, Italy), HU-211 by Pharmos Ltd. (Rehovot, Israel) and specially formulated dantrolene by Lyotropic Therapeutics, Inc. (Ashland, Virginia) are gratefully acknowledged.
In conducting the research described in this report, the investigators adhered to the Guide for the Care and Use of Laboratory Animals by the Institute of Laboratory Animal Resources, National Research Council, in accordance with the stipulations mandated for an AAALAC accredited facility.
The opinions and assertions contained herein are the private views of the authors and are not to be considered as official or as reflecting the views of the Department of the Army or the Department of Defense.
tables
Table 1: Potential Neuroprotective Compounds [go to text reference]
| Drug Class | Examples | Reference |
| NMDA antagonists | MK-801 | Shih, 1990; Sparenborg, 1992. |
| Memantine | McLean, 1992; Koplovitz, 1997; Parsons, 1999; Shih, 1999; Lipton, 2004. | |
| Dexanabinol (HU-211) | Biegon, 1995;Filbert, 1999. | |
| GK-11 Ketamine |
Lallement, 1997; Lallement, 1999; Lepeintre, 2004. | |
| PKC inhibitors | GM1 ganglioside | Manev, 1990; Ballough, 1998. |
| Wild 20 | Tubaro, 1993; present report. | |
| PARP Inhibitors | Benzamide | Meier, 1999; Kamanaka, 2004; Nakajima, 2005. |
| Ryanodine receptor antagonists | Dantrolene | Niebauer, 1999; Ballough, 2003. |
figures
Figure 1. Proposed pathway of neuronal injury in nerve agent-induced brain damage. [go to text reference]
Various sites in this pathway have been targeted. MK-801, memantine, HU-211, GK-11 and ketamine are NMDA receptor antagonists. HU-211 also blocks free radical induced injury. GM1 ganglioside and Wild20 indirectly promote calcium extrusion by blocking PKC translocation (not indicated). Benzamide inhibits PAPR thereby increasing ATP availability. Dantrolene blocks calcium release from intracellular stores.
Figure 2. HU-211 protects against soman-induced neurological damage. [go to text reference]
Microtubule-associated protein 2 (MAP2) staining is neuron-specific. MAP2-negative immunostaining indicates necrosis, except in areas of white matter. Den, dentate gyrus; Pir, piriform cortex; BL, basolateral amygdaloid nuclear group.
references
Abdelkarim GE, Gertz K, Harms C, Katchanov J, Dirnagl U, Szabo C, Endres M. Protective effects of PJ34, a novel, potent inhibitor of poly(ADP-ribose) polymerase (PARP) in in vitro and in vivo models of stroke. Int J Mol Med. 2001; 7:255-60 .
Ballough GP, Martin LJ, Cann FJ, Graham JS, Smith CD, Kling CE, Forster JS, Phann S, Filbert MG. Microtubule-associated protein 2 (MAP-2): A sensitive marker of seizure-related brain damage. J Neurosci Methods. 1995; 61:23-32.
Ballough GPH, Filbert MG. A viable neuroprotection strategy following soman-induced status epilepticus. USAMRICD-TR-03-09, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 2003.
Ballough, GPH, Cann FJ, Smith CD, Forster JS, Kling CE, Filbert MG. GM1 monosialoganglioside pretreatment protects against soman-induced seizure-related brain damage. Molec Chem Neuropathol . 1998; 34:1-23.
Baze WB. Soman-induced morphological changes: an overview in the non-human primate. J Appl Toxicol. 1993; 13:173-7.
Biegon A, Joseph AB. Development of HU-211 as a neuroprotectant for ischemic brain damage. Neurol Res . 1995; 17:275-280.
Bliss TVP, Collingridge GL. A synaptic model of memory: Long-term potentiation in the hippocampus. Nature. 1993; 361:31-39.
Bormann J. Memantine is a potent blocker of N-methyl-D-aspartate (NMDA) receptor channels. Eur J Pharmacol. 1989; 166(3):591-2.
Braitman DJ, Sparenborg S. MK-801 protects against seizures induced by the cholinesterase inhibitor soman. Brain Res Bul. 1989; 23:145-148.
Capacio BR, Shih TM. Anticonvulsant actions of anticholinergic drugs in soman poisoning. Epilepsia. 1991; 32(5), 604-615.
Carafoli E. Calcium signaling: A tale for all seasons. Proc Natl Acad Sci USA. 2002; 99:1115-1122.
Carpentier P, Delamanche IS, Le Bert M, Blanchet G, Bouchaud C. Seizure-related opening of the blood-brain barrier induced by soman: Possible correlation with the acute neuropathology observed in poisoned rats. Neurotoxicology. 1990; 11(3):493-508.
Chaban VV, Li J, Ennes HS, Nie J, Mayer EA, McRoberts JA. N-methyl-D-aspartate receptors enhance mechanical responses and voltage-dependent Ca2+ channels in rat dorsal root ganglia neurons through protein kinase C. Neuroscience. 2004; 128:347-57.
Chang LK, Putcha GV, Deshmukh M, Johnson EM Jr. Mitochondrial involvement in the point of no return in neuronal apoptosis. Biochimie. 2002; 84:223-231.
Chen HS, Pellegrini JW, Aggarwal SK, Lei SZ, Warach S, Jensen FE, Lipton SA. Open-channel block of N-methyl-D-aspartate (NMDA) responses by memantine: Therapeutic advantage against NMDA receptor-mediated neurotoxicity. J Neurosci. 1992; 12:4427-36.
Chen HS, Wang YF, Rayudu PV, Edgecomb P, Neill JC, Segal MM, Lipton SA, Jensen FE. Neuroprotective concentrations of the N-methyl-D-aspartate open-channel blocker memantine are effective without cytoplasmic vacuolation following post-ischemic administration and do not block maze learning or long-term potentiation. Neuroscience. 1998; 86:1121-32.
Choi DW. Ionic dependence of glutamate neurotoxicity. J Neurosci. 1987; 7:369-379.
Choi DW. Calcium-mediated neurotoxicity: relationship to specific channel types and role in ischemic damage. Trends Neurosci . 1988; 11:465-469.
Clement JG, Broxup B. Efficacy of diazepam and avizafone against soman-induced neuropathology in brain of rats. Neurotoxicology. 1993;14:485-504.
Corso TD, Sesma MA, Tenkova TI, Der TC, Wozniak DF, Farber NB, Olney JW. Multifocal brain damage induced by phencyclidine is augmented by pilocarpine. Brain Res. 1997; 752:1-14.
Costa E, Armstrong DM, Guidotti A, Kharlamov A, Kiedrowski L, Manev H, Polo A, Wroblewski JT. Gangliosides in the protection against glutamate excitotoxicity. Prog Brain Res. 1994; 101:357-73.
Darlington CL. Dexanabinol: A novel cannabinoid with neuroprotective properties. IDrugs. 2003; 6:976-9.
DeLorenzo RJ, Waterhouse EJ, Towne AR, Boggs JG, Ko D, DeLorenzo GA,
Brown A, Garnett L. Persistent nonconvulsive status epilepticus after the control of convulsive status epilepticus. Epilepsia. 1998; 39:833-40.
Duchen MR. Mitochondria and calcium: From cell signaling to cell death: topical review. J Physiology. 2000; 529:57-68.
Eliasson MJ, Sampei K, Mandir AS, Hurn PD, Traystman RJ, Bao J, Pieper A, Wang ZQ, Dawson TM, Snyder SH, Dawson VL. Poly(ADP-ribose) polymerase gene disruption renders mice resistant to cerebral ischemia. Nat Med. 1997; 3:1089-95.
Fanzone JF, Levine ES, Hursh SR. Nerve agent bioscavenger pretreatment against chemical warfare agents: Challenge, casualty, and intervention modeling support. Interim Report. Prepared under U.S. Army Medical Research and Materiel Command Contract No. DAMD17-98-D-0022; 2002.
Filbert MG, Forster JS, Smith CD, Ballough GPH. Neuroprotective effects of HU-211 on brain damage resulting from soman-induced seizures. Ann NY Acad Sci . 1999; 890:505-514.
Fix AS, Horn JW, Wightman KA, Johnson CA, Long GG, Storts RW, Farber N, Wozniak DF, Olney JW. Neuronal vacuolization and necrosis induced by the noncompetitive N-methyl-D-aspartate (NMDA) antagonist MK(+)801 (dizocilpine maleate): A light and electron microscopic evaluation of the rat retrosplenial cortex. Exp Neurol. 1993 Oct; 123:204-15.
Fosbraey P, Wetherell JR, French MC. Neurotransmitter changes in guinea-pig brain regions following soman intoxication. J Neurochem . 1990; 54:72-79.
Gilland E, Bona E, Hagnberg H. Temporal changes of regional glucose use, blood flow and microtubule-associated protein 2 immunostaining after hypoxia ischemia in the immature rat brain. J. Cereb.Blood Flow and Metab. 1998: 18:222-28.
Halestrap AP, McStay GP, Clarke SJ. The permeability transition pore complex: Another view. Biochimie. 2002; 84:153-166.
Harris LW, Gennings C, Carter WH, Anderson DR, Lennox WJ, Bowersox SL, Solana RP. Efficacy comparison of scopolamine (SCP) and diazepam (DZ) against soman-induced lethality in guinea pigs. Drug Chem Toxicol. 1994; 17:35-50.
Hayward IJ, Wall HG, Jaax NK, Wade JV, Marlow DD, Nold JB. Decreased brain pathology in organophosphate-exposed rhesus monkeys following benzodiazepine therapy. J Neurol Sci . 1990; 98:99-106.
Hicks RR, Smith DH, MacIntosh TK. Temporal response and effects of excitatory amino acid antagonism on microtubule-associated protein 2 immunoreactivity following experimental brain injury in rats. Brain Res 1995; 678:151-160.
Hirbec H, Gaviria M, Vignon J. Gacyclidine: A new neuroprotective agent acting at the N-methyl-D-aspartate receptor. CNS Drug Rev. 2001; 7:172-98.
Hou ST, MacManus JP. Molecular mechanisms of cerebral ischemic-induced neuronal death. Intern Rev Cytol. 2002; 221:93-149.
Iijima T, Shimase C, Sawa H, Sankawa H. Spreading depressions induces depletion of MAP 2 in area CA3 of the hippocampus in a rat unilateral carotid artery occlusion model. J Neurotrauma 1998; 15:277-84.
Iwashita A, Tojo N, Matsuura S, Yamazaki S, Kamijo K, Ishida J, Yamamoto H, Hattori K, Matsuoka N, Mutoh S. A novel and potent poly(ADP-ribose) polymerase-1 inhibitor, FR247304 (5-chloro-2-[3-(4-phenyl-3,6-dihydro-1(2H)-pyridinyl)propyl]-4(3H)-quinazolinone), attenuates neuronal damage in in vitro and in vivo models of cerebral ischemia. J Pharmacol Exp Ther. 2004; 310:425-36.
Jayakar SS, Dikshit M. AMPA receptor regulation mechanisms: Future target for safer neuroprotective drugs. Int J Neurosci. 2004; 114:695-734.
Kamanaka Y, Kondo K, Ikeda Y, Kamoshima W, Kitajima T, Suzuki Y, Nakamura Y, Umemura K. Neuroprotective effects of ONO-1924H, an inhibitor of poly ADP-ribose polymerase (PARP), on cytotoxicity of PC12 cells and ischemic cerebral damage. Life Sci. 2004; 76:151-62.
Kitagawa K, Matsu;moto M, Ninobe M, Mikoshiba K, Hata R, Ueda H, Handa N, Fukunaga R, Isaka Y, Kimura K, Kamada T. Neurosci 1989; 31:401-411.
Koplovitz I, Schulz S, Shutz M, Railer,R, Smith F, Okerberg C, Filbert M.. Memantine
effects on soman-induced seizures and seizure-related brain damage. Toxicology
Methods 1997; 7:227-239.
Koponen S, Kurkinen K, Akerman KE, Mochly-Rosen D, Chan PH, Koistinaho J. Prevention of NMDA-induced death of cortical neurons by inhibition of protein kinase Czeta. J Neurochem. 2003; 86:442-50.
Krause T, Gerbershagen MU, Fiege M, Weisshorn R, Wappler F. Dantrolene: A review of its pharmacology, therapeutic use, and new developments. Anaesthesia. 2004; 59:364-73.
Krinke GJ, Classen W, Vidotto N, Suter G, Wurmlin CH. Detecting necrotic neurons with fluoro-jade stain. Exp Toxicol Pathol 2001; 53:365-372.
Kulak W, Sobaniec W, Wojtal K, Czuczwar J. Calcium modulation in epilepsy: Review. Polish J Pharmacol. 2004; 56:29-41.
Lallement G, Carpentier P, Collet A, Baubichon D, Pernot-Marino I, Blanchet G. Extracellular acetylcholine changes in rat limbic structures during soman-induced seizures. Neurotoxicology. 1992; 13:557-67.
Lallement G, Carpentier P, Collet A, Pernot-Marino I, Baubichon D, Blanchet G. Effects of soman-induced seizures on different extracellular amino acid levels and on glutamate uptake in rat hippocampus. Brain Res . 1991; 563:234-240.
Lallement G, Clarencon D, Galonnier M, Baubichon D, Burckhart MF, Peoc'h M. Acute soman poisoning in primates neither pretreated nor receiving immediate therapy: Value of gacyclidine (GK-11) in delayed medical support. Arch Toxicol. 1999; 73:115-22.
Lallement G, Clarencon D, Masqueliez C, Baubichon D, Galonnier M, Burckhart MF, Peoc'h M, Mestries JC. Nerve agent poisoning in primates: Antilethal, anti-epileptic, and neuroprotective effects of GK-11. Arch Toxicol. 1998; 72:84-92.
Lallement G, Mestries JC, Privat A, Brochier G, Baubichon D, Carpentier P, Kamenka JM, Sentenac-Roumanou H, Burckhart MF, Peoc'h M. GK 11: Promising additional neuroprotective therapy for organophosphate poisoning. Neurotoxicology. 1997; 18:851-6.
Lallement G, Renault F, Baubichon D, Peoc'h M, Burckhart MF, Galonnier M, Clarencon D, Jourdil N. Compared efficacy of diazepam or avizafone to prevent soman-induced electroencephalographic disturbances and neuropathology in primates: Relationship to plasmatic benzodiazepine pharmacokinetics. Arch Toxicol . 2000; 74:480-486.
Lavie G, Teichner A, Shohami E, Ovadia H, Leker RR. Long term cerebroprotective effects of dexanabinol in a model of focal cerebral ischemia. Brain Res . 2001; 901:195-201.
Lemercier G, Carpentier P, Sentenac-Roumanou H, Morelis P. Histological and histochemical changes in the central nervous system of the rat poisoned by an irreversible anticholinesterase organophosphorus compound. Acta Neuropathol. 1983; 61:123-9.
Lepeintre JF, D'Arbigny P, Mathe JF, Vigue B, Loubert G, Delcour J, Kempf C, Tadie M. Neuroprotective effect of gacyclidine. A multicenter double-blind pilot trial in patients with acute traumatic brain injury. Neurochirurgie. 2004; 50:83-95.
Lipp JA. Effect of benzodiazepine derivative on soman-induced seizure activity and convulsions in the monkey. Arch Int Pharmacodyn Ther. 1973; 202:244-251.
Lipp JA. Effect of diazepam upon soman-induced seizure activity and convulsions. Electroenceph Clin Neurophysiol. 1972; 32:557-560.
Lipton SA. Excitotoxicity. In: M. Bahr (Ed.), Neuroprotection: Models, Mechanisms and Therapies, 2004, pp. 304. Weinheim: WILEY-VCH Verlag GmbH & Co. KGaA.
Mandir AS, Poitras MF, Berliner AR, Herring WJ, Guastella DB, Feldman A, Poirier GG, Wang ZQ, Dawson TM, Dawson VL. NMDA but not non-NMDA excitotoxicity is mediated by poly(ADP-ribose) polymerase. J Neurosci. 2000; 20:8005-11.
Manev H, Favaron M, ViciniS, and Guidotti A. Ganglioside-mediated protection from glutamate-induced neuronal death. Acta Neurobiol Exp 1990; 50:475-488.
Manev H, Guidotti A, Costa E. Protection by gangliosides against glutamate excitotoxicity. Advan. Lipid Res. 1993; 25:269-288.
Mattei V, Garofalo T, Misasi R, Gizzi C, Mascellino MT, Dolo V, Pontieri GM, Sorice M, Pavan A. Association of cellular prion protein with gangliosides in plasma membrane microdomains of neural and lymphocytic cells. Neurochem Res. 2002; 27:743-9.
Mattson MP. Excitotoxic and excitoprotective mechanisms: Review article. Neuromol Med. 2003; 3:65-94.
McDonough JH Jr, Dochterman LW, Smith CD, Shih TM. Protection against nerve agent-induced neuropathology, but not cardiac pathology, is associated with the anticonvulsant action of drug treatment. Neurotoxicology. 1995; 16:123-32.
McDonough JH Jr, Jaax NK, Crowlety RA, Mays M, Modrow HE. Atropine and/or diazepam therapy protects against soman-induced neural and cardiac pathology. Fundam Appl Toxicol . 1989; 13:256-276.
McDonough JH, Shih T-M. Neuropharmacological mechanisms of nerve agent-induced seizure and neuropathology. Neurosci Biobehav Rev. 1997; 21:559-79.
McDonough JH, Shih T-M . Pharmacological modulation of soman-induced seizures. Neurosci Biobehav Rev . 1993; 17:203-215.
McDonough JH, Zoeffel LD, McMonagle J, Copeland TL, Smith CD, Shih T-M. Anticonvulsant treatment of nerve agent seizures: Anticholinergic versus diazepam in soman-intoxicated guinea pigs. Epilepsy Res . 2000; 38:1-14.
McLean MJ, Gupta RC, Dettbarn WD, Wamil AW. Prophylactic and therapeutic efficacy of memantine against seizures produced by soman in the rat. Toxicol Appl Pharmacol. 1992; 112:95-103.
McLeod CG Jr, Singer AW, Harrington DG. Acute neuropathology in soman poisoned rats. Neurotoxicol. 1984; 5:53-7.
Meier HI, Ballough GPH, Forster JS, Filbert MG. Benzamide, a poly(ADP-ribose) polymerase inhibitor, is neuroprotective against soman-induced seizure-related brain damage. Ann NY Acad Sci . 1999; 890:330-335.
Monnet FP, Morin-Surun MP, Leger J, Combettes L. Protein kinase C-dependent potentiation of intracellular calcium influx by sigma1 receptor agonists in rat hippocampal neurons. J Pharmacol Exp Ther. 2003; 307:705-12.
Nakajima H, Kakui N, Ohkuma K, Ishikawa M, Hasegawa T. A newly synthesized poly(ADP-ribose) polymerase inhibitor, DR2313 [2-methyl-3,5,7,8-tetrahydrothiopyrano[4,3-d]-pyrimidine-4-one]: Pharmacological profiles, neuroprotective effects, and therapeutic time window in cerebral ischemia in rats.
J Pharmacol Exp Ther. 2005; 312:472-81.
Nicholls DG. Mitochondrial dysfunction and glutamate excitotoxicity studied in primary neuronal cultures. Cur Molec Med. 2004; 4:149-177.
Nicotera P. Molecular switches deciding the death of injured neurons. Toxicol Sci . 2003; 74:4-9.
Niebauer M, Gruenthal M. Neuroprotective effects of early vs. late administration of dantrolene in experimental status epilepticus. Neuropharm . 1999; 38:1343-1348.
Olney JW, De Gubareff T, Labruyere J. Seizure-related brain damage induced by cholinergic agents. Nature . 1983; 301:520-522.
Olney JW, Labruyere J, Price MT. Pathological changes induced in cerebrocortical neurons by phencyclidine and related drugs. Science. 1989; 244:1360-2.
Olney JW, Labruyere J, Wang G, Wozniak DF, Price MT, Sesma MA. NMDA antagonist neurotoxicity: Mechanism and prevention. Science. 1991; 254:1515-8.
Orrenius S, Burkitt MJ, Kass GE, Dypbukt JM, Nicotera P. Calcium ions and oxidative cell injury. Ann Neurol . 1992; 32:S33-42.
Orrenius S, Nicotera P. The calcium ion and cell death. J Neural Transm Suppl . 1994; 43:1-11.
Otani S, Daniel H, Takita M, Crepel F. Long-term depression induced by postsynaptic group II metabotropic glutamate receptors linked to phospholipase C and intracellular calcium rises in rat prefrontal cortex. J Neurosci. 2002; 22:3434-44.
Parekh AB. Store-operated Ca2+ entry: Dynamic interplay between endoplasmic reticulum, mitochondria and plasma membrane. J Physiol. 2003; 547(Pt 2):333-48.
Park EM, Cho S, Frys K, Racchumi G, Zhou P, Anrather J, Iadecola C. Interaction between inducible nitric oxide synthase and poly(ADP-ribose) polymerase in focal ischemic brain injury. Stroke. 2004; 35:2896-901.
Parsons CG, Danysz W, Quack G. Memantine is a clinically well tolerated N-methyl-D-aspartate (NMDA) receptor antagonist--a review of preclinical data. Neuropharmacology. 1999;38(6):735-67.
Pazdernik TL, Cross R, Giesler M, Nelson S, Samson F, McDonough J Jr. Delayed effects of soman: Brain glucose use and pathology. Neurotoxicol. 1985; 6:61-70.
Petras JM. Neurology and neuropathology of soman-induced brain injury: An overview. J Exp Anal Behav. 1994; 61:319-29.
Petras JM. Soman neurotoxicity. Fundam Appl Toxicol. 1981;1:242.
Philippens IH, Melchers BP, de Groot DM, Wolthuis OL. Behavioral performance, brain histology, and EEG sequela after immediate combined atropine/diazepam treatment of soman-intoxicated rats. Pharmacol Biochem Behav . 1992; 42:711-719.
Randall RD, Thayer SA. Glutamate-induced calcium transient triggers delayed calcium overload and neurotoxicity in rat hippocampal neurons. J Neurosci .1992; 12:1882-1895.
Sharma SS, Munusamy S, Thiyagarajan M, Kaul CL. Neuroprotective effect of peroxynitrite decomposition catalyst and poly(adenosine diphosphate-ribose) polymerase inhibitor alone and in combination in rats with focal cerebral ischemia. J Neurosurg. 2004; 101:669-75 .
Shih T, McDonough JH Jr, Koplovitz I. Anticonvulsants for soman-induced seizure activity. J Biomed Sci. 1999; 6:86-96.
Shih TM, Capacio BR, Cook LA. Effects of anticholinergic-antiparkinsonian drugs on striatal neurotransmitter levels of rats intoxicated with soman. Pharmacol Biochem Behav . 1993; 4:615-622.
Shih TM, Koviak TA, Capacio BR. Anticonvulsants for poisoning by the organophosphorus compound soman: Pharmacological mechanisms. Neurosci Biobehav Rev . 1991; 15:349-362.
Shih TM, McDonough JH Jr. Neurochemical mechanisms in soman-induced seizures.
J Appl Toxicol. 1997; 17:255-64.
Shih TM. Anticonvulsant effects of diazepam and MK-801 in soman poisoning. Epilepsy Res . 1990; 7:105-116.
Shih TM (1999) Memantine? See text, page 6 (this may be a McDonough & Shih ref,
Shih, TM, Duniho SM, McDonough JH. Control of nerve agent-induced seizures is critical for neuroprotection and survival. Toxicol Appl Pharm. 2003; 188:69-80.
Shohami E, Novikov M, Mechoulam R. A nonpsychotropic cannabinoid, HU-211, has cerebroprotective effects after closed head injury in the rat. J Neurotrauma 1993; 10:109-119.
Solberg Y, Belkin M. The role of excitotoxicity in organophosphorous nerve agents central poisoning. Trends Pharmacol Sci. 1997; 18:183-5.
Sparenborg S, Brennecke LH, Beers ET . Pharmacological dissociation of the motor and electrical aspects of convulsive status epilepticus induced by the cholinesterase inhibitor soman. Epilepsy Res . 1993; 14:95-103.
Sparenborg S, Brennecke LH, Jaax NK, Braitman DJ. Dizocilpine (MK-801) arrests status epilepticus and prevents brain damage induced by soman. Neuropharm. 1992; 3 1:357-68.
Szabo C, Dawson VL. Role of poly(ADP-ribose) synthetase in inflammation and ischaemia-reperfusion. Trends Pharmacol Sci. 1998; 19:287-98.
Tonduli LS, Testylier G, Marino IP, Lallement G. Triggering of soman-induced seizures in rats: Multiparametric analysis with special correlation between enzymatic, neurochemical, and electrophysiological data. J Neurosci Res. 1999; 58:464-73.
Tubaro E, Croce C, Cavallo G, Belogi L, Guida G, Santiangeli C, Cifone MG, Santoni A, Mainiero F. In vitro and in vivo impact of a new glycosphingolipid on neutrophils. Agents Actions. 1994; 42:107-113.
Tubaro E, Santiangeli C, Cavallo G, Belogi L, Guida G, Croce C, Modesti A. Effect of a new de-N-acetyl-lysoglycosphingolipid on chemically-induced inflammatory bowel disease: Possible mechanism of action. Naunyn Schmiedebergs Arch Pharmacol. 1993; 348:670-8.
Vaccarino F, Guidotti A, Costa E. Ganglioside inhibition of glutamate-mediated protein kinase C translocation in primary cultures of cerebellar neurons. Proc Natl Acad Sci USA. 1987; 84:8707-11.
Verkhratsky A, Toescu EC. Endoplasmic reticulum calcium homeostasis and neuronal death. Neuroscience Review Series. J Cell Mol Med. 2003; 7:351-361.
Virag L, Szabo C. The therapeutic potential of poly(ADP-ribose) polymerase inhibitors. Pharmacol Rev. 2002; 54:375-429.
Wade JV, Samson FE, Nelson SR, Pazdernik TL. Changes in extracellular amino acids during soman- and kainic acid-induced seizures. J Neurochem . 1987; 49:645-650.
Wagey R, Hu J, Pelech SL, Raymond LA, Krieger C. Modulation of NMDA-mediated excitotoxicity by protein kinase C. J Neurochem. 2001; 78:715-26.
Wang H, Yu SW, Koh DW, Lew J, Coombs C, Bowers W, Federoff HJ, Poirier GG, Dawson TM, Dawson VL. Apoptosis-inducing factor substitutes for caspase executioners in NMDA-triggered excitotoxic neuronal death. J Neurosci. 2004; 24:10963-73.
Watson RE, Wiegand AJ, Clough RW, Hoffman GE. Use of cryoprotectant to maintain long-term peptide immunoreactivity and tissue morphology. Peptides 1986; 7:155-59.
Whalen MJ, Clark RS, Dixon CE, Robichaud P, Marion DW, Vagni V, Graham S, Virag L, Hasko G, Stachlewitz R, Szabo C, Kochanek PM. Traumatic brain injury in mice deficient in poly-ADP(ribose) polymerase: A preliminary report. Acta Neurochir Suppl. 2000; 76:61-4.
Wozniak DF, Dikranian K, Ishimaru MJ, Nardi A, Corso TD, Tenkova T, Olney JW, Fix AS. Disseminated corticolimbic neuronal degeneration induced in rat brain by MK-801:Potential relevance to Alzheimer's disease. Neurobiol Dis. 1998; 5:305-22.
Ying W, Alano CC, Garnier P, Swanson RA. NAD+ as a metabolic link between DNA damage and cell death. J Neurosci Res. 2005; 79:216-23.
Zhang L, Rzigalinski BA, Ellis EF, Satin LS. Reduction of voltage-dependent Mg2+ blockade of NMDA current in mechanically injured neurons. Science. 1996; 274:1921-1923.